Blood system

Embryology

Blood is connective tissue composed of a liquid medium called plasma in which

solid components are suspended.
 Blood cells are produced by a process called “Hemopoiesis”

Hematopoiesis: is the formation of blood process; before and after birth,

which mature blood cells develop from precursor cells.

 It continues continuously throughout embryonic and adult life and as a

result new cells formed in the so-called hematopoietic regions constantly
replace mature blood cells in the circulation.

Sites of Hematopoiesis:
In the embryo Hemopoiesis occurs at different stages in the yolk sac, the
liver, the spleen, lymph nodes and the bone marrow.
Erythrocytes,
granulocytes, bone marrow
In the adult monocytes and platelets
In the lymph nodes, spleen, thymus
the lymphocytes and lymphatic nodules of the
gastrointestinal tract.
Embryo hematopoiesis:
1) In developing embryos, blood formation occurs in aggregates of blood cells in
the yolk sac, called blood islands.
2) As development progresses, blood formation occurs in the spleen, liver, and
lymph nodes.
3) When bone marrow develops, it eventually assumes the task of forming most
of the blood cells for the entire organism.

 Blood cell development begins as early as the seventh day of embryonic life.
Red blood cells are essential in delivering oxygen to tissues and the
development of vascular channels during embryogenesis.

The ontogeny and maturation of these blood cell:

1. The production of primitive erythroid cells (EryP).
2. An expanding population of definitive erythroid cells (EryD) that predominate
subsequently.
Failure of primitive erythropoiesis is fatal to the embryo.
Development
The process of gastrulation begins with the epiblast, a single epithelial cell layer,
transforming into three embryonic germ layers (ectoderm, mesoderm, and
endoderm).
The development occurs in two waves:

First waves:
 Conversion of primitive haematopoiesis into definitive haematopoiesis.
The first wave of primitive hematopoietic and endothelial cell development occurs
via signals to the extraembryonic, endodermal yolk sac within the first two weeks
of gestation, which results primarily in the formation of:
• Primitive erythroid cells (Eryp).
• Megakaryocytes.
• Macrophages.
• Endothelium.

These EryP cells are distinct from their erythroid progenitors in that they are:
1. Larger.
2. Nucleated.
3. Have embryonic globins.
4. Are detected only in the yolk sac.
 EryP help in the formation of structures called blood islands in which the
centrally placed cells give rise to erythroid and myeloid cells while peripherally
placed cells form endothelial cells that form these channels.
 These blood islands fuse to form vascular channels that span throughout the
yolk sac.

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 Through these vascular channels, gradually plasma flows containing EryP
cells and various other primitive cell types, which is stimulated by the developing
heart.
• Once in circulation, the EryP cells are enucleated by the fetal liver and
macrophages clear the nuclei.
 EryP cells continue to form only for a short period.
• Once vascular channels develop in the yolk sac, while the remaining
progenitor cells continue to mature from proerythroblast to orthochromatic
erythroblast to reticulocytes and remain in the bloodstream until at least birth.

 Shortly after the development of primitive hematopoietic cells (EryP), a group

of cells called highly proliferative, multipotent progenitor colony forming cells
(HPP-CFC) arise in the yolk sac.
 These cells initiate the first wave of definitive hematopoiesis and produce
cells of the definitive erythroid lineage.
 These cells are often called erythroid/myeloid progenitors (the transition
between primitive and hematopoietic stem cell (HSC) derived
erythropoiesis).
 These cells will migrate and begin to colonize the liver, which is the next
definitive site of hematopoiesis during gestation.

Second wave
The second wave of definitive hematopoiesis replaces primitive hematopoiesis
and the first wave of definitive hematopoiesis.
Hematopoietic stem cells (HSC) emerge from a specialized hemogenic
endothelium within a limited region of the developing aorta's ventral wall called
the para-aortic splanchnopleure.
• The aorta-gonad-mesonephros (AGM) region develops from the para-aortic
splanchnopleure and produces HSC.
 These cells colonize the fetal liver by the 6th or 7th week of gestation, where
they cycle at a continuous pace and begin to differentiate.
 At this point, the liver becomes a significant source of hematopoietic stem
cell production.
 HSC also colonize the spleen around 10-28 weeks or 20th week and produce
red cells for a brief period.
 A vital organ that HSC starts colonizing around this time is the bone marrow.
HSC seeding (‫ )تنبت‬in the marrow is critical because it is the bone marrow that will
predominate in erythropoiesis as gestation advances (‫)تقدم‬.
 The fetal liver provides the microenvironment needed for expansion and
differentiation of definitive HSCs, from which definitive erythroid cells will
differentiate.
HSC in the fetal liver and spleen produces enucleated erythrocytes (EryD) that
rapidly outnumber (‫ )تفوق عدد‬EryP cells in circulation.

EryD cells express fetal hemoglobin (HbF)

HbF; composed of two γ-globin chains and two adult alpha-globin chains.
 HbF remains the predominant hemoglobin for most of gestation.
 A switch from the HbF to adult hemoglobin (HbA) occurs at about 32 weeks
and continues after birth.
 HbF is completely replaced with HbA in 6 months after the baby is born.
 There is a transcriptional change from gamma- to beta globin, marking the
end of erythroid ontogeny.

Some differenceses between HbF and HbA:

Hb HbF HbA
Globin chains 2 alpha chains and 2 2 alpha chains and 2
gamma chains beta chains.
Oxygen affinity More affinity for binding Less affinity for binding
oxygen oxygen
 Toward the third trimester of development, as skeletal components begin
ossification and bone marrow is developing inside bony cavities, the marrow of
specific bones will become the essential hematopoietic organ.
 Both the liver and spleen at this point cease (stopped) erythropoiesis as
the bone marrow predominates in hematopoietic cell production.
 In postnatal life, definitive erythropoiesis originates from the
marrow (BM) that occurs under normal physiologic conditions.
In All spongy bone and trabecular bone produce RBC.
infants
In RBC production is limited to the vertebra, sternum,
adults ribs, and proximal ends of long bones.

 HSCs in the BM give rise to all mature hematopoietic cells

through a series of intermediate progenitors.
Hematopoietic Growth Factor
 Red and white blood cell production is regulated with great precision in healthy
humans, and the production of granulocytes is rapidly increased during infection.
 Colony-stimulating factors (CSFs) are secreted glycoproteins that bind to
receptor proteins on the surfaces of hematopoietic stem cells, thereby
activating intracellular signaling pathways that can cause the cells to
proliferate and differentiate into a specific kind of blood cell.
Erythrocytes Erythropoietin
Thrombocytes Thrombopoietin
Granulocytes Granulocytes Colony-stimulating factor

Erythropoietin is mainly synthesized in the liver and kidneys:

Organs Adults Fetuses
Kidney ≈ 𝟗𝟎 % ↑ ≈ 𝟏𝟎 % ↓
Liver ≈ 𝟏𝟎 % ↓ ≈ 𝟗𝟎 % ↑

abbreviation term / word

EPO Erythropoietin
CSFs Colony-stimulating factors
Hb Hemoglobin
EryP Primitive erythroid
EryD Definitive erythroid
HSCs Hematopoietic stem cells
MHSCs Multipotent Hematopoietic stem cells
AGM aorta-gonad-mesonephros

7th day Blood cell development beginning

First 2 week First wave of blood cells development
6th or 7th week HSC colonize the fetal liver
10-28 week or 20th week HSC colonize the fetal spleen
After 18th week The marrow of specific bones will become the
essential hematopoietic organ.
Around 32 week switch from the HbF to adult hemoglobin (HbA)
begins
6 months (after birth) HbF is completely replaced with HbA
 The Lymphatic System

Embryology

 The lymphatic system begins to develop at the end of week 5.

 Approximately 2 weeks later than the cardiovascular system.
Theories about lymphatic system development.
1) The lymphatics develop as diverticulae of the endothelium of veins.
 This means that it came out of previous blood vessels, specifically the
veins by sprouting (‫)التبرعم‬, then later grew as an independent vessel.
2) Like other blood vessels they develop from clefts in the mesenchyme that
connect with the venous system secondarily.
 This means that it developed from the venous system, but not by
sprouting, but secondary, according to the following sequence:
Sac  cleft  channel  tubes (lymph vessels)
 Thus, the cells lining the mesenchymal clefts assume an endothelial shape,
and subsequent sprouting of these cells causes the clefts to fuse and form
the lymphatic channels.

IN WEEKS 6-9
 Local dilatations of the lymphatic channels form 6 primary lymph sacs:
Two jugular lymph sacs Pair Near the junction of the subclavian veins
with the anterior cardinals (future internal
jugular vein).
Two iliac lymph sacs Pair near the junction of the iliac veins with the
posterior cardinal veins
One retroperitoneal Single in the root of the mesentery on the posterior
abdominal wall
One cisterna chyli Single Dorsal to the retroperitoneal lymph sac, at
the level of the adrenal glands.
LYMPH VESSELS:
 Grow out from the lymph sacs, along the major
veins to:
head, neck, and arms jugular sacs
lower trunk and legs iliac sacs
gut retroperitoneal and
cisternal sacs

 The cisterna chyli is connected to the jugular lymph sacs by 2 large channels,
the right and left thoracic ducts.
 Both the right and left thoracic ducts join the venous system at the angle of
the subclavian and internal jugular veins at the base of the neck
Duct Left lymphatic (thoracic) duct Right lymphatic duct
1. The caudal portion of the right thoracic duct.
Formed by cranial part of the right
2. The anastomosis.
thoracic duct
3. The cranial portion of the left thoracic duct.

Lymph node development

Except for the upper portion of the cisterna chyli {anterior part}, the lymph sacs
are transformed into groups of lymph nodes during early fetal life, at about month
3.
1. Surrounding mesenchymal cells invade each sac and break it up into
lymphatic channels or sinuses.
2. The mesenchymal cells give rise to the lymph node capsule and the
connective tissue framework of the node.
3. The lymphocytes seen in the node before birth come from the thymus gland.
4. The lymph nodule and germinal centers of lymphocyte production do not
appear in the nodes until just before or after birth.
5. Lymph nodes also develop along the course of other lymph vessels.

 Cisterna chyli; is the only sac that stay as it is, and doesn’t develop into other
new form.
 Cisterna chyli drains lymph from all body parts except right upper
quadrent.
THE SPLEEN development:
Develops from an aggregation of mesenchymal cells in the dorsal mesentery of
the stomach at (15-17) weeks.

THE TONSILS
Palatine Tonsils (≈ 𝟏𝟒 𝒘𝒆𝒆𝒌𝒔). Develop from the second pair of
pharyngeal pouches.
The Tubal (Pharyngo Tympanic) Develop from aggregations of lymph
Tonsils. nodules around the openings of the
auditory tubes.
The Pharyngeal Tonsils (Adenoids). Develop from an aggregation of lymph
nodules in the nasopharyngeal wall.
The Lingual Tonsils. Develop from aggregations of lymph
nodules in the root of the tongue.

 LYMPH NODULES also are seen in the mucosa of the digestive tract and
respiratory tract.
At about the end of week 5 The lymphatic system.
At about the end of week 3 CVS
about WEEKS (6-9) Form 6 primary lymph sacs.
At about month 3. sacs to lymph nodes
At about (15-17) weeks Spleen
at about 14 weeks Palatine Tonsils
 Lymphatic system

Immunity or Resistance

 It is the ability to ward off damage or disease through our defenses

 2 types of immunity

 Lymph not a circulating fluid.

Lymphatic capillaries
 Made of a single layer of squamous epithelial cells
 Slightly larger than blood capillaries
 Cells overlap and act as one-way valves
 Opened by pressure of interstitial fluid
 Anchoring filaments attach cells to surrounding tissue

lymphatic vessels Resemble veins (same 3 layers)

 Found throughout body except:
o Avascular tissues (such as cartilage, the epidermis, and the cornea of
the eye)
o The central nervous system.
o Splenic pulp.
o Bone marrow.
 Capillaries unite to form larger lymph vessels.
 Connect to lymph nodes at various intervals.
 Contain valves.
 Lymphatic vessels join to form lymphatic trunks.

 The principal trunks are the lumbar, intestinal, bronchomediastinal, subclavian,

and jugular trunks.

Channels of Lymphatics:
Lymphatics ultimately deliver lymph into 2 main channels:
Right lymphatic duct Thoracic duct (left)
Drains right side of head & neck, right Drains the rest of the body
arm, right thorax
Empties into the right subclavian vein Empties into the left subclavian vein

Cisterna chyli, it is the main duct for the return of lymph to blood.

Formation of lymph:

 Fluid leaves capillaries by diffusion and filtration to become interstitial fluid

(about 3 liters per day).
 If lymph flow is blocked = tissue swelling or edema.
 Lymphatic vessels return the lost plasma proteins and plasma to the
bloodstream.
 Specialized lymphatic capillaries in vili of small intestine transport lipids - they
are called lacteals, and the fluid is called chyle.

Lymphatic tissues and organs:

Primary (central) lymphatic organs:

 Cells originate or mature
 Red bone marrow
 Thymus gland
Secondary (peripheral) organs:
 Cells are maintained and initiate immune responses.
 Lymph nodes
 Lymph nodules
 Spleen
Lymphatic Tissue:

 No capsule present
Diffuse lymphatic tissue  Found in connective tissue of almost all organs.
 Located where they come in direct contact with
antigens.
 No capsule present
Lymphatic nodules  Oval-shaped masses
 Found singly or in clusters
Lymphatic organs  Capsule present
 Lymph nodes, spleen, thymus

Lymph Node:
 Consists of connective tissue framework & numerous lymphocytes.
 Bean shaped structures placed in pathway of lymphatic
vessels.
 Enclosed by a fibrous capsule.
 Cortex = outer portion
• Germinal centers produce lymphocytes
 Medulla = inner portion
• Medullary cords
 Lymph enters nodes through afferent lymph vessels, flows through sinuses,
exits through efferent lymph vessel.

1.Capsule 2. lymphoid nodule with germinal center

3. subcapsular sinus 4. intermediate sinus
5. medullary cords 6. medullary sinuses 7. trabecula
 Lymph is filtered through lymph nodes.
 Found in clusters;
 Principal groupings in cervical, axillary and
inguinal regions.
 “Waste water treatment plants”
 Site of cancer growth and metastasis.

Cells of Lymph Node:

• Lymphocytes
• Plasma cells
• Reticular cells
• Macrophages and other phagocytic
antigen processing cells
• Lymphatic and vascular endothelial cells

Functions of Lymph Node:

 Filtration of particles and microorganisms to keep them out of general
circulation.
 Interaction of circulating antigens in lymph with lymphocytes to initiate
immune response.
 Activation, proliferation of B lymphocytes and antibody production.
 Activation, proliferation of T lymphocytes.
Lymphatic Nodule:
 Circumscribed concentration of
lymphatic tissue (lymphocytes and
related cells).
 Not surrounded by capsule.
 Scattered throughout lamina propria
(connective tissue) of mucous membranes:
 Lining GI, urinary, reproductive tract.
 Mucosa-associated lymphatic tissue
(MALT) of respiratory tract
 Most small and solitary
 Some larger – tonsils, Peyer’s patches in ileum, appendix.

Spleen:
 Largest lymphoid organ
 Encapsulated
 Located between the stomach & diaphragm In upper left
quadrant of abdomen.
 Structure is similar to a node, But no afferent vessels or
sinuses

Substance is arranged in form of:

Red pulp reddish contains all the components of circulating blood, splenic cords
White pulp basophilic similar to lymphatic nodules

 Supporting Elements:
 Capsule
 Trabeculae
 Trabecular network
 Lymphocytes, macrophages, blood cells.
White pulp: Lymphoid Nodule (Malpighian corpuscle):
1. Germinal center
2. Central artery
 Lymphatic tissue (lymphocytes and macrophages) little
islands, mostly B cells.

Red Pulp of Spleen (Splenic cords: Cords of Billroth):

 Blood-filled venous sinuses and splenic (Bilroth’s) cords –
red blood cells, macrophages, lymphocytes, plasma cells, and
granulocytes.

Functions of Spleen
 Filtration of blood.
 Immune response against antigens circulating in blood.
 Site for production of B & T lymphocytes.
 Formation of all blood cells during fetal life.
 Only lymphocytes and monocytes after birth
 Storage of blood {platelets} (~30%).
 Can contain over one pint (‫ )متر‬of blood
 Site of destruction of aged erythrocytes.
Thymus
Located behind the sternum in the mediastinum.

 Development:
Infant conspicuous
Puberty maximum size
Maturity decreases in size

Function The thymus gland of

infants is sail-shape (‫)شراع‬
• Differentiation and maturation of T cells

Consists of 2 lobes (rt. & lt.), covered by connective tissue capsule.

Septa passing inwards from the capsule subdivide the lobe into a large number of
lobules.

Thymic Lobule:
Lobules supporting stroma made by
epithelioreticular cells
• Cortex: densely packed small
lymphocytes.
• Medulla: Lymphocytes are less densely
packed.
Presence of Hassall’s corpuscles.
groups of epithelial cells within the thymic medulla.

Functions of thymus
• Provides the environment for stem cells where they can divide and mature
into T lymphocytes.
• Thymopoietin induces T cell production & maturation.
• Thymosin supports T cell activities.
Tonsils
– Multiple groups of large lymphatic nodules
Palatine tonsils Posterior-lateral wall of the oropharynx
Pharyngeal tonsil Posterior wall of nasopharynx
Lingual tonsils Base of tongue

Palatine Tonsil:
• Aggregation of lymphatic nodules within
diffuse lymphoid tissue.
• Covered by stratified squamous
epithelium.
• Tonsillar crypts (opening of numerous
mucous glands).

Functions of tonsil
 Production of lymphocytes.
 Immunological response against antigens & organisms coming in contact
with epithelium.
 Physiology, biochemistry and histology

 The blood, lymphatic, and immune systems have separate but interrelated
functions in maintaining a healthy environment within the body (homeostasis).
 Blood is responsible for transporting oxygen (O2) and carbon dioxide
(CO2) and provides cells that defend against disease.
 The lymphatic system is responsible for cellular communication by
delivering nutrients, hormones, and other needed products to body cells
while removing their waste products as it drains tissue fluid back to the
vascular system. It also provides the cells of the immune system needed to
defend the body against disease.
 The immune system defends the body against disease. In its most simple form,
it uses barriers that exclude unwanted substances from entering the body.
 In its most complex form, it uses cells of the lymphatic system to undertake
the complex processes that identify and destroy pathogens and protect the
body against future encounters by these same pathogens.

Major components of circulatory system:

1. Heart.
2. Blood Vessels.
3. Blood.

1) Blood
 Blood is connective tissue composed of a liquid medium
called plasma in which solid components are suspended.
Blood is divided into two main components:

Cells or cells fragments 45 %

Matrix (plasma) 55 %

Although the size of white blood cells is greater than the size of red
blood cells, the density of red blood cells is greater than that of
hemoglobin (each red cell contains about 0.25 million hemoglobin molecules).
The solid components of blood include the following:
1) Red blood cells (erythrocytes)
2) Blood platelets (Thrombocytes)
3) White blood cells (leukocytes)
 Granular leukocytes:

A. Neutrophils {≈60% of WBC}.

B. Eosinophils {≈4% of WBC}.
C. Basophils {<1% of WBC}.

 agranular leukocytes:

i. Lymphocytes = T cells, and B cells {≈27% of WBC}.

ii. Monocytes (macrophages) {≈8% of WBC}.

The buffy coat is the fraction of an anticoagulated blood sample

that contains most of the white blood cells and platelets following
centrifugation.

Hematocrit is the percentage by volume of red cells in your

blood.

Medical notes:
1) The blood components are separated by a centrifuge.
2) The tube in which the sample is placed must contain anticoagulant materials
(practical example: EDTA tubes).
 EDTA tubes contain substances that bind strongly with calcium (it helps in
coagulation greatly) and pull it out so that the sample does not coagulate.
 Compartment Volume of Fluid Fluid Percentage Percentage of Body
(in Liters) of Body Weight
Total Body Fluid 42 100 60
Intracellular Fluid (ICF) 28 67 40
Extracellular Fluid (ECF) 14 33 20
Plasma 3.5 8.3 (25% Of ECF) 5
Interstitial fluid 10.5 25 (75% of ECF) 15

Some Physical Characteristics of Blood:

1) Blood volume:

 Blood makes up (7-9) % of body weight.

 Blood volume (RBCs) varies according to several factors such as size, age,
diseases, and sex:
Adult male 5-6 L
Adult female 4-5 L

 Smoking and testosterone increase the production of red blood cells

(erythropoietin)

2) Viscosity (thickness and stickiness of blood):

 Blood is thicker (more viscous) than water and flows more slowly than water.
 Plasma at 37°C is about 1.8-times more viscous than water; therefore, the
relative viscosity of plasma (compared to water) is about 1.8
 Whole blood viscosity (relative to water) = 4.5-5.5
Increased blood viscosity increases the risk of atherosclerosis

3) pH:

 Slightly alkaline: 7.4 (= Neutral body pH) Major buffer system:

 Ranges from 7.35 to 7.45 HCO3- / H2CO3

4) Color:

Bright Red (rich O2) Arteries

Dull red (poor O2) Venous
5) Osmolarity:

 Plasma osmolarity is about 300 mOsmol/L equal osmolarity of Normal Saline =

0.9% NaCI.
 Under normal conditions, extracellular osmolarity can be roughly estimated as:
Posm = 2 [Na+] ≈ 270-290 mOsm

6) Osmotic pressure:

 Osmotic pressure is the pressure necessary to prevent net movement of water

(in osmosis).
 In other words, osmotic pressure is the pressure developed by solutes
dissolved in water working across a selectively permeable membrane.
 At normal plasma osmolarity of about 300 mOsmol/L, the total plasma osmotic
pressure is about 5540 mmHg.
Fluid Compartments in the body
 Differences between the composition of intracellular and extracellular fluids are caused by
transport mechanisms of cell membranes.
Major differences in composition include the
following:
Extracellular fluid has higher concentrations
of sodium, calcium, chloride, bicarbonate and
glucose compared with intracellular fluid.
Intracellular fluid has higher concentrations
potassium, magnesium, phosphate, sulfate
(SO4-), amino acids, proteins and molecules
derived from lipids. compared with
extracellular fluid.

Composition of blood:
Cells or cells RBCs (4.8 – 6.5 million) 99%
fragments 45 % WBCs (4 – 11 thousands) < 1%
Platelets (150,000 – 450,000)
Water 91.5 %
Plasma protein 7%
Other solutes include:
Matrix (plasma) 55 %
• Electrolytes
• Organic nutrients and wastes 1.5 %
• Respiratory gases
• Vitamins

Note that:

Penia = low number

Cytosis = high number

In childhood the number of

WBCs about 18,000

Polycythemia = erythrocytosis
Anemia = erythropenia
 Plasma proteins:
Plasma proteins: are proteins present in blood plasma.

1. Albumin (58%)
Albumin is the most abundant protein in plasma.

2. Globulins (37%).
𝜶 −Globulins Transport proteins
𝜷 −Globulins Coagulation factors
𝜸 −Globulins Defensive proteins = Immunoglobulins = Antibodies

3. Fibrinogen (4%).
4. Regulatory proteins (<1%)

Functions of Plasma Proteins

Function Properties Responsible Protein
Capillary walls are relatively
Generation of plasma impermeable to the proteins in
colloid osmotic plasma, and the proteins
pressure (oncotic therefore exert an osmotic force Albumin
pressure): of about 25 mm Hg across the
capillary wall (oncotic pressure
that pulls water into the blood.)
Buffering function of The plasma proteins are also All types of plasma
plasma proteins: responsible for 15% of the proteins
buffering capacity of the blood.
Plasma proteins Act as nonspecific carriers for
function as various hormones (e.g., cortisol, Albumin +
nonspecific carriers thyroxin), other solutes (e.g., 𝜶 −Globulins
iron, cupper), and drugs.
Defense: Gamma globulins are antibodies 𝜸 − Globulins
3 Globulins,
Blood clotting -------------- Fibrinogen &
Prothrombin.

If whole blood is allowed to clot

Plasma: whole blood minus cells Then, clot is removed, the

Serum: plasma minus clotting proteins remaining fluid is SERUM
Thus, serum does not contain
coagulation factors
 Blood smear procedure:
 Remember that the cells you see in a blood smear have not been sectioned.
Instead you are seeing whole cells dried down on the glass.
 After the smear is made, it is air-dried and then stained.
 Common stains are Wright’s stain and Giemsa stain.
 The stains generally include two or more dyes, one of them a basic dye (often
methylene blue) and another an acidic dye (usually eosin).
 1) Platelets (thrombocytes)
A) Life Span: ≈ 10 days
B) Shape, size, and origin:
SHAPE biconvex disks
DIAMETER 2-3 𝝁m in diameter
Non-nucleated cell fragments derived from cytoplasm of a very large
cell, the megakaryocyte in BM.

C) LM appearance in smears:
 Small basophilic fragments, often appearing in clusters.
hyalomere Lightly stained peripheral zone.
granulomere Darker-staining central zone rich in granules.

D) Normal platelet counts: 150,000 - 450,000/ 𝝁𝑳 (𝒎𝒎𝟑) of blood.

E) Function: Platelets initiate blood clots (stop bleeding).

F) Ultrastructure of a platelet:
1) Peripheral microtubule bundle: Maintains shape
2) Actin and myosin: clot contraction
Mitochondria for ATP production.
Granules contain clotting factors.
Dense tubular system sequesters
3) Organelles facilitate clotting: Ca+2 for signaling (Similar to SR
in skeletal muscle).
Open canalicular system
facilitates signaling and
secretion.
 2) Red blood cells
1) Life span in blood ≈ 120 days.
 RBCs are the most abundant cells of the blood and are necessary for the
delivery of oxygen to the tissues.
2) Function of RBCs:
1) A major function of RBCs, also known as erythrocytes, is to transport
hemoglobin, which, in turn, carries oxygen from the lungs to the tissues.
 In some animals, including many invertebrates, hemoglobin circulates as
free protein in the circulatory fluids and is not enclosed in RBCs. Therefore,
hemoglobin must remain inside RBCs to perform its functions in humans
effectively.
2) Contain a large quantity of carbonic anhydrase.
 Carbonic anhydrase, an enzyme that catalyzes the reversible reaction
between carbon dioxide (CO2) and water to form carbonic acid (H2CO3),
increasing the rate of this reaction several thousand fold.
 The rapidity of this reaction makes it possible for the water of the
blood to transport enormous quantities of CO2 in the form of
bicarbonate ion (HCO3−) from the tissues to the lungs, where it is
reconverted to CO2 and expelled into the atmosphere as a body waste
product.
3) The hemoglobin in the cells is an excellent acid-base buffer (as is true of most
proteins), so the RBCs are responsible for most of the acid-base buffering power
of whole blood.

3) Shape and Size of Red Blood Cells:

Shape biconcave discs

Diameter 7.5 𝛍𝐦
thickness Thickest part 2 - 2.5 𝝁𝒎
Thinnest part 1 𝝁𝒎
Volume 90 - 95 𝝁𝟑

In males 5,200,000 (±300,000) / 𝝁𝟑

In females 4,700,000 (±300,000) / 𝝁𝟑

 The shapes of RBCs can change remarkably as the cells squeeze through capillaries.
Does not have: nucleus, mitochondria, ribosomes, endoplasmic reticulum or Golgi
apparatus.
 Persons living at high altitudes have greater numbers of RBCs,
 Because the normal cell has a great excess of cell membrane for the quantity
of material inside, deformation does not stretch the membrane greatly and,
consequently, does not rupture the cell.
 The shape is maintained by a cytoskeletal complex inside the plasma
membrane (involving spectrin, actin and other components).
 Spectrin, actin problems cause anemia and spherocytosis.
 The erythrocyte plasmalemma is consists of about 40% lipid, 10%
carbohydrate, and 50% protein.
 Most of the proteins are integral membrane proteins, including: Ion
channels, the anion transporter called band 3 protein, and glycophorin A.
band 3 protein ABO incompatibility
glycophorin A Rh problems

4) LM appearance in smear:
Pink circle with light center (center is thinner
because of the biconcave shape) with NO
NUCLEUS.

5. TEM appearance:
Solid dark gray cytoplasm, because of highly
concentrated hemoglobin.
 Genesis of Blood Cells (hematopoiesis):
In the early weeks of embryonic Primitive nucleated RBCs are produced in the yolk
life: sac.
During the middle trimester of The liver is the main organ for RBC production but
gestation: reasonable numbers are also produced in the spleen
and lymph nodes.
during the last month or so of RBCs are produced exclusively in the bone marrow.
gestation and after birth

 The marrow of essentially all bones produces RBCs until

a person is about 5 years old.
 The marrow of the long bones, except for the proximal
portions of the humeri and tibiae, becomes fatty and
produces no more RBCs after about the age of 20 years.
 Beyond this age, most RBCs continue to be produced in
the marrow of the membranous bones, such as the
vertebrae, sternum, ribs, and ilia.
Even in these bones, the marrow becomes less productive
as age increases.

The blood cells begin their lives in the bone marrow from a single type of cell called the
multipotential hematopoietic stem cell {MHSC}, from which all the cells of the circulating blood
are eventually derived.
The intermediatestage cells are very much like the multipotential stem cells, even though they
have already become committed to a particular line of cells; these are called committed stem
cells.
The hematopoiesis is driven by 2 factors:
1. Growth inducers (factors; e.g., The growth inducers promote growth but not
interleukin-3). differentiation of the ce
Causes one type of committed stem cell to differentiate
2. Differentiation inducers. one or more steps toward a final adult blood cell.

Hematopoiesis responds to changing conditions:

Hypoxia: erythropoiesis
Infection/inflammation: WBC production
Differentiation of Blood Cell
MHSC cells begin to divide, entering one of 3 ways:

First pathway (MHSC):

Exactly like the original MHSC and is retained in the bone marrow to maintain
their supply, although their numbers diminish with age.

Second pathway (Colony-forming unit–spleen (CFU-S)):

As the MHSC become CFU-S will divide in each of the following pathways:
Colony forming unit Abbreviation Differentiation
Colony-forming unit–erythrocytes
Colony-forming unit–blast CFU-B (CFU-E)  production of
erythrocytes
Colony-forming unit– CFU-GM Production of granulocytes and
granulocytes, monocytes monocytes
Colony-forming unit– CFU-M Production of megakaryocytes 
megakaryocytes platelets

Third pathway lymphoid stem cell (LSC):

Differentiation into either:

T-Lymphocytes TH: = CD4

TC = CD8
B-lymphocytes Plasma cells
Memory cells
Nk- cells
Differentiation of red blood cells:
1) Under appropriate stimulation, large numbers of proerythroblast cells are
formed from the CFU-E stem cells.
2) Once the proerythroblast has been formed, it divides multiple times, eventually
forming many mature RBCs.

Very detailed stages:

It contains a nucleus and does not contain hemoglobin or
organelles.
proerythroblast
 From here begins the synthesis of hemoglobin or
in polychromatophil erythroblasts
basophil Organelles begin to form and are called basophil
erythroblasts erythroblasts because they stain with basic dyes.
polychromatophil From here begins the appears / synthesis of
erythroblasts hemoglobin and becomes the cell color (blue-red).
Orthochromatic The cells become filled with hemoglobin to a concentration of
erythroblast about 34%, the nucleus condenses to a small size, and its
final remnant is absorbed or extruded from the cell.
Called a reticulocyte because it still contains a small amount
reticulocyte of basophilic material, consisting of remnants of the Golgi
apparatus, mitochondria, and a few other cytoplasmic
organelles.
During this reticulocyte stage, the cells pass from the bone
marrow into the blood capillaries.
 The remaining basophilic material in the reticulocyte
Erythrocytes normally disappears within 1 to 2 days, and the cell is then
a mature erythrocyte.
 Because of the short life of the reticulocytes, their
concentration among all the RBCs is normally slightly less
than 1%.

Note: It is normal for

the reticulocyte rate
to be 1%, but in cases
of bleeding, the bone
marrow excretes it, so
it rises to about 3.4%
Regulation of Red Blood Cell Production:
The total mass of RBCs in the circulatory system is regulated within narrow
limits, and thus for:
Maintain adequate oxygen carrying capacity.
Avoid excessive blood viscosity.
If the bone marrow is damaged or if demand for erythropoiesis is extreme, other
parts of the bone marrow may become hyperplastic, or extramedullary
hematopoiesis may occur.

Tissue Oxygenation—Essential Regulator of Red Blood Cell

Production.
Conditions that decrease the quantity of oxygen transported to the tissues
ordinarily increase the rate of RBC production.
In this case, it is not the concentration of RBCs in the blood that controls
RBC production but the amount of oxygen transported to the tissues in
relation to tissue demand for oxygen.

Factors that decrease oxygenation:

Anemia as a result of hemorrhage
The quantity of oxygen in the air is greatly decreased,
At very high altitudes. insufficient oxygen is transported to the tissues, and
RBC production is greatly increased.
Destruction of major especially by x-ray therapy, causes hyperplasia of the
portions of the bone remaining bone marrow in an attempt to supply the
marrow. body’s need for RBCs
Prolonged diseases Heart failure.
Lung disease.
Low blood volume
Low hemoglobin
Poor blood flow
Deficiency of: B12 vitamin, iron and folic acid
Athletes
Erythropoietin (Epo):
 The principal stimulus for RBC production in a low oxygen state is a circulating
hormone called erythropoietin,
 Circulating hormone, mw ≈ 34,000
 Necessary for erythropoiesis in response to hypoxia
 ≈ 90% made in the kidney and the remainder is formed mainly in the liver.
 In the absence of erythropoietin, hypoxia has little or no effect to stimulate
RBC production. However, when the erythropoietin system is functional, hypoxia
causes a marked increase in erythropoietin production and the erythropoietin, in
turn, enhances RBC production until the hypoxia is relieved.
Some studies have suggested that erythropoietin is secreted mainly by:

Fibroblast-like interstitial cells Where much of the kidney’s oxygen

surrounding the tubules in the cortex consumption occurs.
and outer medulla.
Renal epithelial cells Secrete erythropoietin in response to
hypoxia.

Renal tissue hypoxia leads to increased tissue levels of hypoxia-inducible factor-

1 (HIF-1).
 HIF-1 serves as a transcription factor for a large number of hypoxia
inducible genes, including the erythropoietin gene.
 HIF-1 binds to a hypoxia response element in the erythropoietin gene,
inducing transcription of messenger RNA and, ultimately, increased
erythropoietin synthesis.
In organs other than kidney there some nonrenal sensor that sends an additional
signal to the kidneys to produce erythropoietin.
norepinephrine and epinephrine and several of the prostaglandins

 When both kidneys are removed from a person (anephric person), or when the
kidneys are destroyed by renal disease, the person invariably becomes very
anemic.
 This is because the 10% of the normal erythropoietin formed in other
tissues (mainly in the liver) is sufficient to cause only one third to half the
RBC formation needed by the body.
 Hematocrit (packed cell volume) ≈ 23–25% rather than 40–45%
When an animal or person is placed in an atmosphere of low oxygen, erythropoietin
begins to be formed within minutes to hours, and it reaches maximum production within
24 hours.

 Yet, almost no new RBCs appear in the circulating blood until about 5 days later.
 The rapid production of cells continues as long as the person remains in a low
oxygen state or until enough RBCs have been produced to carry adequate
amounts of oxygen to the tissues despite the low level of oxygen; at this time, the
rate of erythropoietin production decreases to a level that will maintain the
required number of RBCs but not an excess.
In the absence of erythropoietin, few RBCs are formed by the bone marrow.

 At the other extreme, when large quantities of erythropoietin are formed, and if
plenty of iron and other required nutrients are available, the rate of RBC production
can rise to perhaps 10 or more times normal.

Maturation of Red Blood Cells

Certain elements are essential for RBC formation and maturation:

Amino acids: formation of globin in haemoglobin

Iron: formation of haemoglobin
Vit B12 (Cyanocobalamin) Synthesis of nucleoprotein
Vitamins: and Folic acid (Vit B9)
Other : Vit B6, Riboflavin, nicotinic acid,
biotin, Vit C, Vit E
Essential elements
Hormones

 As would be expected, their maturation and rate of production are affected greatly by a
person’s nutritional status.
 Deficiency of amino acids, iron or vitamins caused:
1) Anemia.
2) Vit B12 and Folic acid are essential for synthesis of DNA because each, in a different
way, is required for formation of thymidine triphosphate, one of the essential building
blocks of DNA.
3) Lack of vitamin B12 or folic acid causes abnormal and diminished DNA and,
consequently, failure of nuclear maturation and cell division.
4) Furthermore, the erythroblastic cells of the bone marrow, in addition to failing to
proliferate rapidly, produce mainly larger than normal RBCs called macrocytes, which
have a flimsy (‫ )رقيق‬membrane and are often irregular, large, and oval instead of the usual
biconcave disc.
 These poorly formed cells (macrocytes,), after entering the circulating blood, are
capable of carrying oxygen normally, but their fragility causes them to have a
short life, half to one-third normal. Therefore, deficiency of vitamin B12 or folic
acid causes maturation failure in the process of erythropoiesis.

Molecules Optimum Absorption site

Iron Duodenum
Folic acid (B9) Jejunum
Cobalamin (B12) Ileum

Vitamin B12
 Is a water-soluble vitamin that is naturally present in
some foods, added to others, and available as a
dietary supplement and a prescription medication.
 Because vitamin B12 contains the mineral cobalt,
compounds with vitamin B12 activity are collectively
called “Cobalamins”.
 The main dietary source is liver, kidney, red meat,
eggs, shellfish and dairy products.
 Vitamin B12 is relatively stable and little is lost during
cooking.

The metabolically active forms of vitamin B12 are:

1. Methylcobalamin
2. 5-deoxyadenosylcobalamin
However, two others forms, hydroxycobalamin and cyanocobalamin, become
biologically active after they are converted to methylcobalamin or 5-
deoxyadenosylcobalamin.

The role of vitamin B12 within the body:

 Vitamin B12 is required for:
1. the development
2. Myelination
3. Function of the central nervous system
4. Heme synthesis
5. DNA synthesis
Cobalamin enzymes, which are present in most organisms, catalyze three types of
reactions:
1. Intramolecular rearrangements.
2. Methylations, as in the synthesis of methionine.
3. The reduction of ribonucleotides to deoxyribonucleotides.

Vitamin B12 is required as coenzyme for two metabolic reaction:

A. Isomerization of L-methylmalonyl CoA to succinyl CoA. L=R isoform
 Succinyl CoA is an important substrate in Heme synthesis.

B. Methylation of homocystine to methionine.

 Synthesis of purines and thymine.
Methylation of homocystine to methionine is especially important because
methionine is required for the generation of coenzymes that participate in the
synthesis of purines and thymine, which are needed for nucleic acid synthesis.
 A common cause of RBC maturation failure is failure to absorb vitamin B12
from the gastrointestinal tract.
 This situation often occurs in the disease pernicious anemia.
 In which the basic abnormality is an atrophic gastric mucosa that fails to
produce normal gastric secretions.
 The parietal cells of the gastric glands secrete a glycoprotein called intrinsic
factor, which combines with vitamin B 12 in food and makes the B12 available for
absorption by the gut.

Absorption of vitamin B12 in Gut mechanism (active process):

1. Intrinsic factor binds tightly with the vitamin B12 (1:1 ratio).
In this bound state, vitamin B12 is protected from digestion by the
gastrointestinal secretions.
2. Still in the bound state, intrinsic factor binds to specific receptor sites on the
brush border membranes of the mucosal cells in the ileum.
This process requires the presence of calcium ions and neutral pH.
3. Vitamin B12 is then transported into the blood (portal circulation) during the
next few hours (𝟔 𝒉𝒓) by the process of pinocytosis, carrying intrinsic factor and
the vitamin together through the membrane.
4. Once vitamin B12 has been absorbed from the gastrointestinal tract, it is first
stored in large quantities in the liver (3-4 𝒎𝒈) and then released slowly as needed
by the bone marrow.

Lack of intrinsic factor, therefore, decreases availability of vitamin B12 because

of faulty absorption of the vitamin.
 The minimum amount of vitamin B12 required each day to maintain normal
RBC maturation (daily losses in urine or feces) is only 1 to 4 𝝁𝒈, and the normal
storage in the liver and other body tissues is about 1000 times this amount.
 Therefore, 3 to 4 years of defective vitamin B12 absorption are usually
required to cause maturation failure anemia.
 If normally there is no consumption of B12 within the body; the daily
requirement matches the daily lose.
 Vitamin B12 Transport
There are three vitamin B12 transport proteins
normally present in the plasma, which are
known as Transcobalamines (TCI-TCII-TCIII).

The physiologically active is TCII which

complex in a 1:1 ratio with vitamin B12.
The complex is then binds to specific surface
receptors on developing blood cells in the bone
marrow. Vitamin B12 is then released by
hydrolysis.
The TCII is not reutilized.

 The plasma half-life of TCII is 12 hours and

congenital absence of it causes megaloblastic
anaemia within weeks of birth.

 Transcobalamines I and III are a-globulins

synthesized by granulocytes and known as R-
binders that are found in a wide range of body
fluids.
 TCI&III do not readily release vitamin B12 to the
developing tissues.
 The plasma half-life is 9-12 days and
congenital absence of them causes no
physiological impairment.

Activation Vitamin B12

 Methyl-THF converts vitamin B12 to methyl B12
(methylcobalamin).
 Methyl-B12 converts homocysteine, in a reaction
catalyzed by homocysteine methyltransferase, to
methionine.
 Causes of Vitamin B12 Deficiency:
A) Inadequate dietary intake.
 This is uncommon for three main reasons:

1. Vitamin B12 is present in a wide range of readily available foodstuffs.

2. Vitamin B12 is relatively heat-stable.
3. Body stores of vitamin B12 are sufficient to meet the requirements for at least three years
following complete cessation of dietary intake or intestinal absorption.

B) Intestinal malabsorption.
 The most common cause of the deficiency, which could be due to:

1) Lack of intrinsic factor:

 Pernicious anaemia.

2) Gastrointestinal disease:
Total gastrectomy: the anaemia is developed after depletion of the body stores, which is usually,
occur within 5 years. This is sever when accompanied with iron deficiency anaemia.

Partial gastrectomy: (stagnant or blind loop syndrome) Partial removal of the stomach, and
refashioning the junction with the gut. The sterile duodenal part will colonized with bacteria, which
will consume huge amount of the vitamin.

Ileal resection or ileostomy: Involve removal of the vitB12 receptor.

3) Drug-induced Malabsorption:
 Anticonvulsant, phenytoin.
 Antimicrobial, neomycin.
 Antigout, colchicine.
 Alcohol.

C) Increased Requirements.
The requirements are increased during pregnancy. The increase is not sufficient to cause deficiency
unless the pregnant was previously borderline body stores of the vitamin.

D) Failure of Vitamin B12 Transport.

Congenital deficiency of transcobalamin II develops megaloblastic anaemia in the first weeks of life,
despite the presence of normal vitamin B12 concentration in the serum. Early diagnosis prevents
neurological damages.
Folic Acid (Pteroylglutamic Acid) (vitamin B9)
Folic acid is a normal constituent of green vegetables, some fruits, yeasts and meats
(especially liver). However, it is easily destroyed during cooking.
Also, people with gastrointestinal absorption abnormalities, such as the frequently
occurring small intestinal disease called sprue, often have serious difficulty
absorbing both folic acid and vitamin B12.
 Daily folate losses are about 100 µg/day, mainly in the faeces, urine, and sweat and
skin cells.
 In order to maintain body stores, the total daily requirement must match
losses.
 Manufactured folic acid, which is converted into folate in the body.
 Humans are incapable to synthesize it so the only source is diet.
The active form of folate is tetrahydrofolic acid (THF).

 THF serves in single-carbon donor-acceptors.

These reactions are involved in synthesis of nucleotides and amino acids.
DHF can be converted intoTHF dihydrofolic acid reductase (DHFR) enzyme as the
following reaction:
DHFR is a ubiquitous enzyme that catalyzes the NADPH-dependent conversion of
7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF).

The role of folate within the body:

1. Amino acids synthesis.
The amino acids requiring folic acid for metabolism are:
Methionine, Histidine, Cysteine, Serine and Glycine.

Essential amino acids

The process begins with glycolytic precursors where 3-
phosphoglycerate precursor plays a major role.

Vitamin B12 as co-factor

 Synthesis of methionine:
By donation of methyle group from N-5-methyl-tetrahydrofolate and requires
vitamin B12 as a coenzyme.
 Conversion of serine into glycine. Glycine is obtained either through food or from
a neighboring reaction.
 Histidine catabolism.
2. Pyrimidine synthesis Pyrimidine: six-membered ring) and
includes cytosine, thymine, and uracil.
The rate limiting step in DNA synthesis.
Purine: 2 rings and includes adenine and guanine
3. Purine synthesis.

 Methionine, as S-adenylmethionine (SAM), serves as the donor of one carbon

units to methylate sites within DNA, RNA, proteins, and phospholipids.

4. Folic acid is required for erythropoiesis.

Thus folic acid deficiency leads to megaloblastic anemia.

5. DNA repair and normal cell growth.

Typical body stores of folate in a normal, healthy adult are about 10 mg and are
located in liver.
Thus, if dietary folate intake or intestinal absorption ceased, the body stores
would become exhausted in about 3-4 months.

Folate absorption and transport

 Folates are absorbed maximally from the upper jejunum.
 Absorbed folates are converted into N-5- methyltetrahydrofolate and released
into portal blood stream.
 Plasma folates circulate freely or loosely bound to a variety of specific plasma
proteins.
 There is some evidence that a specific folate transport protein exists and that
its concentration is increased by folate deficiency but its physiological
significance is unknown.
 Folic acid deficiency:
1- Inadequate diet:

 The ideal diet contains 700 𝝁𝒈 of folate of which about half is absorbed.
 Folate is very labile (‫ )حساس‬to heat; cooking can destroy up to 90% of folate in it.
 Body stores are only sufficient for 3 months when dietary intake stop.

2- Intestinal malabsorption.

Coelic disease: Villous atrophy, which decreases iron and folate absorption.
Tropical sprue: Similar to coelic disease.
Crohn disease: Generalized malabsorption in the intestine.

3- Increased requirement.

 Pregnancy; the daily requirement for folate can rise to 500μg in the 3ed trimester of pregnancy.
 More than 60% of pregnant women have subnormal folate concentration.
 This recently, demonstrated to be associated with neural tube defects.
 The anaemia of chronic haemolytic conditions such as sickle cell anaemia frequently is
exacerbated by folate deficiency. Sever haemolytic conditions increases the rate of haemopoiesis
by a factor of 10, which cannot be met by dietary sources.

4- Failure of utilization the absorbed vitamin.

 A number of rare enzyme deficiencies have been reported which cause impairment of folate
metabolism. Most of these are associated with megaloblastic changes and mental retardation.

5- Drug-induced folate deficiency:

 Some drugs are demonstrated to inhibit folate absorption such as:

 Long-term therapy with anticonvulsant, phenyton.

 Alcohol.
 The cytotoxic drugs methotrexate, which inhibit the enzyme dihydrofolate reducates and
cause depletion of thymidin and purine nucleotides.
6- Neurological manifestations:

 Degeneration of the dorsal and lateral columns of the spinal cord are typical findings in sever
megaloblastic anemia due to deficiency of vitamin B12. The mechanism is not known yet.
 Folic acid deficiency in pregnancy is associated with the incidence of neural tube defects such as
spina bifida and is also believed to lead to mild dementia and impairment of intellectual function.
 Hemoglobin (the oxygen-binding protein of red blood cells)
 Hemoglobin (Hb) is a major hemoprotein of human body.

Heme prosthetic group

Globin protein part

What are hemoproteins (Metalloproteins)?

Hemoproteins are heme containing proteins and enzymes of human body.

 Tetramer, made up of four subunits.

Haemoglobin  Quaternary structure of Protein.
 Reversibly combine with oxygen & transports oxygen.
 Monomer, Structure is similar to Hb.
Myoglobin  Present in muscles.
 Tertiary structure of Protein.
 Serve to Storage of oxygen.
 Components of respiratory chain in mitochondria &
Cytochromes transport electron.
 Cyto- P450 , takes part in microsomal hydroxylation.
 Present in animals.
Catalase
 It acts on hydrogen peroxide.
Peroxidase  Present mainly in plants.
 It acts on hydrogen peroxide.
Tryptophan
 It acts on tryptophan.
Pyrrolase

Hb Function:

1) Hb is a characteristic of aerobic life very important for survival.

2) Hb serves in transport and exchange of gases (O2 and CO2) between lungs and tissues.

 Oxygen has a higher affinity for Haemoglobin than carbon Dioxide.

Respiration mechanism:

Hb majorly transports oxygen (97% -100%) The remnant is transported by

Hb Minorly transports carbon dioxide (15% -25 %) diffusion

Hemoglobin in the body is to combine with oxygen in the lungs and then to
release this oxygen readily in the peripheral tissue capillaries, where the
gaseous tension of oxygen is much lower than in the lungs.
3) Hemoglobin Plays Role as Buffer:

 (Hb/Hb-H+) in the Erythrocytes.

 Imidazole group of amino acid Histidine of Hb molecule.

Amount of Hb
Adult males: 13.5–17.5 𝒈/𝒅𝑳
Adult females: 12.5–16.5 𝒈/𝒅𝑳
Infant: 14–19 𝒈/𝒅𝑳

Each RBC contain about (250– 300) million hemoglobin molecules.

Hemoglobin makes up about one third (1/3) of the RBC volume.
90-95% of the dry weight of RBC is by Hb.

Hb Biosynthesis:
 6.25 g Hb/day
 Produced during stages of erythropoiesis in bone marrow.
 Synthesis of Hb begins in Proerythroblast

Hb structure:
 Tetrameric (quaternary) structure.
 Hb consists of 4 polypeptide subunits.
 Globular protein contain 4 allosteric sites.

 The synthesis of hemoglobin begins in polychromatophil erythroblasts and

continues even into the reticulocyte stage of the RBCs. Therefore, when reticulocytes
leave the bone marrow and pass into the blood stream, they continue to form minute
quantities of hemoglobin for another day or so until they become mature
erythrocytes.

There are several slight variations in the different subunit hemoglobin chains,
depending on the amino acid composition of the polypeptide portion.
The different types of chains are designated as alpha (α) chains, beta (β) chains, (γ)
gamma chains, and (δ) delta chains.
 The most common form of hemoglobin in adults, hemoglobin A, is a combination
of two alpha chains and two beta chains (MW 64,458).
 Both alpha and beta subunits of hemoglobin consist primarily of α-helical
Secondary structure and each subunit has a non-polypeptide component,
called heme with an iron atom that binds with oxygen.
Each of these hemes can bind loosely with one molecule of oxygen, making a
total of four molecules of oxygen (or eight oxygen atoms) that can be transported
by each hemoglobin molecule.
The types of hemoglobin chains in the hemoglobin molecule determine the
binding affinity of the hemoglobin for oxygen.
Abnormalities of the chains can alter the physical characteristics of the
hemoglobin molecule as well, for example; sickle cell anemia.

In Hb 4 polypeptide chains are visualized as:

Two identical dimers, (αβ)1 and (αβ)2.
 Two dimers (αβ - αβ) are linked to each other
by weak polar bonds- movement at the
interface of these two occurs more freely.

 Two polypeptide chains (α - β) within a dimer

are held together tightly by: Ionic bonds and
hydrophobic interactions, which prevent
their movement relative to each other.

Globin Subunits:
Adult Hemoglobin has 4 Polypeptide chains 2α and 2β (identical pair).
globin chains Alpha globin chains Beta Globin chains
Composition 141 amino acids. 146 amino acids
MW 15,126 Daltons 15,866 Daltons
Biosynthesis-Expression α Globin gene on β Globin gene on
16th Chromosome. 11th chromosome.

2α (282 amino acid residues). 2β (292 amino acid residues).

Total 574 amino acids are present in one Hemoglobin molecule.
 Hemoglobin Combines Reversibly With Oxygen
 The most important feature of the hemoglobin
molecule is its ability to combine loosely and
reversibly with oxygen.
 Oxygen does not combine with the two positive
bonds of the iron in the hemoglobin molecule.
Instead, it binds loosely with one of the so-
called coordination bonds of the iron atom.
 Furthermore, the oxygen does not become ionic
oxygen but is carried as molecular oxygen
(composed of two oxygen atoms) to the tissues,
where, because of the loose, readily reversible
combination, it is released into the tissue fluids
still in the form of molecular oxygen rather than
ionic oxygen.

Hemoglobin and Hematocrit

Normal hematocrit (packed cell volume) is 40– 45% (slightly lower in women).
Normal hemoglobin concentration is 34 g per 100 mL of packed cells.
Thus normal hemoglobin is 14–15 g per 100 mL of blood.
O2 carrying capacity is 1.34 mL/g Hgb, or 19–20 mL O2/100 mL blood.
Sex Males Females
normal hemoglobin 15 g/100 mL of blood 14 g/100 mL of blood
O2 carrying capacity in saturation 20 mL O2/100 mL blood 19 mL O2/100 mL blood

The concentration of hemoglobin does not rise above this value because this is
the metabolic limit of the cell’s hemoglobin-forming mechanism.
Furthermore, in normal people, the percentage of hemoglobin is almost always
near the maximum in each cell. However, when hemoglobin formation is deficient,
the percentage of hemoglobin in the cells may fall considerably below this value,
and the volume of the RBC may also decrease because of diminished hemoglobin
to fill the cell.
 In the case of lack of oxygen, new red blood cells are produced, not
hemoglobin.
What Is Heme? Metalloporphyrins are inhibitors of the rate-
limiting enzyme, heme oxygenase, in the
Prosthetic group of Hemoproteins. pathway of heme degradation leading to
Red color pigment. bilirubin production.

Metalloporphyrin.
Chemically heme is a Ferroprotoporphyrin Ferroprotoporphyrin is an iron-containing
cofactor.
 Heme is a derivative of the porphyrin.
Porphyrins are cyclic compounds formed by fusion of 4 pyrrole rings
linked by methenyl bridges.
 Most common porphyrin in humans is heme.

Iron in Heme:
 Iron content of Hb: 3.4 mg / g of Hb

 Functional form iron in heme is:

 Ferrous form (Fe+2)

 Reduced state
 Fe+2 located centrally in Protoporphyrin ring system.

 Fe of Heme is Hexavalent:
Fe of heme forms 6 coordinated bonds to satisfy its six
valencies:
4 bonds linked with each nitrogen of 4 pyrrole rings.
5th bond linked with Proximal Histidine.
6th bond is with Oxygen.

Iron-protoporphyrin-IX
Heme synthesishemoglobin:
 The major sites of heme biosynthesis are the liver, which synthesizes a
number of heme proteins (particularly the CYP proteins}, and the erythrocyte-
producing cells of the bone marrow, which are active in Hb synthesis.
 Over 85% of all heme synthesis occurs in erythroid tissue.
 Mature red blood cells (RBC) lack mitochondria and are unable to
synthesize heme.

In the liver The rate of heme synthesis is highly variable caused by fluctuating
demands for hemeproteins.
in erythroid cells Relatively constant and is matched to the rate of globin synthesis.

 The formation of porphyrins occurs along 8 steps:

Initial and last three steps Mitochondria
The rest Cytosol

Sep 1 and step 2 are allosterically regulated;

Steps Step 1 Step 2
feedback inhibitor Heme and hematin Lead

 Heme is repressing transcription of the ALA Synthase gene in most cells.

Genes involved in the chemical pathway for making heme:

ALAD aminolevulinic acid, δ-, dehydratase (deficiency causes ala-
dehydratase deficiency porphyria).
ALAS1 aminolevulinate, δ-, synthase 1

Synthesis of heme:
 Liver.
 Bone Morrow.
 No heme synthesis in RBC; don’t have mitochondria
 Substrates mainly include succinyl-CoA, glycine, Fe2+.
 Almost all the tissues can synthesize heme.
 Major sites of synthes is liver and bone marrow
bone - marrow heme production equal to globin synthesis.
liver Variable dependent on heme pool balance.

ALA Synthase is the committed step (2 substeps) of the heme synthesis pathway,
& is usually rate limiting for the overall pathway.
Regulation occurs through control of gene transcription.

Mechanism
A) In the mitochondria Combination of Succinyl-CoA + Glycine in the process of
forming 𝜶-Amino-𝜷 −ketoadipate (Schiff base).
B) By decarboxylation using ALA synthase to produce ALA.

 Condensation with succinyl-CoA takes place while the amino

group of glycine is in Schiff base linkage to the Pyridoxal
phosphate aldehyde.
 Pyridoxal phosphate (PLP) {V.B6 derivate} serves as
coenzyme for d-Aminolevulinate Synthase (ALA Synthase).

C) ALA is transported to cytosol.

D) Two ALA are combined (releasing 2 H2O) via ALA dehydratase to produce
porphobilinogen.
 porphobilinogen contain acetyl and propionyl side chains
 High ALA is thought to cause some of the neurological effects of lead
poisoning, although Pb+2 also may directly affect the nervous system.
ALA is toxic to the brain, perhaps due to:
 Similar ALA & neurotransmitter GABA (g-aminobutyric acid) structures.
 ALA autoxidation generates reactive oxygen species (oxygen radicals).

Regulation ALA synthase:

1. When there is excess of free heme without globin chains to bind with, the Fe+2
is oxidized to Fe+3 forming hematin. Hematin will inhibit ALA synthase to
prevent excessive unwanted production of heme.

2. Hematin will also inhibit the translocation of ALA synthase from the cytoplasm
into the mitochondria where its substrate, succinyl CoA is formed. thus heme
synthesis is inhibited till there are sufficient globin chains to bind with.

3. Lack of Vit B6 will decrease the synthesis of ALA.

 Drugs like INH (isonicotinic acid hydrazide) that decrease the
availability of pyridoxal phosphate may also affect heme synthesis.

Regulation ALA dehydratase:

Heme synthesis may be inhibited by heavy metals. The steps catalyzed by ALA
dehydratase and ferrochelatase are inhibited by lead.
1) succinyl-CoA, which is formed in the Krebs
metabolic cycle binds with glycine to form a
pyrrole molecule.
2) In turn, four pyrroles combine to form
protoporphyrin IX, which then combines with
iron to form the heme molecule.
3) Each heme molecule combines with a long
polypeptide chain, a globin synthesized by
ribosomes, forming a subunit of hemoglobin
called a hemoglobin chain.
 Each chain has a molecular weight of
about 16,000; four of these chains, in turn,
bind together loosely to form the whole
hemoglobin molecule.
 Heme Degradation
 Degradation begins inside macrophages of the spleen.

 In the first step:

heme is converted to biliverdin by the enzyme heme oxygenase
(HO).
 NADPH is used as the reducing agent.
 Molecular oxygen enters the reaction, carbon monoxide
(CO) is produced, which acts as a cellular messenger and
functions in vasodilation.
 The iron is released from the molecule as the ferrous ion
(Fe2+).
Heme degradation increase by increase oxidative stress.
Briefly, when cells are exposed to free radicals, there is a rapid induction of the
expression of the stress-responsive heme oxygenase-1 (HMOX1) isoenzyme that
catabolizes heme.
 The reason why cells must increase exponentially their capability to
degrade heme in response to oxidative stress remains unclear, but this
appears to be part of a cytoprotective response that avoids the deleterious
effects of free heme.
 When large amounts of free heme accumulates, the heme detoxification
/degradation systems get overwhelmed, enabling heme to exert its damaging
effects.
In the second reaction:
Biliverdin is converted to bilirubin by biliverdin reductase (BVR).

In the third reaction:

 Bilirubin is transported into the liver by facilitated diffusion bound to a protein
(serum albumin), where it is conjugated with glucuronic acid to become more
water-soluble.
The reaction is catalyzed by the enzyme UDP-glucuronosyltransferase.

 This form of bilirubin is excreted from the liver in bile.

 Excretion of bilirubin from liver to biliary canaliculi is an active, energy-
dependent and rate-limiting process.
 The intestinal bacteria deconjugate bilirubin diglucuronide and convert
bilirubin to urobilinogens.
 Some urobilinogen is absorbed by intestinal cells and transported into the
kidneys and excreted with urine (urobilin, which is the product of oxidation
of urobilinogen, and is responsible for the yellow colour of urine).
 The remainder travels down the digestive tract and is converted to
stercobilinogen.
 This is oxidized to stercobilin, which is excreted and is responsible for the
brown color of feces.
Heme in Health and Disease:
 Under homeostasis, the reactivity of heme is controlled by its insertion into the
“heme pockets” of hemoproteins.
 Under oxidative stress however, some hemoproteins, e.g. hemoglobin, can
release their heme prosthetic groups.
 The non-protein-bound (free) heme produced in this manner becomes highly
cytotoxic, most probably due to the iron atom contained within its protoporphyrin
IX ring, which can act as a Fenton's reagent to catalyze in an unfettered manner
the production of free radicals.
 It catalyzes the oxidation and aggregation of proteins, the formation of
cytotoxic lipid peroxide via lipid peroxidation and damages DNA through
oxidative stress.
 Due to its lipophilic properties, it impairs lipid bilayers in organelles such as
mitochondria and nuclei.

Porphyria:
 Porphyria is a name given to a group of metabolic disorders.
 These disorders cause the individual to accumulate "porphyrins" or "porphyrin
precursors" in their body.
 Which in turn causes an abundance of the porphyrins.
 In porphyria, the cells do not convert porphyrins to heme in a normal manner,
which will result in the accumulation of the heme synthesis intermediate
products, related to as Porphyria.
 The types that affect the nervous system are also known as acute porphyria, as
symptoms are rapid in onset and short in duration.
 Symptoms of an attack include abdominal pain, chest pain, vomiting,
confusion, constipation, fever, high blood pressure, and high heart rate.

Porphyrias are genetic diseases in which activity of one of the enzymes involved
in heme synthesis is decreased (e.g., PBG Synthase, Porphobilinogen
Deaminase, etc…).
They are dominate, recessive autosomal and X-linked genes.
Symptoms of porphyria vary depending on:
 The enzyme.
 The severity of the deficiency (partial or complete).
 Whether heme synthesis is affected primarily in liver or in developing
erythrocytes.

2) Photosensitivity:
 Is another common symptom of porphyria.
 Formation of superoxide radicals.
 Skin damage may result from exposure to light.
 This is attributable to elevated levels of
light-absorbing pathway intermediates and
their degradation products.

When Porphyrins present in very high levels, they

cause the urine to have a port yellow color.
 Porphyrias can be grouped into erythropoietic porphyria and hepatic porphyria:
 Hepatic can be acute or chronic.
 Caused by hereditary or acquired defects in heme synthesis:
 Genetic diseases: the enzymes of heme synthesis.
 Liver dysfunction, lead poisoning.

Common symptom of Porphyrias:

Occasional episodes of severe neurological symptoms.
 Permanent nerve damage and even death can result, if not treated promptly.

Acute hepatic porphyrias:

 Acute Intermittent porphyria is a result of a deficiency (Uroporphyrinogen I
synthase) in the heme biosynthesis pathway.
 These deficiencies result in an accumulation of the precursors of
porphyrins in the liver (delta-aminolevulinic acid, ALA and
porphobilinogen, PBG).
 variagate porphyria (Protoporphyrinogen oxidase).
Hereditary coproporphyria (Coproporphyrinogen oxidase), an accumulation of
porphyrins resulting in cutaneous manifestations.

Porphyria cutanea tarda:

 A chronic porphyria in liver and erythroid tissues.
 Deficiency in uroporphyrinogen decarboxylase.
 Often no symptoms until 4th or 5th decade.
Clinical expression determined by many factors:
 Hepatic iron overload.
 Exposure to sunlight.
 Hepatitis B or C.
Symptoms include:
 Cutaneous rashes, blisters.
 Urine that is red to brown in natural light, or pink to red in UV light.
Erythropoietic porphyrias:
Congenital erythropoietic porphyria (uroporphyrinogen III synthase).
erythropoietic protoporphyria (ferrochelatase).

Symptoms include:
 Skin rashes and blisters early in childhood.
 cholestatic liver cirrhosis and progressive liver failure.

Treatment for Porphyrias:

 Medical support for vomiting and pain.
 Hemin, decreases ALA synthase synthesis.
 Avoidance of sunlight and precipitating drugs, factors.

 “Monkey children” type of acquired porphyria.

Lead poisoning
 Inhibition of ferrochelatase, ALA dehydratase.
Displaces Zn+2 at enzyme active site.
Children:
Developmental defects.
Drop in IQ.
Hyperactivity.
Insomnia.
Many other health problems.
Adults:
Severe abdominal pain.
Mental confusion.
Many other symptoms.
Prehepatic (hemolytic) jaundice:
 Results from excess production of bilirubin (beyond the livers ability to
conjugate it) following hemolysis.
 Excess RBC lysis is commonly the result of autoimmune disease; hemolytic
disease of the newborn (Rh- or ABO incompatibility); structurally abnormal RBCs
(Sickle cell disease).
 High plasma concentrations of unconjugated bilirubin (normal concentration
~0.5 mg/dL).

Intrahepatic jaundice
 Impaired uptake, conjugation, or secretion of bilirubin.
 Reflects a generalized liver (hepatocyte) dysfunction.
 In this case, hyperbilirubinemia is usually accompanied by other abnormalities
in biochemical markers of liver function.

Posthepatic jaundice:
 Caused by an obstruction of the biliary tree.
 Plasma bilirubin is conjugated, and other biliary metabolites, such as bile acids
accumulate in the plasma.
 Characterized by pale colored stools (absence of fecal bilirubin or urobilin),
and dark urine (increased conjugated bilirubin).
 In a complete obstruction, urobilin is absent from the urine.

Neonatal Jaundice:
 Common, particularly in premature infants.
 Transient (resolves in the first 10 days).
 Due to immaturity of the enzymes involved in bilirubin conjugation.
 High levels of unconjugated bilirubin are toxic to the newborn – due to its
hydrophobicity it can cross the blood-brain barrier and cause a type of
mental retardation known as kernicterus.
 If bilirubin levels are judged to be too high, then phototherapy with UV light
is used to convert it to a water soluble, non-toxic form.
IRON METABOLISM:
 The body cannot get rid of iron except in two cases:
1) Shedding the epithelium.
2) The menstrual cycle; therefore, women are more susceptible to iron deficiency.
 The total quantity of iron in the body averages 4 to 5 grams, about 65% of
which is in the form of hemoglobin.
4% myoglobin
1% heme compounds
0.1% combined with the protein transferrin in the blood
Stored for later mainly in the reticuloendothelial system
15% to 30% and liver parenchymal cells, principally in the form of
ferritin.

Iron two forms:

Ferric Fe+3 Can’t be absorbed
Ferrus Fe+2 Can be absorbed

The iron according to the sites:

Blood Serum iron
Tissue Ferritin

Transport and Storage of Iron:

 When iron is absorbed from the small intestine, it immediately combines in the
blood plasma with a beta globulin, apotransferrin, to form transferrin, which is then
transported in the plasma.

 The iron is loosely bound in the transferrin and, consequently, can be released to
any tissue cell at any point in the body.
 Excess iron in the blood is deposited especially in the liver hepatocytes and less
in the reticuloendothelial cells of the bone marrow.
 In the cell cytoplasm, iron combines mainly with a protein, apoferritin, to form
ferritin.
 Apoferritin has a molecular weight of about 460,000, and varying quantities of iron
can combine in clusters of iron radicals with this large molecule; therefore, ferritin
may contain only a small or a large amount of iron. This iron stored as ferritin is
called storage iron.
Smaller quantities of the iron in the storage pool are in an extremely insoluble
form called hemosiderin.
 This is especially true when the total quantity of iron in the body is more
than the apoferritin storage pool can accommodate.
Hemosiderin large particles can be observed microscopically
ferritin So small particles Can be seen in the cell cytoplasm only
with an electron microscope.

How does iron enter RBCs?

1) When the quantity of iron in the plasma falls low, some of the iron in the ferritin
storage pool is removed easily and transported in the form of transferrin in the
plasma to the areas of the body where it is needed.
2) The transferrin molecule binds strongly with receptors in the cell membranes
of erythroblasts in the bone marrow.
3) Then, along with its bound iron, it is ingested into the erythroblasts by
endocytosis.
4) There the transferrin delivers the iron directly to the mitochondria, where heme
is synthesized.
In people who do not have adequate quantities of transferrin in their blood, failure
to transport iron to the erythroblasts in this manner can cause severe
hypochromic anemia (i.e., RBCs that contain much less hemoglobin than normal).

Recycling of RBCs:
About 90% of the red blood cells are recycled by macrophages within the spleen,
liver and lymph nodes.
 When RBCs have lived their life span of about 120 days and are destroyed,
the hemoglobin released from the cells is ingested by monocyte-
macrophage cells.
 There, iron is liberated and is stored mainly in the ferritin pool to be used as
needed for the formation of new hemoglobin.
man woman
An average excretes about 0.6 mg of iron Additional menstrual loss of
each day, mainly into the feces. Additional blood brings long-term iron loss
quantities of iron are lost when bleeding to an average of about 1.3
occurs mg/day.
Absorption of Iron from the Intestinal Tract
 Iron absorption from the intestines is extremely slow, at a maximum rate of
only a few milligrams per day. This slow rate of absorption means that even when
tremendous quantities of iron are present in the food, only small proportions can
be absorbed.
 Iron is absorbed from all parts of the small intestine, mostly by the following
mechanism.
1) The liver secretes moderate amounts
of apotransferrin into the bile, which
flows through the bile duct into the
duodenum.
2) The apotransferrin binds with free iron
and also with certain iron compounds,
such as hemoglobin and myoglobin
(monomers) from meat, two of the most
important sources of iron in the diet.
 This combination is called
transferrin.
3) In turn, it is attracted to and binds with
receptors in the membranes of intestinal
epithelial cells.
4) By pinocytosis, the transferrin
molecule, carrying its iron store, is
absorbed into the epithelial cells and
later released into the blood capillaries
beneath these cells in the form of plasma
transferrin.

Regulation of Total Body Iron by Controlling Absorption Rate.

Total body iron is regulated mainly by altering the rate of absorption.
When the body becomes saturated
with iron  so that essentially all when the iron stores become depleted,
apoferritin in the iron storage areas is
already combined with iron.
The rate of additional iron absorption The rate of absorption can probably
from the intestinal tract markedly accelerate five or more times normal.
decreases.
Mature RBCs do not have a nucleus, mitochondria, or endoplasmic reticulum,
they do have cytoplasmic enzymes that are capable of metabolizing glucose and
forming small amounts of adenosine triphosphate.
These enzymes also do the following:
(1) Maintain pliability (‫ )مرونة‬of the cell membrane.
(2) Maintain membrane transport of ions.
(3) Keep the iron of the cells’ hemoglobin in the ferrous form rather than the ferric
form.
(4) Prevent oxidation of the proteins in the RBCs.
Even so, the metabolic systems of old RBCs become progressively less active,
and the cells become more and more fragile, presumably because their life
processes wear out.
 Once the RBC membrane becomes fragile, the cell ruptures during passage
through some tight spot of the circulation.
 Many of the RBCs self-destruct in the spleen, where they squeeze through the
red pulp of the spleen.
 There, the spaces between the structural trabeculae of the red pulp, through
which most of the cells must pass, are only 3 micrometers wide, in
comparison with the 8-micrometer diameter of the RBC.
 When the spleen is removed, the number of old abnormal RBCs circulating in
the blood increases considerably.

Destruction of Hemoglobin by Macrophages.

 When RBCs burst and release their hemoglobin, the hemoglobin is
phagocytized almost immediately by macrophages in many parts of the body, but
especially by the Kupffer cells of the liver and macrophages of the spleen and
bone marrow.
 During the next few hours to days, the macrophages release iron from the
hemoglobin and pass it back into the blood to be carried by transferrin
either to the bone marrow for production of new RBCs or to the liver and
other tissues for storage in the form of ferritin.
The porphyrin portion of the hemoglobin molecule is converted by the
macrophages, through a series of stages, into the bile pigment bilirubin, which is
released into the blood and later removed from the body by secretion through the
liver into the bile.
 How Significant Is The Presence Of Hemoglobin To Human Body?
1) Presence of Hb in blood gives less load to heart.

 Without hemoglobin; the heart would have to pump 140 liters per
minute Instead of normally 4 liters per minute.

 Body cells requires approximate 500 g/day of molecular oxygen.

 Molecular oxygen is sparingly (‫ )قليل‬soluble in body fluids.
 This limits the oxygen transport in blood < 30 g/day.
In fact if the body had to depend upon dissolved oxygen in the plasma to supply
oxygen to the cells (diffusion).
 Presence of Hb in blood facilitates the blood to dissolve approximate 70 times
more oxygen than the plasma without Hb can do.

2) Oxygen transported by Hb and reached to every cell is used up in

mitochondrial oxidative Phosphorylation: ATP production.

Oxygenation/Loading of Oxygen
 Hemoglobin gets oxygenated:
 At lungs
 At increased pO2 concentration (100-120 mm Hg) and decreased pCO2

Normal ranges of pO2:

Vessels Arteries of the lung Veins of the tissues
Pressure 100 – 120 mmHg 35 – 40 mmHg

Vessels Arterial Blood-Oxy Hb Venous blood- Deoxy Hb

Saturation with oxygen 97% when it leaves the Lungs. 75%
The degree of saturation with oxygen is related to:
 Oxygen tension (pO2).
 Oxygen requirement for metabolic use at cellular level.

Deoxygenation/Unloading or Off loading of Oxygen

Hemoglobin gets deoxygenated:
 At Tissues.
 With increased pCO2 and decrease pO2.

 Globin chains move closer when Hb is oxygenated.

 Globin chains are pulled apart (‫ )تتفكك‬when Hb is deoxygenated.

 Deoxy Hb has 2,3-Bis PhosphoGlycerate

(2,3BPG) within it located centrally.
 2,3-BPG is pushed out of the Deoxy Hb
molecule during oxygenation.
 When the oxygen exits, the affinity
(2,3BPG) of hemoglobin increases and it binds
to the center between the two chains (𝛃𝟏 −
𝛃𝟐), which leads to a change in the active site
of oxygen binding.
 2,3-BPG reduces the HB affinity to Hb and
thus enhances the cooperatively between the
Hb subunits.

Fetal Hb has gamma subunit. This Hb doss NOT bind 2,3BPG.

Fetal hemoglobin’s “gamma – 72% same beta” chains have serine in place of
histidine 143 (adult beta chains).
 This serine is near the binding site for 2,3-BPG and reduces the affinity of
fetal Hb for 2,3-BPG  The result of this change and reduced affinity for 2,3-
BPG is an increased affinity for oxygen.
 Therefore, a fetus can effectively draw oxygen across the placental membrane
especially at low pO2.
Oxygen binds to hemoglobin with Cooperative Mechanism
 Hb is an allosteric oxygen binder with
cooperative mechanism.
 Cooperative binding mechanism is due to
tetrameric structure of Hb.
 The binding of the first O2 to one subunit of Hb
enhances the binding of further O2 molecules to
remaining subunits of Hb with greater affinities
(Positive cooperativity).
Fourth Oxygen molecule binds to fourth subunit
of Hb 300 times rapidly and tightly as that of first
oxygen bound to first subunit.
 When a First Oxygen binds to Fe in Heme of
Hb, the Heme Fe is drawn into the plane of
the Porphyrin ring.
 This initiates a series of small
conformational changes that are transmitted
to adjacent Globin subunits.
 This induces more of the R-state, allowing
for more oxygen to bind.

During deoxygenation hemoglobin releases its bound oxygen.

 As soon as the first oxygen molecule drops off, the Hemoglobin starts
changing its shape.
 This prompts the remaining three Oxygen molecules to be quickly released.
In this positive cooperative way
 Hemoglobin picks up the largest possible load of oxygen in the lungs and
delivers the oxygen where and when needed.
The cooperative binding of oxygen by hemoglobin enables it to deliver 1.7 times
as much oxygen as it would if the sites were independent.
The homotropic regulation of hemoglobin by its ligand oxygen dramatically
increases its physiological oxygen-carrying capacity.
Max Perutz revealed the structure of hemoglobin in various formconformationals.
Because of cooperativity between O2 binding sites, hemoglobin delivers more O2
to tissues than would myoglobin or any noncooperative protein, even one with
optimal O2 affinity.

Let us consider how the cooperative behavior indicated by the sigmoid curve
leads to efficient oxygen transport.
 In the lungs, hemoglobin becomes nearly saturated with oxygen such that 98%
of the oxygen-binding sites are occupied.
 When hemoglobin moves to the tissues and releases O2, the saturation
level drops to 32%.
 Thus, a total of (98 – 32) = 66% of the potential oxygen-binding sites
contribute to oxygen transport.
 If myoglobin were employed for oxygen transport, it would be 98% saturated in
the lungs, but would remain 91% saturated in the tissues.
 Only (98 – 91) = 7% of the sites would contribute to oxygen transport.
 The most oxygen that could be transported from a region in which pO2 is
100 torr to one in which it is 20 torr is (63 – 25) = 38%.

Effectors may be positive or negative;

Homotropic (when it is the pysiological ligand [substarte]).
Heterotropic effectors.
 Oxygen is a homotropic positive effector.
 Globin genes and molecular biology of globin synthesis
There must be 2 Alpha and 2 non alpha for oxygen carrying function, which
differs at different stages of foetal and early neonatal life.
Normal adult hemoglobin (HbA) consists of four polypeptide chains α2-β2
Normally in adult blood, small quantities of two other varieties of hemoglobins i.e.
HbA2 and HbF are found.

Embryonic Hb:
 Hbε Gower-1 (ζ2ε2) Primary Hb in embryonic life ≈ 8 wks.
Portland-1 (ζ2γ2) is the ζ-substituted counterpart of fetal HbF (α2γ2) (By 12 weeks
of gestation, embryonic Hb changes into HbF.
 Hb Portland-2 (ζ2β2) since it occurs only in cases of an extreme type of α-
thalassemia.

Fetal hemoglobin (α2γ2):

Lifespan of RBC containing HBF is decreased to 80 days only.
  HbF can take up large volume of oxygen at lower PO2.
 At pO2 around 28mmHg, 50% of HbA is saturated but
nearly 80% of the HbF are saturated at same pO2.

Hemoglobin and allosteric effectors:

 A variety of molecules affect the O2 binding by Hb (and
their binding is in turn affected by O2 binding).

 These are allosteric effectors of Hb binding of O2.

 Some of these molecules are also transported by Hb.
A. O2 - positively affects binding of O2 (positive cooperativity).  {Homotropic}
This results from the breakage of salt bridges between Hb subunits that help stabilize
the "tense" deoxy forms of the subunits.

 When oxygen binding leading to conformational shape  relaxing form.

B. H+ negatively affects binding of O2.  {Heterotropic}

 For example, H+ is bound by a histidine residue in the beta chain that forms a salt
bridge with an aspartate residue only in the deoxy form.
 These salt bridges stabilize the deoxy form.
 This histidine has a higher pKa in the deoxy form than it does in the oxy form of
Hb.
 As a result, more H+ is bound to the deoxy form than to the oxy form.
 H+ is preferentially carried from the tissues to the lungs.

C. CO2 negatively affects binding of O2.  {Heterotropic}

 CO2 is bound by Hb in a carbamate linkage to the N-terminus of the beta chain
and is carried from the tissues to the lungs  stabilizing T form of Hb.

D. 2,3-Bisphosphoglycerate (BPG) negatively affects binding of O2.  {Heterotropic}

 Changes in BPG concentration are associated with adaptation to altitude.
Smokers also have elevated BPG because the oxygen carrying capacity of their
blood is reduced due to the inhalation of CO in tobacco smoke.

Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen: The Bohr Effect

The Bohr Effect describes hemoglobin's lower affinity for oxygen secondary to increases in the partial
pressure of carbon dioxide and/or decreased blood pH. This lower affinity, in turn, enhances the
unloading of oxygen into tissues to meet the oxygen demand of the tissue
 According to local regulations (allosteric) there are two states:

1) R-state: 2) T-state:
Active Relaxed. Inactive Tense.

 Allosterically Regulated Enzymes Do Not Follow Michaelis-Menten

Kinetics.
 Binding of the O to hemoglobin would lead to allosteric changes switching
from the T (tight state-not active) to the R-(relaxed more active) state,
favoring more binding of oxygen molecules.
 This is referred to as Cooperative binding.
 Yet the shape of the rate of the reaction is not typical MM shape, but rather
has a sigmoidal shape (S-shape), repressing both states of the enzyme, the
T and R states.
 When in T state (inhibited) need higher concentration of S to reverse the
action of inhibition by the inhibitor.
 Rate of reaction (velocity) increasing with more S bound to E, leading
exponential increase in the rate, until reaching a Vmax plateau.

 The T conformation of hemoglobin is unstable and needs to be stabilized.

 On the other hand link O2 will encourage beyond R and in this situation the O2
can be released.
 Therefore, an additional mechanism is needed to stabilize the various
conformations. This mechanism was discovered when comparing the O2 binding
to hemoglobin in blood cells versus isolated hemoglobin.
1) Isolated hemoglobin binds the O2 much more tightly. It turned out that the red
blood cells contain 2,3BPG (not exist in fetus).
2) It is found in a red blood cell at a concentration similar to hemoglobin ~ 2mM.
2,3BPG decreases the affinity of hemoglobin for O2.
 BPG binds in the middle of the hemoglobin molecule between all the subunits
only when the protein is in the T state.
Animals at heights (mountains) have more T in order to release more O2 in the
tissues.
In the transition from T to R, the bonds between BPG and hemoglobin are broken
and this occurs when most sites are occupied by O2. Therefore in this state (R),
O2 can be released.
The Bohr Effect:
 The regulation of oxygen binding by hydrogen ions and carbon dioxide.
 Two heterotopic factors effectors of hemoglobin that affect oxygen release in
tissue.
 The heterotopic regulation of hemoglobin by hydrogen ions and carbon
dioxide further increases the oxygen-transporting
efficiency of this magnificent allosteric protein.
 CO2 and H+, which are released at high PO2 in the lungs.
 In tissue, needy for oxygen, the pH (7.2) is lower than in
lung (7.4) due to large amount of CO2 and H+, in the cells, in
addition to low pO2 pressure.
 The oxygen affinity of hemoglobin decreases as pH
decreases from a value of 7.4 leading to releasing 77% of
loaded oxygen.
 At high CO2 concentration in tissue, oxygen is further
released / replaced by CO2.
 At pCO2 of nearly 40 torr, the amount of oxygen
released is nearly 90%.

Haemoglobin VS leghaemoglobin
Haemoglobin Red colored protein present in Alpha = chromosome 16
red blood cells (erythrocytes). Beta = chromosome 11
leghaemoglobin Red pigment present in Alpha = chromosome 16
the root nodules of The others chains encoded by
leguminous plants and assists chromosome 11
in nitrogen fixation.

Hemoglobin electrophoresis
 Is a blood test that can detect different types of hemoglobin.
 The test can detect hemoglobin S, the form associated with sickle cell disease,
as well as other abnormal types of hemoglobin, such as hemoglobin C.
 It can also be used to investigate thalassemias.
Procedure:
 The test uses the principles of gel electrophoresis to separate out the various types of
hemoglobin and is a type of native gel electrophoresis.
 After the sample has been treated to release the hemoglobin from the red cells, it is
introduced into a porous gel (usually made of agarose or cellulose acetate) and
subjected to an electrical field, most commonly in an alkaline medium.
 Different hemoglobins have different charges, and according to those charges, they
move at different speeds in the gel and eventually form discrete bands.
 A quality control sample containing hemoglobins A, F, S, and C is
run along with the patient sample to aid in identifying the different
bands.
The relative amounts of each type of hemoglobin can be estimated
by measuring the optical density of the bands, though this method is
not reliable for hemoglobins that are present in low quantities.

Because hemoglobins exhibit different migration patterns depending

on the pH level, testing the same sample at both an acid and an
alkaline pH can help to identify some abnormal hemoglobins that
would otherwise be impossible to distinguish from others.

Clinical significance:
Adult human blood normally contains three types of hemoglobin:
 Hemoglobin A, which makes up approximately 95% of the total.
 Hemoglobin A2, which accounts for less than 3.5%; and a minute amount of
hemoglobin F.
 If abnormal hemoglobin variants such as hemoglobin S, C or E are present, they will
appear as unexpected bands on electrophoresis (provided they do not migrate to the
same place as other hemoglobins).
 Hemoglobin A2 levels are typically elevated in beta-thalassemia minor and
hemoglobin F may be slightly increased.
 In beta-thalassemia major, hemoglobin A is decreased (or in some cases absent) and
hemoglobin F is markedly elevated; A2 levels are variable.
 In hemoglobin H disease, a form of alpha-thalassemia, an abnormal band of
hemoglobin H can be detected, and sometimes a band of Hemoglobin Barts; but in the
milder alpha-thalassemia trait, electrophoresis results are effectively normal
 2) White blood cells (leucocytes)
Classification of WBCs:
1) Granular leukocytes (polymorphnucleated):

 Neutrophils {≈60% of WBC}.

 Eosinophils {≈4% of WBC}.
 Basophils {<1% of WBC}.

2) agranular leukocytes:

 Lymphocytes = T cells, B cells and natural killer {≈27% of WBC}.

 Monocytes (macrophages) {≈8% of WBC}.
Neutrophil (polymorphonuclear leukocyte):
1. Life Span: < 1 week.

2. Granulocyte with specific and non-specific granules.

Specific granules Non-specific/AZUROPHILIC granules

Type IV collagenase (aids migration) Lysozyme
Lactoferrin (sequesters iron). Acid hydrolase
Phospholipase A (leukotriene synthesis). Myeloperoxidase
Lysozyme (digests bacterial cell wall). Elastase

3. LM appearance in smear:

 About 9-12 𝝁𝒎 in diameter (thus larger than

RBC).
 Nucleus long and multi-lobed (usually 3-5 lobes).

4. Function:

 Primarily antibacterial.
 Neutrophils leave the blood and follow
chemotaxis signals to sites of wounding or other
inflammation, and phagocytose foreign agents
such as bacteria.
 Pus is composed largely of dead neutrophils.
Eosinophil:
1. Life Span: < 2 weeks.

2. Granulocyte with specific and non-specific granules.

Specific granules Non-specific/AZUROPHILIC granules

Major basic protein Lysozyme
Eosinophilic cationic protein Acid hydrolase
Neurotoxin Myeloperoxidase
Histaminase Elastase

2. LM appearance in smear:

 About 10-14 𝝁𝒎 in diameter.

 Bi-lobed nucleus.
 The cytoplasm has prominent pink/red
specific granules (stained with eosin
dye).

3. Function:

 Anti-parasitic activity.
 Mediators of inflammatory/allergic
responses in tissues.
Basophil
Lifespan: 1-2 years.

2. Granulocyte with specific and non-specific granules.

Specific granules Non-specific/AZUROPHILIC granules

Histamine Lysozyme
Heparin Acid hydrolase
Eosinophil chemotactic factor Myeloperoxidase
Phospholipids for synthesis of leukotrienes, e.g. Elastase
slow-reacting substance of anaphylaxis ( SRS-A)

3. LM appearance in smear:

 About 8-10 𝝁𝒎 in diameter.

 The cytoplasm contains large,
purple/black specific granules
(stained with the basic dye)
that are larger but not as numerous
as those of eosinophils.
 The nucleus is usually bilobed, but
usually is partially obscured by
granules, which can lie
over it.

4. Function:

 Allergies and anaphylaxis

(hypersensitivity reaction).

5. Similarity to tissue mast cells:

 Tissue mast cells also have IgE

receptors and similar (though not
identical) granule content.
 Mast cells and basophils have a
common precursor in bone marrow.
 Lymphocyte:
1. Life Span: variable (few days to several years).

2. LM appearance in smear:

 Small lymphocyte (about 90% of lymphocytes you

will see) are ~8 𝝁𝒎.
in diameter with a narrow rim of cytoplasm around
the nucleus, and
when well stained is pale blue.
 Large lymphocytes may be up to about 15 𝝁𝒎.
 Round, dense nucleus (abundant
heterochromatin).
 T-lymphocytes and B-lymphocytes cannot be
distinguished in a smear.

3. Function:

B-lymphocytes (B-cells): T-lymphocytes (T-

cells):
humoral immunity Cellular immunity
May differentiate into Cytotoxic T cells and
memory or tissue plasma helper T cells.
cells which make antibodies.
Monocyte:
1. Life Span: few days in blood, several months in connective tissue.

2. LM appearance in smears:

 About 16 𝝁𝒎 in smears (the largest

leukocyte).
 Large, eccentric nucleus either oval,
kidney-shaped or horseshoe-shaped, with
delicate chromatin that is less dense than that
of lymphocytes (heterochromatin).
 Pale cytoplasm, often grayish, may contain
occasional stained granules (lysosomes =
azurophilic granules).
 Large lymphocytes may resemble
monocytes, but the lymphocyte nucleus is
usually more dense.

3. Function:

 Migrate into tissues and constitute mononuclear phagocyte system that

help destroy foreign bodies and maintain or remodel tissues and mediate
inflammatory response.
 Tissue macrophages Kupfer cells (liver), Osteoclasts (bone), Dust cells
(lungs) and Microglia (brain).
 Antigen presenting cells: Dendritic Cells, Langerhans cells.
 CHAPTER 34

Resistance of the Body to Infection: I. Leukocytes,

UNIT VI
Granulocytes, the Monocyte-Macrophage System,
and Inflammation

Our bodies are exposed continually to bacteria, viruses, eosinophils (polymorphonuclear), basophils (polymorpho-
fungi, and parasites, all of which occur normally and to nuclear), monocytes, lymphocytes and, occasionally, plasma
varying degrees in the skin, mouth, respiratory passage- cells. In addition, there are large numbers of platelets, which
ways, intestinal tract, lining membranes of the eyes, and are fragments of another type of cell similar to the WBCs
even the urinary tract. Many of these infectious agents found in the bone marrow, the megakaryocyte. The first three
are capable of causing serious abnormal physiological types of cells, the polymorphonuclear cells, all have a granu-
function or even death if they invade deeper tissues. We lar appearance, as shown in cell numbers 7, 10, and 12 in
are also exposed intermittently to other highly infectious Figure 34-1, and for this reason they are called granulocytes.
bacteria and viruses besides those that are normally pres- The granulocytes and monocytes protect the body
ent, and these agents can cause acute lethal diseases such against invading organisms by ingesting them (by phago-
as pneumonia, streptococcal infection, and typhoid fever. cytosis) or by releasing antimicrobial or inflammatory
Our bodies have a special system for combating the dif- substances that have multiple effects that aid in destroy-
ferent infectious and toxic agents. This system is composed ing the offending organism. The lymphocytes and plasma
of blood leukocytes (white blood cells [WBCs]) and tissue cells function mainly in connection with the immune
cells derived from leukocytes. These cells work together system, as discussed in Chapter 35. Finally, the function
in two ways to prevent disease: (1) by actually destroying of platelets is specifically to activate the blood-clotting
invading bacteria or viruses by phagocytosis; and (2) by mechanism, discussed in Chapter 37.
forming antibodies and sensitized lymphocytes that may
destroy or inactivate the invader. This chapter discusses the
first of these methods, and Chapter 35 discusses the second. Concentrations of Different White Blood Cells in
Blood. An adult human has about 7000 WBCs per mi-
croliter of blood (in comparison with 5 million red blood
LEUKOCYTES (WHITE BLOOD CELLS)
cells [RBCs] per microliter). Of the total WBCs, the nor-
The leukocytes, also called white blood cells, are the mobile mal percentages of the different types are approximately
units of the body’s protective system. They are formed the following:
partially in the bone marrow (granulocytes and monocytes • Neutrophils: 62.0%
and a few lymphocytes) and partially in the lymph tissue • Eosinophils: 2.3%
(lymphocytes and plasma cells). After formation, they are • Basophils: 0.4%
transported in the blood to different parts of the body • Monocytes: 5.3%
where they are needed. • Lymphocytes: 30.0%
The real value of WBCs is that most of them are specif- The number of platelets, which are only cell fragments,
ically transported to areas of serious infection and inflam- in each microliter of blood is normally between 150,000
mation, thereby providing a rapid and potent defense and 450,000, averaging about 300,000.!
against infectious agents. As we see later, the granulo-
cytes and monocytes have a special ability to “seek out
GENESIS OF WHITE BLOOD CELLS
and destroy” a foreign invader.
Early differentiation of the multipotential hematopoi-
etic stem cell into the different types of committed stem
GENERAL CHARACTERISTICS OF
cells was shown in Figure 33-2 in the previous chapter.
LEUKOCYTES
Aside from the cells committed to form RBCs, two major
Types of White Blood Cells. Six types of WBCs are nor- lineages of WBCs are formed, the myelocytic and lym-
mally present in the blood: neutrophils (polymorphonuclear), phocytic lineages. The left side of Figure 34-1 shows the

449
UNIT VI Blood Cells, Immunity, and Blood Coagulation

Genesis of Myelocytes Genesis of Lymphocytes

1

3
2

13

4 8 11

14

5
Figure 34-1. Genesis of white blood
cells. The different cells of the myelo- 9
cyte series are shown: 1, myeloblast;
15
2, promyelocyte; 3, megakaryocyte; 4,
6
neutrophil myelocyte; 5, young neutro-
phil metamyelocyte; 6, band neutrophil
metamyelocyte; 7, neutrophil; 8, eo-
sinophil myelocyte; 9, eosinophil meta- 10 12 16
myelocyte; 10, eosinophil; 11, basophil 7
myelocyte; 12, basophil; 13–16, stages
of monocyte formation.

myelocytic lineage, beginning with the myeloblast; the needed. In times of serious tissue infection, this total life
right side shows the lymphocytic lineage, beginning with span is often shortened to only a few hours because the
the lymphoblast. granulocytes proceed even more rapidly to the infected
The granulocytes and monocytes are formed only area, perform their functions, and in the process, are
in the bone marrow. Lymphocytes and plasma cells are themselves destroyed.
produced mainly in the various lymphogenous tissues— The monocytes also have a short transit time, 10 to 20
especially the lymph glands, spleen, thymus, tonsils, and hours in the blood, before wandering through the cap-
various pockets of lymphoid tissue elsewhere in the body, illary membranes into the tissues. Once in the tissues,
such as in the bone marrow and in Peyer’s patches under- they swell to much larger sizes to become tissue macro-
neath the epithelium in the gut wall. phages and, in this form, they can live for months unless
The WBCs formed in the bone marrow are stored in destroyed while performing phagocytic functions. These
the marrow until they are needed in the circulatory sys- tissue macrophages are the basis of the tissue macrophage
tem. Then, when the need arises, various factors cause system (discussed in greater detail later), which provides
them to be released (these factors are discussed later). continuing defense against infection.
Normally, about three times as many WBCs are stored in Lymphocytes enter the circulatory system continually,
the marrow as circulate in the entire blood. This quantity along with drainage of lymph from the lymph nodes and
represents about a 6-day supply of these cells. other lymphoid tissue. After a few hours, they pass out
The lymphocytes are mostly stored in the various lym- of the blood back into the tissues by diapedesis/extrava-
phoid tissues, except for a small number that are tempo- sation. Then, they re-enter the lymph and return to the
rarily being transported in the blood. blood again and again; thus, there is continual circulation
As shown in Figure 34-1, megakaryocytes (cell 3) are of lymphocytes through the body. Lymphocytes have life
also formed in the bone marrow. These megakaryocytes spans of weeks or months, depending on the body’s need
fragment in the bone marrow and the small fragments, for these cells.
known as platelets (or thrombocytes), then pass into the The platelets in the blood are replaced about once
blood. They are very important in the initiation of blood every 10 days. In other words, about 30,000 platelets are
clotting.! formed each day for each microliter of blood.!

LIFE SPAN OF WHITE BLOOD CELLS NEUTROPHILS AND MACROPHAGES

DEFEND AGAINST INFECTIONS
The life of the granulocytes after being released from the
bone marrow is normally 4 to 8 hours circulating in the It is mainly the neutrophils and tissue macrophages that
blood and another 4 to 5 days in tissues where they are attack and destroy invading bacteria, viruses, and other

450
 Chapter 34 Resistance of the Body to Infection

inflamed area are formed (Video 34-1). They include the

following: (1) some of the bacterial or viral toxins; (2) de-
generative products of the inflamed tissues; (3) several
reaction products of the complement complex (discussed
in Chapter 35) activated in inflamed tissues; and (4) sev-
Increased Margination Diapedesis

UNIT VI
permeability
eral reaction products caused by plasma clotting in the
inflamed area, as well as other substances.
As shown in Figure 34-2, chemotaxis depends on the
concentration gradient of the chemotactic substance. The
concentration is greatest near the source, which directs
the unidirectional movement of the WBCs. Chemotaxis
Chemotaxis is effective up to 100 micrometers away from an inflamed
source
tissue. Therefore, because almost no tissue area is more
than 50 micrometers away from a capillary, the chemo-
tactic signal can easily move hordes of WBCs from the
capillaries into the inflamed area.!

Chemotactic substance PHAGOCYTOSIS

Figure 34-2. Movement of neutrophils by diapedesis or extravasa-
A major function of the neutrophils and macrophages
tion through capillary pores and by chemotaxis toward an area of
tissue damage. is phagocytosis, which means cellular ingestion of the
offending agent. Phagocytes must be selective of the
material that is phagocytized; otherwise, normal cells and
harmful agents. The neutrophils are mature cells that structures of the body might be ingested. Whether phago-
can attack and destroy bacteria, even in the circulating cytosis will occur especially depends on three selective
blood. Conversely, the tissue macrophages begin life as procedures (Figure 34-3).
blood monocytes, which are immature cells while still in First, most natural structures in the tissues have
the blood and have little ability to fight infectious agents smooth surfaces, which resist phagocytosis. However,
at that time. However, once they enter the tissues, they if the surface is rough, the likelihood of phagocytosis is
begin to swell—sometimes increasing their diameters as increased.
much as fivefold—to as great as 60 to 80 micrometers, Second, most natural substances of the body have pro-
a size that can barely be seen with the naked eye. These tective protein coats that repel the phagocytes. Conversely,
cells are now called macrophages, and they are extremely most dead tissues and foreign particles have no protective
capable of combating disease agents in the tissues. coats, which makes them subject to phagocytosis.
Third, the immune system of the body (described in
White Blood Cells Enter the Tissue Spaces by Diape- Chapter 35) develops antibodies against infectious agents
desis. Neutrophils and monocytes can squeeze through such as bacteria. The antibodies then adhere to the bacterial
gaps between endothelial cells of the blood capillaries and membranes and thereby make the bacteria especially sus-
postcapillary venules by diapedesis. Although the inter- ceptible to phagocytosis. To do this, the antibody molecule
cellular gaps are much smaller than a cell, a small portion also combines with the C3 product of the complement cas-
of the cell slides through the gap at a time; the portion cade, which is an additional part of the immune system dis-
sliding through is momentarily constricted to the size of cussed in the next chapter. The C3 molecules, in turn, attach
the gap, as shown in Figure 34-2 (also see Figure 34-6).! to receptors on the phagocyte membrane, thus initiating
phagocytosis. This process whereby a pathogen is selected
White Blood Cells Move Through Tissue Spaces by for phagocytosis and destruction is called opsonization.
Ameboid Motion. Both neutrophils and macrophages can
move through the tissues by ameboid motion, described in Phagocytosis by Neutrophils. The neutrophils enter-
Chapter 2. Some cells move at velocities as great as 40 µm/ ing the tissues are already mature cells that can imme-
min, a distance as great as their own length each minute.! diately begin phagocytosis. On approaching a particle to
be phagocytized, the neutrophil first attaches itself to the
White Blood Cells Are Attracted to Inflamed Tissue particle and then projects pseudopodia in all directions
Areas by Chemotaxis. Many different chemical sub- around the particle. The pseudopodia meet one another
stances in the tissues cause both neutrophils and mac- on the opposite side and fuse. This action creates an en-
rophages to move toward the source of the chemical. This closed chamber that contains the phagocytized particle.
phenomenon, shown in Figure 34-2, is known as chemo- Then, the chamber invaginates to the inside of the cyto-
taxis. When a tissue becomes inflamed, at least a dozen plasmic cavity and breaks away from the outer cell mem-
different products that can cause chemotaxis toward the brane to form a free-floating phagocytic vesicle (also called

451
UNIT VI Blood Cells, Immunity, and Blood Coagulation

Microbe Both neutrophils and macrophages contain an abun-

Antibodies dance of lysosomes filled with proteolytic enzymes espe-
cially geared for digesting bacteria and other foreign
protein matter. The lysosomes of macrophages (but not of
neutrophils) also contain large amounts of lipases, which
PHAGOCYTOSIS
digest the thick lipid membranes possessed by some bac-
teria, such as the tuberculosis bacillus.!
Receptors
Neutrophils and Macrophages Can Kill Bacteria.
Pseudopod Phagosome In addition to the digestion of ingested bacteria in phago-
somes, neutrophils and macrophages contain bactericidal
agents that kill most bacteria, even when the lysosomal
enzymes fail to digest them. This characteristic is espe-
Lysosome cially important because some bacteria have protective
coats or other factors that prevent their destruction by
Phagolysosome
digestive enzymes. Much of the killing effect results from
several powerful oxidizing agents formed by enzymes in
EXOCYTOSIS
the membrane of the phagosome or by a special organelle
called the peroxisome. These oxidizing agents include
Digestion large quantities of superoxide (O2−), hydrogen peroxide
of microbe (H2O2), and hydroxyl ions (OH−), which are lethal to most
bacteria, even in small quantities. Also, one of the lyso-
Microbial
debris somal enzymes, myeloperoxidase, catalyzes the reaction
between H2O2 and chloride ions to form hypochlorite,
Figure 34-3. Phagocytosis of pathogens, such as bacteria, by a
phagocytic cell, such as a macrophage. Antibodies coat the bacteria, which is exceedingly bactericidal.
making them more susceptible to phagocytosis by the macrophage Some bacteria, notably the tuberculosis bacillus,
that engulfs the bacterium, bringing it into the cell and forming a have coats that are resistant to lysosomal digestion and
phagosome. Lysosomes then attach to the phagosome to form a also secrete substances that partially resist the killing
phagolysosome, which digests the invading pathogen. The phago-
effects of the neutrophils and macrophages. These bac-
cytic cell then releases the digested products by exocytosis.
teria are responsible for many chronic diseases, such as
tuberculosis.!
a phagosome) inside the cytoplasm. A single neutrophil
can usually phagocytize 3 to 20 bacteria before the neu-
MONOCYTE-MACROPHAGE CELL
trophil becomes inactivated and dies.!
SYSTEM (RETICULOENDOTHELIAL
SYSTEM)
Phagocytosis by Macrophages. Macrophages are the
end-stage product of monocytes that enter the tissues In the preceding paragraphs, we described the mac-
from the blood. When activated by the immune system, rophages mainly as mobile cells capable of wandering
as described in Chapter 35, they are much more powerful through the tissues. However, after entering the tissues
phagocytes than neutrophils, often capable of phagocyt- and becoming macrophages, another large portion of
izing as many as 100 bacteria. They also have the abil- monocytes becomes attached to the tissues and remains
ity to engulf much larger particles, even whole RBCs or, attached for months or even years until they are called
occasionally, malarial parasites, whereas neutrophils are on to perform specific local protective functions. They
not capable of phagocytizing particles much larger than have the same capabilities as the mobile macrophages to
bacteria. Also, after digesting particles, macrophages can phagocytize large quantities of bacteria, viruses, necrotic
extrude the residual products and often survive and func- tissue, or other foreign particles in the tissue. In addition,
tion for many more months.! when appropriately stimulated, they can break away from
their attachments and, once again, become mobile mac-
Once Phagocytized, Most Particles Are Digested by rophages that respond to chemotaxis and all the other
Intracellular Enzymes. Once a foreign particle has been stimuli related to the inflammatory process. Thus, the
phagocytized, lysosomes and other cytoplasmic granules body has a widespread monocyte-macrophage system in
in the neutrophil or macrophage immediately come into virtually all tissue areas.
contact with the phagocytic vesicle, and their membranes The total combination of monocytes, mobile macro-
fuse, thereby dumping many digestive enzymes and bac- phages, fixed tissue macrophages, and a few specialized
tericidal agents into the vesicle. Thus, the phagocytic vesi- endothelial cells in the bone marrow, spleen, and lymph
cle now becomes a digestive vesicle, and digestion of the nodes is called the reticuloendothelial system. However,
phagocytized particle begins immediately. all or almost all these cells originate from monocytic stem

452
 Chapter 34 Resistance of the Body to Infection

Afferent lymphatics Hepatocytes Endothelial cells Space of Disse

Primary
nodule Capsule

Subcapsular

UNIT VI
sinus

Valve

Lymph in
medullary
sinuses
Germinal
center
Hilus Medullary cord

Efferent lymphatics
Figure 34-4. Functional diagram of a lymph node.

cells; therefore, the reticuloendothelial system is almost Kupffer cells

synonymous with the monocyte-macrophage system. Figure 34-5. Kupffer cells lining the liver sinusoids, showing phago-
Because the term reticuloendothelial system is much bet- cytosis of India ink particles into the cytoplasm of the Kupffer cells.
ter known in medical literature than the term monocyte-
macrophage system, it should be remembered as a the lungs. Large numbers of tissue macrophages are pre-
generalized phagocytic system located in all tissues, espe- sent as integral components of the alveolar walls. They
cially in the tissue areas where large quantities of particles, can phagocytize particles that become entrapped in the
toxins, and other unwanted substances must be destroyed. alveoli. If the particles are digestible, the macrophages can
also digest them and release the digestive products into
Tissue Macrophages in Skin and Subcutaneous Tissues the lymph. If the particle is not digestible, the macrophag-
(Histiocytes). The skin is mainly impregnable to infectious es often form a giant cell capsule around the particle until
agents, except when it is broken. When infection begins in a such time—if ever—that it can be slowly dissolved. Such
subcutaneous tissue and local inflammation ensues, local tis- capsules are frequently formed around tuberculosis bacilli,
sue macrophages can divide in situ and form still more mac- silica dust particles, and even carbon particles.!
rophages. Then, they perform the usual functions of attack-
ing and destroying the infectious agents, as described earlier.! Macrophages (Kupffer Cells) in Liver Sinusoids. An-
other route whereby bacteria invade the body is through
Macrophages in Lymph Nodes. Essentially no particu- the gastrointestinal tract. Large numbers of bacteria from
late matter that enters the tissues, such as bacteria, can be ingested food constantly pass through the gastrointestinal
absorbed directly through the capillary membranes into mucosa into the portal blood. Before this blood enters the
the blood. Instead, if the particles are not destroyed locally general circulation, it passes through the liver sinusoids,
in the tissues, they enter the lymph and flow to the lymph which are lined with tissue macrophages called Kupffer
nodes located intermittently along the course of the lymph cells, shown in Figure 34-5. These cells form such an ef-
flow. The foreign particles are then trapped in these nodes fective particulate filtration system that almost none of
in a meshwork of sinuses lined by tissue macrophages. the bacteria from the gastrointestinal tract pass from the
Figure 34-4 illustrates the general organization of portal blood into the general systemic circulation. Indeed,
the lymph node, showing lymph entering through the videos of phagocytosis by Kupffer cells have demonstrat-
lymph node capsule via afferent lymphatics, then flowing ed phagocytosis of a single bacterium in less than 0.01
through the nodal medullary sinuses, and finally pass- second.!
ing out the hilus into efferent lymphatics that eventually
empty into the venous blood. Macrophages of Spleen and Bone Marrow. If an invad-
Large numbers of macrophages line the lymph sinuses ing organism succeeds in entering the general circulation,
and if any particles enter the sinuses by way of the lymph there are other lines of defense by the tissue macrophage
the macrophages phagocytize them and prevent general system, especially by macrophages of the spleen and bone
dissemination throughout the body.! marrow. In both these tissues, macrophages become en-
trapped by the reticular meshwork of the two organs, and
Alveolar Macrophages in Lungs. Another route where- when foreign particles come into contact with these mac-
by invading organisms frequently enter the body is through rophages, they are phagocytized.

453
UNIT VI Blood Cells, Immunity, and Blood Coagulation

system (described in Chapter 35), reaction products of the

blood clotting system, and multiple substances called lym-
phokines that are released by sensitized T cells (part of the
immune system; also discussed in Chapter 35). Several of
these substances strongly activate the macrophage system,
Pulp
and within a few hours, the macrophages begin to devour
Capillaries the destroyed tissues. At times, however, the macrophages
may also further injure the still-living tissue cells.
Venous sinuses
Walling-Off Effect of Inflammation. One of the first re-
Vein sults of inflammation is to wall off the area of injury from
Artery the remaining tissues. The tissue spaces and the lymphat-
ics in the inflamed area are blocked by fibrinogen clots so
that after a while, fluid barely flows through the spaces.
This walling-off process delays the spread of bacteria or
Figure 34-6. Functional structures of the spleen. toxic products.
The intensity of the inflammatory process is usually
The spleen is similar to the lymph nodes, except that proportional to the degree of tissue injury. For example,
blood, instead of lymph, flows through the tissue spaces of when staphylococci invade tissues, they release extremely
the spleen. Figure 34-6 shows a small peripheral segment lethal cellular toxins. As a result, inflammation develops
of spleen tissue. Note that a small artery penetrates from rapidly—indeed, much more rapidly than the staphylo-
the splenic capsule into the splenic pulp and terminates in cocci can multiply and spread. Therefore, local staphy-
small capillaries. The capillaries are highly porous, allow- lococcal infection is characteristically walled off rapidly
ing whole blood to pass out of the capillaries into cords and prevented from spreading through the body. Strep-
of red pulp. The blood then gradually squeezes through tococci, in contrast, do not cause such intense local tissue
the trabecular meshwork of these cords and eventually destruction. Therefore, the walling-off process develops
returns to the circulation through the endothelial walls slowly over many hours, while many streptococci repro-
of the venous sinuses. The trabeculae of the red pulp and duce and migrate. As a result, streptococci often have a
venous sinuses are lined with vast numbers of macro- far greater tendency to spread through the body and cause
phages. This peculiar passage of blood through the cords death than staphylococci, even though staphylococci are
of the red pulp provides an exceptional means of phago- far more destructive to the tissues.!
cytizing unwanted debris in the blood, including espe-
cially old and abnormal RBCs.!
MACROPHAGE AND NEUTROPHIL
RESPONSES DURING INFLAMMATION
INFLAMMATION: ROLE OF NEUTRO-
Tissue Macrophages Provide First Line of Defense
PHILS AND MACROPHAGES
Against Infection. Within minutes after inflammation
begins, the macrophages already present in the tissues,
INFLAMMATION
whether histiocytes in the subcutaneous tissues, alveolar
When tissue injury occurs, whether caused by bacteria, macrophages in the lungs, microglia in the brain, or oth-
trauma, chemicals, heat, or any other phenomenon, mul- ers, immediately begin their phagocytic actions. When
tiple substances are released by the injured tissues and activated by the products of infection and inflammation,
cause dramatic secondary changes in the surrounding the first effect is rapid enlargement of each of these cells.
uninjured tissues. This entire complex of tissue changes is Next, many of the previously sessile macrophages break
called inflammation. loose from their attachments and become mobile, form-
Inflammation is characterized by the following: (1) ing the first line of defense against infection during the
vasodilation of the local blood vessels, with consequent first hour or so. The numbers of these early mobilized
increased local blood flow; (2) increased permeability of macrophages often are not great, but they are lifesaving.!
the capillaries, allowing leakage of large quantities of fluid
into the interstitial spaces; (3) often, clotting of the fluid Neutrophil Invasion of the Inflamed Area Is a Sec-
in the interstitial spaces because of increased amounts of ond Line of Defense. Within the first hour or so after
fibrinogen and other proteins leaking from the capillar- inflammation begins, large numbers of neutrophils begin
ies; (4) migration of large numbers of granulocytes and to invade the inflamed area from the blood. This invasion
monocytes into the tissue; and (5) swelling of the tissue is caused by inflammatory cytokines (e.g., tumor necrosis
cells. Some of the many tissue products that cause these factor and interleukin-1) and other biochemical products
reactions are histamine, bradykinin, serotonin, prostaglan- produced by the inflamed tissues that initiate the following
dins, several different reaction products of the complement reactions:

454
 Chapter 34 Resistance of the Body to Infection

Rolling adhesion Tight binding Diapedesis Migration

Receptors
Neutrophil

UNIT VI
Endothelial cell

Selectin ICAM-1

Cytokines
Inflamed tissue

Figure 34-7. Migration of neutrophils from the blood into inflamed tissue. Cytokines and other biochemical products of the inflamed tissue
cause increased expression of selectins and intercellular adhesion molecule-1 (ICAM-1) on the surface of endothelial cells. These adhesion mol-
ecules bind to complementary molecules or receptors on the neutrophil, causing it to adhere to the wall of the capillary or venule. The neutrophil
then migrates through the vessel wall by diapedesis or extravasation toward the site of tissue injury.

1. They cause increased expression of adhesion mol- neutrophils in the blood sometimes increases fourfold
ecules, such as selectins and intercellular adhesion to fivefold—from a normal of 4,000 to 5,000 to 15,000 to
molecule-1 (ICAM-1) on the surface of endothelial 25,000 neutrophils/µl. This is called neutrophilia, which
cells in the capillaries and venules. These adhesion means an increase in the number of neutrophils in the
molecules, reacting with complementary integrin blood. Neutrophilia is caused by products of inflamma-
molecules on the neutrophils, cause the neutro- tion that enter the blood stream, are transported to the
phils to stick to the capillary and venule walls in bone marrow, and act there on the stored neutrophils of
the inflamed area. This effect is called margination the marrow to mobilize these into the circulating blood.
and is shown in Figure 34-2 and in more detail in This makes even more neutrophils available to the in-
Figure 34-7. flamed tissue area.!
2. They also cause the intercellular attachments be-
tween the endothelial cells of the capillaries and Second Macrophage Invasion Into the Inflamed
small venules to loosen, allowing openings large Tissue Is a Third Line of Defense. Along with the in-
enough for neutrophils to crawl through the capil- vasion of neutrophils, monocytes from the blood enter
laries by diapedesis into the tissue spaces. the inflamed tissue and enlarge to become macrophages.
3. They then cause chemotaxis of the neutrophils to- However, the number of monocytes in the circulating
ward the injured tissues, as explained earlier. The blood is low. Also, the storage pool of monocytes in the
entire process of neutrophil (or other substances bone marrow is much less than that of neutrophils. There-
and cells such as monocytes) translocation through fore, the buildup of macrophages in the inflamed tissue
the capillaries into the tissues surrounding them is area is much slower than that of neutrophils, requiring
called extravasation; the specific passage of blood several days to become effective. Furthermore, even after
cells through the intact walls of the capillaries is invading the inflamed tissue, monocytes are still immature
called diapedesis, although this term is often used cells, requiring 8 hours or more to swell to much larger siz-
interchangeably with extravasation when discussing es and develop tremendous quantities of lysosomes. Only
blood cell movement through the capillaries into then do they acquire the full capacity of tissue macrophages
tissues. for phagocytosis. After several days to several weeks, the
Thus, within several hours after tissue damage begins, macrophages finally come to dominate the phagocytic
the area becomes well supplied with neutrophils. Because cells of the inflamed area because of greatly increased bone
the blood neutrophils are already mature cells, they are marrow production of new monocytes, as explained later.
ready to begin their scavenger functions of killing bacteria As already noted, macrophages can phagocytize far
and removing foreign matter immediately.! more bacteria (about five times as many) and far larger
particles, including even neutrophils and large quantities
Acute Increase in the Number of Neutrophils in of necrotic tissue, than can neutrophils. Also, the macro-
Blood—Neutrophilia. Also, within a few hours after phages play an important role in initiating development of
the onset of acute severe inflammation, the number of antibodies, as discussed in Chapter 35.!

455
UNIT VI Blood Cells, Immunity, and Blood Coagulation

INFLAMMATION colony-stimulating factors; one of these, GM-CSF, stim-

Activated ulates both granulocyte and monocyte production; the
macrophage other two, G-CSF and M-CSF, stimulate granulocyte and
monocyte production, respectively. This combination
of TNF, IL-1, and colony-stimulating factors provides a
TNF powerful feedback mechanism that begins with tissue
IL-1 inflammation and proceeds to formation of large num-
bers of defensive WBCs that help remove the cause of the
Endothelial cells,
inflammation.!
fibroblasts,
TNF lymphocytes Formation of Pus
IL-1
GM-CSF
When neutrophils and macrophages engulf large num-
G-CSF bers of bacteria and necrotic tissue, essentially all the
GM-CSF
M-CSF
G-CSF neutrophils and many, if not most, of the macrophages
M-CSF eventually die. After several days, a cavity is often exca-
vated in the inflamed tissues. This cavity contains varying
portions of necrotic tissue, dead neutrophils, dead macro-
phages, and tissue fluid. This mixture is commonly known
Bone marrow
as pus. After the infection has been suppressed, the dead
cells and necrotic tissue in the pus gradually autolyze
over a period of days, and the end products are eventually
Granulocytes absorbed into the surrounding tissues and lymph until
Monocytes/macrophages most of the evidence of tissue damage is gone.!
Figure 34-8. Control of bone marrow production of granulocytes
and monocyte-macrophages in response to multiple growth fac-
tors released from activated macrophages in an inflamed tissue. G- EOSINOPHILS
CSF, Granulocyte colony-stimulating factor; GM-CSF, granulocyte-
monocyte colony-stimulating factor; IL-1, interleukin-1; M-CSF,
The eosinophils normally constitute about 2% of all the
monocyte colony-stimulating factor; TNF, tumor necrosis factor. blood leukocytes. Eosinophils are weak phagocytes, and
they exhibit chemotaxis, but in comparison with neutro-
Increased Production of Granulocytes and Mono- phils, it is doubtful that eosinophils are significant in pro-
cytes by Bone Marrow Is a Fourth Line of Defense. tecting against the usual types of infection.
The fourth line of defense is greatly increased production Eosinophils, however, are often produced in large
of granulocytes and monocytes by the bone marrow. This numbers in people with parasitic infections, and they
action results from stimulation of the granulocytic and migrate into tissues diseased by parasites. Although most
monocytic progenitor cells of the marrow. However, it parasites are too large to be phagocytized by eosinophils
takes 3 to 4 days before newly formed granulocytes and or any other phagocytic cells, eosinophils attach them-
monocytes reach the stage of leaving the bone marrow. If selves to the parasites by way of special surface molecules
the stimulus from the inflamed tissue continues, the bone and release substances that kill many of the parasites. For
marrow can continue to produce these cells in large quan- example, one of the most widespread infections is schis-
tities for months and even years, sometimes at a rate 20 to tosomiasis, a parasitic infection found in as many as one -
50 times normal.! third of the population of some developing countries in
Africa, Asia, and South America. An estimated 85% to
Feedback Control of Macrophage and 90% of the world’s cases of schistosomiasis are in Africa.
Neutrophil Responses The schistosome parasitic worms can invade any part
Although more than two dozen factors have been impli- of the body. Eosinophils attach themselves to the juvenile
cated in control of the macrophage response to inflam- forms of the parasite and kill many of them. They do so
mation, five of these are believed to play dominant roles. in several ways: (1) by releasing hydrolytic enzymes from
They are shown in Figure 34-8 and consist of the fol- their granules, which are modified lysosomes; (2) prob-
lowing: (1) tumor necrosis factor (TNF); (2) interleukin-1 ably also by releasing highly reactive forms of oxygen that
(IL-1), (3) granulocyte-monocyte colony-stimulating fac- are especially lethal to parasites; and (3) by releasing from
tor (GM-CSF); (4) granulocyte colony-stimulating factor the granules a highly larvicidal polypeptide called major
(G-CSF); and (5) monocyte colony-stimulating factor (M- basic protein.
CSF). These factors are formed by activated macrophage In a few areas of the world, another parasitic disease
cells in the inflamed tissues and in smaller quantities by that causes eosinophilia is trichinosis. This disease results
other inflamed tissue cells. from invasion of the body’s muscles by the Trichinella
The cause of increased production of granulocytes parasite (pork worm) after a person eats undercooked
and monocytes by the bone marrow is mainly the three infested pork.

456
 Chapter 34 Resistance of the Body to Infection

Eosinophils also have a special propensity to collect in always find bacteria on the surfaces of the eyes, urethra,
tissues in which allergic reactions occur, such as in the and vagina. Any decrease in the number of WBCs imme-
peribronchial tissues of the lungs in people with asthma diately allows invasion of adjacent tissues by bacteria that
and in the skin after an allergic skin reaction. This action are already present.
is caused at least partly by the fact that many mast cells Within 2 days after the bone marrow stops producing

UNIT VI
and basophils participate in allergic reactions, as dis- WBCs, ulcers may appear in the mouth and colon, or some
cussed in the next paragraph. The mast cells and baso- form of severe respiratory infection might develop. Bacte-
phils release an eosinophil chemotactic factor that causes ria from the ulcers rapidly invade surrounding tissues and
eosinophils to migrate toward the inflamed allergic tis- the blood. Without treatment, death often ensues in less
sue. The eosinophils are believed to detoxify some of the than 1 week after acute total leukopenia begins.
inflammation-inducing substances released by the mast Irradiation of the body by x-rays or gamma rays, or
cells and basophils and probably also phagocytize and exposure to drugs and chemicals that contain benzene or
destroy allergen-antibody complexes, thus preventing anthracene nuclei, is likely to cause aplasia of the bone
excess spread of the local inflammatory process.! marrow. Some common drugs such as chloramphenicol
(an antibiotic), thiouracil (used to treat thyrotoxicosis),
and even various barbiturate hypnotics on rare occasions
BASOPHILS
cause leukopenia, thus setting off the entire infectious
The basophils in the circulating blood are similar to the sequence of this disorder.
large tissue mast cells located immediately outside many After moderate irradiation injury to the bone marrow,
of the capillaries in the body. Both mast cells and baso- some stem cells, myeloblasts, and hemocytoblasts may
phils liberate heparin into the blood. Heparin is a sub- remain undestroyed in the marrow and are capable of
stance that can prevent blood coagulation. regenerating the bone marrow, provided sufficient time
The mast cells and basophils also release histamine, as is available. A patient properly treated with transfusions,
well as smaller quantities of bradykinin and serotonin. It is plus antibiotics and other drugs to ward off infection, usu-
mainly the mast cells in inflamed tissues that release these ally develops enough new bone marrow within weeks to
substances during inflammation. months for blood cell concentrations to return to normal.!
The mast cells and basophils play an important role
in some types of allergic reactions because the type of
LEUKEMIAS
antibody that causes allergic reactions, immunoglobulin
E (IgE), has a special propensity to become attached to Uncontrolled production of WBCs can be caused by can-
mast cells and basophils. Then, when the specific anti- cerous mutation of a myelogenous or lymphogenous cell.
gen for the specific IgE antibody subsequently reacts This process causes leukemia, which is usually character-
with the antibody, the resulting attachment of antigen ized by greatly increased numbers of abnormal WBCs in
to antibody causes the mast cell or basophil to release the circulating blood.
increased quantities of histamine, bradykinin, sero- There are two general types of leukemia, lymphocytic
tonin, heparin, slow-reacting substance of anaphylaxis and myelogenous. The lymphocytic leukemias are caused
(a mixture of three leukotrienes), and several lysosomal by cancerous production of lymphoid cells, usually begin-
enzymes. These substances cause local vascular and ning in a lymph node or other lymphocytic tissue and
tissue reactions that mediate many, if not most, of the spreading to other areas of the body. The second type of
allergic manifestations. These reactions are discussed in leukemia, myelogenous leukemia, begins by cancerous
greater detail in Chapter 35.! production of young myelogenous cells in the bone mar-
row and then spreads throughout the body so that WBCs
are produced in many extramedullary tissues—especially
LEUKOPENIA
in the lymph nodes, spleen, and liver.
A clinical condition known as leukopenia, in which In myelogenous leukemia, the cancerous process occa-
the bone marrow produces very few WBCs, occasion- sionally produces partially differentiated cells, resulting in
ally occurs. This condition leaves the body unprotected what might be called neutrophilic leukemia, eosinophilic
against many bacteria and other agents that might invade leukemia, basophilic leukemia, or monocytic leukemia.
the tissues. More frequently, however, the leukemia cells are bizarre
Normally, the human body lives in symbiosis with and undifferentiated and not identical to any of the nor-
many bacteria because the mucous membranes of the mal WBCs. Usually, the more undifferentiated the cell, the
body are constantly exposed to large numbers of bac- more acute is the leukemia, often leading to death within a
teria. The mouth almost always contains various spiro- few months if untreated. With some of the more differen-
chetal, pneumococcal, and streptococcal bacteria, and tiated cells, the process can be chronic, sometimes devel-
these same bacteria are present to a lesser extent in the oping slowly over 10 to 20 years. Leukemic cells, especially
entire respiratory tract. The distal gastrointestinal tract is the very undifferentiated cells, are usually nonfunctional
especially loaded with colon bacilli. Furthermore, one can for providing normal protection against infection.

457
UNIT VI Blood Cells, Immunity, and Blood Coagulation

Effects of Leukemia on the Body Hallek M, Shanafelt TD, Eichhorst B: Chronic lymphocytic leukaemia.
Lancet 391:1524, 2018.
The first effect of leukemia is metastatic growth of leuke- Honda M, Kubes P: Neutrophils and neutrophil extracellular traps in
mic cells in abnormal areas of the body. Leukemic cells the liver and gastrointestinal system. Nat Rev Gastroenterol Hepa-
from the bone marrow may reproduce so much that they tol 15:206, 2018.
Lemke G: How macrophages deal with death. Nat Rev Immunol 19:
invade the surrounding bone, causing pain and, eventu-
539, 2019.
ally, a tendency for bones to fracture easily. Liew PX, Kubes P: The neutrophil’s role during health and disease.
Almost all leukemias eventually spread to the spleen, Physiol Rev 99:1223, 2019.
lymph nodes, liver, and other vascular regions, regardless Medzhitov R: Origin and physiological roles of inflammation. Nature
of whether the leukemia originated in the bone marrow or 454:428, 2008.
Ng LG, Ostuni R, Hidalgo A: Heterogeneity of neutrophils. Nat Rev
lymph nodes. Common effects in leukemia are the develop-
Immunol 19:255, 2019.
ment of infection, severe anemia, and a bleeding tendency Papayannopoulos V: Neutrophil extracellular traps in immunity and
caused by thrombocytopenia (lack of platelets). These effects disease. Nat Rev Immunol 18:134, 2018.
result mainly from displacement of the normal bone mar- Phillipson M, Kubes P: The healing power of neutrophils. Trends
row and lymphoid cells by the nonfunctional leukemic cells. Immunol 2019 May 31. pii: S1471-4906(19)30103-30106.
Pinho S, Frenette PS: Haematopoietic stem cell activity and interac-
Finally, an important effect of leukemia on the body
tions with the niche. Nat Rev Mol Cell Biol 20:303, 2019.
is excessive use of metabolic substrates by the growing Russell DG, Huang L, VanderVen BC: Immunometabolism at the
cancerous cells. The leukemic tissues reproduce new interface between macrophages and pathogens. Nat Rev Immunol
cells so rapidly that tremendous demands are made on 19:291, 2019.
the body reserves for foodstuffs, specific amino acids, Short NJ, Rytting ME, Cortes JE: Acute myeloid leukaemia. Lancet
392:593, 2018.
and vitamins. Consequently, the energy of the patient is
Spivak JL: Myeloproliferative neoplasms. N Engl J Med 376:2168,
greatly depleted, and excessive utilization of amino acids 2017.
by leukemic cells causes especially rapid deterioration of Watanabe S, Alexander M, Misharin AV, Budinger GRS: The role of
the normal protein tissues of the body. Thus, while the macrophages in the resolution of inflammation. J Clin Invest 129:
leukemic tissues grow, other tissues become debilitated. 2619, 2019.
Werner S, Grose R: Regulation of wound healing by growth factors
After metabolic starvation has continued long enough,
and cytokines. Physiol Rev 83:835, 2003.
this factor alone is sufficient to cause death.

Bibliography
David BA, Kubes P: Exploring the complex role of chemokines and
chemoattractants in vivo on leukocyte dynamics. Immunol Rev
289:9, 2019.
DeNardo DG, Ruffell B: Macrophages as regulators of tumour immu-
nity and immunotherapy. Nat Rev Immunol 19:369, 2019.

458
 CHAPTER 37

UNIT VI
Hemostasis and Blood Coagulation

HEMOSTASIS EVENTS fragment into the minute platelets in the bone marrow or
soon after entering the blood, especially as they squeeze
The term hemostasis means prevention of blood loss. through capillaries. The normal concentration of platelets
Whenever a vessel is severed or ruptured, hemostasis is in the blood is between 150,000 and 450,000/µl.
achieved by several mechanisms: (1) vascular constric- Platelets have many functional characteristics of whole
tion; (2) formation of a platelet plug; (3) formation of a cells, even though they do not have nuclei and cannot
blood clot as a result of blood coagulation; and (4) even- reproduce. In their cytoplasm are the following: (1) actin
tual growth of fibrous tissue into the blood clot to close and myosin molecules, which are contractile proteins
the hole in the vessel permanently. similar to those found in muscle cells, and still another
VASCULAR CONSTRICTION contractile protein, thrombosthenin, that can cause
the platelets to contract; (2) residuals of both the endo-
Immediately after a blood vessel has been cut or rup- plasmic reticulum and Golgi apparatus that synthesize
tured, the trauma to the vessel wall causes smooth muscle various enzymes and especially store large quantities of
in the wall to contract; this instantaneously reduces the calcium ions; (3) mitochondria and enzyme systems that
flow of blood from the ruptured vessel. The contraction are capable of forming adenosine triphosphate (ATP) and
results from the following: (1) local myogenic spasm; (2) adenosine diphosphate (ADP); (4) enzyme systems that
local autacoid factors from the traumatized tissues, vas- synthesize prostaglandins, which are local hormones that
cular endothelium, and blood platelets; and (3) nervous cause many vascular and other local tissue reactions; (5)
reflexes. The nervous reflexes are initiated by pain nerve an important protein called fibrin-stabilizing factor, which
impulses or other sensory impulses that originate from the we discuss later in relation to blood coagulation; and (6)
traumatized vessel or nearby tissues. However, even more a growth factor that causes vascular endothelial cells, vas-
vasoconstriction probably results from local myogenic cular smooth muscle cells, and fibroblasts to multiply and
contraction of the blood vessels initiated by direct dam- grow, thus causing cellular growth that eventually helps
age to the vascular wall. And, for the smaller vessels, the repair damaged vascular walls.
platelets are responsible for much of the vasoconstriction On the platelet cell membrane surface is a coat of glyco-
by releasing a vasoconstrictor substance, thromboxane A2. proteins that repulses adherence to normal endothelium
The more severely a vessel is traumatized, the greater and yet causes adherence to injured areas of the vessel
the degree of vascular spasm. The spasm can last for many wall, especially to injured endothelial cells and even more
minutes or even hours, during which time the processes so to any exposed collagen from deep within the vessel
of platelet plugging and blood coagulation can take place.! wall. In addition, the platelet membrane contains large
amounts of phospholipids that activate multiple stages in
FORMATION OF THE PLATELET PLUG
the blood-clotting process, as discussed later.
If the cut in the blood vessel is very small—many very Thus, the platelet is an active structure. It has a half-life
small vascular holes develop throughout the body each in the blood of only 8 to 12 days, so over several weeks its
day—the cut is often sealed by a platelet plug rather than functional processes run out; it is then eliminated from
by a blood clot. To understand this process, it is important the circulation mainly by the tissue macrophage system.
that we first discuss the nature of platelets themselves. More than half of the platelets are removed by macro-
phages in the spleen, where the blood passes through a
Physical and Chemical Characteristics latticework of tight trabeculae.!
Platelets (also called thrombocytes) are minute discs 1 to
4 micrometers in diameter. They are formed in the bone Mechanism of Platelet Plug Formation
marrow from megakaryocytes, which are extremely large Platelet repair of vascular openings is based on several
hematopoietic cells in the marrow; the megakaryocytes important functions of the platelet. When platelets
477
UNIT VI Blood Cells, Immunity, and Blood Coagulation

Figure 37-1. Formation of a platelet plug

in a severed blood vessel. Endothelial in-
jury and exposure of the vascular extracel- Shape Granule Recruitment
lular matrix facilitates platelet adhesions Adhesion change release
and activation, which changes their shape (ADP, PAF, TXA2)
and causes release of adenosine diphos-
phate (ADP), thromboxane A2 (TXA2),
Gplb Aggregation
and platelet-activating factor (PAF). These
platelet-secreted factors recruit additional
platelets (aggregation) to form a hemo-
static plug. Von Willebrand factor (vWF)
serves as an adhesion bridge between sub- Endothelium vWF
endothelial collagen and the glycoprotein Basement Damaged blood
Ib (GpIb) platelet receptor. membrane vessel wall

when platelet
come in contact with a damaged vascular surface,
especially with collagen fibers in the vascular wall, the
platelets rapidly change their own characteristics dras-
tically (Figure 37-1). They begin to swell, they assume
irregular forms with numerous irradiating pseudo- 1. Severed vessel 2. Platelets agglutinate
pods protruding from their surfaces, their contractile
proteins contract forcefully and cause the release of
granules that contain multiple active factors, and they
become sticky so that they adhere to collagen in the
tissues and to a protein called von Willebrand factor 3. Fibrin appears 4. Fibrin clot forms
(vWF), which leaks into the traumatized tissue from
the plasma. The platelet surface glycoproteins bind to
vWF in the exposed matrix below the damaged endo-
thelium. The platelets then secrete increased quanti-
ties of ADP and platelet- activating factor (PAF), and
their enzymes form thromboxane A2. Thromboxane is 5. Clot retraction occurs
a vasoconstrictor and, along with ADP and PAF, acts on Figure 37-2. Clotting process in a traumatized blood vessel. (Modi-
nearby platelets to activate them as well; the stickiness fied from Seegers WH: Hemostatic Agents. Springfield, IL: Charles C
of these additional activated platelets causes them to Thomas, 1948.)
adhere to the original activated platelets.
Therefore, at the site of a puncture in a blood vessel
wall, the damaged vascular wall activates successively BLOOD COAGULATION IN THE RUPTURED
increasing numbers of platelets that attract more and VESSEL
more additional platelets, thus forming a platelet plug. The third mechanism for hemostasis is formation of the
This plug is loose at first but is usually successful in block- blood clot. The clot begins to develop in 15 to 20 seconds
ing blood loss if the vascular opening is small. Then, dur- if the trauma to the vascular wall is severe and in 1 to 2
ing the subsequent process of blood coagulation, fibrin minutes if the trauma is minor. Activator substances from
threads form. These threads attach tightly to the platelets, the traumatized vascular wall, from platelets, and from
thus constructing an unyielding plug. blood proteins adhering to the traumatized vascular wall
Importance of Platelet Mechanism for Closing initiate the clotting process. The physical events of this
Vascular Holes. The platelet- plugging mechanism is process are shown in Figure 37-2; Table 37-1 lists the
extremely important for closing minute ruptures in most important clotting factors.
very small blood vessels that occur many thousands of Within 3 to 6 minutes after rupture of a vessel, the
times daily. Indeed, multiple small holes through the entire opening or broken end of the vessel is filled with
endothelial cells themselves are often closed by plate- clot if the vessel opening is not too large. After 20 to 60
lets actually fusing with the endothelial cells to form minutes, the clot retracts, which closes the vessel still
additional endothelial cell membranes. Literally thou- further. Platelets also play an important role in this clot
sands of small hemorrhagic areas develop each day un- retraction, as discussed later.!
der the skin (petechiae, which appear as purple or red
FIBROUS ORGANIZATION OR
dots on the skin) and throughout the internal tissues
DISSOLUTION OF BLOOD CLOTS
of a person who has few blood platelets. This phenom-
enon does not occur in persons with normal numbers Once a blood clot has formed, it can follow one of two
of platelets.! courses: (1) it can become invaded by fibroblasts, which

478
 Chapter 37 Hemostasis and Blood Coagulation

Table 37-1 Clotting Factors in Blood and Their Prothrombin

Synonymsa
Prothrombin Ca2+
Clotting Factor Synonym(s) activator
Fibrinogen Factor I
Thrombin
Prothrombin Factor II

UNIT VI
Tissue factor Factor III; tissue thromboplastin
Fibrinogen Fibrin monomer
Calcium Factor IV
Ca2+
Factor V Proaccelerin; labile factor; Ac-
globulin (Ac-G) Fibrin fibers

Factor VII Serum prothrombin conversion Thrombin Activated

accelerator (SPCA); proconvertin; fibrin-stabilizing
factor
stable factor
Factor VIII Antihemophilic factor (AHF); Cross-linked fibrin fibers
antihemophilic globulin (AHG);
Figure 37-3 Schema for conversion of prothrombin to thrombin and
antihemophilic factor A
polymerization of fibrinogen to form fibrin fibers.
Factor IX Plasma thromboplastin component
(PTC); Christmas factor;
antihemophilic factor B in the blood vessels. However, when a vessel is ruptured,
Factor X Stuart factor; Stuart-Prower factor procoagulants from the area of tissue damage become
Factor XI Plasma thromboplastin antecedent activated and override the anticoagulants, and then a clot
(PTA); antihemophilic factor C does develop.
Factor XII Hageman factor Clotting takes place in three essential steps:
Factor XIII Fibrin-stabilizing factor 1. In response to rupture of the vessel or damage to
the blood itself, a complex cascade of chemical re-
Prekallikrein Fletcher factor
actions occurs in the blood involving more than 12
High-molecular- Fitzgerald factor; high-molecular-
blood coagulation factors. The net result is the for-
weight kininogen weight kininogen (HMWK)
mation of a complex of activated substances collec-
Platelets
tively called prothrombin activator.
aThese are listed here mainly for historical interest. 2. The prothrombin activator catalyzes the conversion
of prothrombin into thrombin.
subsequently form connective tissue all through the clot; 3. The thrombin acts as an enzyme to convert fibrino-
or (2) it can dissolve. The usual course for a clot that forms gen into fibrin fibers that enmesh platelets, blood
in a small hole of a vessel wall is invasion by fibroblasts, cells, and plasma to form the clot.
beginning within a few hours after the clot is formed, We will first discuss the mechanism whereby the blood
which is promoted at least partially by growth factor clot is formed, beginning with conversion of prothrombin
secreted by platelets. This process continues to complete to thrombin, and then come back to the initiating stages
organization of the clot into fibrous tissue within about 1 in the clotting process whereby prothrombin activator is
to 2 weeks. formed.!
Conversely, when excess blood has leaked into the
CONVERSION OF PROTHROMBIN TO
tissues, and tissue clots have formed where they are not
THROMBIN
needed, special substances in the clot usually become
activated. These substances function as enzymes to dis- 1. Prothrombin activator is formed as a result of rup-
solve the clot, as discussed later in the chapter.! ture of a blood vessel or as a result of damage to
special substances in the blood.
2. Prothrombin activator, in the presence of sufficient
MECHANISM OF BLOOD COAGULATION
amounts of ionic calcium (Ca2+), causes conver-
sion of prothrombin to thrombin (Figure 37-3 and
GENERAL MECHANISM
37-4).
More than 50 important substances that cause or affect 3. Thrombin causes polymerization of fibrinogen
blood coagulation have been found in the blood and in molecules into fibrin fibers within another 10 to 15
the tissues—some that promote coagulation, called pro- seconds.
coagulants, and others that inhibit coagulation, called Thus, the rate-limiting factor in causing blood coagula-
anticoagulants. Whether blood will coagulate depends on tion is usually the formation of prothrombin activator and
the balance between these two groups of substances. In not the subsequent reactions beyond that point because
the blood stream, the anticoagulants normally predomi- these terminal steps normally occur rapidly to form the
nate, so the blood does not coagulate while it is circulating clot.

479
UNIT VI Blood Cells, Immunity, and Blood Coagulation

Prothrombin Thrombin
Cross-linked
Figure 37-4. Coagulation cascade after Fibrinogen Fibrin
Platelet fibrin
vascular injury. Exposure of blood to the
Release of phospholipid
vascular wall causes release of tissue factor
tissue factor complex
(also called factor III or thromboplastin) from
endothelial cells, phospholipid expression,
activation of thrombin, which then acts on
fibrinogen to form fibrin, and fibrin polym-
erization to form a meshwork that stabilizes
the platelet plug. Endothelium Fibrin clot

Platelets also play an important role in the conversion Action of Thrombin on Fibrinogen to Form Fibrin.
of prothrombin to thrombin because much of the pro- Thrombin is a protein enzyme with weak proteolytic
thrombin first attaches to prothrombin receptors on the capabilities. It acts on fibrinogen to remove four low-
platelets that are already bound to the damaged tissue. molecular-weight peptides from each molecule of fi-
brinogen, forming one molecule of fibrin monomer that
Prothrombin and Thrombin. Prothrombin is a plasma
has the automatic capability to polymerize with other fi-
protein, an α2-globulin, having a molecular weight of
brin monomer molecules to form fibrin fibers. Therefore,
68,700. It is present in normal plasma in a concentration
many fibrin monomer molecules polymerize within sec-
of about 15 mg/dl. It is an unstable protein that can split
onds into long fibrin fibers that constitute the reticulum of
easily into smaller compounds, one of which is thrombin,
the blood clot.
which has a molecular weight of 33,700, almost half that
In the early stages of polymerization, the fibrin mono-
of prothrombin.
mer molecules are held together by weak noncovalent
Prothrombin is formed continually by the liver, and it
hydrogen bonding, and the newly forming fibers are not
is continually being used throughout the body for blood
cross-linked with one another. Therefore, the resultant
clotting. If the liver fails to produce prothrombin, in a day
clot is weak and can be broken apart with ease. However,
or so prothrombin concentration in the plasma falls too
another process occurs during the next few minutes that
low to provide normal blood coagulation.
greatly strengthens the fibrin reticulum. This process
Vitamin K is required by the liver for normal activa-
involves a substance called fibrin stabilizing factor that
tion of prothrombin, as well as a few other clotting fac-
is present in small amounts in normal plasma globulins
tors. Therefore, lack of vitamin K or the presence of liver
but is also released from platelets entrapped in the clot.
disease that prevents normal prothrombin formation can
Before fibrin stabilizing factor can have an effect on the
decrease the prothrombin to such a low level that a bleed-
fibrin fibers, it must be activated. The same thrombin that
ing tendency results.!
causes fibrin formation also activates the fibrin stabiliz-
ing factor. This activated substance then operates as an
enzyme to form covalent bonds between more and more
CONVERSION OF FIBRINOGEN TO
of the fibrin monomer molecules, as well as multiple
FIBRIN—FORMATION OF THE CLOT
cross-linkages between adjacent fibrin fibers, thus adding
Fibrinogen Formed in the Liver Essential for Clot For- tremendously to the three-dimensional strength of the
mation. Fibrinogen is a high-molecular-weight protein fibrin meshwork.!
(molecular weight ≈340,000) that occurs in the plasma in
Blood Clot. The clot is composed of a meshwork of fi-
quantities of 100 to 700 mg/dl. Fibrinogen is formed in
brin fibers running in all directions and entrapping blood
the liver, and liver disease can decrease the concentration
cells, platelets, and plasma (see Figure 37-4). The fibrin
of circulating fibrinogen, as it does the concentration of
fibers also adhere to damaged surfaces of blood vessels;
prothrombin, noted earlier.
therefore, the blood clot becomes adherent to any vascu-
Because of its large molecular size, little fibrinogen
lar opening and thereby prevents further blood loss.!
normally leaks from the blood vessels into the intersti-
tial fluids, and because fibrinogen is one of the essential Clot Retraction and Expression of Serum. Within a few
factors in the coagulation process, interstitial fluids ordi- minutes after a clot is formed, it begins to contract and
narily do not coagulate. Yet, when the permeability of the usually expresses most of the fluid from the clot within
capillaries becomes pathologically increased, fibrinogen 20 to 60 minutes. The fluid expressed is called serum be-
does leak into the tissue fluids in sufficient quantities to cause all its fibrinogen and most of the other clotting fac-
allow clotting of these fluids in much the same way that tors have been removed; in this way, serum differs from
plasma and whole blood can clot.! plasma and cannot clot because it lacks these factors.

480
 Chapter 37 Hemostasis and Blood Coagulation

Platelets are necessary for clot retraction to occur. (1) Tissue trauma
Therefore, failure of clot retraction is an indication that the
number of platelets in the circulating blood might be low.
Electron micrographs of platelets in blood clots show that
they become attached to the fibrin fibers in such a way that Tissue factor

UNIT VI
they actually bond different fibers together. Furthermore,
platelets entrapped in the clot continue to release proco-
agulant substances, one of the most important of which (2) Vll VIIa
is fibrin stabilizing factor, which causes more and more
cross-linking bonds between adjacent fibrin fibers. In addi- X Activated X (Xa)
tion, the platelets contribute directly to clot contraction
by activating platelet thrombosthenin, actin, and myosin Ca2+
molecules, which are all contractile proteins in the plate- V Ca2+
lets; they cause strong contraction of the platelet spicules
(3) Prothrombin
attached to the fibrin. This action also helps compress the activator
Platelet
fibrin meshwork into a smaller mass. The contraction is phospholipids
activated and accelerated by thrombin and by calcium ions
Prothrombin Thrombin
released from calcium stores in the mitochondria, endo-
plasmic reticulum, and Golgi apparatus of the platelets.
As the clot retracts, the edges of the broken blood ves- Ca2+
sel are pulled together, thus contributing still further to Figure 37-5. Extrinsic pathway for initiating blood clotting.
hemostasis.!

POSITIVE FEEDBACK OF CLOT Prothrombin activator is generally considered to be

FORMATION formed in two ways, although, in reality, the two ways
interact constantly with each other: (1) by the extrinsic
Once a blood clot starts to develop, it normally extends
pathway that begins with trauma to the vascular wall and
within minutes into the surrounding blood—that is, the
surrounding tissues; and (2) by the intrinsic pathway that
clot initiates a positive feedback to promote more clot-
begins in the blood.
ting. One of the most important causes of this clot pro-
In both the extrinsic and the intrinsic pathways, a
motion is that the proteolytic action of thrombin allows
series of different plasma proteins called blood-clotting
it to act on many of the other blood-clotting factors in
factors plays a major role. Most of these proteins are inac-
addition to fibrinogen. For example, thrombin has a direct
tive forms of proteolytic enzymes. When converted to the
proteolytic effect on prothrombin, tending to convert it
active forms, their enzymatic actions cause the successive,
into still more thrombin, and it acts on some of the blood-
cascading reactions of the clotting process.
clotting factors responsible for formation of prothrombin
Most of the clotting factors listed in Table 37-1 are
activator. (These effects, discussed in subsequent para-
designated by Roman numerals. To indicate the activated
graphs, include acceleration of the actions of factors VIII,
form of the factor, a small letter “a” is added after the
IX, X, XI, and XII and aggregation of platelets.) Once a
Roman numeral, such as factor VIIIa to indicate the acti-
critical amount of thrombin is formed, a positive feedback
vated state of factor VIII.
develops that causes still more blood clotting and more
and more thrombin to be formed; thus, the blood clot Extrinsic Pathway for Initiating Clotting
continues to grow until blood leakage ceases.!
The extrinsic pathway for initiating the formation of
prothrombin activator begins with a traumatized vas-
INITIATION OF COAGULATION:
cular wall or traumatized extravascular tissues that
FORMATION OF PROTHROMBIN
come in contact with the blood. This condition leads
ACTIVATOR
to the following steps, as shown in Figure 37-4 and
Now that we have discussed the clotting process, the Figure 37-5:
more complex mechanisms that initiate clotting in the 1. Release of tissue factor. Traumatized tissue releases
first place will be described. These mechanisms are set a complex of several factors called tissue factor or
into play by the following: (1) trauma to the vascular wall tissue thromboplastin. This factor is composed es-
and adjacent tissues; (2) trauma to the blood; or (3) con- pecially of phospholipids from the membranes of
tact of the blood with damaged endothelial cells or with the tissue plus a lipoprotein complex that functions
collagen and other tissue elements outside the blood ves- mainly as a proteolytic enzyme.
sel. In each case, this leads to the formation of prothrom- 2. Activation of factor X—role of factor VII and tissue
bin activator, which then causes prothrombin conversion factor. The lipoprotein complex of tissue factor fur-
to thrombin and all the subsequent clotting steps. ther complexes with blood coagulation factor VII

481
UNIT VI Blood Cells, Immunity, and Blood Coagulation

and, in the presence of calcium ions, acts enzymati- Intrinsic Pathway for Initiating Clotting
cally on factor X to form activated factor X (Xa). The second mechanism for initiating formation of pro-
3. Effect of Xa to form prothrombin activator—role thrombin activator, and therefore for initiating clotting,
of factor V. The activated factor X combines im- begins with trauma to the blood or exposure of the blood
mediately with tissue phospholipids that are part to collagen from a traumatized blood vessel wall. Then the
of tissue factors or with additional phospholipids process continues through the series of cascading reac-
released from platelets, as well as with factor V, tions shown in Figure 37-6.
to form the complex called prothrombin activator. 1. Blood trauma causes (1) activation of factor XII and
Within a few seconds, in the presence of Ca2+, pro- (2) release of platelet phospholipids. Trauma to the
thrombin is split to form thrombin, and the clot- blood or exposure of the blood to vascular wall col-
ting process proceeds as already explained. At first, lagen alters two important clotting factors in the
the factor V in the prothrombin activator complex blood: factor XII and the platelets. When factor XII
is inactive, but once clotting begins and thrombin is disturbed, such as by coming into contact with
begins to form, the proteolytic action of thrombin collagen or with a wettable surface such as glass, it
activates factor V. This activation then becomes an takes on a new molecular configuration that con-
additional strong accelerator of prothrombin ac- verts it into a proteolytic enzyme called activated
tivation. Thus, in the final prothrombin activator factor XII. Simultaneously, the blood trauma also
complex, activated factor X is the actual protease damages the platelets because of adherence to col-
that causes splitting of prothrombin to form throm- lagen or to a wettable surface (or by damage in other
bin. Activated factor V greatly accelerates this pro- ways); this releases platelet phospholipids that con-
tease activity, and platelet phospholipids act as a tain the lipoprotein called platelet factor 3, which
vehicle that further accelerates the process. Note also plays a role in subsequent clotting reactions.
especially the positive feedback effect of thrombin, 2. Activation of factor XI. The activated factor XII also
acting through factor V, to accelerate the entire pro- acts enzymatically on factor XI to activate this fac-
cess once it begins.! tor, which is the second step in the intrinsic path-

Blood trauma or
contact with collagen

(1) XII Activated XII (XIIa)

(HMW kininogen, prekallikrein)

(2) XI Activated XI (XIa)

Ca2+

(3) IX Activated IX (IXa)

VIII
Thrombin
VIIIa Ca2+

(4) X Activated X (Xa)

(5) Platelet
phospholipids
Thrombin Ca2+

Prothrombin
activator
Platelet
phospholipids
Prothrombin Thrombin

Figure 37-6. Intrinsic pathway for initiating

blood clotting. HMW, High-molecular weight. Ca2+

482
 Chapter 37 Hemostasis and Blood Coagulation

way. This reaction also requires high-molecular- the blood. With severe tissue trauma, clotting can occur
weight kininogen and is accelerated by prekallikrein. in as little as 15 seconds. The intrinsic pathway is much
3. Activation of factor IX by activated factor XI. The slower to proceed, usually requiring 1 to 6 minutes to
activated factor XI then acts enzymatically on fac- cause clotting.!
tor IX to activate this factor as well.
Intravascular Anticoagulants Prevent Blood

UNIT VI
4. Activation of factor X—role of factor VIII. The acti-
vated factor IX, acting in concert with activated fac- Clotting in the Normal Vascular System
tor VIII and the platelet phospholipids and factor III Endothelial Surface Factors. Probably the most impor-
from the traumatized platelets, activates factor X. It tant factors for preventing clotting in the normal vas-
is clear that when either factor VIII or platelets are cular system are the following: (1) the smoothness of the
in short supply, this step is deficient. Factor VIII is endothelial cell surface, which prevents contact activation
the factor that is missing in a person who has clas- of the intrinsic clotting system; (2) a layer of glycocalyx on
sic hemophilia, so it is called antihemophilic factor. the endothelium (glycocalyx is a mucopolysaccharide ad-
Platelets are the clotting factor that is lacking in the sorbed to the surfaces of the endothelial cells), which repels
bleeding disease called thrombocytopenia. clotting factors and platelets, thereby preventing activation
5. Action of activated factor X to form prothrombin of clotting; and (3) a protein bound with the endothelial
activator—role of factor V. This step in the intrinsic membrane, thrombomodulin, which binds thrombin. Not
pathway is the same as the last step in the extrinsic only does the binding of thrombin with thrombomodulin
pathway. That is, activated factor X combines with slow the clotting process by removing thrombin, but the
factor V and platelet or tissue phospholipids to form thrombomodulin-thrombin complex also activates a plas-
the complex called prothrombin activator. The pro- ma protein, protein C, that acts as an anticoagulant by inac-
thrombin activator, in turn, initiates the cleavage tivating activated factors V and VIII.
of prothrombin to form thrombin within seconds, When the endothelial wall is damaged, its smoothness
thereby setting into motion the final clotting pro- and glycocalyx-thrombomodulin layer are lost, which
cess, as described earlier.! activates both factor XII and the platelets, thus setting off
the intrinsic pathway of clotting. If factor XII and platelets
Role of Calcium Ions in the Intrinsic and
come into contact with the subendothelial collagen, the
Extrinsic Pathways
activation is even more powerful.
Except for the first two steps in the intrinsic pathway, cal- Intact endothelial cells also produce other substances
cium ions are required for promotion or acceleration of such a prostacyclin and nitric oxide (NO) that inhibit
all the blood-clotting reactions. Therefore, in the absence platelet aggregation and initiation of blood clotting. Pros-
of calcium ions, blood clotting by either pathway does not tacyclin, also called prostaglandin I2 (PGI2), is a member
occur. of the eicosanoid family of lipids and is a vasodilator, as
In the living body, the calcium ion concentration well as an inhibitor of platelet aggregation. As discussed
seldom falls low enough to affect blood-clotting kinet- in Chapter 17, NO is a powerful vasodilator released from
ics significantly. However, when blood is removed from healthy vascular endothelial cells throughout the body,
someone, it can be prevented from clotting by reducing and it is an important inhibitor of platelet aggregation.
the calcium ion concentration below the threshold level When endothelial cells are damaged, their production of
for clotting by deionizing the calcium by causing it to prostacyclin and NO is greatly diminished.!
react with substances such as citrate ion or by precipitat-
ing the calcium with substances such as oxalate ion.! Antithrombin Action of Fibrin and Antithrombin III.
Among the most important anticoagulants in the blood
Interaction Between Extrinsic and are those that remove thrombin from the blood. The most
Intrinsic Pathways—Summary of Blood- powerful of these are the following: (1) the fibrin fibers
Clotting Initiation that are formed during the process of clotting; and (2)
It is clear from the schemas of the intrinsic and extrinsic an α globulin called antithrombin III or antithrombin-
systems that after blood vessels rupture, clotting occurs heparin cofactor.
by both pathways simultaneously. Tissue factor initiates While a clot is forming, about 85% to 90% of the throm-
the extrinsic pathway, whereas contact of factor XII and bin formed from the prothrombin becomes adsorbed to
platelets with collagen in the vascular wall initiates the the fibrin fibers as they develop. This adsorption helps
intrinsic pathway. prevent the spread of thrombin into the remaining blood
An especially important difference between the and, therefore, prevents excessive spread of the clot.
extrinsic and intrinsic pathways is that the extrin- The thrombin that does not adsorb to the fibrin
sic pathway can be explosive; once initiated, its speed fibers soon combines with antithrombin III. This further
of completion to the final clot is limited only by the blocks the effect of thrombin on the fibrinogen and then
amount of tissue factor released from the traumatized also inactivates thrombin itself during the next 12 to 20
tissues and by the quantities of factors X, VII, and V in minutes.!

483
UNIT VI Blood Cells, Immunity, and Blood Coagulation

tPA
Heparin. Heparin is another powerful anticoagulant but, converts plasminogen to plasmin, which in turn removes
because its concentration in the blood is normally low, the remaining unnecessary blood clot. In fact, many small
it has significant anticoagulant effects only under special blood vessels in which blood flow has been blocked by
physiological conditions. However, heparin is used widely clots are reopened by this mechanism. Thus, an especially
as a pharmacological agent in medical practice in much important function of the plasmin system is to remove
higher concentrations to prevent intravascular clotting. minute clots from millions of tiny peripheral vessels that
The heparin molecule is a highly negatively charged eventually would become occluded were there no way to
conjugated polysaccharide. By itself, it has little or no clear them.!
anticoagulant properties, but when it combines with
antithrombin III, the effectiveness of antithrombin III
for removing thrombin increases by a hundredfold to a CONDITIONS THAT CAUSE EXCESSIVE
thousandfold and thus acts as an anticoagulant. There- BLEEDING IN HUMANS
fore, in the presence of excess heparin, the removal of free Excessive bleeding can result from a deficiency of any of
thrombin from the circulating blood by antithrombin III the many blood-clotting factors. Three particular types of
is almost instantaneous. bleeding tendencies that have been studied to the greatest
The complex of heparin and antithrombin III removes extent are discussed here—bleeding caused by (1) vitamin
several other activated coagulation factors in addition to K deficiency, (2) hemophilia, and (3) thrombocytopenia
thrombin, further enhancing the effectiveness of antico- (platelet deficiency).
agulation. The others include activated factors IX through
XII. DECREASED PROTHROMBIN, FACTOR VII,
Heparin is produced by many different cells of the FACTOR IX, AND FACTOR X CAUSED BY
body, but the largest quantities are formed by the baso- VITAMIN K DEFICIENCY
philic mast cells located in the pericapillary connec-
tive tissue throughout the body. These cells continually With few exceptions, almost all the blood-clotting fac-
secrete small quantities of heparin that diffuse into the tors are formed by the liver. Therefore, diseases of the
circulatory system. The basophil cells of the blood, which liver such as hepatitis, cirrhosis, and acute yellow atrophy
are functionally almost identical to the mast cells, release (degeneration of the liver caused by toxins, infections, or
small quantities of heparin into the plasma. other agents) can sometimes depress the clotting system
Mast cells are abundant in tissue surrounding the cap- so much that the patient develops a severe tendency to
illaries of the lungs and, to a lesser extent, capillaries of the bleed.
liver. It is easy to understand why large quantities of hepa- Another cause of depressed formation of clotting
rin might be needed in these areas because the capillaries factors by the liver is vitamin K deficiency. Vitamin K
of the lungs and liver receive many embolic clots that have is an essential factor to a liver carboxylase that adds a
formed in slowly flowing venous blood; sufficient produc- carboxyl group to glutamic acid residues on five of the
tion of heparin prevents further growth of the clots.! important clotting factors—prothrombin, factor VII, fac-
tor IX, factor X, and protein C. On adding the carboxyl
PLASMIN CAUSES LYSIS OF BLOOD CLOTS group to glutamic acid residues on the immature clot-
ting factors, vitamin K is oxidized and becomes inactive.
The plasma proteins contain a euglobulin called plas-
Another enzyme, vitamin K epoxide reductase complex 1
minogen (profibrinolysin) that when activated, becomes a
(VKORC1), reduces vitamin K back to its active form. In
substance called plasmin (fibrinolysin). Plasmin is a pro-
the absence of active vitamin K, subsequent insufficiency
teolytic enzyme that resembles trypsin, the most impor-
of these coagulation factors in the blood can lead to seri-
tant proteolytic digestive enzyme of pancreatic secretion.
ous bleeding tendencies.
Plasmin digests fibrin fibers and some other protein
Vitamin K is continually synthesized in the intestinal
coagulants, such as fibrinogen, factor V, factor VIII, pro-
tract by bacteria, so vitamin K deficiency seldom occurs
thrombin, and factor XII. Therefore, whenever plasmin
in healthy persons as a result of the absence of vitamin K
is formed, it can cause lysis of a clot by destroying many
from the diet (except in neonates, before they establish
of the clotting factors, thereby sometimes even causing
their intestinal bacterial flora). However, in persons with
hypocoagulability of the blood.
gastrointestinal disease, vitamin K deficiency often occurs
Activation of Plasminogen to Form Plasmin, Then as a result of poor absorption of fats from the gastrointes-
Clot Lysis. When a clot is formed, a large amount of plas- tinal tract because vitamin K is fat-soluble and is ordinar-
minogen is trapped in the clot, along with other plasma ily absorbed into the blood along with the fats.
proteins. This will not become plasmin or cause lysis of One of the most prevalent causes of vitamin K defi-
the clot until it is activated. The injured tissues and vascu- ciency is failure of the liver to secrete bile into the gas-
lar endothelium very slowly release a powerful activator trointestinal tract, which occurs as a result of obstruction
called tissue plasminogen activator (t-PA); a few days later, of the bile ducts or of liver disease. Lack of bile prevents
after the clot has stopped the bleeding, t-PA eventually adequate fat digestion and absorption and, therefore,

484
 Chapter 37 Hemostasis and Blood Coagulation

depresses vitamin K absorption as well. Thus, liver dis- disease with somewhat different characteristics, called
ease often causes decreased production of prothrombin von Willebrand disease, results from loss of the large
and some other clotting factors because of poor vitamin component.
K absorption and because of the diseased liver cells. As When a person with classic hemophilia experiences
a result, vitamin K is injected into surgical patients with severe prolonged bleeding, almost the only therapy that is

UNIT VI
liver disease or with obstructed bile ducts before the truly effective is injection of purified factor VIII or factor
surgical procedure is performed. Ordinarily, if vitamin IX. Both these clotting factors are now available as recom-
K is given to a deficient patient 4 to 8 hours before the binant proteins, although they are expensive and their
operation and the liver parenchymal cells are at least half- half-lives are relatively short; therefore, these products are
normal in function, sufficient clotting factors will be pro- not readily available for many patients with hemophilia,
duced to prevent excessive bleeding during the operation.! especially in economically disadvantaged countries.!

HEMOPHILIA THROMBOCYTOPENIA
Hemophilia is a bleeding disease that occurs almost Thrombocytopenia means the presence of very low num-
exclusively in males. In 85% of cases, it is caused by an bers of platelets in the circulating blood. People with
abnormality or deficiency of factor VIII; this type of hemo- thrombocytopenia have a tendency to bleed, as do hemo-
philia is called hemophilia A or classic hemophilia. About philiacs, except that the bleeding is usually from many
1 of every 10,000 males in the United States has classic small venules or capillaries, rather than from larger ves-
hemophilia. In the other 15% of patients with hemophilia sels, as in hemophilia. As a result, small punctate hem-
B, the bleeding tendency is caused by deficiency of factor orrhages occur throughout all the body tissues. The skin
IX. Both these factors are transmitted genetically by way of such a person displays many small petechiae, red or
of the female (X) chromosome and are recessive in their purplish blotches, giving the disease the name throm-
inheritance. Therefore, a woman will rarely have hemo- bocytopenic purpura. As noted, platelets are especially
philia because at least one of her two X chromosomes will important for the repair of minute breaks in capillaries
have the appropriate genes. If one of her X chromosomes and other small vessels.
is deficient, she will be a hemophilia carrier; her male off- Platelet counts below 30,000/µl, compared with the
spring will have a 50% chance of inheriting the illness, and normal value of 150,000 to 450,000/µl, increase the risk
her female offspring will have a 50% chance of inheriting for excessive bleeding after surgery or injury. Spontane-
the carrier status. ous bleeding, however, will not ordinarily occur until the
Although female carriers have one normal allele and number of platelets in the blood falls below 30,000/µl.
usually do not develop symptomatic hemophilia, some Levels as low as 10,000/µl are frequently lethal.
may experience a mild bleeding trait. It is also possible Even without determining specific platelet counts in
for female carriers to develop mild hemophilia due to the blood, sometimes one can suspect the existence of
loss of part or all of the normal X chromosome (as in thrombocytopenia if the person’s blood clot fails to retract.
Turner syndrome) or inactivation (lyonization) of the As noted earlier, clot retraction is normally dependent
X-chromosomes. For a female to inherit full-blown on release of multiple coagulation factors from the large
symptomatic hemophilia A or B, she must receive two numbers of platelets entrapped in the fibrin mesh of the
deficient X-chromosomes, one from her carrier mother clot.
and the other from her father, who must have hemophilia. The major causes of thrombocytopenia include the
Most cases of hemophilia are inherited, but approxi- following: (1) decreased platelet production in the bone
mately one-third of hemophilia patients do not have a marrow due to infections or sepsis, nutrient deficiencies,
family history of the disease, which appears to be caused or myelodysplastic disorders, which usually also reduce
by novel mutation events. production of other cells (red blood cells [RBCs] and
The bleeding trait in hemophilia can have various white blood cells); (2) peripheral platelet destruction by
degrees of severity, depending on the genetic deficiency. antibodies; (3) sequestration (pooling) of platelets in the
Bleeding usually does not occur except after trauma, but spleen, especially in individuals with portal hypertension
in some patients, the degree of trauma required to cause and excessively large spleens (splenomegaly); (4) con-
severe and prolonged bleeding may be so mild that it is sumption of platelets in thrombi; and (4) dilution of the
hardly noticeable. For example, bleeding can often last for blood from fluid resuscitation or massive transfusion.
days after extraction of a tooth. Most people with thrombocytopenia have the disease
Factor VIII has two active components, a large compo- known as idiopathic thrombocytopenia, which means
nent with a molecular weight in the millions and a smaller thrombocytopenia of unknown cause. In most of these
component with a molecular weight of about 230,000. people, it has been discovered that, for unknown reasons,
The smaller component is most important in the intrinsic specific antibodies have formed and react against the plate-
pathway for clotting, and it is deficiency of this part of fac- lets to destroy them. Relief from bleeding for 1 to 4 days
tor VIII that causes classic hemophilia. Another bleeding can often be effected in a patient with thrombocytopenia

485
UNIT VI Blood Cells, Immunity, and Blood Coagulation

by giving fresh whole blood transfusions that contain large artery is blocked, death may not occur, or the embolism
numbers of platelets. Also, splenectomy may be helpful, may lead to death a few hours to several days later because
sometimes resulting in an almost complete cure because of further growth of the clot in the pulmonary vessels.
the spleen normally removes large numbers of platelets However, again, t-PA therapy can be a lifesaver.!
from the blood.!
DISSEMINATED INTRAVASCULAR
COAGULATION
THROMBOEMBOLIC CONDITIONS
Occasionally, the clotting mechanism becomes activated
Thrombi and Emboli. An abnormal clot that develops in widespread areas of the circulation, giving rise to the
in a blood vessel is called a thrombus. Once a clot has de- condition called disseminated intravascular coagulation
veloped, continued flow of blood past the clot is likely to (DIC). This condition often results from the presence
break it away from its attachment and cause the clot to of large amounts of traumatized or dying tissue in the
flow with the blood; such freely flowing clots are known body that releases great quantities of tissue factor into
as emboli. Also, emboli that originate in large arteries or the blood. Frequently, the clots are small but numerous,
in the left side of the heart can flow peripherally and plug and they plug a large share of the small peripheral blood
arteries or arterioles in the brain, kidneys, or elsewhere. vessels. This process occurs especially in patients with
Emboli that originate in the venous system or in the right widespread septicemia, in which circulating bacteria or
side of the heart generally flow into the lungs to cause pul- bacterial toxins—especially endotoxins—activate the clot-
monary arterial embolism.! ting mechanisms. The plugging of small peripheral vessels
greatly diminishes delivery of oxygen and other nutrients
Causes of Thromboembolic Conditions. The causes of to the tissues, a situation that leads to or exacerbates cir-
thromboembolic conditions in people are usually twofold: culatory shock. It is partly for this reason that septicemic
(1) a roughened endothelial surface of a vessel—as may be shock is lethal in 35% to 50% of patients.
caused by arteriosclerosis, infection, or trauma—is likely A peculiar effect of disseminated intravascular coagu-
to initiate the clotting process; and (2) blood often clots lation is that the patient, on occasion, begins to bleed. The
when it flows very slowly through blood vessels, where reason for this bleeding is that so many of the clotting fac-
small quantities of thrombin and other procoagulants are tors are removed by the widespread clotting that too few
always being formed.! procoagulants remain to allow normal hemostasis of the
Use of Tissue Plasminogen Activator in Treating In- remaining blood.!
travascular Clots. Genetically engineered tissue plas-
minogen activator (t-PA) is available. When delivered ANTICOAGULANTS FOR CLINICAL USE
through a catheter to an area with a thrombus, it is effec-
tive in activating plasminogen to plasmin, which in turn In some thromboembolic conditions, it is desirable to
can dissolve some intravascular clots. For example, if used delay the coagulation process. Various anticoagulants
within the 1 or 2 hours after thrombotic occlusion of a have been developed for this purpose. The ones most
coronary artery, the heart is often spared serious damage.! clinically useful are heparin and the coumarins.

FEMORAL VENOUS THROMBOSIS AND HEPARIN—INTRAVENOUS ANTICOAGULANT

MASSIVE PULMONARY EMBOLISM Commercial heparin is extracted from several differ-
Because clotting almost always occurs when blood flow ent animal tissues and prepared in almost pure form.
is blocked for many hours in any vessel of the body, the Injection of relatively small quantities, about 0.5 to 1
immobility of patients confined to bed, plus the practice mg/kg of body weight, causes the blood-clotting time
of propping the knees up with pillows, often causes intra- to increase from a normal of about 6 minutes to 30 or
vascular clotting because of blood stasis in one or more more minutes. Furthermore, this change in clotting time
of the leg veins for hours at a time. Then the clot grows, occurs instantaneously, thereby immediately preventing
mainly in the direction of the slowly moving venous or slowing further development of a thromboembolic
blood, sometimes growing the entire length of the leg condition.
veins and occasionally even up into the common iliac vein The action of heparin lasts about 1.5 to 4 hours. The
and inferior vena cava. About 10% of the time, a large part injected heparin is destroyed by an enzyme in the blood
of the clot disengages from its attachments to the vessel known as heparinase.!
wall and flows freely with the venous blood through the
COUMARINS AS ANTICOAGULANTS
right side of the heart and into the pulmonary arteries to
cause massive blockage of the pulmonary arteries; this is When a coumarin, such as warfarin, is given to a patient,
called a massive pulmonary embolism. If the clot is large the amounts of active prothrombin and factors VII, IX,
enough to occlude both pulmonary arteries at the same and X, all formed by the liver, begin to fall. Warfarin
time, immediate death ensues. If only one pulmonary causes this effect by inhibiting the enzyme VKORC1. As

486
 Chapter 37 Hemostasis and Blood Coagulation

discussed previously, this enzyme converts the inactive, polymerized into glucose or metabolized directly for
oxidized form of vitamin K to its active, reduced form. energy. Consequently, 500 milliliters of blood that has
By inhibiting VKORC1, warfarin decreases the avail- been rendered noncoagulable by citrate can ordinarily be
able active form of vitamin K in the tissues. When this transfused into a recipient within a few minutes, without
decrease occurs, the coagulation factors are no longer car- dire consequences. However, if the liver is damaged, or

UNIT VI
boxylated and are biologically inactive. Over several days, if large quantities of citrated blood or plasma are given
the body stores of the active coagulation factors degrade too rapidly (within fractions of a minute), the citrate ion
and are replaced by inactive factors. Although the coagu- may not be removed quickly enough, and the citrate can,
lation factors continue to be produced, they have greatly under these conditions, greatly depress the level of cal-
decreased coagulant activity. cium ion in the blood, which can result in tetany and con-
After administration of an effective dose of warfarin, vulsive death.!
the coagulant activity of the blood decreases to about 50%
of normal by the end of 12 hours and to about 20% of
BLOOD COAGULATION TESTS
normal by the end of 24 hours. In other words, the coagu-
lation process is not blocked immediately but must await
BLEEDING TIME
the degradation of the active prothrombin and the other
affected coagulation factors already present in the plasma. When a sharp-pointed knife is used to pierce the tip of the
Normal coagulation usually returns 1 to 3 days after dis- finger or earlobe, bleeding ordinarily lasts for 1 to 6 min-
continuing coumarin therapy.! utes. This time depends largely on the depth of the wound
and degree of hyperemia in the finger or earlobe at the
PREVENTION OF BLOOD COAGULATION time of the test. Lack of any one of several of the clotting
OUTSIDE THE BODY factors can prolong the bleeding time, but it is especially
prolonged by lack of platelets.!
Although blood removed from the body and held in a
glass test tube normally clots in about 6 minutes, blood CLOTTING TIME
collected in siliconized containers often does not clot for
Many methods have been devised for determining blood-
1 hour or more. The reason for this delay is that preparing
clotting time. The one most widely used is to collect
the surfaces of the containers with silicone prevents con-
blood in a chemically clean glass test tube and then to
tact activation of platelets and factor XII, the two princi-
tip the tube back and forth about every 30 seconds until
pal factors that initiate the intrinsic clotting mechanism.
the blood has clotted. By this method, the normal clot-
Conversely, untreated glass containers allow contact acti-
ting time is 6 to 10 minutes. Procedures using multiple
vation of the platelets and factor XII, with the rapid devel-
test tubes have also been devised for determining clotting
opment of clots.
time more accurately.
Heparin can be used for preventing coagulation of
Unfortunately, the clotting time varies widely, depend-
blood outside the body, as well as in the body. Heparin is
ing on the method used for measuring it, so it is no lon-
especially used in surgical procedures in which the blood
ger used in many clinics. Instead, measurements of the
must be passed through a heart-lung machine or artificial
clotting factors themselves are made, using sophisticated
kidney machine and then back into the patient.
Various substances that decrease the concentration of chemical procedures.!
calcium ions in the blood can also be used for preventing PROTHROMBIN TIME AND
blood coagulation outside the body. For example, a solu- INTERNATIONAL NORMALIZED RATIO
ble oxalate compound mixed in a very small quantity with
a sample of blood causes precipitation of calcium oxalate The prothrombin time indicates the concentration of
from the plasma and thereby decreases the ionic calcium prothrombin in the blood. Figure 37-7 shows the rela-
level so much that blood coagulation is blocked. tionship of prothrombin concentration to prothrombin
Any substance that deionizes the blood calcium will time. The method for determining prothrombin time is
prevent coagulation. The negatively charged citrate ion is the following.
especially valuable for this purpose; it is mixed with blood Blood removed from the patient is immediately oxa-
usually in the form of sodium, ammonium, or potassium lated so that none of the prothrombin can change into
citrate. The citrate ion combines with calcium in the thrombin. Then, a large excess of calcium ion and tis-
blood to produce a nonionized calcium compound, and sue factor is quickly mixed with the oxalated blood. The
the lack of ionic calcium prevents coagulation. Citrate excess calcium nullifies the effect of the oxalate, and the
anticoagulants have an important advantage over the oxa- tissue factor activates the prothrombin to thrombin reac-
late anticoagulants because oxalate is toxic to the body, tion by means of the extrinsic clotting pathway. The time
whereas moderate quantities of citrate can be injected required for coagulation to take place is known as the
intravenously. After injection, the citrate ion is removed prothrombin time. The shortness of the time is determined
from the blood within a few minutes by the liver and is mainly by the prothrombin concentration. The normal

487
UNIT VI Blood Cells, Immunity, and Blood Coagulation

100 Tests similar to that for prothrombin time and INR have
been devised to determine the quantities of other blood-
clotting factors. In each of these tests, excesses of calcium
Concentration (percent of normal)
ions and all the other factors in addition to the one being
tested are added to oxalated blood all at once. Then, the time
required for coagulation is determined in the same manner
as for prothrombin time. If the factor being tested is defi-
50.0 cient, the coagulation time is prolonged. The time itself can
then be used to quantitate the concentration of the factor.

25.0 Bibliography
12.5 Becker RC, Sexton T, Smyth SS: Translational implications of platelets
6.25 as vascular first responders. Circ Res 122:506, 2018.
0 Furie B, Furie BC: Mechanisms of thrombus formation. N Engl J Med
0 10 20 30 40 50 60 359:938, 2008.
Gupta S, Shapiro AD: Optimizing bleed prevention throughout the
Prothrombin time
(seconds) lifespan: womb to tomb. Haemophilia 24 Suppl 6:76, 2018.
Hess CN, Hiatt WR: Antithrombotic therapy for peripheral artery dis-
Figure 37-7. Relationship of prothrombin concentration in the blood ease in 2018. JAMA 319:2329, 2018.
to prothrombin time. Hunt BJ: Bleeding and coagulopathies in critical care. N Engl J Med
370:847, 2014.
Koupenova M, Clancy L, Corkrey HA, Freedman JE: Circulating plate-
prothrombin time is about 12 seconds. In each laboratory, lets as mediators of immunity, inflammation, and thrombosis. Circ
a curve relating prothrombin concentration to prothrom- Res 122:337, 2018.
bin time, such as that shown in Figure 37-7, is drawn for Kucher N: Clinical practice. Deep-vein thrombosis of the upper ex-
the method used so that the prothrombin in the blood can tremities. N Engl J Med 364:861, 2011.
Leebeek FW, Eikenboom JC: Von Willebrand’s disease. N Engl J Med
be quantified. 375:2067, 2016.
The results obtained for prothrombin time may vary Luyendyk JP, Schoenecker JG, Flick MJ: The multifaceted role of fi-
considerably, even in the same individual if there are brinogen in tissue injury and inflammation. Blood 133:511, 2019.
differences in activity of the tissue factor and the ana- Maas C, Renné T: Coagulation factor XII in thrombosis and inflamma-
lytical system used to perform the test. Tissue factor tion. Blood 131:1903, 2018.
McFadyen JD, Schaff M, Peter K: Current and future antiplatelet
is isolated from human tissues, such as placental tis- therapies: emphasis on preserving haemostasis. Nat Rev Cardiol
sue, and different batches may have different activity. 15:181, 2018.
The international normalized ratio (INR) was devised Mohammed BM, Matafonov A, Ivanov I, et al: An update on factor XI
as a way to standardize measurements of prothrombin structure and function. Thromb Res 161:94, 2018.
time. For each batch of tissue factor, the manufacturer Nachman RL, Rafii S: Platelets, petechiae, and preservation of the vas-
cular wall. N Engl J Med 359:1261, 2008.
assigns an international sensitivity index (ISI), which Negrier C, Shima M, Hoffman M: The central role of thrombin in bleed-
indicates the activity of the tissue factor with a stan- ing disorders. Blood Rev 2019 May 22. pii: S0268-960X(18)30097-3.
dardized sample. The ISI usually varies between 1.0 https://www.doi.org/10.1016/j.blre.2019.05.006
and 2.0. The INR is the ratio of the person’s prothrom- Peters R, Harris T: Advances and innovations in haemophilia treat-
bin time (PT) to a normal control sample raised to the ment. Nat Rev Drug Discov 17:493, 2018.
Samuelson Bannow B, Recht M, Négrier C, et al: Factor VIII: long-
power of the ISI: established role in haemophilia A and emerging evidence beyond
ISI haemostasis. Blood Rev 35:43, 2019.
INR =  PTtest

Tillman BF, Gruber A, McCarty OJT, Gailani D: Plasma contact factors
PT normal

as therapeutic targets. Blood Rev 32:433, 2018.
The normal range for INR in a healthy person is 0.9 to van der Meijden PEJ, Heemskerk JWM: Platelet biology and functions:
1.3. A high INR level (e.g., 4 or 5) indicates a high risk of new concepts and clinical perspectives. Nat Rev Cardiol 16:166, 2019.
Wells PS, Forgie MA, Rodger MA: Treatment of venous thromboem-
bleeding, whereas a low INR (e.g., 0.5) suggests that there bolism. JAMA 311:717, 2014.
is a chance of having a clot. Patients undergoing warfarin Weyand AC, Pipe SW: New therapies for hemophilia. Blood 133:389,
therapy usually have an INR of 2.0 to 3.0. 2019.

488
 Lymphoreticular system

Physiology Lecture 5+6:

Blood Types; Transfusion, and Tissue and
Organ Transplantation
Chapter 36
Dr. Reham Khalaf-Nazzal, MD, PhD
Copyright
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by Saunders,
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Learning Resources

Guyton and Hall, Textbook

of Medical Physiology, 14th
Edition, unit VI- Chapter 33

Fourteenth Edition

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Intended Learning Outcomes

After reviewing the PowerPoint presentation and the associated

learning resources, the student should be able to:

Describe the ABO and Rhesus blood group systems

Recognize agglutinogens on the surface of the RBCs
Recognize agglutinins in the plasma
Describe grouping, crossmatching & typing with antisera
List precautions taken in preparing blood for transfusion and storage.
Define blood transfusion and list its uses
Describe the hazards of incompatible blood transfusion reactions.
Define hemolytic disease of newborn, describe its pathophysiology and
outline its prevention

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Early Transfusions

Red cell agglutination and lysis

Severe transfusion reactions, often
fatal
In other cases, well-tolerated and
beneficial
Led to the discovery of red blood cell
antigens and the practice of cross-
matching
>30 common antigens, many rare
ones
Rowlinson BRANCH:
Britain, Representation and Nineteenth-Century History. Ed. Dino Franco Felluga. Extension of Romanticism
and Victorianism on the Net. Web. [Here, add your last date of access to BRANCH]

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The ABO System

Red blood cell surface antigens:

glycolipids or glycoproteins

Present on all cells in the body, not

just blood cells

Agglutinogens: surface antigens (A,B)

Genes: A, B, O (maternal, paternal
alleles)
Genotypes: OO, OA, OB, AA, BB,
AB
Agglutinins (immunoglobulins): anti-A,
anti-B

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Genetic Determination of ABO Antigens

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Agglutinins

Antibodies, mostly
IgM and IgG
Begin developing
age 2 8 months,
peak ~age 10 years
Response to A and
B antigens in foods,
bacteria; initial
exposures are
environmental
Figure 36-1

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
 Blood Groups

Table 36-1

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Transfusion Reactions

Red cells agglutinate

Clumps plug small vessels
Physical distortion, phagocytic
attack

In some cases, immediate,

complement-dependent
hemolysis (depends on Ig
IgM

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
The Rh (rhesus) Antigens

Requires prior exposure to incompatible blood

Six common antigens ( Rh factors )
C, D, E, c, d, e
Each person is CDE, CDe, Cde, CdE, cDE, cDe, or cde
D ( Rh positive ) is prevalent (85% EA, 100% Africans)
and particularly antigenic
C and E can also cause transfusion reactions, generally
milder

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Anti-Rh Transfusion Reactions (1 of 2)

Rh positive blood into Rh negative person:

sensitization to further Rh+ transfusion
agglutinins peak 2 4 months after exposure

50% of Rh neg are sensitized by a single exposure

20% after a second exposure
30% are non-responders

Rh matching between donor and recipient to prevent

immunization

85 % of caucasians , 95% of black Americans, of chinese

and nearly 100 99 % % of black Africans are Rh+

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Anti-Rh Transfusion Reactions (2 of 2)

Naïve Rh negative recipient

Usually no reaction initially
Within 2 4 weeks sufficient Ig for agglutination
Delayed reaction, usually mild hemolysis within
tissue macrophages
Any subsequent transfusion with Rh positive
blood
Potentially severe transfusion reaction

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Agglutinins

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Rh Blood Types

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Universal Donor; Suitable for all?

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Blood Transfusion

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Hemolytic Disease of the Newborn
(Erythroblastosis Fetalis)
ABO incompatibility
O mother and A or B fetus
Most anti-A is IgM which does not cross placenta
ABO antigens are not well developed in fetus.

Rh incompatibility
Rh (D) positive fetus and Rh negative mother
Immunization due to fetal-maternal bleeding during delivery. Mother develops Anti-
D agglutinins
Usually not a problem with first pregnancy
Worse with subsequent pregnancies (3% EF second pregnancy, 10% with third)

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Hemolytic Disease of the Newborn-
Overview

During birth, there is often a leakage of the

baby's red blood cells into the mother's
circulation. But the first pregnancy passes without
any problems. ??

If the baby is Rhpositive (having inherited the

trait from its father) and the mother Rhnegative,
these red cells will cause her to develop
antibodies (IgG class) against the antigen.

In 2 nd RhD child, hemolytic disease of the

newborn may develop causing hemolysis of the

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Hemolytic Disease of the Newborn
(Erythroblastosis Fetalis) Pathophysiology

Maternal antibodies cross the placenta and cause

agglutination and lysis of fetal erythrocytes
Fetal macrophages convert hemoglobin to bilirubin

Anemic at birth; continued hemolysis 1 2 months

Hepato-splenomegaly from extramedullary
erythropoiesis
May have permanent neurologic damage from
deposition of bilirubin in neural tissues (kernicterus)

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Hemolytic Disease of the Newborn
(Erythroblastosis Fetalis) Treatment

Repetitive removal of Rh positive blood,

replacement with Rh negative (400 mL
exchange over 90 minutes)
May be done several times over a few
weeks
Maternal antibodies disappear over 1 2
Rh positive cells
cease to be a target.
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Hemolytic Disease of the Newborn-
Complications

Hemolytic anemia:
If severe: treated with exchange
transfusion: Replace baby blood with
Rh- ve RBC (several times)

Kernicterus (mental retardation due

to bilirubin deposition in the brain).

Hydrops fetalis (death in utero)

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Hemolytic Disease of the Newborn
(Erythroblastosis Fetalis) Prevention

Provide exogenous anti-D antibodies to the

mother in late pregnancy and just after birth.

These bind to D antigenic sites on fetal

erythrocytes that enter the circulation,
preventing an immune response.

The routine administration of such treatment

to Rh ve mothers after the delivery of Rh+ve
baby has reduced the incidence of disease by
>90%.

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Blood Transfusion

Single donation is 450 mL.

Processed into components
a. Packed Red Cell
Stored for ~30 40 days
b. Plasma (clotting factors); Frozen
c. Platelets; Stored for 8 10 days
d. White blood cells; Rarely used

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Requirements Prior to Blood
Transfusion

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Typing and Cross-Matching Blood

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Transfusion Reactions

Occur because of mismatched blood

Recipient antibodies react against donor antigens
Either immediate or delayed agglutination and
hemolysis
Fever, chills, shortness of breath; potentially shock,
renal shutdown
Macrophages produce bilirubin.

ml blood hemolyzed in <1 day.

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Acute Renal Failure After
Transfusion Reaction

Products of hemolysis cause powerful renal

vasoconstriction
Immune-mediated circulatory shock
Free hemoglobin can leak through
glomerular membranes into tubules.

May require acute or even chronic

hemodialysis
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Extra slides for multidisciplinary

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Transplantation of Solid Tissues
and Organs

Autograft Transplantation to self (e.g., skin,

hair, tendon)
Isograft Between genetically identical donor
and recipient
Allograft Between out-bred individuals of
same species
Xenograft Across species

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
 Graft Acceptance/Rejection

Autografts and isografts Obstacles are mechanical

attachment and adequate blood supply
Allografts Antigenic matching is important
Xenografts Almost always rejected with tissue death 1 day to
5 weeks after grafting
Successful allografts: skin, kidney, heart, liver, pancreas, bone
marrow, lung, lasting up to 15 years or more

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Rejection

Hyperacute rejection Days

Acute rejection Weeks

Chronic rejection Months to years

Rejection is mainly due to activated T cells

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Avoidance or Suppression of
Rejection

Tissue typing
Blood type
HLA (MHC) antigens

Immunosuppressive drugs

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HLA Antigens

Encoded by the MHC

Six classes, total of >150 antigens expressed
on all nucleated cells
MHC Class I: HLA-A, -B, and -C
MHC Class II: HLA-DP, -DQ and DR

Seek the best match possible among the

closest relatives possible

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Histocompatibility Testing

Originally panels of anti-sera from multiparous

women
Workshops to define common antigens
Now monoclonal antibodies or genetic testing
Transplant success is directly related to quality
of histocompatibility matching.

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Immunosuppressive Drugs

Glucocorticoids Suppress growth of lymphoid cells;

reduce production of antibodies and activated T Cells
Drugs that are toxic for lymphocytes e.g.,
azathioprine
*T cell-specific agents Anti-lymphocyte globulin,
Cyclosporin, FK506 (tacrolimus), mycophenylate
mofetil
* Transformative, preventing graft rejection while leaving
much of the immune system intact

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Challenges with Immunosuppression

Overwhelming infection, particularly viral

infection

Reduced tumor surveillance and increased

incidence of cancer

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
Thank you

Dr. Reham Khalaf-Nazzal, MD, PhD Copyright © 2021 by Saunders, an imprint of Elsevier Inc.
 Pathology and microbiology

1) Associated with RBCs:

Erythropoiesis (RBC production):
Week 3-8 3-8: yolk sac
Week 6-birth: liver
Week 10-28: spleen
After week 18: red bone marrow

 After birth: red bone marrow:

In children: diaphyses of long bones
In adults: Mainly plain bones (pelvis, cranium, vertebrae, sternum)
and metaphyses of long bones.

Regulation:
Primarily EPO (Endothelial cells in the peritubular capillaries of the Kidneys and
hepatocytes).
Stages:
Hematopoietic stem cell (erythroid progenitor cell):
 Proerythroblast
In bone  Erythroblast
marrow  Normoblast.
 Reticulocyte:
acidophilic cytoplasm with granules composed of cytoplasmic
libosomal RNA (enabling reticulocytes to be stained with methylene
blue)
Reticulocytes are released into the blood.
 The peripheral reticulocyte count reflects erythropoietic activity.
In  Increased reticulocytes indicate increased erythropoiesis (e.g.
blood due to hemolysis).
 Decreased reticulocytes indicate decreased erythropoiesis (e.g.,
due to aplastic anemia).
Reticulocytes mature into erythrocytes.
MCV vs MCH
Mean cell volume (MCV): Mean cell hemoglobin (MCH):
The average volume per red cell, the average mass of hemoglobin per
expressed in femtoliters (𝝁𝒎𝟑 ). red cell, expressed in picograms

A) Anemia
Decrease in hemoglobin concentration. RBC count, and/or hematocrit.
General principles:
1) Reduction in circulating red blood cell (RBC) mass.
2) Presents with signs and symptoms of hypoxia (general features of anemia):
 Weakness, fatigue, and dyspnea.
 Pale conjunctiva and skin.
 Headache and light headedness.
 Angina, especially with preexisting coronary artery disease.
 Dizziness, difficulty of concentration and insomnia.
3) Hemoglobin (Hb), hematocrit (Hct), and RBC count are used as surrogates for
RBC mass, which is difficult to measure.
Men: < 𝟏𝟑. 𝟓 𝒈/𝒅𝒍
Women: < 𝟏𝟐 𝒈/𝒅𝒍
Children: 6 - 59 months < 𝟏𝟏 𝒈/𝒅𝒍
The fetus may reach 20-22 within 30 5 -11 years < 𝟏𝟏. 𝟓 𝒈/𝒅𝒍
days. 12 -14 years < 𝟏𝟐 𝒈/𝒅𝒍

Grading of anemia:
Grades Hemoglobin count
Normal 𝒉𝒊𝒈𝒉𝒆𝒓 𝒐𝒓 𝒆𝒒𝒖𝒂𝒍 − 𝟏𝟏
Mild 𝟗. 𝟓 – 𝟏𝟏
Moderate 𝟖 – 𝟗. 𝟓
Serious or sever 𝟔. – 𝟖
Life threatening 𝒍𝒆𝒔𝒔 𝒕𝒉𝒂𝒏 − 𝟔
Classification of anemia:
 Anemia may be classified into several subtypes based on the following methods:
 Morphology/size of RBCs (the classification most widely used).
 Time course: acute vs. chronic
 Inheritance: inherited vs. acquired
 Etiology: primary vs. secondary.
 RBC proliferation: hypoproliferative (decreased RBC production) vs.
hyperproliferative (increased RBC destruction or blood loss).

Anemia Microcytic anemia normocytic macrocytic

MCV < 𝟖𝟎 𝝁𝒎𝟑 𝟖𝟎 – 𝟏𝟎𝟎 𝝁𝒎𝟑 > 𝟏𝟎𝟎 𝝁𝒎𝟑
Mechanism Insufficient Decreased Blood Defective DNA
hemoglobin volume or / and synthesis or
production erythropoiesis repair.
MCV and MCH low Normal High
 Blood loss (acute).
Common cause  Iron deficiency.  Haemolysis.  Megaloblastic
 Thalassaemia.  Chronic disease. anemias
 Marrow infiltration.

MICROCYTIC ANEMIAS
A. Anemia with MCV < 𝟖𝟎 𝝁𝒎𝟑 .
B. Microcytic anemias are due to decreased production of hemoglobin.
 RBC progenitor cells in the bone marrow are large and normally divide
multiple times to produce smaller mature cells (MCV = 80-100 µm3).
 Microcytosis is due to an "extra" division which occurs to maintain
hemoglobin concentration.
C. A decrease in heme, globin, iron or protoporphyrin. leads to microcytic
anemia.
D. Microcytic anemias include;

1) Defective heme synthesis:

 Iron deficiency anemia (the most common).
 Anemia of chronic disease (late phase). Erythrocytes with different sizes
 sideroblastic anemia. (anisocytosis).
 Lead poisoning. Abnormal shapes (poikilocytosis).

2) Defective globin chain:

 Thalassemia.
 1) IRON DEFICIENCY ANEMIA
A. Due to decreased levels of iron, heme or globin.
B. Most common type of anemia.
C. Iron is consumed in heme (meat-derived) and non-heme (vegetable-derived)
forms.
Absorption occurs in the duodenum.
Stored intracellular iron is bound to ferritin, which prevents iron from
forming free radicals via the Fenton reaction.
D. Laboratory measurements of iron status.
 Serum iron-measure of iron in the blood.
 Total iron-binding capacity (TIBC) - measure of transferrin molecules in the
blood.
 % saturation - percentage of transferrin molecules that are bound by iron
(normal is 33%).
 Serum ferritin - reflects iron stores in macrophages and the liver.
E. Iron deficiency is usually caused by dietary lack or blood loss.
Infants  Exclusive intake nonfortified cow's milk
‫حليب االبقار ال يحتوي‬
 Exclusive breast-feeding after 6 months of age ‫الحديد كما يحتاج الطفل‬
Children  Exclusive intake nonfortified cow's milk. ‫ شهور الخذ الحديد‬6 ‫بعد‬
 Malnutrition. ‫من مصادر خارج‬
Adults (20-50 peptic ulcer disease in males and menorrhagia or .‫الرضاعة الطبيعية‬
years) pregnancy in females
Elderly > 𝟓𝟎 colon polyps/carcinoma

F. Stages of iron deficiency

Storage iron is depleted Ferritin (↓) TIBC (↑)
Serum iron is depleted serum iron (↓) % saturation (↓)
Normocytic anemia Bone marrow makes fewer, but normal-sized, RBCs.
Microcytic, hypochromic anemia Bone marrow makes smaller and fewer RBCs.

H. Laboratory findings include

 Microcytic, hypochromic RBCs with (↑) red cell distribution width (RDW).
 (↓) ferritin; (↓) serum iron; (↓)% saturation and (↑)TIBC.
 (↑) Free erythrocyte protoporphyrin (FEP).
Pathophysiology:
Iron deficiency  (↓) binding of iron to protoporphyrin (last reaction in heme
synthesis)  (↓) production of hemoglobin.

Epidemiology
 Children up to 5 years of age.
 Young women of child-bearing age (due to menstrual blood loss).
 Pregnant women

Iron deficiency mechanisms

1) Iron losses (Bleeding- the most common cause)
A) Menorrhagia: .‫دم نراه بالعين وال نراه بالمجهر‬
B) Gastrointestinal bleeding:
 Occult (‫ )مخفي‬gastrointestinal malignancy.
 Hookworm infestation (e.g., Ancylostoma duodenale and Necator americanus).
 Peptic ulcer disease.
 Increased risk with NSAID use - NSAID inhibit cox pathway.

2) Decreased intake:
 Chronic undernutrition.
 Cereal-based diet (‫)النظام الغذائي القائم على الحبوب‬.

3) Decreased iron absorption:

 Achlorhydria / hypochlorhydria.
 Inflammatory bowel disease, celiac disease )‫(حساسية القمح‬.
 Surgical resection of the duodenum.
 Bariatric surgery )‫(قص المعدة‬.

4) Increased demand
 Pregnancy.
 Growth spurt (‫)النمو السريع جدا‬.
 Erythropoietin (EPO) therapy.
 Clinical features:
 Fatigue, lethargy.
Signs and
symptoms of  Pallor (primarily seen in highly vascularized mucosa, e.g., the
anemia: conjunctiva).

 Tachycardia, angina, dyspnea on exertion, pedal edema, and

cardiomyopathy in severe cases.
nails  Brittle.

 koilonyclia (spoon-like nail deformity).

Pica Craving and chewing substances that have no nutritional value —
such as ice.
Angular cheilitis inflammation and Assuring of the comers of the mouth
Atrophic glossitis Erythematous, edematous, painful tongue with loss of tongue
papillae (smooth, bald appearance).
Hair Hair lose

IDA can be associated with Plummer-Vinson syndrome (PVS):

 Iron deficiency anemia dysphagia, and upper esophageal webs.
 Associated with an increased risk of esophageal squamous cell carcinoma and glossitis

Treatment:
1) Dietary modifications:

 All patients:
 Encourage consumption of iron-rich foods.
 prevent patients from taking non supplements to avoid the following substances
that reduce non absorption:
Food: tea, cereals, daily products
Drugs: calcium, antacids, protein pump inhibitors {PPIs}

 Infants < 1 year old: Avoid cow's milk.

2) Iron therapy - Supplemental iron (ferrous sulfate).

 2) ANEMIA OF CHRONIC DISEASE
 Anemia associated with chronic inflammation (e.g., endocarditis or autoimmune
conditions) or cancer; most common type of anemia in hospitalized patients.

 Epidemiology: Notes about hepcidin:

Second most common anemia.
A) Hepcidin sequesters iron in
storage sites by:
 Pathophysiology:
(1) Limiting iron transfer from
Inflammation  increase in cytokines (esp. IL-6) and macrophages to erythroid
hepcidin  results in the following outcomes: precursors.
 Reduced iron release from macrophages in the (2) Suppressing erythropoietin
reticuloendothelial system and reduced intestinal iron (EPO) production; aim is to
absorption  reduced iron available systemically. prevent bacteria from
 Reduced response (of production) to erythropoietin accessing iron, which is
(EPO) and relative reduction of EPO levels  reduced necessary for their survival.
RBC synthesis.
 Reduced erythrocyte survival.

 Etiology
 Inflammation (e.g., rheumatoid arthritis, systemic lupus erythematosus).
 Malignancy.
 Chronic infections.

 Diagnostics:
Normocytic anemia (early phase)  microcytic anemia (late phase).
(↓) available iron → (↓) heme → (↓) hemoglobin → microcytic anemia.

 Laboratory findings include:

High Serum Ferritin and Free erythrocyte protoporphyrin (FEP)
Low TIBC, serum iron, reticulocyte count and % iron saturation

 Treatment:
 Treat the underlying cause.
 Blood transfusion if required.
 EPO in chronic incurable diseases (e.g., chronic kidney disease).
 3) SIDEROBLASTIC ANEMIA
 Defective heme metabolism, which leads to iron trapping inside
the mitochondria.

 Laboratory findings include:

High Serum iron, ferritin and % saturation

Low TIBC

4) THALASSEMIA
 Genetic disorders resulting from decreased biosynthesis of globin chains of
hemoglobin.
 Due to mutations in and around the globin genes.
 Decreased production of one or more of the globin chains.
 Result in an imbalance in the relative amounts of the 𝛂- and non 𝛂-chains.
 Altered α /non- α ratio.
 As a consequences of thalassemia there is excess production of the other chains,
and a decreased over-all hemoglobin synthesis.
 Thalassemia provides partial resistance against malaria.

Epidemiology:
Beta thalassemia: most commonly seen in people of Mediterranean descent
Alpha thalassemia: most commonly seen in people of Asian and African descent

Etiology:
General cause; gene mutations.

Beta  Usually due to point mutations in promoter sequences or

thalassemia: splicing sites.
 𝜷-globin locus - short aim of chromosome 11
Alpha  Usually due to deletion of at least one out of the four existing
thalassemia: alleles.
 The 𝜶-globin gene cluster is located on chromosome 16

Pathophysiology:
 Increased hemolysis: erythrocyte instability with hemolysis.
 Anemia → ↑ erythropoietin → bone marrow hyperplasia and skeletal
deformities.
 Beta thalassemia:
In a normal cell: The (𝜷-globin chains are coded by a total
of two alleles.
Beta thalassemia minor (trait): one defective allele
Beta thalassemia major (Cooley Two defective alleles.
anemia):
A combination of one defective (𝜷 -globin
Sickle cell beta thalassemia: allele and one defective HbS allele.

Pathophysiology of Beta thalassemia minor and major:

 Faulty (‫𝛃 )تعطل‬-globin chain synthesis  decrease 𝛃 𝐜𝐡𝐚𝐢𝐧𝐬  increase
𝜸 𝒂𝒏𝒅 𝜹 𝒄𝒉𝒂𝒊𝒏𝒔  increased HbF and HbA2.
 HbF protects infants up to the age of 6 months, after which HbF production
declines and symptoms of anemia appear.

Clinical features of Beta thalassemia:

1) Minor variant:

Unremarkable symptoms.

2) Major variant:

 Severe hemolytic anemia that often requires transfusions; secondary iron

overload due to hemolysis transfusion, or both:  secondary hemochromatosis.
 Hepatosplenomegaly.
Dark black stools
 Growth retardation. Dark orange urine

 Skeletal deformities (enlargement of upper jaw, bossing of skull

and tendency of bone fractures).
 Transient aplastic crisis (secondary to infection with parvovirus B19).

3) Sickle cell beta thalassemia:

Features of sickle cell disease.

Severity depends on the amount of 𝜷-globin synthesis.
 Alpha thalassemia
𝜶-globin chains are coded by a total of four alleles (𝜷 𝒃𝒚 𝒕𝒘𝒐).
Silent carrier one defective allele (-𝜶 / 𝜶𝜶)
(minima form):
Two defective alleles (-𝜶 /-𝜶 or -- / 𝜶𝜶)
Alpha thalassemia
trait (minor form) Children of parents with a two-gene deletion in cis are at
higher risk of developing Hb Bart.
Hemoglobin H three defective alleles (--/-𝜶)
disease
(intermedia form): Made of four beta chains.
Hemoglobin Bart four defective alleles (--/--)
disease
(major form): consists of four y-chams (y-tetramers)

Pathophysiology of Alpha thalassemia:

Intermedia (HbH Faulty (‫𝛂 )تعطل‬-globin chain synthesis  decrease 𝛂 𝐜𝐡𝐚𝐢𝐧𝐬  impaired pairing
disease) and major of 𝛂 -chahis with 𝜷-chains and 𝜸-chains  increase free 𝜸 𝒂𝒏𝒅 𝜷 chains 
(Bart disease) increased HbH and Hb bart’s.
Minor and minima Production of the affected chain is reduced, but enough is produced to prevent severe anemia.

Clinical features of alpha thalassemia:

1) Silent carrier (minima form):
 Asymptomatic.

2) Alpha thalassemia trait (minor form):

 Mild hemolytic anemia with normal RBC and RDW.

3) Hemoglobin H disease (intermedia form):

 Jaundice and anemia at birth.
 Hepatosplenomegaly.
 Chronic hemolytic anemia that may require transfusions  secondary iron overload due to
hemolysis, transfusion, or both  secondary hemochromatosis.

4) Hb-Bart's hydrops fetalis syndrome:

 Most severe variant of alpha thalassemia.
 Incompatible with life (death in utero or shortly after birth).
Diagnostics of thalassemia:
1) Initial investigations;

A) CBC:
 Microcytic hypochromic anemia.

B) Hemolysis evaluation:
 Nonimmune-mediated hemolytic anemia.
 ↓ Haptoglobin, ↑ LDH ↑ reticulocytes. HALT:
 Hyperbilirubinemia (indirect). H: Hemoglobin H
C) Peripheral blood smear findings include: A: Asplenia
 Target cells. L: Liver diseases
 Teardrop cells (highly in beta thalassemia).
 Anisopoikilocytosis. T: Thalassemia

2) Confirmatory diagnostic studies;

A) Detection of hemoglobin variants:

 Hb-electrophoresis.
 Findings (vary depending on the subtype); hemoglobin A2 values are helpful
to determine the diagnosis:
Beta thalassemia minor should be strongly suspected if HbA2 is > 3.5%.

Differentiate Thalassemia minor/trait from Iron deficiency anemia by CBC:

IDA Thalassemia
RDW high RDW typically normal / low
RBC count low RBC count Higher than IDA
MCV Low MCV (Hb is < 10 𝒈/𝒅𝑳) Lower than IDA

 Low ferritin suggests Iron deficiency anemia and patients should receive iron
supplementation.
 Suspect thalassemia if there is no significant response after three months.
Complications of thalassemia:
A. Iron overload disease:
o All patients receiving transfusion therapy should be periodically
evaluated for non-overload disease and subsequent organ damage.
B. Hepatobiliary complications Cholelithiasis
C. Hematologic complications Hypercoagulable states Hemolytic crisis.
D. Extramedullary hematopoietic pseudotumors.
E. Cardiovascular complications.
F. Chronic leg ulcers.
G. Mental health complications.

Management of thalassemia:
Thalassemia minor:

 Usually no treatment required.

Thalassemia major and intermedia:

 Transfusion therapy (erythrocyte concentrates).

 Surveillance (‫ )مراقبة‬and treatment of complications.

Iron overload diseases:

 Chelating agents, e.g., deferasirox.

Teardrop-shaped erythrocytes, known as

dacrocytes, are found in conditions that
involve extramedullary hematopoiesis
(e.g., myelofibrosis, thalassemia, and
splenomegaly).
 MACROCYTIC ANEMIA
 Impaired DNA synthesis and/or repair with hypersegmented neutrophils.
 Anemia with MCV > 100 µm3; most commonly due to folate or vitamin B12
deficiency.
Macrocytic anemia can be:

Megaloblastic anemia Non-megaloblastic anemia

Large immature RBCs Large mature RBCs
Hypersegmented neutrophil (more than 5 lobes) No Hypersegmented neutrophil

Macrocytic anemia causes:

1) Vitamin B12 deficiency.

2) Folate deficiency:

 Drugs enhanced folate deficiency; “metabolic inhibitors”

 Phenytoin.
 Sulfa drugs.
 Trimethoprim.
 Hydroxyurea.
 MTX 6-mercaptopurine.

3) Fanconi anemia

Fanconi anemia is a rare disease passed down through families (inherited), that
mainly affects the bone marrow.
It results in decreased production of all types of blood cells. This is the most
common inherited form of aplastic anemia.

4) Orotic aciduria

Orotic aciduria (AKA hereditary orotic aciduria) is a disease caused by an enzyme

deficiency resulting in a decreased ability to synthesize pyrimidines.

Non-megaloblastic anemia: Diamond-Blackfan anemia is a disorder that

primarily affects the bone marrow. People with
 Liver disease (Alcohol use). this condition often also have physical
 Diamond-Blackfan anemia. abnormalities affecting various parts of the body.
 Myelodysplastic syndrome. Myelodysplastic syndromes are a group of
 Multiple myeloma. cancers in which immature blood cells in the
 Hypothyrodism. bone marrow do not mature or become healthy
 Pregnancy. blood cells.
Megaloblastic anemia
I. Cobalamin (vit B12) deficiency:

Decreased ingestion: Vegetarians.

Impaired absorption: Small intestinal disease.
Impaired utilization.

II. Folate deficiency:

Decreased ingestion: Prolonged parenteral feeding, alcoholism.

Impaired absorption: Small intestinal disease.
Impaired utilization: Drug induced eg: sulfa drugs, methotrexate, phenytoin...
Increased requirement: Pregnancy, hemolysis.
Increased loss: Through urine.

III. Drugs:

 Metabolic inhibitors.

IV. Miscellaneous (‫)متنوع‬:

 Inborn errors.
 Unexplained disorders.

 Lack of folate or vitamin B12 impairs synthesis of DNA precursors.

I. Impaired division and enlargement of RBC precursors leads to megaloblastic
anemia.
II. Impaired division of granulocytic precursors leads to hypersegmented
neutrophils.
III. Megaloblastic change is also seen in rapidly-dividing (e.g., intestinal) epithelial
cells.

 Folate and vitamin B12 are necessary for synthesis of DNA precursors.
A. Folate circulates in the serum as methyltetrahydrofolate (methyl THF); removal of
the methyl group allows for participation in the synthesis of DNA precursors.
B. Methyl group is transferred to vitamin B12 (cobalamin).
C. Vitamin B12 then transfers it to homocysteine, producing methionine.
Clinical features for anemia in general:
1) Asymptomatic:
2) One or more of the following symptoms.
 Pallor (e.g., on mucous membranes, conjuiictivae).
 Exertional dyspnea and fatigue.
 Pica (craving for ice or dirt).
 Jaundice (in hemolytic anemia).
 Muscle cramps.
 Growth impairment (chronic anemia).
 Worsening of angina pectoris.
 Features of hyperdynamic state.
 Bounding pulses (‫)االحساس بنبض قوي‬.
 Tachycardia/palpitatioiis.
 Flow murmur.
 Pulsatile sound in the ear.
 Possibly heart failure (anemia-induced heart failure).
 Features of extramedullary hematopoiesis may be present in certain
severe, chronic forms of anemia (e.g., thalassemia, myelofibrosis).
 Hepatosplenomegaly.
 Paravertebral mass.
 Widening of diploic spaces of the skull.

General Treatment of macrocytic:

1) Blood transfusion with RBCs for severe anemia:

 Hb < 7 g/dL  whatever his condition, we give him blood.

 Hb < 8 g/dL  if the patient either has a preexisting cardiovascular disease or
is undergoing cardiac or orthopedic surgery.

2) Consider hospital admission or observation in:

 Acutely symptomatic anemia, actively bleeding

patient, as clinically indicated, Patients requiring
blood transfusion.

3) Bone marrow transplantation may be indicated in

certain cases (e.g., aplastic anemia).
LABORATORY INVESTIGATIONS of macrocytic anemia:
1- Complete Blood Count &Blood Film:

Macrocytosis increased MCV, Neutropenia, Thrombocytopenia

Neutrophil hyper segmentation Reticulocvtopenia

2- Bone Marrow Aspirate &Biopsy:

 Hypercellular, Megaloblastic morphology, Giant bands & metamyelocytes.

3- Indirect Hyperbilirubinemia, elevated LDH.

4- Decrease serum cobalamin Normal: normal, 200-900 pg/mL

5- Decrease Serum folate Normal: 2.5-20 ng/mL

6- Schilling test for diagnosing the cause of Cobalamine malabsorption.

7- Biopsy:

Gastric biopsy For pernicious anemia (cobalamine deficiency).

Small Intestinal Biopsy For malabsorption.

8- Anti-Intrinsic factor Ab& Anti-Parietal cell Ab in Pernicious anemia

(Cobalamine deficiency).

9- Elevated serum Methylmalonic acid in Cobalamine deficiency.

TREATMENT OF MEGALOBLASTIC ANEMIA
 Hydroxycobalamine 1 mg daily for 10 days, then once
every one month for life.
Cobalamin  Iron is added for slow response.
 Hypokalemia may develop during therapy.
Deficiency:
 Reticulocytosis at day 7 will indicate response.
 Blood is given for severely symptomatic patients.
Folate Oral Folic acid 5mg/day for 3 weeks then weekly as
maintenance.
Deficiency:

NOTES FOR MEGALOBLASTIC ANEMIA:

 Always consider vitamin B12 deficiency when evaluating patients with
dementia.
 Dementia is a term used to describe a group of symptoms affecting
memory, thinking and social abilities severely enough to interfere with your
daily life.
 Folate deficiency also leads to low levels of THF, resulting in megaloblastic
anemia.
 Starting folate treatment before excluding vitamin B12 deficiency may collect
anemia, but it can worsen neuropathy.
 In contrast to vitamin B12 deficiency, folic acid deficiency is generally not
associated with neurological symptoms.
 Folic acid deficiency: Dining fetal development: nucleotide synthesis
impairment  neural tube defects.
 If folate deficiency is suspected, always exclude the possibility of vitamin B12
deficiency.
 Megaloblastic anemia is referred to a group of panhypoplastic disorders, which
are characterized by retardation of DNA synthesis but RNA synthesis proceeds at
a normal rate.
 The resulting asynchrony (‫ )عدم التزامن الناتج‬between nuclear and cytoplasm
maturation in developing cells is responsible for the distinctive morphological
and biochemical features of the megaloblastic anaemias.
 PERNICIOUS ANEMIA
 The most of common cause of cobalamin malabsorption.
 Autoimmune destruction (atrophy) of parietal cells (body of stomach) leads to intrinsic
factor deficiency.

 Histological appearance of gastric mucosa; infiltration of plasma cells and lymphocytes

 suggestive of autoimmune disorder.
 There is a positive family history for about 30% of patients.
 This condition is especially common among the elderly, with an observed prevalence
of up to 1.9%.
 The disease is more common in women than in men and is associated with blood
group A.
 Some studies clime that remission of Pernicious anemia occurs after corticosteroids
therapy.
 About 90% of patients have cytotoxic IgG directed against gastric parietal cells or intrinsic
factor demonstrated in serum.

 In about 75% of these the antibody is demonstrated in gastric juice.

 Polyclonal IgG or IgA are demonstrated in serum and gastric juice in 50% of patients with
pernicious anaemia, this acts in one of two ways:

Type 1 antibody Prevents the binding of vitamin B12 to intrinsic factor.

Type 2 antibody Inhibit the absorption of VitB12-IF complex.

 Pernicious anaemia is associated with an increased incidence with congenital deficiency of

autoimmune thyroid disease, rheumatoid arthritis and gastric carcinoma.
 A rare type of pernicious anaemia is associated with congenital deficiency of intrinsic
factor or the synthesis of dysfunctional variant of intrinsic factor.
 NORMOCYTIC ANEMIA
Anemia with normal-sized RBCs (MCV = 80-100 µm3).
Due to increased peripheral destruction or underproduction;
 Reticulocyte count helps to distinguish between these two etiologies.

Reticulocyte count and corrected reticulocyte count

According to reticulocyte count the normocytic anemia divides into two parts:
1) Reticulocytes ≤ 𝟐% 𝒄𝒂𝒍𝒍𝒆𝒅 "𝐍𝐨𝐧 − 𝐡𝐞𝐦𝐨𝐥𝐲𝐭𝐢𝐜 𝐚𝐧𝐞𝐦𝐢𝐚":
 Anemia of chronic disease, B12 and B9 deficiency (early phase).
 Aplastic anemia.
 Chronic kidney disease.
 Blood lose.

2) Reticulocytes ≥ 𝟐% 𝒄𝒂𝒍𝒍𝒆𝒅 "𝐡𝐞𝐦𝐨𝐥𝐲𝐭𝐢𝐜 𝐚𝐧𝐞𝐦𝐢𝐚":

A) Extrinsic hemolytic anemia:
Membrane defects Hereditary spherocytosis.
Paroxysmal nuctumal.
Enzyme deficiency G6DP deficiency.
Pyruvate kinase deficiency.
Hemoglobinopathies Sickle cell anemia.
Hemoglobin C,E,S,SC..

B) Intrinsic hemolytic anemia:

Autoimmune (cold or warm).
Microangiopathic.
Macroangiopathic.
Infections.
Mechanical destruction.
 Non-hemolytic anemia

Aplastic anemia
 Damage to hematopoietic stem cells, resulting in pancytopenia (anemia,
thrombocytopenia, and leukopenia) with low reticulocyte count.
 Should not be confused with aplastic crisis, a condition in which erythropoiesis
is temporarily suppressed (e.g., due to parvovirus B19 infection hi patients with
hemolytic anemias).

 Etiologies:
Idiopathic > 𝟓𝟎% of cases [Possibly immune-mediated]
Medication side Carbamazepine, metliimazole. NSAIDs, chloramphenicol.,
effects: propylthiouracil sulfa drugs, cytostatic drugs (esp.
alkylating agents and antimetabolites)
Toxins: benzene
Viruses: HBV, EBV, CMV and HIV
Fanconi anemia This is the most common inherited form of aplastic anemia.
Ionizing
radiation

 Clinical features:
 Fatigue, malaise.
 Pallor.
 Infection.
 Purpura, petechiae, mucosal bleeding.

As a result of platelets deficiency.

 Diagnostics:

1) CBC:
 Pancytopenia.
 Normocytic or macrocytic anemia (funconi anemia)
Reticulocytes EPO
Low High

2) bone marrow biopsy:

 Hypocellular fat-filled marrow (dry bone
marrow tap).
 RBCs normal morphology.

Treatment:
1) Cessation of the causative agent.
2) Supportive therapy: Myelophthisic anemia is a
 Treatment of infections. normocytic, normochromic anemia
that occurs when normal marrow
 Blood transfusion.
space is infiltrated and replaced by
 Platelet transfusion.
nonhematopoietic or abnormal cells.
3) Bone marrow stimulants: Causes include tumors, granulomatous
disorders, lipid storage diseases, and
 GM-CSF. primary myelofibrosis.
4) Immunosuppressive therapy:
Cyclosporine, Antithymocyte globulin (ATG), hematopoietic cell transplantation
(HCT) in young patients.
Basophilic stippling
Distinctive property of pure
Pure red cell aplasia:
Normocytic, normochromic anemia characterized by a
severe reduction in circulating reticulocytes and marked
reduction or absence of erythroid precursors in the bone
marrow.

The major difference between PRCA and aplastic anemia is that, in PRCA, only the red
blood cell line is affected, while the white blood cells and platelets remain at normal
levels. In aplastic anemia, all three blood cell types are typically affected.
 Hemolytic Anemia
 The term given to a large group of anemias that are caused by the premature
destruction / hemolysis of circulating red blood cells (RBCs).

Epidemiology:
 5% of all cases of anemia.
 Hereditary causes present early in life.
 Autoimmune hemolytic anemia (AIHA) is more common in
middle-aged and older adults.
 Males are predominantly affected by X-linked hereditary
spherocytosis.

Classification:
According to the cause of hemolysis: According the location of hemolysis:
intrinsic or extrinsic Intravascular or extravascular

Intrinsic hemolytic Abnormal membrane structure or shape of the

anemia RBCs.
Membrane defect: Hereditary spherocytosis and paroxysmal
nocturnal hemoglobinuria (PNH)
Enzyme deficiency: G6PD deficiency Pyruvate kinase deficiency
Hemoglobinopathy: Sickle cell anemia, Thalassemia, hemoglobin C
disease, and hemoglobin Zurich

Extrinsic hemolytic Abnormal breakdown of normal RBCs.

anemia (coated RBCs)
Autoimmune hemolytic anemia
Mechanical destruction in small vessels (MAHA) - e.g., HUS, TTP, DIC, HELLP syndrome
Mechanical destruction in large vessels; prosthetic heart valves
ABO incompatibility, Rhesus incompatibility.
Drugs (e.g., rifampin nonsteroidal anti-inflammatory drugs, penicillin).
Infections (malaria), gas gangrene Hypersplenism.
 Intravascular hemolytic Increased destruction of RBCs within the blood
anemia vessels.
Toxins (e.g., snake bites) and oxidizing agents (e.g., copper
poisoning).
G6PD deficiency
Antibody-mediated Transfusion ABO incompatibility, hemolytic anemia
hemolysis of the newborn, cold agglutinin disease.
Complement-mediated PNH
hemolysis
Macroangiopathic mechanical destruction by, e.g., prosthetic cardiac
anemia valves
Microangiopathic TTP, HUS, disseminated intravascular coagulation,
anemia HELLP syndrome, systemic lupus erythematosus.

Extravascular Increased destruction of RBCs by the

hemolytic anemia reticuloendothelial system (primarily the spleen).
RBC defects Sickle cell disease, spherocytosis, pyruvate kinase
deficiency.
antibody-mediated warm and cold agglutinin disease
hemolysis

Pathophysiology
1) Extravascular hemolysis:
 Normal breakdown of RBCs occurs in the spleen every 120 days.

 An increased splenic clearance occurs in:

 IgG / IgM tagging of RBCs in auto-immune conditions.

 Intrinsically defective RBCs: sickle cell, spherocytosis, G6PD deficiency.

 Increased breakdown results in increased heme which is converted into bilirubin.

 Excessive bilirubin → unconjugated bilirubinemia and jaundice.

 Iron from the breakdown also begins to accumulate in the spleen.

 Excessive functioning and clearance results in splenomegaly

2) Intravascular hemolysis:
 RBCs become fragmented when passing through narrowed vessel lumen
(microangiopathic) or prosthetic valves (macroangiopathic).

 Splitting of the RBCs results in schistocytes in the smear.

 Hemoglobin in the bloodstream binds to haptoglobin.

 Excessive heme also presents in the urine as hematuria /hemoglobinuria.
  Fragmented red blood cells.
schistocytes  Characteristic feature of microangiopathic hemolytic
anemia.
 Is a protein made by your liver.
 Detect whether you have hemolytic anemia or another type
haptoglobin of anemia.
 Help to determine the exact cause of increased red blood
cell destruction.

Clinical features
 Jaundice.
 Pigmented gallstones.
1) Signs of hemolysis  Splenomegaly.
 Back pain and dark urine in severe
hemolysis with hemoglobinuria.

2) Signs of increased Mostly in severe chronic anemias, e.g.,

hematopoiesis thalassemia.
3) Bone marrow expansion: Widening of the diploic space of the skull,
biconcave deformity of the vertebral bodies.
4) Cortical thinning and Increase risk of pathologic fractures.
softening of bone:
Extramedullary hepatosplenomegaly
hematopoiesis:

General Diagnostics of hemolytic anemia

 ↓ Hb, N-MCV, N-MCH.
 Normocytosis: typical finding for most hemolytic anemias; consider other
anemia of chronic disease.
 ↑ MCV: Can be due to severe reticulocytosis consider other (e.g.,
megaloblastic anemia).
 Microcytosis: consider thalassemia and/or iron deficiency.
 WBC count: can be elevated due to inflammation or malignancy.
 Platelets: decreased in MAHA and in Evan syndrome.
 Reticulocytosis (≤ 3%), may be normal.
 Decreased hematocrit.
 Increased lactate dehydrogenase.
 Decreased haptoglobin.
 Increased unconjugated bilirubin.
Intravascular hemolysis:
 Hemoglobinuria / hemosiderinuria: hemoglobin that
exceeds haptoglobin binding (most sensitive).
 Schistocytes on blood film.
 Indirect Coombs test (positive for complements).

Extravascular hemolysis
 Direct Coombs test (positive for IgM / IgG).
 Blood film: spherocytes, sickle cell anemia,
agglutination (in IgM Autoimmune hemolytic anemia
{AIHA}).

Investigation of underlying causes:

Hb Abnormal Hb patterns, e.g., in thalassemia.
electrophoresis:
Flow cytometry  Absence of CD55 and CD59 on RBC surface in PNH.
 Detection of CD19, CD20, and CD23, light chain restriction
(kappa or lambda) in CLL.
Genetic  Mutations in congenital hemolytic anemia.
analysis:
 Rarely used in the workup of anemia due to its invasiveness.
Bone marrow  Indication: pancytopenia, presence of abnormal cells (e.g.,
biopsy blasts) in CBC/peripheral blood smear.
 Pathologic findings are most common in malignancies that
replace bone marrow (e.g., CLL).

Findings for: Intravascular hemolysis (I) Extravascular hemolysis (E)

Peripheral smear Schistocyles Spherocytes
Haptoglopain Decrease / absent Mild decrease
Urine hemoglobin / Positive Negative
hemosiderin.
Reticulocytes
Unconjugated bilirubin Increase Increase
LDH
IMMUNE HEMOLYTIC ANEMIA (IHA):
A) Antibody-mediated (IgG or IgM) destruction of RBCs.

B) IgG-mediated disease usually involves extravascular hemolysis.

 IgG binds RBCs in the relatively warm temperature of the central body
(warm agglutinin); membrane of antibody-coated RBC is consumed by splenic
macrophages, resulting in spherocytes.
 Associated with SLE (most common cause), chronic lymphocytic
leukemia {CLL}, and certain drugs (classically, penicillin and cephalosporins).
 Drug may attach to RBC membrane (e.g., penicillin) with subsequent
binding of antibody to drug-membrane complex.
 Drug may induce production of autoantibodies (e.g., α-methyldopa)
that bind self-antigens on RBCs.
 Treatment involves cessation of the offending drug, steroids, Intravenous
immune globulin (IVIG), and, if necessary, splenectomy.

C) IgM-mediated disease can lead to intravascular hemolysis.

1. IgM binds RBCs and fixes complement in the relatively cold temperature of
the extremities (cold agglutinin).
2. RBCs inactivate complement, but residual C3b serves as an opsonin for
splenic macrophages resulting in spherocytes; extreme activation of
complement can lead to intravascular hemolysis.
3. Associated with Mycoplasma pneumoniae and infectious mononucleosis.
4. Maybe found reticulocytes.
D) Coombs test is used to diagnose IHA; testing can be direct or indirect.
Direct Coombs test Indirect Coombs test
 Transfusion medicine
Clinical Workup of autoimmune (workup and prevention of
applications hemolytic anemia transfusion reactions).
 Perinatal care (workup and
prevention of HDFN).
Location of
antibodies RBC surface Serum (free)
detected
Coombs serum The patient's purified The patient's purified serum
added to: RESCs which has been mixed with
test RBCs.
 AHGs in Antibodies and/or The patient's antibodies which
Coombs serum complement already are bound to test RBCs.
bind to: coating the patient's RBCs.
 When anti-IgG/complement
is added to patient RBCs,
confirms the presence of agglutination occurs if RBCs
Direct Coombs antibody- or complement- are already coated with IgG or
test coated RBCs. complement.
 This is the most important
test for IHA.
 Anti IgG and test RBCs are
Indirect Coombs Confirms the presence of mixed with the patient serum;
test antibodies in patient serum. agglutination occurs if serum
antibodies are present.

Laboratory investigations for antibody-mediated hemolysis:

AIHA can be cold or warm:
 Direct Coombs test.
 Features of extravascular hemolysis.
Warm AIHA Cold AIHA
The direct antiglobulin (DAT) Positive for IgG. positive for C3d.
Peripheral blood smear (PBS) Spherocytes. RBC agglutination.

Suggested additional investigations of direct coombs test:

Infections: HIV test, HBsAg, HCV antibody.
Autoimmune disorders: Antinuclear antibody (ANA).
Lymphoproliterative diseases: Serum protein electrophoresis.
Immune deficiency syndromes (e.g., CVID): Serum immunoglobulins and electrophoresis.
Malignancy: CT chest, abdomen, pelvis or bone marrow biopsy.

Hemolytic transfusion reactions (Indirect Coombs test):

 Features of intravascular hemolysis.
Laboratory studies PBS Suggested additional investigations
DAT: positive for IgG ± C3d. Spherocytes  Indirect antiglobulin test {IAT}
 Repeat blood typing and crossmatching.
Mechanical destruction of RBCs
1. Microangiopathic hemolytic anemia.
2. Macroangiopathic hemolytic anemia.
Hemolysis.

Kidney disease.

Microangiopathic hemolytic anemia: Thrombocytopenia.

Etiology:
Primary  Thrombotic thrombocytopenic purpura.
MAHA  Hemolytic uremic syndrome.
 Autoimmune disease (e.g., SLE).
 Hemolysis, Elevated Liver enzymes and Low Platelets {HELLP} syndrome.
Secondary
 Hypertensive emergency.
MAHA
 Disseminated intravascular coagulation (DIC).
 Drug induced (e.g., quinine, trimethoprim /sulfamethoxazole, cyclosporine).

Pathophysiology:
 Systemic microthrombi plug small vessels  physical intravascular shearing
of RBCs that pass through the small vessels  intravascular hemolysis,
schistocytes, and (↑) free Hb.

Clinical features:
 Features of anemia.
 Organ dysfunction due to microthrombi formation (e.g., renal dysfunction,
altered mental status).

Diagnostics: Because it is not an autoimmune

hemolytic anemia type.
 CBC showing anemia and thrombocytopenia.
 Laboratory evidence of hemolysis with a negative DAT.
 PBS showing abundant schistocytes.
Macroangiopathic hemolytic anemia:
Etiology:
 Aortic stenosis; Heart valve replacement usually resolves the anemia.
 In other patient groups: Prosthetic heart valves can cause anemia.
 Exertional hemoglobinuria (“March hemoglobinuria”).

Pathophysiology:
RBC destruction in the systemic circulation (large vessels) due to mechanical forces
applied to RBC membrane  intravascular hemolysis, schistocytes, ↑ free Hb.

Pyruvate kinase deficiency

Etiology:
 Autosomal recessive defect of pyruvate kinase.

Pathophysiology:
 Glucose is the only energy source in RBCs.
 Pyruvate kinase catalyzes the last step of glycolysis (i.e., irreversibly converts
phosphoenolpyruvate into pyruvate).
 Absence of pyruvate kinase → ATP deficiency in RBC.
 ATP deficiency disrupts the cation gradient along the RBC membrane  rigid
RBCs  (↑) hemolysis (extravascular - tissues).
 Accumulation of 2,3-bisphosphoglycerate  (↑) release of O2 from Hb  masks
symptoms of anemia.

Clinical symptoms:
 Usually asymptomatic.
 Typically newborn jaundice due to hemolysis and history of exchange transfusions.
 Splenomegaly.
 Pallor, fatigue, weakness.
 In rare cases: hydrops fetalis. Severe anemia  the child may die
during birth or a few days after birth.
 Diagnosis of pyruvate kinase deficiency:
(↓) Pyruvate kinase enzyme activity, PKLR gene mutation, Blood smear: burr
cells.

Therapy of pyruvate kinase deficiency:

1. Phototherapy and/or exchange transfusions.
2. In the case of severe anemia or excessively enlarged spleen: splenectomy.

Hemoglobin Zurich
Pathophysiology:
Replacement of distal histidine in the beta-globin
chain with arginine  enlargement of the ligand-
binding space around iron  increased affinity for
carbon monoxide  increased carboxyhemoglobin
levels (≥ 3%)  Oxidative stress  Formation of
Heinz body and hemolysis.

Normal level of
carboxyhemoglobin is 1-2 %
Hemoglobin C disease
Hemoglobin Occurs in individuals who are homozygous for the hemoglobin C
C disease mutation (HbCC).
Hemoglobin Occurs in individuals who are heterozygous carriers of the
C trait hemoglobin C mutation (HbAC).

Pathophysiology:
 β-globin mutation (glutamate replaced by lysine).
 HbC precipitates as crystals  (↑) RBC rigidity and
(↓) deformability  extravascular hemolysis.
 HbC is less soluble than HbA and tends to form
hexagonal crystals, which lead to RBC dehydration (↑)
MCHC).
 RBCs have reduced oxygen-binding capacity and a shorter lifespan.
 Glucose-6-phosphate dehydrogenase deficiency (Favism)
Epidemiology:
 G6PD deficiency is the most common human enzyme deficiency.
 Prevalence: ∼ 400 million worldwide.
 Affects primarily males of African, Mediterranean, and Asian descent.

Pathophysiology:
 X-linked recessive inheritance.
 G6PD is the rate-limiting enzyme of the pentose phosphate pathway (also
known as the hexose monophosphate shunt).
 This pathway yields NADPH, which is essential for converting oxidized
glutathione back to its reduced form.
 Reduced glutathione is capable of neutralizing reactive oxygen species
(ROS) and free radicals and therefore protecting RBCs from oxidative
damage.
 In the absence of reduced glutathione (e.g., due to G6PD deficiency), RBCs
become susceptible to oxidative stress that can damage erythrocyte membranes,
resulting in intravascular and extravascular hemolysis.

Causes of increased oxidative stress are triggers of hemolytic crisis and include:
Antimalarial drugs (e.g., chloroquine, primaquine), sulfa
Drugs: drugs (e.g., trimethoprim-sulfamethoxazole),
nitrofurantoin, isoniazid, dapsone, NSAIDs, ciprofloxacin,
‫االسباب‬
chloramphenicol.
‫التي تزيد‬
‫حدة التفول‬ Bacterial and viral Severe enzymatic deficiency can inhibit respiratory burst
‫وليست‬ infections (most activity due to reduced NADPH production in phagocytes.
‫االسباب‬ common cause):
.‫التي تسببه‬ Inflammation: During an inflammatory reaction free radicals are
produced and can diffuse into RBCs.
Metabolic acidosis
Fava beans (‫)الفول‬.
Clinical features of G6DP deficiency:
1) Most patients are asymptomatic.
2) Recurring hemolytic crises may occur, especially following triggers:
 Arise within 2–3 days after increased oxidative stress.
 Sudden onset of back or abdominal pain.
 Jaundice.
 Dark urine.
3) Transient splenomegaly.
4) Recurrent severe infections causing symptoms of chronic granulomatous
disease (autoimmune  recurrent abcess).

Diagnostics = Blood smear:

Heinz bodies: Oxidative stress causes hemoglobin denaturation 
Hemoglobin then precipitates as small inclusions within the
erythrocytes.
Bite cells: due to macrophages selectively removing the denatured
hemoglobin inclusions of RBCs.

Signs of intravascular hemolysis

 Normocytic anemia.
 Hemoglobinuria
Increase LDH, Unconjugated bilirubin and Reticulocyte count.
Decrease Haptoglobin

Confirmatory test:
 Quantitative G6PD enzyme analysis.
 Tests should be performed during remission  (nearly about 2-3 weeks
after crisis).
 Hereditary spherocytosis
Epidemiology:
 Incidence: 1/5000 in the US.
 Most common inherited hemolytic disease among individuals of Northern
European descent.

Etiology:
 Congenital RBC membrane protein defect (mainly spectrin).
 Inheritance pattern;
Autosomal dominant 75% of cases
Autosomal recessive 25% of cases
Often positive for relatives who required splenectomy
Family history and/or developed cholelithiasis (gall bladder stones) at
a young age.

Frequently affected proteins:

 Spectrin.
 Ankyrin.
 Band 3.
 Protein 4.2.

Pathophysiology:
1) Genetic mutation → defects in RBC membrane proteins (especially spectrin and/or
ankyrin) responsible for tying the inner membrane skeleton with the outer lipid bilayer.
2) Continuous loss of lipid bilayer components → decreased surface area of RBCs in
relation to volume.
3) sphere-shaped RBCs with decreased membrane stability → inability to change form
while going through narrowed vessels.

Entrapment within splenic vasculature → splenomegaly.

Destruction via splenic macrophages → extravascular hemolysis.
Clinical features of hereditary spherocytosis:
1) Presentation is variable:

Mild HS 20–30% often asymptomatic

Moderate HS 60–75% onset of symptoms in infancy or childhood
Severe HS 5% Onset of symptoms in newborns or even in utero
(hydrops fetalis).

2) Anemia and pallor.

3) Jaundice (due to ↑ unconjugated bilirubin).
4) Splenomegaly with left upper quadrant pain.
5) Black pigment gallstones (made of calcium bilirubinate), may lead to cholecystitis.

Diagnostics:
 MCV within normal range (80-100 fL) or slightly decreased

1) Laboratory findings;
Increased RDW, MCHC, reticulocytes, Unconjugated bilirubin and LDH.
Decreased Haptoglobin.

2) Laboratory tests
A) Eosin-5-maleimide binding test (EMA):

 Test of choice.
 Decreased binding between dye (eosin-5-maleimide) and RBC membrane proteins.
 Binding is quantified using flow cytometry, which shows decreased mean
fluorescence.

B) Coombs test:

 Both negative and positive.

C) Positive osmotic fragility test:

 Measures the ability of RBCs to resist hemolysis when exposed to different

degrees of salt dilution.
 RBCs are more fragile and more vulnerable to osmotic stress.

3) Blood smear:
 Spherocytosis (small round cells without central pallor)
Treatment of Spherocytosis:
1) Non-surgical treatments:
Phototherapy and/or exchange May be necessary in neonates (e.g., to avoid
transfusions kernicterus).
Blood transfusions May be required in cases of aplastic or
hemolytic crisis.
Folic acid supplementation To maintain erythropoiesis.

 Kernicterus: is a type of brain damage that can result from high levels of
bilirubin in a baby's blood.
 Neonates: refers to the first 28 days after birth.

2) Splenectomy:
 Sole definitive treatment (‫)العالج النهائي الوحيد‬.
 Prior to splenectomy, vaccinate against Streptococcus pneumoniae,
Haemophilus influenzae type B, and Neisseria meningitides.

Complications:
Hemolytic crisis: esp. as a result of viral infection.
Aplastic crisis: following infection with parvovirus B19 (erythema infectiosum);
characterized by a low reticulocyte count (< 0.1% of total RBC count).
Megaloblastic anemia: folate and vitamin B12 deficiency may develop due to
chronic hemolysis and high RBC turnover.
Megaloblastic crisis: due to folate deficiency (although uncommon in developed
countries, it might still be seen among pregnant women).
Bilirubinate gallstone formation, possibly leading to cholecystitis, cholangitis,
and pancreatitis.
Growth retardation and skeletal abnormalities due to bone marrow expansion.
 Hereditary elliptocytosis
 An asymptomatic condition characterized by the
presence of elliptocytes in the blood.
 Caused by mutations in genes encoding RBC
membrane proteins (e.g., spectrin, protein 4.1).

Hemoglobinopathies
An inherited mutation o the globin genes leading to a qualitative or quantitative
abnormality of globin synthesis.
a substitution of AA by another in the globin chain rate of synthesis of a globin chain is reduced:
sickle hemoglobin (HbS) Thalassemia:
hemoglobin C β-Thalassemia- reduced beta chain
hemoglobin E synthesis.
hemoglobin D α-Thalassemia- reduced alpha chain
hemoglobin O synthesis.
Hemoglobin Zurich.

Remember there are 8 genes

dedicated to the manufacture of
heme with 7 chromosomes.
 Name Composition Description
Hb A 𝟐𝛂 + 𝟐 𝛃 Adult Hb
Hb A2 𝟐𝛂 + 𝟐 𝛅 Minor adult Hb
Hb F 𝟐𝛂 + 𝟐 𝛄 Fetal Hb
Hb Barts 𝟒𝛄 Abnormal Hb
Hb H 𝟒𝛃 Abnormal Hb

1) Zeta gene is expressed only during the first few weeks of embryogensis.
 Thereafter, the alpha globin genes take over.
2) The epsilon gene is expressed initially during embryogensis.
 The gamma gene is expressed during fetal development.
 The combination of two alpha genes and two gamma genes forms fetal
hemoglobin, or hemoglobin F.

3) Around the time of birth, the production of gamma globin declines in concert
with a rise in beta globin synthesis.
 The combination of two alpha genes and two beta genes comprises the normal
adult hemoglobin, hemoglobin A.

4) The delta gene, produces a small amount of delta globin in children and adults.
 The combination of two alpha genes and two delta genes forms A2 hemoglobin

Chromosome 11 Chromosome 16
Epsilon Zeta 1
Gamma Zeta 2
Delta Alpha 1
Beta Alpha 2
Hemoglobinopathies Genetic structural disorder
 Due to mutation in the globin gene of hemoglobin.
 Mostly autosomal recessive inheritance.
 Result in hemoglobin variants with altered structure and function.
1) Altered functions include:
 Reduced solubility.
 Altered oxygen affinity- increased or decreased.
 Methemoglobin formation (Fe 2+ Fe +3 (oxidized state) less oxygen
transport).
2) Decrease, lack of, or abnormal globin:
 May be severe hemolytic anemia.
 Abnormal Hb with low functionality.
 Mutation may be deletion, substitution, elongation.

Hb electrophoresis may be helpful.

Types of Mutation in Hemoglobin:

Point mutation: A change of a single nucleotide base in a DNA giving rise to
altered amino acids in the polypeptide chains (e.g., Hb S, Hb C).
Deletions and Addition and deletion of one or more bases in the globin genes.
additions:
Unequal As in Hb-lepore and Hb-antilepore associated with β-thalassemia.
crossing over:

 Hb Lepore is one type of hemoglobin disorder in which there is structurally

abnormal hemoglobin (Hb) that results from in-frame fusion between the 5’ end of
the 𝜹-globin gene and the 3’ end of the β-globin gene,.
Geographical distribution of common Hb Variants:
Variant NO. AA AA conversion Occurrence predominantly in:
Hb S 𝜷−𝟔 Glutamate  valine Africa, Arabia. Black Americans
Hb O 𝜷 − 𝟏𝟐𝟏 Glutamate  valine Turkey and Bulgury
Hb C 𝜷−𝟔 Glutamate  lysine West Africa. China
Hb E 𝜷 − 𝟐𝟔 Glutamate  lysine South East Asia
Hb D 𝜷 − 𝟏𝟐𝟏 Glutamate  Glutamine Asia
Zurich Glutamate  arginine
 Sickle Cell Anemia:
 Hereditary (autosomal recessive) hemoglobinopathies.
 Partially intravascular hemolysis and mainly extravascular.
 First molecular disease to be recognized.
 𝜶 chains are normal and basic abnormality is in the 𝜷 chain due to a point
mutation.
sickle cell disease Homozygous HbSS
Sickle cell trait heterozygous earners (HbSA)

 Homozygous sickle cell anemia is the most

common variant of the sickle cell syndromes and
occurs predominantly in individuals of African and
East Mediterranean descent.
 Other rare variants of sickle cell syndrome occur hi
individuals with one HbS allele and one other allele
(HbC or Hb-𝜷 thalassemia).

Epidemiology:
 Predominantly affects individuals of African and East Mediterranean descent.
 HbS gene is carried by 8% of the African American population.
 Sickle cell anemia is the most common form of intrinsic hemolytic anemia
worldwide.

Pathophysiology:

 Point mutation in the (𝜷-globin gene (chromosome 11)  glutamic acid

replaced with valine.
 2 𝜶-globin and 2 mutated (𝜷-globin subunits)  create pathological
hemoglobin S (HbS).
 Hemoglobin SC disease:
 Heterozygosity for hemoglobin S and hemoglobin C.
 Results in a phenotype more severe than sickle cell trait but not as severe as sickle cell
disease (e.g., fewer acute sickling events).

Pathomechanism:
 HbS polymerizes when deoxygenated, causing deformation of erythrocytes
(“sickling”).
 This can be triggered by any event associated with reduced oxygen
tension.
 GLU is polar AA, and thus hydrophilic, whereas the Val is
hydrophobic…leading to stickness of the hemoglobin in the deoxyform.
 Hypoxia (e.g., at high altitudes).
In homozygotes, up to 100% of the hemoglobin molecules are affected, leading to
sickle cell formation under minimally decreased oxygen tension.
 Infections.
 Dehydration.
 Sudden changes in temperature.
 Stress.
 Sickle cells lack elasticity and adhere to vascular endothelium, which disrupts
microcirculation and causes vascular occlusion and subsequent tissue
infarction.
 Extravascular hemolysis and intravascular hemolysis are common and result
in anemia.
 The body increases the production of fetal hemoglobin (HbF) to compensate
for low levels of HbA in sickle cell disease.

Hemoglobin Normal Sickle cell trait Sickle cell disease

HbA (95-98) % 60 % 0%
HbS 0% 40 % (75-95) %
HbF < 2% <2% (5-25) %
 Sickle cell trait:
1. Often asymptomatic.
2. Painless gross hematuria due to renal papillary necrosis (isosthenuria): often
the only symptom.
3. Renal medullary carcinoma.
4. The sickle cell trait confers a small but highly significant degree of protection
against the most lethal form of malaria.
5. The Sickle Cell trait can be identified through DNA testing  yields three
fragments for a normal β gene, but only two for the sickle-cell gene.

 Sickle cell disease:

A) Onset:
 Manifests after 6 months of age as the production of HbF decreases and
HbS levels increase.
B) Acute symptoms:

1) Acute hemolytic crisis (severe anemia);

 Splenic sequestration crisis “Splenic sequestration crisis”.

2) Aplastic crisis;
 Severe drop in hemoglobin and associated reticulocytopenia due to an
infection with parvovirus B19.
 Can temporarily suppress bone marrow erythropoiesis.
C) Infection:
 Pneumonia.
 Osteomyelitis; most common cause: Salmonella.
 Sepsis; most common cause: Streptococcus pneumoniae.

D) Vaso-occlusive events:
Vaso-occlusive crises (painful episodes, painful crisis): recurrent episodes of
severe deep bone pain and dactylitis {‫ }التهاب نهايات األصابع‬ most common
symptom in children and adolescents (‫)المراهقين‬.
Acute chest syndrome.
Priapism (‫)الم في القضيب نتيجة االنتصاب لفترة طويلة‬.
Stroke.
Infarctions of virtually any organ (particularly spleen) and avascular necrosis
with corresponding symptoms.
Chronic symptoms:
 Chronic hemolytic anemia: fatigue, weakness, pallor: usually well-tolerated.
 Chronic pain.
 Cholelithiasis (pigmented stones).
 Symptoms of other forms of sickle cell syndrome (HbSC disease and HbS/beta-
thalassemia) are similar to sickle cell anemia but less severe.

Diagnosis of sickle cell anemia:

1) Neonatal screening (mandatory in all states):
If positive: Repeat hemoglobin electrophoresis (gold standard) confirms the
diagnosis and distinguishes between heterozygotes and homozygotes and other
forms of sickle cell syndrome (e.g., HbSC disease).
 Migration towards the anode (-ve charge): HbA > HbF > HbS > HbC.

2) Older children and adults:

 Peripheral blood smear:
Sickle cells: crescent-shaped RBCs.
Howell-Jolly bodies.
 Imaging:
X-ray of the skull shows hair-on-end (“crew
cut”) sign due to periosteal reaction to
erythropoietic bone marrow hyperplasia
(also present in thalassemia).

 Sickle cell disease with drepanocytes (sickle cell) and target cells
morphologies in sickle cell disease.
Complications:
1) Organ damage:
 Recurrent vascular occlusion and disseminated infarctions lead to progressive
organ damage and loss of function.
homozygotes High morbidity and mortality (‫)ارتفاع االغتالل والوفيات‬.
heterozygotes Organ damage is very rare.

2) Functional asplenia.
3) Renal papillary necrosis.
4) Avascular osteonecrosis.
5) Recurrent strokes.
6) Priapism.
7) Chronic lung disease, acute chest syndrome.
8) Cardiomyopathy (cardiomegaly) Heart failure.
9) Acute sickle hepatic crisis.
10) Hand-foot syndrome (in small, i.e., around age of 3y);
Treatment {Long term management}:
1) Prevent infections:
 Pneumococcal vaccines.
 Meningococcal vaccines.
 Daily penicillin prophylaxis; at least until the age of 5 years.
 If sepsis is suspected, treat with IV third-generation cephalosporin (e.g., ceftriaxone).

2) Prevent vaso-occlusive crises and manage anemia:

A) Hydroxyurea: first-line treatment (‫)لمدى الحياه‬.
 Indications:
 Frequent, acute painful episodes or other vaso-occlusive events.
 Severe symptomatic anemia.
 Effect:
 Stimulates erythropoiesis and increases fetal hemoglobin.
B) If the response to hydroxyurea alone is not adequate:
 Blood transfusions.

Management of acute sickle cell crisis:

1) Prompt and adequate supportive treatment:
 Hydration.
 Pain management.
 Nasal oxygen.
2) Exchange transfusions (erythrocytapheresis):

 Automated removal of erythrocytes containing HbS and simultaneous

replacement with HbS free erythrocytes.
 Used in; acute vaso-occlusive crisis (stroke, acute chest syndrome, acute multi
organ failure).

Curative therapy:
Allogeneic bone marrow transplantation:
Used in; homozygotes, children <16 years with severe disease.
Hemoglobin C Disease:
Is an inherited (autosomal recessive) blood disorder that that may cause a person
to have mild anemia (low blood count).
Is a common hemoglobin variant that has a single amino acid substitution (lysine
substituted for the glutamate) in the sixth position of the beta-globin chain.
hemoglobin C trait (HbAC) Hemoglobin C disease (HbCC)
phenotypically normal May have chronic hemolytic anemia.

Hemoglobin E (HbE)
 An abnormal hemoglobin with a single point mutation in the β chain. At
position 26 there is a change in the amino acid, from glutamic acid to lysine
(E26K).
 E26K;  E = Glutamate, 26 = NO.AA. K = Lysine
 Is a common but minor blood abnormality.
 The blood disorder is often identified because there are slight abnormalities in
the size and appearance of the red blood cells.

Hemoglobin E genetics and detection:

 The βE mutation affects β-gene expression creating an alternate splicing site in
the mRNA at codons 25-27 of the β-globin gene.
Through this mechanism, there is a mild deficiency in normal β mRNA and
production of small amounts of anomalous β mRNA.
 The reduced synthesis of β chain may cause β-thalassemia.
 Also, this hemoglobin variant has a weak union between α- and β-globin,
causing instability when there is a high amount of oxidant.

 HbE can be detected on electrophoresis.

Variant NO. AA (𝛃) AA conversion Occurrence predominantly in:
Hb S 𝟔 Glutamate  valine Africa, Arabia. Black Americans
Hb O 𝟏𝟐𝟏 Glutamate  valine Turkey and Bulgari
Hb C 𝟔 Glutamate  lysine West Africa. China
Hb E 𝟐𝟔 Glutamate  lysine South East Asia
Hb D 𝟏𝟐𝟏 Glutamate  Glutamine Asia
Zurich Glutamate  arginine
 https://youtu.be/YClJciaGe6s

2) Bleeding disorders:
 A group of disorders characterized by defects in hemostasis, which leads to an
increased susceptibility to bleeding.
 Caused either by:
 Platelet disorders (primary hemostasis defect).
 Coagulation defects (secondary hemostasis defect):
 Combination of both.
 Coagulation defects may be further divided into intrinsic or extrinsic defects
according to the pathway of the coagulation cascade affected.

Etiology

A) Disorders of primary hemostasis:

1) Platelet deficiency.
2) Platelet dysfunction (thrombocytopathy): disorders that lead to dysfunctional
adhesion or aggregation of platelets.
Inherited Acquired
 Von Willebraiid disease.  Drug-induced: e.g. Aspirin. NSAID, clopidogrel.
 Bemard-Soulier syndrome.  Immune thrombocytopenic purpura (ITP).
 Glanzmann thrombasthenia.  Chronic kidney disease.

3) Disorders affecting the vessel wall:

 Thrombotic microangiopathy;
 Hemolytic Uremic Syndrome (HUS).
 Thrombotic Thrombocytopenia Purpura (TTP).
 B) Disorders of secondary hemostasis:
1) Intrinsic pathway:
Factor VIII deficiency (hemophilia A).
Factor IX deficiency (hemophilia B).
Factor XI deficiency (hemophilia C).

2) Extrinsic pathway:
Factor VII deficiency.

3) Both pathways:
A) Deficiency or inhibition of vitamin K-dependent coagulation factors (II, VII, IX, and X)
--- {1972}.

Vitamin K deficiency: Vitamin K antagonist therapy

 Malabsorption syndrome.
 Depletion of gut flora (e.g., following antibiotic administration).  warfarin
 Vitamin K deficiency bleeding of the newborn.

B) Inhibition of coagulation factors by autoantibodies (most commonly anti-factor VIII).

C) Disseminated intravascular coagulation (DIC).
D) Impaired hepatic production of coagulation factors (e.g., cirrhosis).

Clinical feature:
1) Blood in urine or stool.
2) Frequent, large bruises.
3) Heavy bleeding after giving birth.
4) Excessive bleeding: that does not stop with pressure and may start spontaneously,
such as with nosebleeds, or bleeding after a cut, dental procedure, or surgery.
5) Heavy menstrual bleeding: which includes menstrual bleeding that often lasts longer
than seven days or requires changing sanitary pads or tampons more than every hour.
6) Petechiae: bleeding under the skin causing tiny purple, red, or brown spots.
7) Redness, swelling, stiffness, or pain from bleeding into muscles or joints, which is
particularly common with inherited hemophilia.
8) Umbilical stump bleeding: that lasts longer than what is typical for newborns— about
one to two weeks after the umbilical cord is cut— or that does not stop.
Complications:
 Severe bleeding disorders can cause serious and life-threatening problems,
including:
Bleeding in the CNS hemorrhagic stroke
Bleeding in the throat Swelling and block the windpipe.
Bleeding into the abdomen Inflammation and damage to nerves.
From bleeding into joints overtime, especially
Damaged joints for people who have inherited hemophilia 
This can also cause chronic pain.
Hard masses in the bones From pooled blood.
Miscarriages ‫اإلجهاض‬

 Impaired secondary hemostasis in lupus erythematosus  diffuse ecchymoses

are visible on the radial aspect of the forearm.

Diagnosis - Bleeding Disorders

A. Medical history.
B. Physical examination.
C. Diagnostic tests and procedures:

1) Complete blood count (CBC):

If the number of platelets is too low, you may have a platelet disorder instead of a
clotting factor disorder.

2) Extrinsic and intrinsic tests:

Determine whether certain clotting factors are involved

Intrinsic pathway Extrinsic pathway
Partial thromboplastin time (PTT) or activated PTT (aPTT) Prothrombin time (PT).
1– 2 – 5 – 8 – 9 – 10 – 11 – 12 10 – 7 – 5 – 2 –1

4) Mixing test:

Determine whether the bleeding disorder is caused by antibodies blocking the

function of clotting factors, such as with autoimmune disorders or acquired
hemophilia.
Diagnosis - Bleeding Disorders “con’t”
5) Von Willebrand factor (vWF) tests:

Measure the amount of von Willebrand factor, whether the factors are working
correctly, or which type of VWD you have.

6) Clotting factor tests {factor assays or a coagulation panel}:

Determine whether certain clotting factors are missing or show up at lower levels
than normal, which can indicate the type and severity of the bleeding disorder.
 For example, if you have very low levels of clotting factor VIII, you may
have hemophilia A.

7) Bethesda test:

Look for antibodies to factor VIII or IX.

8) Factor XIII antigen and activity assays:

Look for factor XIII deficiency.

9) Genetic testing:

Determine if particular genes may be causing the bleeding disorder.

D. Peripheral blood smear:

Identification Suggests
Platelet clumping: pseudothrombocytopenia
Schistocytes or other fragmented cells: MAHA (e.g., TTP)
blasts: hematologic malignancies

Clinical features according to platelet count:

Mild 𝟕𝟎, 𝟎𝟎𝟎 − 𝟏𝟒𝟗, 𝟎𝟎𝟎  no abnormal bleeding, i.e., asymptomatic thrombocytopenia
 Prolonged bleeding following surgery or trauma.
Moderate 𝟐𝟎, 𝟎𝟎𝟎 – 𝟕𝟎, 𝟎𝟎𝟎  Easy bruising.
 petechiae and purpura.
 Spontaneous bruising (ecchymoses), petechiae, and purpura.
Severe 𝒍𝒆𝒔𝒔 𝒕𝒉𝒂𝒏 𝟐𝟎, 𝟎𝟎𝟎  Bleeding from the mucosa (e.g., bleeding gums) or after
minimal trauma.
 Increased risk of spontaneous, life-threatening bleeding.
 https://www.youtube.com/watch?v=EwXVGpBJdlQ

Von Willebrand disease

 Most common congenital bleeding disorder.
 A bleeding disorder characterized by a deficiency or dysfunction of von
Willebrand factor (vWF).
 In the vast majority of cases, vWD is an inherited disorder caused by mutations
in the vWF gene.
 vWF is involved in platelet adhesion and prevents degradation of factor VIII.
 VIII  aPTT should be increased.

Variants of von Willebrand disease:

Inherited (autosomal recessive) Acquired
VWD caused by mutations in the vWF gene. VWF deficiency that occurs secondary to other
medical conditions.

Inherited von Willebrand disease (autosomal recessive) types:

Types Percent Mechanism

Type –1 𝟖𝟎 – 𝟖𝟓 % Mild to moderate deficiency of vWF and factor VIII
Type –2 𝟏𝟓 – 𝟐𝟎 % Dysfunctional vWF
Type –3 ≈𝟑% Complete absence of vWF and factor VIII.

 The most common type is type 1.

 The most severe form is type 3.

Etiology of Acquired von Willebrand disease (aVWD)

 Lymphoproliferative and myeloproliferative diseases (e.g., multiple myeloma,
monoclonal gammopathies, lymphoma, essential thrombocythemia).
 Autoimmune diseases (e.g., SLE).
 Cardiovascular defects (e.g., ventricular septal defect, aortic stenosis).
 Side effects of certain drugs (e.g., valproic acid).
The mechanism of acquired
von Willebrand disease (aVWD)
is unknown.
Pathophysiology:
Deficiency or dysfunction of vWF leads to:
 Dysfunctional platelet adhesion → impaired primary hemostasis
 Reduced binding of factor VIII → increased degradation → ↓ factor VIII activity
→ impaired intrinsic pathway of secondary hemostasis

Clinical features
 Type 1 and avWD usually manifest more mildly; type 3 is the most severe form.

1) Often asymptomatic.
2) Symptomatic individuals may develop the following symptoms:
A) Bleeding after surgical procedures or tooth extraction
B) GI bleeding (can be caused by angiodysplasia)
C) Menorrhagia (affects up to 92% of women with vWD) [5]
D) Severe cases: large hematomas, hemarthrosis, life-threatening bleeding
(e.g., during childbirth).
E) Mucocutaneous bleeding:
 Ecchymoses, easy bruising
 Epistaxis
 Bleeding of gingiva and gums
 Petechiae
 Prolonged bleeding from minor injuries

Diagnostics

1) History:
 Recurrent episodes of bleeding since childhood
 Often positive family history.

2) Laboratory studies:
 High bleeding time.
 High or normal aPTT.
Ristocetin: drug aid
 Normal PT and platelet count.
platelet aggregation.
 Decrease in vWF and VIII factor.
 Ristocetin cofactor level < 30 IU/dL.
Treatment of VWFDs:

 Treatment is only indicated if symptoms occur or as prophylaxis before

surgical procedures.

1) Inherited von Willebrand disease:

Desmopressin (DDAVP): stimulates vWF release from endothelial cells.

 Best initial treatment for mild or moderate symptoms (typically type 1 and,
sometimes, type 2).
 Not effective for type 3.

2) Acquired von Willebrand syndrome:

 Treatment of the underlying cause.

Platelet aggregation inhibitors (e.g., aspirin, NSAIDs, clopidogrel) and

intramuscular injections are contraindicated in von Willebrand disease because
they further increase the risk of bleeding!
 https://www.osmosis.org/learn/Hemophilia

Hemophilia
 Disorders of blood clotting and consequently may lead to serious bleeding. In
the majority of cases, these disorders are hereditary.
 Hemophilia is caused by an X-linked recessive defect (inherited or
spontaneous mutation) or antibody production against clotting factors.

Etiology:
Type Deficiency Percent
Hemophilia A Factor VIII 80 %
Hemophilia B Factor IX 20 %
very rare (increased frequency in the Ashkenazi
Hemophilia C Factor XI Jewish population); caused by an autosomal
recessive defect

1) Spontaneous bleeding or delayed-onset bleeding (joints, muscular and soft tissue, and
mucosa) in response to different degrees of trauma:

 Repeated hemarthrosis (e.g., knee joint)  hemophilic arthropathy.

 Recurrent bruising or hematoma formation.
 Oral mucosa bleeding, epistaxis, excessive bleeding following small
procedures (e.g., dentist procedures).
Hemophilia C does not typically manifest with spontaneous bleeding,
hemarthrosis, or deep tissue bleeding.
2) Further sites/symptoms of hemorrhage:

 Melena (black stool).

Hemarthrosis
Diagnostics:
1) Patient and family history.
2) Screening.
PT aPTT Platelet count
Normal Usually prolonged Normal

 If aPTT prolonged  mixing study.

 If mixing study is positive (or if patient/family history are strongly positive) →
quantitative assessment of factor activity levels.

Treatment
A) Substitution of clotting factors:

for hemophilia A Factor VIII

for hemophilia B Factor IX
for hemophilia C Factor XI

B) Desmopressin; for mild hemophilia A.

C) Emicizumab: liver Produced all clotting

factor except (III, IV, XII)
 Humanized monoclonal bispecific antibody.
 Used for hemophilia A.
 Mechanism of action:
Bridges activated factor IX and factor X by binding to both factors (thereby replacing the
deficient factor VIII).

‫كأنه بقفز عن خطوة‬

‫العامل الثامن‬
 https://www.osmosis.org/learn/Disseminated_intravascular_coagulation

Disseminated intravascular coagulation (DIC)

Syndrome characterized by thrombosis, hemorrhage, and organ dysfunction
caused by systemic activation of the clotting cascade, which leads to platelet
consumption and exhaustion of clotting factors.

Etiology:
Infections Sepsis (esp. G –Ve)
Trauma  Acute traumatic coagulopathy.
 Burns.
Obstetric  Amniotic fluid embolism.
complications  Abruptio placenta
Organ failure  Acute pancreatitis.
 Acute respiratory distress syndrome (ARDS).
Acute promyelocytic leukemia (APL), acute myelocytic leukemia.
Malignancies Solid tumors, e.g.:
 Pancreatic, Ovarian, Gastric and Non-small cell lung cancer.
Toxins Snake bites
 Acute hemolytic transfusion reaction (AHTR).
Immunologic  Transplant reaction (e.g., graft-versus-host disease).
 Extracorporeal procedures (e.g., dialysis).
Vascular Aortic aneurysms.
malformations
Dilution Massive transfusion
 Nephrotic syndrome.
Other  New thrombus formation.
 Hemolysis.
 Acidosis.
Drug reactions -----------------------------------------------------------------------------------------

Pathophysiology:

1) Underlying disease  (↑) tissue factor (III) (TF) presentation (e.g., due to increased
expression after trauma)  (↑) activation of thrombin  generation of fibrin 
consumption of natural anticoagulants (e.g., antithrombin, thrombin-antithrombin
complex (TAT), protein C).

 Platelet activation  hypercoagulable state.

 ↓ Fibrinolysis  (↑) intravascular fibrin  obstruction of the microvasculature 
organ dysfunction and multi-organ failure.

2) Consumption of platelets, clotting factors, and fibrinogen  thrombocytopenia and lack of

clotting factors  severe bleeding.
Nonsymptomatic Mild fibrinolysis and/or mild hypercoagulation
type DIC
Hyperfibrinolysis (excessive plasmin activity  increased fibrin
Bleeding type
DIC degradation  thrombi become unstable and dissolve shortly after
forming).
Massive Hypercoagulation and hyperfibrinolysis ((↑) plasmin) 
bleeding type
DIC consumption of platelets and all coagulation factors  bleeding
diathesis.
(↑) Cytokines  (↑) plasminogen activator inhibitor-I (PAI-I) and (↑)
Organ failure
type DIC neutrophil extracellular traps (NETs)  hypercoagulation with
In sever stages hypofibrinolysis  platelet and fibrin-rich microthrombi  impaired
perfusion and tissue necrosis.

Clinical features:
1) Bleeding:
 Hematemesis, hematochezia.
 Hematuria.
 Oozing of blood from surgical wounds or intravenous lines.
 Petechiae, purpura, ecchymoses.

2) Massive hemorrhage: collection of blood in body cavities (hemoperitoneum,

hemothorax).

3) Organ failure: primarily due to hypercoagulation:

Microangiopathic hemolytic anemia

Acute renal failure oliguria
Hepatic dysfunction jaundice
ARDS dyspnea, rales
Pulmonary thromboembolism: dyspnea, chest pain, hemoptysis
Deep vein thrombosis lower limb edema
Neurological dysfunction altered mental status, stroke
Purpura fulminans: DIC with extensive skin necrosis
Waterhouse Friderichsen syndrome adrenal infarcts → adrenal insufficiency
Signs of shock
Diagnostics
1) Coagulation panel: Monitor frequently (e.g., every 6–8 hours or until stable or
improving).
Increase in: PT, aPTT, bleeding time and D-dimer.
Decrease in: Fibrinogen, factor (V + VIII).

2) CBC and PBS: D-dimer

(↓) Platelet count. A fibrin degradation product released when plasmin cleaves
(↓) Hct. crosslinked fibrin. Increased serum concentrations of D-dimer
Schistocytes. indicate recent intravascular coagulation and/or fibrinolysis
(e.g., from deep vein thrombosis, pulmonary embolism,
disseminated intravascular coagulation).

Treatment
1) Treatment of the underlying disease is the core of management.

2) Blood products:

RBCs with active bleeding or 𝑯𝒃 < 𝟕 𝒈𝒓/𝒅𝑳

Fresh frozen PT or aPTT > 𝟏. 𝟓 times the normal value.
plasma (FFP)
Cryoprecipitate: For bleeding and 𝒇𝒊𝒃𝒓𝒊𝒏𝒐𝒈𝒆𝒏 𝒍𝒆𝒗𝒆𝒍𝒔 < 𝟏𝟓𝟎 𝒎𝒈/𝒅𝑳 despite FFP.
Active bleeding or high risk of bleeding (e.g, planned invasive
Platelets procedure): 𝒑𝒍𝒂𝒕𝒆𝒍𝒆𝒕 𝒄𝒐𝒖𝒏𝒕 < 𝟓𝟎, 𝟎𝟎𝟎
No bleeding: platelet count < 10,000-20,000

3) Antifibrinolytic therapy: Fresh frozen plasma (FFP)

Tranexamic acid; not routinely recommended. A blood product containing
all coagulation factors.
4) Anticoagulation:

 Prophylactic heparin.
 Therapeutic heparin.
 Other coagulation inhibitors: Consider in individual cases in consultation with
a specialist.

Cryoprecipitate
A product obtained from frozen blood
plasma via centrifuge.
 Inherited platelet disorders
 Platelet membrane glycoproteins and their function: An overview:
Platelet membrane glycoproteins are surface glycoproteins found on platelets
(thrombocytes) which play a key role in hemostasis. When the blood vessel wall
is damaged, platelet membrane glycoproteins interact with the extracellular
matrix.

Membrane glycoproteins:
 Glycoprotein Ib-IX-V complex (GPIb-IX-V):
The deficiency in glycoprotein Ib-IX-V complex synthesis leads to Bernard-
Soulier syndrome.

 Glycoprotein VI (GPVI):
Is an important collagen receptor involved in collagen-induced platelet activation
and adhesion.
 It plays a key role in then procoagulant activity and subsequent thrombin
and fibrin formation.

 Glycoprotein la /Ila complex (GPIa / IIa = integrin 𝜶𝟐𝜷𝟏)

Interaction with collagen leads to stabilization of the platelets.

 Glycoprotein lIb / IlIa complex (GPIIb / IlIa = integrin 𝜶𝑰𝑰𝑰𝒃 𝜷𝟑 )

Plays an important role in platelet aggregation and adhesion to endothelial
surfaces.

 GPV / IlIa (GPV / IIa = integrin 𝜶𝟓 𝜷𝟏 ):

Main function is in the adhesion of cells to the extracellular matrix components.
Glanzmann thrombasthenia Bernard-Soulier syndrome
Pathophysiology Deficiency or dysfunction of Deficiency or dysfunction of
the platelet GP (IIb / IIla) the platelet Gp (Ib / V / IX)
complex complex
Affected platelet function Aggregation Adhesion
Genetic mutation 17q21 chromosome, GPIb𝜶, GPIb𝜷, and GPIX
ITGA2B or ITGBS genes. genes.
 Bernard-Soulier syndrome

 BSS is a rare autosomal recessive platelet dysfunction that is characterized by a low

levels, absence, or dysfunction of the Gp (Ib / V / IX) complex on the platelet surface.

Pathophysiology:
 There are specific GP receptors on the platelet membrane, which function in platelet
adhesion, activation, and aggregation.
 The GP (Ib-IX-V) receptor complex is responsible for platelet adhesion through its
interaction with von Willebrand factor on the exposed subendotlielium.
 The GP (Ib-IX-V) receptor complex is composed of four transmembrane polypeptide
subunits-disulfide-linked alpha and beta subunits of GPIb, and noncovalently bound
subunits GPIX and GPV.
 The platelets of BSS cases lack or have a dysfunctional GPIb-IX-V receptor.
 This results in defective adhesion to the subendotlielium.

Clinical manifestations:
 Manifest with a tendency to bleed in early childhood.
 Mucocutaneous bleeding is seen predominantly.
 Easy bruising, purpura, epistaxis, bleeding gums, menorrhagia, and excessive bleeding
after surgery or trauma are common symptoms.

Diagnosis:
 Although thrombocytopenia is generally observed in BSS, the number of platelets is
variable.
 Giant platelets are seen in peripheral blood smear.
 Bleeding time found to be prolonged. Routine coagulation tests should be found normal.
 In vitro platelet aggregation studies characteristically indicate that aggregation with
ristocetin failed and responded slowly with low doses of thrombin.
 Flow cytometric analysis of platelet: defective binding with CD42a (GPIX), CD42b (GP
lb), CD42c (GP lb), and CD42d (GPV).
 Treatment:
1) BSS treatment is generally supportive.
2) Platelet transfusion is used to treat when
surgery is needed or when there is a risk of
life-threatening bleeding.
 The patient may develop antiplatelet
antibodies due to the presence of
glycoproteins Ib/IX/V, which are
present
on the transfused platelets but absent
from the patient’s own platelets.

If the above treatments do not work, apply the following:

 Antifibrinolytic agents such as p-aminocaproic acid or tranexamic acid may be useful
for mucosal bleeding.
 Desmopressin has been found to shorten bleeding episodes.
 Recombinant activated factor VII (rFVIIa) has been reported to reduce bleeding times.
 Stem cell transplantation.

Glanzmann thrombasthenia
 GT is an autosomal recessive congenital bleeding disorder characterized by a lack of
platelet aggregation due to defect and/or deficiency of (𝜶Ilb𝜷3) integrin.
 Patients with this disorder often experience lifelong bleeding episodes involving
mucocutaneous membranes.

Etiology:
GT is usually caused by decreased or absent expression of 𝜶Ilb or 𝜷3, abnormalities in
protein folding, transport of the integrin subunit causing post-translational defective
processing or decreased surface expression, or abnormalities affecting protein function.

Pathophysiology:

The main mechanism in the pathophysiology of GT is the qualitative or quantitative

disorder of the autosomal recessive platelet surface receptor of GPIIb/IIIa (ITG 𝜶llb𝜷3).
As a result, it results in erroneous platelet aggregation and reduced clot retraction.
Clinical manifestations
 The most common symptoms of bleeding are purpura, nosebleeds (60-80%). gingival
bleeding (20-60%), and menorrhagia (60-90%).
 Gastrointestinal bleeding in the form of melena or hematochezia is found in 10-20% and
intracranial hemorrhage is developed in 1-2%.
 Mucocutaneous bleeding may occur spontaneously or following minimal trauma.
 Epistaxis is the most common cause of severe bleeding especially in children.
 Menorrhagia is quite common in affected women, and there is a higher risk of serious
bleeding during menarche (‫ )اول دورة‬due to the prolonged estrogenic effect on the
proliferative endometrium that occurs during anovulatory cycles.

Diagnosis:
The diagnosis of GT is often not noticed, because many platelet disorders share common
clinical and laboratory features.

 Complete blood count:

In the evaluation of peripheral blood smear with light microscopy, normal platelet count
and normal granular size should be. If the bleeding is severe and/or chronic, patients may
have a red cell distribution width that increases with low hemoglobin, microcytosis, and
secondary iron deficiency.

 Coagulation screening tests:

Prothrombin time (PT), activated thromboplastin time (aPTT), and fibrinogen values are
usually normal unless a patient is evaluated in a significant acute bleeding environment
and there is no evidence of consumption coagulopathy.

Treatment:
 A gradual treatment standard is applied in GT treatment. The first treatment for mild
bleeding is local measures including local compression, cauterization, stitching, or ice
therapy.
 The treatment applied in case of unresponsiveness to these treatments or in heavier
bleeding is antifibrinolytic therapy first, followed by platelet transfusion, and recombinant
active factor VII (rFVIIa) if bleeding persists.
 Immune thrombocytopenia (Immune thrombocytopenic purpura)
Primary immune thrombocytopenia Secondary immune thrombocytopenia
An autoimmune disorder characterized An autoimmune hematologic disorder
by isolated thrombocytopenia causing isolated thrombocytopenia
(< 𝟏𝟎𝟎, 𝟎𝟎𝟎/𝒎𝒎𝟑) with no known that is secondary to an identifiable
precipitating cause. trigger.

Newly diagnosed ITP:

All cases within the first 3 months of diagnosis;

Persistent ITP Chronic ITP

ITP lasting 𝟑 − 𝟏𝟐 months ITP lasting > 𝟏𝟐 months

Etiology:
 Primary ITP: idiopathic (most common).

 Secondary ITP associated with:

Autoimmune disorders: SLE, Antiphospholipid syndrome.

Malignancy: Lymphoma, leukemia (particularly CLL).
Infection: HIV HCV.
quinine, beta-lactam antibiotics, carbamazepine,
Drugs: heparin, vaccines, linezolid, sulfonamides,
vancomycin, TMP-SMX, antiepileptics

Pathophysiology:
Antiplatelet antibodies (mostly IgG directed against, e.g., GpIIb/IIIa, GpIb/IX) bind
to surface proteins on platelets  sequestration by spleen and liver  (↓) platelet
count  bone marrow megakaryocytes and platelet production increase
in response (in most cases).
Clinical features:
 Most commonly:
 A Symptomatic.
 Splenomegaly is typically absent.

 Minor mucocutaneous bleeding (less common):

 Subcutaneous: e.g., bruising, petechiae, purpura.
 Mucosal: e.g., mild epistaxis, gingival bleeding.

 Other types of bleeding (rare):

 Gastrointestinal: e.g, melena.
 Genitourinary: e.g., hematuria, menorrhagia.
 CNS: e.g., features of intracranial hemorrhage.
 Prolonged or excessive traumatic or surgical bleeding.
Splenomegaly is very unusual in ITP and makes other diagnoses more likely.
There should be suspicion for ITP in a child with thrombocytopenia and petechiae
following a viral illness.

Diagnostics:
ITP is a diagnosis of exclusion; patients typically have a low platelet count with no other
abnormalities.

Laboratory studies Additional investigations

(↓) platelet count (< 100,000/𝒎𝒎𝟑 ) All adults: HIV and HCV screening
Bleeding time: may be prolonged Bone marrow biopsy:
Findings: normal or (↑) megakaryocytes
Peripheral blood smear: normal to Additional testing as required:
large platelets.

An excessive number of abnormally

large or small platelets may indicate
an inherited platelet disorder rather
than ITP.
Treatment of ITP:
Age First therapy
Adult Corticosteroid
Children IV-Ig
 Thrombotic thrombocytopenic purpura (TTP)
 Is a thrombotic microangiopathy, a condition in which microthrombi,
consisting primarily of platelets, form and occlude the microvasculature (i.e., the
arterioles and capillaries').
 The other main thrombotic microangiopathy is hemolytic uremic syndrome
(HUS).
 Although TTP and HUS share similarities in both pathophysiological
findings and clinical features, these conditions differ in etiology.

Etiology:
1) ADAMTS13 deficiency/inhibition:

Acquired TTP (∼ 95%): Congenital TTP (∼ 5%):

autoantibodies against ADAMTS13 gene mutations resulting in deficiency of ADAMTS13

A zinc-containing metalloprotease that

2) Risk factors: cleaves von Willebrand factor, a large protein
involved in blood clotting, which results in
 Drugs decreased enzyme activity. Deficiency (e.g., in
 Pregnancy patients wth thrombotic thrombocytopenic
 Systemic disease: purpura) causes small-vessel platelet-rich
Cancer, HIV, SLE, infections thrombus deposition with subsequent
hemolytic anemia, thrombocytopenia, and
possible organ damage.
Pathophysiology:
1. Autoantibodies or gene mutations → deficiency of ADAMTS13 (a
metalloprotease that cleaves von Willebrand factor)
2. (↓) Breakdown of vWF multimers → vWF multimers accumulate
on endothelial cell surfaces
3. Platelet adhesion and microthrombosis
4. Microthrombi → fragmentation of RBCs with schistocyte formation
→ hemolytic anemia
5. Arteriolar and capillary microthrombosis → end-organ ischemia and damage,
especially in the brain and kidneys (potentially resulting in acute kidney
injury or stroke)

ADAMTS13 deficiency  excess vWF  microthrombus formation

 blockage of small vessels  RBC fragmentation (hemolysis)
and end-organ damage.
 Clinical features:
 TTP patients are typically previously healthy adults.
 Fever.
 Neurological signs and  Altered mental status, delirium.
symptoms:  Seizure, focal defects, stroke.
 Headache, dizziness.
 Low platelet count  Petechiae, purpura
Hemolytic
(thrombocytopenia):  Mucosal bleeding
syndrome
 Prolonged bleeding after minor cuts
 Microangiopathic hemolytic  Fatigue, dyspnea, and pallor
anemia  Jaundice
 Impaired renal function  Hematuria, proteinuria
 Oliguria, anuria

Diagnostics:
 Hematology

(↓) Platelets
(↓) Hemoglobin
(↑) Reticulocytes
(↓) Haptoglobin
Normal or mildly prolonged prothrombin time (PT) and activated partial
thromboplastin time (aPTT)
Negative Coombs test.

 Peripheral blood smear

Large number of schistocytes (up to 10% of RBCs).

Low number of platelets.

 Serum chemistry

(↑) LDH, (↑) indirect bilirubin (hemolytic anemia)

(↑) BUN and (↑) creatinine (impaired renal function).

 ADAMTS13 activity and inhibitor testing

(↓) ADAMTS13 activity.

 Identification of secondary causes (e.g., tests for pregnancy, SLE, HIV, malignancy)
Treatment:
1) Monitoring and correction;
 Electrolyte disturbances.
 RBC transfusions.
2) Prompt initiation of plasma exchange therapy (PEX).
3) Glucocorticoids.
4) Rituximab is reserved for severe cases.

Neonatal thrombocytopenia
 Neonatal thrombocytopenia is defined as a platelet count <150,000/ 𝝁L.
 Although severe neonatal thrombocytopenia (defined as a platelet count <50,000/𝝁L) is
uncommon in the general healthy newborn population, the risk increases for infants
admitted to the neonatal intensive care unit (NICU) especially for the extremely preterm
infants (birth weight [BW] <1000 g or gestational age [GA] < 28 weeks).

Presentation
 The clinical presentation of neonatal thrombocytopenia includes patients detected
incidentally (‫ )بالصدفة‬by a low platelet count from a complete blood count (CBC) obtained
for other reasons, at-risk patients identified by a screening CBC, and symptomatic
infants with evidence of bleeding (eg. petechiae, large ecchymoses, cephalohematoma,
or oozing from the umbilical cord or puncture sites).

Diagnostic evaluation
The diagnostic evaluation is focused on determining and, if possible, directing specific
therapy to the underlying cause of neonatal thrombocytopenia.
However, establishing a diagnosis can be challenging because of the overlap of the
clinical presentation among different conditions, and because frequently there may be
multiple potential causes.
The diagnostic approach to neonatal thrombocytopenia is centered on the timing of
presentation (early within the first 72 hours of life or later), the severity of
thrombocytopenia, the infant's clinical condition, and the maternal and neonatal history,
including labor and delivery.
 Pathophysiology:
The major mechanism is impaired platelet production.

Kinetic Mechanisms Pathophysiologic Categories

1) Decreased platelet production Immune-mediated.
2) Accelerated platelet destruction. or Infectious.
sequestration. Genetic.
3) Combination of above two mechanisms. Drug-related.
DIC.

Management approach:
A) In the majority of cases, neonatal thrombocytopenia resolves without intervention.
B) Specific therapy, if available, should be given to patients in whom an etiology has
been identified (e.g., sepsis).
C) Platelet transfusion:
Indications; Most platelet transfusions in neonates are given prophylactically to patients
without evidence of bleeding.
Indications for Platelet Transfusion in Neonates:

Clinical Condition Platelet Count

Stable neonate < 𝟐𝟓, 𝟎𝟎𝟎/𝒎𝒄𝑳
Unstable neonate < 𝟓𝟎, 𝟎𝟎𝟎/𝒎𝒄𝑳
Neonate on ECMO < 𝟏𝟎𝟎, 𝟎𝟎𝟎/𝒎𝒄𝑳
ECMO - extracorporeal membrane oxygenation

In extracorporeal membrane oxygenation (ECMO), blood is pumped outside of your body to a heart-
lung machine that removes carbon dioxide and sends oxygen-filled blood back to tissues in the body

Neonatal Immune-Mediated Thrombocytopenia:

Neonatal Alloimmune Thrombocytopenia
 Healthy newborns present with petechiae and low platelet counts.
 The pathogenesis of neonatal alloimmune thrombocytopenia is analogous to Rh hemolytic
disease of the newborn. Mothers of affected infants have normal platelet counts.
 The fetus carries an antigen that is foreign to maternal platelets and sensitizes the mother.
 Transplacental antibodies produced by the mother in the second trimester destroy fetal platelets,
resulting in thrombocytopenia.
 Unlike Rh disease, a first pregnancy can be affected. Intracranial hemorrhage, which can occur
prior to birth, is found in about 10% to 15% of affected neonates.

Neonatal Autoimmune Thrombocytopenia

 In autoimmune thrombocytopenia, the mother has idiopathic thrombocytopenia or systemic
lupus erythematosus and is thrombocytopenic.
 Maternal antiplatelet antibodies cross the placenta and destroy fetal platelets.
 Post-transfusion purpura (PTP)

 Delayed adverse reaction to a blood transfusion or platelet transfusion that

occurs when the body has produced alloantibodies to the allogeneic transfused
platelets' antigens.
 These alloantibodies destroy the patient's platelets leading to
thrombocytopenia, a rapid decline in platelet count.
 PTP usually presents 5-12 days after transfusion, and is a potentially fatal
condition in rare cases.

Mechanism:
 PTP is rare, but usually occurs in women who have had multiple pregnancies
or in people who have undergone previous transfusions.
 The precise mechanism leading to PTP is unknown.
 The most commonly occurs in individuals whose platelets lack the HFA-1a
antigen (old name: PLA1).
 The patient develops antibodies to the HPA-1a antigen leading to platelet
destruction.

Treatment:
1) Symptoms are usually sudden in onset and self-limiting, most often resolving
within 2 weeks.
2) IVIG therapy is the primary treatment.
3) Additionally, PTP is an American Society for Apheresis Category III indication
for plasmapheresis.
 Hypercoagulable disorders
The main causes of thrombosis:
1. Turbulence flow (Stasis).
2. Endothelial injury.
3. Hypercogulation.
 Endothelium, the inner most single layer of cells lining the blood vessels,.
Endothelium functions:
1) Surface for thrombosis formation and critically regulates blood fluidity and
homeostasis.
2) Barrier which separates blood clotting factors from exposure to subendothelial
prothrombotic extracellular matrix components.
3) Secretes or expresses vasoactive factors that modulate platelet reactivity,
coagulation, fibrinolysis, and vascular contractility, all of which contribute to
thrombotic formation.
 Such factors include nitric oxide, prostacyclin, Von Willebrand factor
(VWF), thrombomodulin, endothelin, etc.
Antithrombin III, protein C, and protein S. naturally occurring
anticoagulant proteins
protein C and protein S Antithrombin III
Inhibiting the action of the cofactors factor inhibits the serine proteases (factors II, IX,
Va and factor VIlla. X, XI, and XII)

 Antithrombin III action is dramatically enhanced by heparin.

Hypercoagulable states (Thrombophilia, Hypercoagulability)

Thrombophilia: Thromboembolism:
A predisposition to increased The formation and/or migration of blood clots
coagulation that typically in different locations of the venous or arterial
manifests as recurrent vasculature that can occlude or impair the
thromboembolism. pulmonary or systemic circulation.

Venous thromboembolism (VTE) Arterial thromboembolism

blood clots that form within the Blood clots that form within the
venous vascular system arterial vascular system
Travel to pulmonary arteries via the Travel to distal systemic
right heart arteries and arterioles.

Clinical features:
Venous thromboembolism Arterial thromboembolism
Deep venous thrombosis (DVT),
thromboembolism pulmonary embolism (PE), portal Ischemia.
vein thrombosis (PTV), cerebral Acute coronary syndrome.
venous thrombosis (CVT).
 Onset at age <50 years of
either of the following:
 Unprovoked VTE.
 VTE associated with only
thrombophilia weak risk factors. In a young patient with no
 Unusual thrombus localization. cardiovascular risk factors.

 Strong family history of VTE.

 Recurrent VTE or multiple
VTE.
Etiology:
 Traditional risk factors for thromboembolic disease:
Venous thromboembolism Arterial thromboembolism
 Trauma, fractures, major orthopedic surgery,  Smoking.
oncological surgery, immobilization combined  Obesity.
with other risk factors.  Hyperlipidemia.
 Diabetes mellitus.
 Exogenous estrogen (e.g., Oral contraceptive  Hypertension.
pills (OCPs) or hormone replacement therapy
(HRT)); pregnancy.

 Hereditary causes of hypercoagulability:

1) Predisposing to venous thrombosis:
 Factor V Leiden (autosomal dominant inheritance): most common genetic
cause of hypercoagulability in white populations.
 Protein C deficiency.
 Prothrombin G2Q210A mutation.
 Hyperhomocysteinemia.
 Sickle cell anemia.
 Antithrombin III deficiency.
 Autosomal dominant inheritance.
 Occasionally acquired:
 Renal failure.
 Nephrotic syndrome (urinary loss of antithrombin).

2) Predisposing to both venous and arterial thrombosis

 Protein S deficiency.

Acquired causes of hypercoagulability:

Predisposing to venous thrombosis Predisposing to both venous and arterial
thrombosis
 Malignancy.  Antiphospholipid syndrome.
 Nephrotic syndrome.  Heparin-induced thrombocytopenia.
 Systemic lupus erythematosus.
 Pathophysiology:
Hereditary thrombophilia: Acquired thrombophilia:
Typically caused by mutations of Varying underlying mechanisms that
proteins and enzymes involved in include stasis, endothelial injury, changes
the coagulation cascade. in elements of the coagulation cascade, as
 Like; protein S, C and well as the formation, release, or exposure
antithrombin III. to additional procoagulant substances.

 Pathophysiology of hereditary thrombophilia :

1) Activated protein C resistance (APC-R) – the most common cause Factor V
Leiden:

Prevalence
Heterozygosity: Homozygosity:
∼ 5% < 1%

Normal Pathological
 Activated protein C (APC)  In such patients, Gln506-Va is resistant
inactivates factor V in the clotting to cleavage by APC → factor V remains
cascade → decreases the activation active → activates prothrombin →
of thrombin. increases thrombotic events (e.g.,
peripheral and cerebral vein thrombosis,
 A DNA point mutation substitutes recurrent pregnancy loss).
guanine for adenine → corresponding
mRNA codon forms glutamine in  Risk of thromboembolism is several
place of arginine on position 506 times higher in patients with homozygous
(Arg506Gln mutation) near the mutations than in those with heterozygous
polypeptide cleavage site of factor V. mutations.

2) Elevated factor VIII:

Pathophysiology Prevalence
In combination with factor IXa, factor VIIIa activates factor X → ∼ 5%
increases thrombotic events.
 3) Prothrombin mutation:

Normal Pathological Prevalence

Mutation (G20210A) in the noncoding
Prothrombin is activated three prime untranslated region (3'-
to thrombin (factor II) UTR) of the prothrombin gene → ∼ 3%
which cleaves fibrinogen increased expression of prothrombin
to form fibrin. → increased prothrombin serum levels
→ (↑) thrombotic events.

4) Protein S and Protein C deficiency:

Normal Pathological Prevalence

Endothelial cells express A deficiency of protein S
thrombomodulin, which binds activated or protein C results in Protein S:
thrombin → activates protein C → overactivity of factors V ∼ 1%
complexes with protein S to inhibit factor and VIII (factor Va and
Va and factor VIIIa (both procoagulant factor VIIIa) → increases Protein C:
factors in the clotting cascade). thrombotic events. < 1%

5) Antithrombin III deficiency:

Normal Pathological Prevalence

Antithrombin III binds to and Deficiency leads to
inactivates thrombin and factor X → decreased inhibition and ∼ 1%
inhibits coagulation. elevated thrombin and factor
Heparin normally increases PTT. X.
Decrease in PTT
No other direct effects on PT, PTT, or thrombin time

6) Hyperhomocysteinemia:
  Pathophysiology of acquired thrombophilia
Etiology Pathophysiology
 Extended immobilization during procedure → blood stasis.
Surgery
 Vessel instrumentation → endothelial damage.
 Results in decreased venous blood flow, immobilization (blood
Trauma stasis), and release of tissue factor (hypercoagulability) → increased
clotting.
 Cancers excrete procoagulant factors (e.g., tissue factor and cancer
Malignancy procoagulant).
 The risk of thromboembolism is highest during first hospitalization
and initiation of chemotherapy.
Immobilization  Prolonged immobilization (e.g., extended travel, hospitalization, bed
rest) → increased venous stasis.
 Causes endothelial damage.
Smoking  The risk is significantly higher in women who also use oral
contraceptives.
 Leads to chronic systemic inflammation and impaired fibrinolysis.
Obesity  The risk of thromboembolism increases with increasing Body Mass
Index (BMI).
Acquired antibodies directed against plasma proteins bound to
phospholipids (e.g., lupus anticoagulant, anti-cardiolipin, beta2-
Antiphospholipid glycoprotein I antibodies) → aggregation of plasma proteins (e.g.,
syndrome clotting factors) → induces venous and arterial clotting →
miscarriages, DVTs, portal vein thrombosis, and strokes.
Associated with SLE and rheumatoid arthritis.
Nephrotic syndrome  Loss of plasma antithrombin in urine and an increase in blood
viscosity due to extravasation of fluid from albumin loss in urine.
Oral contraceptive
pills (OCPs) or  Increased estrogen and progestin → increase in prothrombin and
hormone fibrinogen and a decrease in protein S.
replacement therapy
(HRT)
Heparin-induced  Antibodies against platelet factor 4 (PF-4) → increased activation of
thrombophilia platelets (hypercoagulability) and a depletion of platelets
 Clotting factors increase (hypercoagulability).
Pregnancy  Protein C and protein S decrease.
 Venous stasis as the uterus enlarges.
 Progressive endothelial damage.
 Increase in pro-clotting factors without a concomitant increase in
Advanced age protein C.
 Increase in other pro-clotting comorbidities (e.g., malignancy).
 Decreased physical activity.

The role of platelet factor 4 in platelet aggregation induced by the

antibodies implicated in heparin-induced thrombocytopenia
 Diagnostics
1) Routine investigations
CBC: Cell counts may be abnormal in Myeloproliferative
neoplasms (MPNs).
Basic metabolic panel (BMP): can suggest nephrotic syndrome
Liver function tests (LFTs): abnormalities can suggest liver disease, e.g, cirrhosis
Coagulation studies: (↑) aPTT in antiphospholipid antibody syndrome
ESR: can be elevated in malignancy or SLE
Human chorionic gonadotropin (β-hCG): Sensitive and specific for undiagnosed pregnancy.

2) Thrombophilia testing
Hereditary thrombophilia Acquired thrombophilia
 Activated protein C resistance assay.  Antiphospholipid antibody panel:
 Prothrombin G20210A mutation testing.  Lupus anticoagulants.
 Activity assays for protein C, protein S, and  anticardiolipin antibodies).
antithrombin.

Treatment
1) Standard management of thromboembolic diseases:

Arterial thromboembolism Venous thromboembolism

Acute management can include Anticoagulant administration.
revascularization, fibrinolytic, and Consider bridging anticoagulation.
anticoagulation depending on the Consider an inferior vena cava filter if
location and extent of thromboembolism anticoagulant therapy is contraindicated.
and patient factors.
 Treatment “con’t"
2) Acute management of specific thrombophilia

Can lead to heparin resistance and may require

Antithrombin III deficiency: antithrombin concentrate in addition to heparin in
order to be effective.
Protein C deficiency and protein To avoid warfarin-induced skin necrosis, bridge
S deficiency: oral anticoagulation with heparin.
lifelong anticoagulation usually required:
VTE: VKAs (e.g., warfarin)
Protein C deficiency and protein
with heparin bridging
S deficiency:
Arterial High-dose VKA OR
thromboembolism: standard-dose VKA
combined with ASA
Antiphospliolipid syndrome

Prevention:
Standard VTE prophylaxis is indicated in select circumstances regardless of
thrombophilia status (e.g., postoperative status, prolonged immobilization or
hospitalization, active malignancy).

Management of asymptomatic thrombophilia:

Obesity: Recommend weight loss.

Tobacco use: Encourage smoking cessation.
Avoid estrogens OCPs
High-risk Primary prophylaxis (compression stockings, chemical
situations: prophylaxis with a Low molecular weight heparin
(LMWH) or direct oral anticoagulant) may be appropriate.
Surgery: Consider pneumatic stockings, early physiotherapy, and
hydration in addition to the above measures.

Compression stockings: Stockings (‫ )جوارب‬that compress the legs to prevent thrombosis.

 https://www.osmosis.org/learn/Acute_leukemia

Acute leukemia
 Acute leukemia are malignant neoplastic diseases that arise from either the
lymphoid or myeloid cell line.
 In acute leukemia, the peripheral blood has decreased mature forms and
increased immature forms “blasts”, which have immature chromatin with
nucleoli.
 The bone marrow has increased immature cells (blasts).
 Acute symptoms are secondary to marrow failure, which can produce
decreased erythrocytes (causing anemia and fatigue), decreased leukocytes
(permitting infections and fever), and decreased platelets (inducing bleeding).
 There are two main types of acute leukemia:
Acute lymphoblastic leukemia (ALL) acute myeloid leukemia (AML)
most common childhood malignancy Primarily affects adults.

Etiology of Acute leukemia:

ALL AML
Most cases No identifiable cause
Environmental Alkylating chemotherapy, benzene or ionizing radiation
factors:
Genetic or Down syndrome Down syndrome
chromosomal Ataxia telangiectasia Fanconi anemia
factors
Pre-existing hematopoietic disorder (most
Adult T-cell common identifiable cause):
Other factors leukemia/lymphoma  Myelodysplastic syndromes.
is linked to infection  Aplastic anemia.
with HTLV.  Myeloproliferative disorders (e.g.,
osteomyelofibrosis; CML).

 HTLV: Human T-cell lymphotropic virus

 Ataxia telangiectasia: Walking problems + a network of capillaries in the eye.

aplastic anemia Myelodysplastic syndromes

mature leukopenia Immature leukopenia

 Down syndrome: The risk of AML is, like that of ALL, 10–20 times higher in
patients with Down syndrome compared to the general population.
Classification of ALL:
 French-American-British (FAB).
 Based on morphologic and genetic factors.
 Based on the origin (B cell or T cell) and maturity of the leukemic cells.

1) French-American-British (FAB) historical classification of ALL:

Type Characteristics Prevalence
L1 small cells Intermediate (~[𝟐𝟎 − 𝟑𝟎] %)
L2 heterogeneous large cells The most common (~𝟕𝟎%)
L3 Large (homo) cells (Burkitt lymphoma) The least common (~[𝟏 − 𝟐] %)

2) The current WHO Classification (2016):

 Classifies ALL into subtypes of precursor lymphoblastic leukemia/lymphoma
based on morphologic and genetic factors:

A))) Precursor T lymphoblastic leukemia/lymphoma.

B))) B lymphoblastic leukemia with recurrent genetic abnormalities:

 ALL with BCR-ABL (Philadelphia chromosome).
 Hypodiploid
 Hyperdiploid >50. Hyperdiploid
 ALL with t(v;11q23).
 ALL with t(1;19)(q23;p13.3). A descriptor for having greater than the normal
 ALL with t(12;21)(p13;q22). diploid number of chromosomes (46).

Prognosis of hyperdiploid is better than hypodiploid

3) Immunophenotype classification of ALL: based on the origin (B cell or T cell)

and maturity of the leukemic cells:
B Cells T Cells
Prevalence ∼ 80–85% of cases ∼ 15–20% of cases
Early (pro-B) ALL.
Early (pro-T) ALL.
Example Common ALL. Intermediate-T ALL.
Precursor B-ALL. Mature T-cell ALL.
Mature B-cell ALL (also known as Burkitt leukemia).
 B-cell acute lymphoblastic leukemia

 The large basophilic cell in the center of each photomicrograph is a

lymphoblast, identifiable by its large spherical nucleus surrounded by a thin rim
of cytoplasm (high nucleocytoplasmic ratio).
 The nucleus of the lymphoblasts is often indented (‫ )ممزقة‬and shows fine loose
(noncondensed) chromatin.
 Lymphocytes smaller in size with smaller nuclei, condensed chromatin, and
more cytoplasm.

Classification of AML:
 The French-American-British (FAB) - according to the histopathological
appearance of the cells:
For myoblast in general M0-AML Acute myeloblastic leukemia without maturation
M1-AML Acute myeloblastic leukemia with minimal granulocyte
For granulocytes maturation.
M2-AML Acute myeloblastic leukemia with granulocyte maturation.
M3-AML Acute promyelocytic leukemia (APL).
For monocytes M4-AML Acute myelomonocytic leukemia.
M5-AML Acute monocytic leukemia.
For erythrocytes M6-AML Acute erythroid leukemia.
For thrombocytes M7-AML Acute megakaryoblastic leukemia.

 The most common type of AML is M2.

 The highest nonspecific esterase (NSE); is M5.
 NSE; is used to identify normal and leukemic mononuclear phagocytes.
cytochemically.
 Acute monocytic leukemia (M5-AML)
The large basophilic cell visible in each photomicrograph is a monocyte,
identifiable by its large size, folded or lobed nucleus with fine chromatin and
prominent nucleoli and abundant granular cytoplasm with cytoplasmic vacuoles.

Pathophysiology

 Acquired somatic mutations (chromosomal translocations and other genetic

abnormalities) in early hematopoietic precursors  clonal proliferation of a lymphoid or
myeloid stem cell line and arrest in cell differentiation and maturation in early stages
of hematopoiesis  rapid proliferation of abnormal and dysfunctional blasts (with
impaired apoptosis pathways)  accumulation of leukemic white blood cells in the
bone marrow  disrupted normal hematopoiesis  leukopenia (↑ risk of
infections), thrombocytopenia (↑ bleeding), and anemia.

 Immature blasts enter the bloodstream  infiltration of other organs (particularly

the CNS, testes, liver, and skin).

Clinical features:
Clinical features are either related to bone marrow failure, infiltration of organs by
leukemic cells, or a combination of both.
 Sudden onset of symptoms and rapid progression (days to weeks).
 Anemia: fatigue, pallor, weakness.
 Thrombocytopenia: epistaxis, bleeding gums, petechiae, purpura.
 Immature leukocytes: frequent infections, fever.
 Hepatosplenomegaly (caused by leukemic infiltration).
 Oncologic emergencies can be the first sign of leukemia, e.g., an elderly
patient presenting with priapism or DIC may have leukostasis (more common
in AML than ALL): See oncologic emergencies for further details.
Clinical features of ALL:
1) Painless lymphadenopathy.
2) Bone pain (presenting as limping or refusal to bear weight in children).
3) Airway obstruction (stridor, difficulty breathing) due to mediastinal or thymic
infiltration (primarily in T-cell ALL).
4) Features of SVC syndrome (SVC compression).
5) Meningeal leukemia (or leukemic meningitis); headache, neck stiffness, visual
field changes, or other CNS symptoms (caused by CNS involvement).
6) Testicular enlargement (rare finding).
7) B symptoms: Fever, night sweats, unexplained weight loss.

Clinical features of AML:

1) Signs of CNS involvement, e.g., headache, visual field changes (uncommon in
comparison with ALL).
2) Gingival hyperplasia (AML subtype M4 and M5).
3) No fever.
4) Leukemia cutis (or myeloid sarcoma): Multiple
erythematous papules and nodules are seen on the face and
neck of this patient as a result of infiltrating malignant
neoplastic cells accumulating in the dermis.
Complications of leukemia:

1) Tumor lysis syndrome:

 A potentially life-threatening oncologic emergency resulting from the rapid

destruction of tumor cells, which leads to a massive release of intracellular
components, e.g., potassium (K+), phosphate (PO4-), and uric acid.

Pathophysiology:

1) Tumor cell lysis  intracellular component (K+, PO4-, nucleic acid) released
into blood  (↑) nucleic acid  conversion to uric acid  hyperuricemias 
urate nephropathy and risk of acute kidney injury.
2) (PO4-) binds with calcium  decrease calcium (ca+2)  hypocalcemia 
neuronal excitability  risk of seizures.
3) Hyperkalemia  hyperpolarization  cardiac arrhythmias.

Clinical features and diagnosis:

 Cardiac arrhythmias.
 Seizures, epilepsy.
 Kidney injuries.
 Hypocalcemia.
 Hyperkalemia, hyperuricemia and (↑)𝑷𝑶𝟒−𝟐.

2) Leukostasis:
 A medical emergency characterized by tissue hypoxia and hypercoagulability
due to an excessive number of immature leukocytes causing microvascular
obstruction.
 Diagnosis of leukemia:

1) Routine laboratory studies:

 CBC:
Leukocytes: The white blood cell count (WBC) may be elevated, normal, or
low and is not a reliable diagnostic marker.
Platelets: Typically mild to severe thrombocytopenia.
Hemoglobin: Typically anemia.

 Peripheral blood smear: Presence of blasts (immature WBCs).

 Liver chemistries and renal function tests: May be abnormal (e.g., secondary to disease infiltration).
Comprehensive metabolic panel Often abnormal due to increased cell lysis
Coagulation studies: Mild coagulopathy may be present.
Sodium, Potassium, Calcium and Phosphate.
Common findings include derangements ↑ LDH.
↑ Uric acid.

‫ممكن تنخفض وتزيد هاي‬

‫االشياء بسرطان الدم‬
2) Confirmatory diagnostic tests:

Blasts (in bone marrow or peripheral blood)

ALL AML
> 20% myeloblasts.

> 20% lymphoblasts  Presence of recurrent genetic abnormalities, regardless

of the blast percentage:

 AML: t(8;21), inv(16), or t(16;16).

 APL: t(15;17).

Cell morphology
ALL AML
Size Small- to intermediate-sized blasts. Large blasts (2–4 times the size of an RBC).
Nucleus Blasts with large, irregular nuclei Blasts with round or kidney-shaped nuclei that
(high nuclear-cytoplasmic ratio). contain more cytoplasm.
Clarity Inconspicuous nucleoli. Prominent nucleoli.
Granules Coarse granules. Fine granules.
 Some subtypes (especially M3, or APL)
exhibit Auer rods:
Pink-red, rod-shaped granular inclusion
Auer rods No Auer rods. bodies in malignant immature myeloblasts
or promyelocytes..
Myeloperoxidase (MPO) positive.
Leukemic hiatus: the presence of blasts and
Others ------------------------------------------------ mature leukocytes but no intermediate forms.
 3) Specialized studies:
A) Immunophenotype:
Immunohistochemistry
ALL AML
MPO Negative. positive
Terminal deoxynucleotidyl transferase (TdT) Positive. negative
Periodic acid-Schiff (PAS): often positive negative

It rises Flow cytometry (NOT IMPORTANT)

with the ALL AML
height of
immature
T and B
cells.

Most common in
chronic leukemia
B) Genetic studies

Cytogenetics (karyotype, FISH)

ALL AML
 Philadelphia translocation:
 Present in ∼ 20–30% of adults.  t(15:17), especially in
 Only ∼ 5% of children with ALL. acute promyelocytic
 Childhood B ALL: leukemia (M3 AML).
 t(12;21): most common specific abnormality.
 Trisomy 4 and 10: associated with a good prognosis.  Philadelphia
 Hyperdiploidy is common in pre-B-ALL translocation (rare)

Cytogenetics (karyotype, FISH)

ALL AML
 BCR-ABL1 in confirmed or suspected B-ALL. FLT3-ITD: associated with a poor
 Potential findings in childhood B-ALL include: prognosis.
 t(12;21)(p13.2;q22.1). PML-RARA in patients with APL
 ETV6-RUNX1.
 Auer rods

Prognosis:
5-year survival rate following treatment; Five-year life expectancy for a patient
with leukemia.
 ALL generally higher compared to AML.
 AML 30% (80% for elderly people and 20% for children).

Unfavorable prognostic factors

Favorable prognostic factors

Treatment:
1) Pretreatment:
All patients should undergo a thorough evaluation, including baseline laboratory studies,
ECG, and, if appropriate, a pregnancy test and an assessment for fertility preservation.
2) Chemotherapy:

Chemotherapy The regimen of choice is based on individual patient and disease

factors.
Intrathecal (Commonly used); Consider adding for patients with or at high risk
chemotherapy of CNS infiltration.
Targeted Consider adding for leukemia with specific immunophenotype
chemotherapy andgenetic profiles, e.g., Philadelphia translocation.

3) Adjunctive treatment, e.g., radiation therapy, immunotherapy, or stem cell

transplantation (SCT).
4) Supportive care.
5) Optimize nutrition and hydration.
6) Manage chemotherapy-induced nausea and vomiting.
7) Start general measures infection control measures.
8) Provide psychosocial support as needed.
9) Management of complications:
 Relapse or refractory leukemia:
 Consider re-induction chemotherapy, autologous SCT, or enrollment (‫ )التسجيل‬in a
clinical trial in consultation with hematologist-oncologist.
 Systemic chemotherapy:
 Regimens vary depending on the subtype of leukemia, the age of the patient, and
immunophenotype and genetic study results.
 Induction therapy:
 High doses to induce a massive reduction of tumor cell count.
 Average duration for an adult with ALL: 4-8 weeks.
 Reinduction therapy may be required in case of relapse or failure of primary
induction.
Consolidation therapy:
 Medium doses to destroy any remaining tumor cells after induction.
 Average duration for an adult with ALL: 4-8 months.
 Maintenance therapy:
 Low doses to maintain remission
 Average duration for an adult with ALL: 2-3 years.
 Lymphoma
Lymphadenopathy is lymph node enlargement due to reactive conditions or
neoplasia.
Acute nonspecific lymphadenitis produces tender enlargement of lymph nodes;
focal involvement is seen with bacterial lymphadenitis.
o Microscopically, there may be neutrophils within the lymph node.
o Generalized involvement of lymph nodes is seen with viral infections.
Chronic nonspecific lymphadenitis causes nontender enlargement of lymph
nodes.
Follicular hyperplasia involves B lymphocytes and may be seen with rheumatoid
arthritis.
Paracortical lymphoid hyperplasia involves T cells and may be seen with viruses.

Neoplasia usually causes nontender enlargement of lymph nodes.

 The most common tumor to involve lymph nodes is metastatic cancer
(e.g.,breast, lung, malignant melanoma, stomach and colon carcinoma), which is
initially seen under the lymph node capsule.
 Other important causes of lymphadenopathy are malignant lymphoma and
infiltration by leukemias.

Lymphoma
 Is a cancer of the lymphatic system, which is part of the body's germ-fighting
network.
 The lymphatic system includes the lymph nodes (lymph glands), spleen,
Thymus gland and bone marrow.
 Lymphoma can affect all those areas as well as other organs throughout
the body.
The main subtypes of lymphoma:
 Hodgkin's lymphoma (formerly called Hodgkin's disease).
 Non-Hodgkin's lymphoma.
 Hodgkin lymphoma (Lymphogranulomatosis)
 Hodgkin lymphoma (HL) is a malignant lymphoma that is typically of B-cell
origin.
 The incidence of HL has a bimodal age distribution, with peaks in the 3rd and
6th–8th decades of life.

 The WHO classifies HL into two types:

 Classical HL (CHL).
 Nodular lymphocyte-predominant HL (NLPHL).

CHL is further divided into four subtypes:

 Nodular sclerosis (most common).
 Mixed cellularity.
 lymphocyte-depleted.
 lymphocyte-rich CHL.

Etiology
 The exact causes are unknown, but several risk factors have been associated
with HL:
 Strong association with Epstein-Barr virus (EBV).
 Immunodeficiency: e.g., organ or cell transplantation, immunosuppressant,
HIV infection , chemotherapy.
 Autoimmune diseases (e.g., rheumatoid arthritis, sarcoidosis).
Clinical features
1) Painless lymphadenopathy

Cervical lymph nodes axillary lymph nodes inguinal lymph nodes

∼ 60–70% ∼ 25–35% ∼ 8–15%

2) Involvement of a single group of lymph nodes

3) Contiguous pattern of lymph node spread

Mediastinal mass chest pain, dry cough, and shortness of breath

hepatoSplenomegaly May occur if the spleen or liver are involved.

4) B symptoms

 Night sweats, weight loss > 10% in the past 6 months, fever > 38°C (100.4°F)
 Can occur in a variety of diseases In the case of confirmed HL, the presence of a
single B symptom suffices for a positive diagnosis of B symptoms.

5) Pel-Ebstein fever:

Intermittent fever with periods of high temperature for 1–2 weeks, followed by a
febrile periods for 1–2 weeks.
 Relatively rare but very specific for HL.

6) Alcohol-induced pain:

Pain in involved lymph nodes after ingestion of alcohol.

 Relatively rare but highly specific for HL.

7) Pruritus (focal or generalized)

Diagnostics
Diagnosis of HL is primarily based on medical history and clinical features (B
symptoms, localization of lymph node involvement) and is confirmed with lymph
node biopsy.

 Elevated or decreased WBC count.

Complete blood count  Anemia.
 Eosinophilia
 (↑) LDH
Serum chemistry  Hypercalcemia: most commonly due to
paraneoplastic production of 1,25-dihydroxyvitamin
D.
Histology
Obligatory diagnostic step.
 Lymph node excision by core needle aspiration {not FNA}.
1) Reed-Sternberg cells (RSCs)
 Tumor cells that are pathognomonic of HL
 Originate from B cells
 Large cells with binuclear/bilobed nuclei with dark centers of chromatin and
pale halos, which results in an owl-eye appearance on histopathologic
examination.
 Positive for: CD15 and CD30.
2) Hodgkin cells:
 Mononuclear, malignant B lymphocytes
 Inflammatory background containing the following cell types in varying numbers:
lymphocytes, neutrophils, eosinophils, macrophages/ histiocytes, plasma cells, and
fibroblasts.
 Granuloma formation
 Classical Hodgkin lymphoma (95%)
Lymphoma Mixed-cellularity Lymphocyte-rich Lymphocyte-
Nodular classical HL classical HL (LRHL) depleted
sclerosing (MCHL) classical HL
classical HL (LDHL)
(NSHL)
Commonly Very rare (< 1%),
Most common found in
subtype (> 60%). immunocompromised Rare  Commonly
Characteristics patients (e.g., found in
HIV-positive immunocompromised
individuals). patients.
Localization Mediastinal and abdominal and cervical and axillary below the
cervical. splenic diaphragm
Prognosis Good (but
Good slightly worse Very good Poor
than NSHL)

 Nodules of
Reed-Sternberg  Nodules with  Presence of  Numerous
cells within numerous Reed- Reed-Sternberg Reed-Sternberg
lacunae, Sternberg cells, cells. cells.
Pathology separated by
collagenous  Histiocytes,  Reactive  Decreased
tissue with eosinophils, and lymphocytosis number of
 sclerosing plasma cells. that causes lymphocytes.
appearance. distortion of the
 Lymphocyte lymph node
rich. architecture.

Nodular lymphocyte predominant HL (NLPHL) {5%}

Characteristics Rare (5%).
Localization: Neck, axillary, and inguinal.
Prognosis Very good {slightly worse than LRHL }
 Presence of popcorn cells: a variant of a Reed-Sternberg
cell characterized by polylobulated nuclei that resemble
Pathology popcorn.
 Lymphocyte predominant (LP) cells express CD20, CD79a,
and CD45.
 Unlike in classical Hodgkin lymphoma, tumor cells are
negative for CD15 and CD30.
Treatment
The most widely used chemotherapy approach is ABVD: adriamycin
(doxorubicin), bleomycin, vinblastine, dacarbazine.
 Early stage (I and II):
 Combination of chemotherapy and radiation therapy
 Advanced stage (III and IV and often II with bulky disease):
 Combination chemotherapy with radiation therapy in select cases
 Primary refractory or relapsed disease:
 Trial of alternative chemotherapy or consideration of high-dose
chemotherapy and autologous stem cell transplantation
 Three possible treatment approaches are commonly considered:
1) ABVD
Stanford V: doxorubicin, vinblastine, mechlorethamine, vincristine, bleomycin,
etoposide, prednisone
BEACOPP: bleomycin, etoposide, adriamycin (doxorubicin), cyclophosphamide,
oncovin (vincristine), procarbazine, prednisone.

Prognosis
 Good prognosis:

• Best prognosis: lymphocyte-rich classical HL (LRHL) and nodular

lymphocyte predominant HL (NLPHL).
• Prognosis is largely determined by disease stage

 ∼ 10–20% of patients will develop secondary malignancies (especially

lung cancer; related to radiation therapy and chemotherapy).

 Unfavorable factors for Hodgkin lymphoma (relevant when selecting a

treatment regimen)
 High ESR
 High LDH
Differential diagnoses of lymphadenopathy
 Examining lymph nodes can yield important diagnostic clues.
 Generalized lymphadenopathy is usually a sign of systemic illness, such as
HIV, mycobacterial infection (e.g., tuberculosis), infectious mononucleosis,
systemic lupus erythematodes, or serum sickness.

 Signs of malignancy include; rapid growth, painlessness,

hardness/coarseness, and being fixed to underlying or surrounding tissue.
 Most often, though, there is no discernible cause or pathology.
 Non-Hodgkin's lymphoma (NHL)
 Non-Hodgkin lymphomas (NHL) comprise a diverse group of hematologic
malignancies that are variously derived from B cell progenitors, T cell
progenitors, mature B cells, mature T cells, or (rarely) natural killer cells.
 NHL is seen in patients of all ages, races, and socioeconomic status { ‫الوضع‬
‫االجتماعي واالقتصادي‬.}.
 Diagnosis and classification of NHL requires an adequate biopsy specimen and
expert pathologic review because the clinical manifestations, prognosis, and
management of lymphomas vary widely according to the type of lymphoma.

Etiology
1) Various viruses have been attributed to different types of NHL.
Epstein-Barr virus, a endemic variant of Burkitt lymphoma
DNA virus
Human T-cell leukemia adult T-cell lymphoma
virus type 1 (HTLV-1)
Hepatitis C virus (HCV) Clonal B-cell expansions.
Helicobacter Increased risk of gastric mucosa-associated lymphoid
pylori infection tissue (MALT) lymphomas, a primary gastrointestinal
lymphoma.

2) Drugs like;
Phenytoin, digoxin, TNF antagonist are also associated with Non-Hodgkin
lymphoma.
3) Environmental factors:
Aromatic hydrocarbons (e.g., benzene, associated for AML), radiation.
4) Congenital immunodeficiency: increased risk of
 Wiskott-Aldrich syndrome.
 Severe combined immunodeficiency disease (SCID).
 Patients with AIDS (Acquired immunodeficiency syndrome) can have
primary CNS lymphoma.
5) The autoimmune disorders:
Like Sjögren syndrome, rheumatoid arthritis, and Hashimoto thyroiditis are
associated with an increased risk of NHL.
B-cell lymphomas (85% of all NHLs):
Indolent (low-grade) Aggressive (high-grade)
 Hairy cell leukemia.  Burkitt lymphoma.
 Follicular lymphoma.  Precursor B-cell lymphoblastic
 Marginal zone B-cell lymphomas (MZLs). lymphoma.
 Waldenstrom macroglobulinemia.  Diffuse large B-cell lymphoma.
 Small lymphocytic lymphoma (SLL).  Mantle cell lymphoma.

Indolent (low-grade)

BRAF gene are

Hairy cell leukemia (low grade) associated with
 Pathology: mature B-cell tumor; BRAF mutations are common melanoma, non-
Hodgkin
lymphoma,
 Clinical features of Hairy cell leukemia papillary thyroid
Symptomatic pancytopenia, Massive splenomegaly, No lymphadenopathy, carcinoma
B symptoms are rare.

 Diagnostics of Hairy cell leukemia

 Usually tartrate-resistant acid phosphatase; (TRAP) stain positive
 Flow cytometry (preferred over TRAP stain): CD11c marker
 CBC: Leucopenia is common but up to 20% of patients have leukocytosis.
 Peripheral blood smear: Hairy cells have irregular cytoplasmic projections that
cause the characteristic “hairy” appearance.
 Bone marrow aspiration: often yields a dry tap due to bone marrow
involvement with subsequent fibrosis.
Follicular lymphoma (low grade)
 Most common low-grade lymphoma in adults.
 Slowly progressive and painless course with
an alternating (waxing and waning) pattern of
lymphadenopathy and splenomegaly.
 Translocation t(14;18), which involves the
heavy-chain Ig (chromosome 14) and Bcl-2
gene (chromosome 18) → overexpression of
Bcl-2 → dysregulation of apoptosis (normally
inhibited by Bcl-2)
 Centrocyte: nodular, small cells with cleaved nuclei.

Marginal zone B-cell lymphomas (MZLs):

 A group of lymphomas that arise from postgerminal center B cells.
 Gastric MALT lymphoma: associated with translocation t(11;18) (q21;q21) and
H. pylori infection.
 Associated with autoimmune diseases (e.g., Sjogren syndrome, Hashimoto
thyroiditis).
Hashimoto thyroiditis: The most common form of autoimmune thyroiditis and the
leading cause of hypothyroidism.

Sjogren syndrome: A chronic inflammatory autoimmune disease characterized by

inflammatory destruction of the lacrimal and salivary glands.

Small lymphocytic lymphoma (SLL):

SLL shares histological features with chronic lymphocytic leukemia (CLL);
however, the neoplastic cells are primarily located in the lymphatic tissue rather
than circulating in peripheral blood.

Waldenstrom macroglobulinemia:
IgM antibodies
 Aggressive (high-grade)

Diffuse large B-cell lymphoma

{DLBCL}:
 Most common NHL in adults.
 Caused by mutations in Bcl-2, Bcl-6, and
p53.
 Primary CNS lymphoma (subtype of DLBCL).
 Associated with EBV and AIDS.

Mantle cell lymphoma (aggressive and low grade):

 Most common in adult men.
 Translocation t(11;14) involving cyclin D1
(chromosome 11) and heavy-chain Ig (chromosome
14) → increased levels of cyclin D1 → promotes the
transition of cells to S phase.
 CD5+

Burkitt lymphoma
 Most common in children.
 Translocation t(8;14) in 75% of cases.
 Starry sky pattern
 Microscopic finding that resembles a starry sky

 Immunodeficiency-associated: e.g. HIV infection.

Tingible body macrophages (containing many phagocytized tumor cells): are

scattered diffusely within a sheet of uniform neoplastic cells (lymphocytes).

Forms of Burkitt lymphoma:

Sporadic: typically located in the abdomen or pelvis
Endemic: Associated with EBV is typically located in the maxillary and mandibular bones.
 Burkitt lymphoma
Seven-year-old boy from Nigeria
A massive swelling with central
ulceration and peripheral scaling
is visible above the right mandible

Precursor B-cell lymphoblastic lymphoma (aggressive grade)

 Most common in adolescents and young adults.

T-cell lymphomas (15% of all NHL)

1) Mycosis fungoides:
 Most common form of cutaneous T-cell lymphoma (a type of lymphoma
characterized by malignant T-cell infiltration of the skin).

2) Sezary syndrome:

Leukemic form of cutaneous T-cell lymphoma.

3) Adult T-cell lymphoma.

4) Aggressive NK-cell leukemia. Aggressive

5) Angioimmunoblastic T-cell lymphoma.

Precursor T-cell lymphoblastic lymphoma.

Cutaneous T-cell lymphoma; Sézary Cell: convoluted or
“brain-like” (cerebriform) dark
Mycosis fungoides purple nucleus.

The neoplastic lymphocytes are small with

round nuclear contours and clumped
chromatin.
Slightly less mature cells (prolymphocytes)
have a bit more abundant cytoplasm and
distinct nucleoli (arrows).
In preparation of the smear, some of the
neoplastic lymphocytes are mechanically
damaged and appear as ‘smudge’ cells
(arrowhead)
Clinical features of NHL
1) Nodal disease:
Typically painless lymphadenopathy associated with fatigue and weakness
(multiple noncontiguous lymph nodes may be involved)
 High grade
 Rapidly growing mass/nodes
 Constitutional symptoms or B symptoms (i.e., weight loss, fever, night
sweats)
 Low grade
 Slow-growing or undulant lymphadenopathy (over months or years)
 Hepatosplenomegaly
 Cytopenia: Patients may present with anemia or bleeding, or have an
increased susceptibility to infections.

2) Extranodal disease (primary or secondary):

The symptoms are highly dependent on the affected tissue; B symptoms are
common.
GI tract: early satiety {‫}تخمة‬, GI bleeding
Neurological involvement headache, focal neurologic symptom
Primary cutaneous NHL or rash, plaques, tumors, ulcers
secondary skin infiltration
Thyroid involvement (rare) Nodules, goiter (abnormal enlargement of the
thyroid gland).

3) Oncologic emergencies/paraneoplastic syndromes

Diagnostics
• CBC: may show anemia, thrombocytopenia; WBC count may be high or low
(commonly leukopenia, lymphocytosis)
• BMP: may show abnormal renal function.
• Serum calcium: may show hypercalcemia
• Liver chemistries: may be abnormal in patients with liver infiltration or
primary hepatic lymphoma
• Markers of disease activity
• Uric acid: usually elevated
• LDH: usually elevated
• Serum β2-microglobulin: may be elevated
• Others: CRP, ESR
• Viral serologies
HIV screening
Additional studies can be suggestive of the underlying etiology (e.g., hepatitis B
and C, EBV, HTLV-1).

Confirmatory diagnostics tests

Selection of biopsy sample
 Nodal disease:
Select the most appropriate node for biopsy (e.g., a node with significant,
progressive, and persistent enlargement).
Techniques:
Preferred: excisional lymph node biopsy or core needle biopsy
Alternative: incisional lymph node biopsy.
Avoid fine-needle aspiration biopsy.

 Extranodal disease:
 Excisional tissue biopsies are recommended.
Histopathology and specialized studies
These studies help determine the subtype of NHL.
 Histopathology:
Provides a detailed morphology of individual proliferating cells and a description of the
pattern of lymph node (or tissue) infiltration (e.g., nodular, diffuse).
 Immunophenotype (e.g., flow cytometry, immunohistochemistry)
 Detects surface antigens,
Determines the specific cell type (B cell/T cell), and identifies specific markers Possible
findings include:
 B-cell lymphomas: CD20 positive
 T-cell lymphomas: CD3 positive
 Genetic studies
• Cytogenetics (karyotype, FISH): can identify chromosomal abnormalities.

Treatment
 Most patients will receive treatment with systemic chemotherapy and/or radiotherapy.
Selection of treatment: based on the subtype of NHL, staging, and prognosis;

Low-grade NHL (initial stages): Consider radiotherapy

Low-grade NHL (advanced stages): usually palliative chemotherapy
High-grade NHL: usually chemotherapy

 Systemic chemotherapy: Regimens usually include combinations of chemotherapeutic

agents, steroids, and immunotherapy.
 Anti-cancer drugs {CHOP}.

• high-dose methotrexate ; for primary CNS lymphoma

• cyclophosphamide (C)
• doxorubicin/hydroxydaunorubicin (H)
• vincristine/oncovin (V/O)
• prednisolone (P)
• rituximab (R)
Prognosis of NHL
Typically, the prognosis of NHL is worse than that of Hodgkin lymphoma.
Low-grade lymphomas: median survival of 6–10 years
High-grade lymphomas: survival typically several months (years in less
aggressive variants)
Indicators of poor prognosis: Old age, number of involved nodal and
extranodal sites, ↑ LDH, ↑ beta2 macroglobulin.
 https://www.osmosis.org/learn/Chronic_leukemia

Chronic lymphocytic leukemia (CLL)

 CLL is low-grade B-cell lymphoma with lymphocytic leukocytosis.

 Sex: ♂ > ♀ (∼ 2:1)
 Age: The median age at the time of diagnosis is 70–72 years (incidence of CLL
increases with age).

 Risk factors
 Advanced age
 Environmental factors: organic solvents
 Family history

Classification of CLL (Rai staging system):

Pathophysiology
 Acquired mutations in hematopoietic stem cells → increased proliferation of
leukemic B cells with impaired maturation and differentiation in the bone marrow,
resulting in:
 Suppression of the proliferation of normal blood cells
 Immunosuppression.
 Hypogammaglobulinemia.
 Granulocytopenia.
 Thrombocytopenia.
 Anemia.
 Infiltration of the lymph nodes, liver, and spleen.
Clinical features
About half of cases of CLL remain asymptomatic for a long period, resulting in
late or incidental diagnosis.
1) Weight loss, fever, night sweats, fatigue (B symptoms)
2) Painless lymphadenopathy
3) Hepatomegaly and/or splenomegaly may occur.
4) Repeated infections
 Severe bacterial infections (e.g., necrotic erysipelas)
 Mycosis (candidiasis)
 Viral infections (herpes zoster)
 Symptoms of anemia and thrombocytopenia
5) Dermatologic symptoms
Leukemia cutis.
Chronic pruritus.
Chronic urticaria.

Complications
1) Immunosuppression with subsequent infections (most common cause of
death)
2) Secondary malignancies
3) Hyperviscosity syndrome
3) Autoimmune hemolytic anemia (of both the warm and cold agglutinin type)
4) Richter transformation or Richter syndrome: transformation into a high-grade
NHL (usually diffuse large B cell lymphoma)
 Occurrence: ∼ 5% of cases
 Diagnostic indicators:
 Rapidly progressive lymphadenopathy → lymph node biopsy required.
 New onset of B symptoms.
 ↑ LDH.
Diagnostics
1) CBC:
Persistent lymphocytosis with a high percentage of small mature lymphocytes
Findings that indicate suppression of normal myelopoiesis:
 Granulocytopenia
 Low RBC count (due to autoimmune hemolysis)
 Low platelet count

2) Bone marrow aspiration

 Bone marrow aspiration is not necessary to confirm the diagnosis but may be
helpful in investigating cytopenia of unknown origin, for instance, during later
stages of the disease.

3) Flow cytometry:
Detection of B-CLL immunophenotype (CD5, CD19, CD20, CD23), light chain
restriction (kappa or lambda)

4) Serum antibody electrophoresis:

Antibody deficiency (decreased γ globulin fraction)

5) Blood smear:
Smudge cells (Gumprecht shadows) – mature lymphocytes that rupture easily
and appear as artifacts on a blood smear
Principles of treatment
1) Asymptomatic CLL: observe and monitor disease progression
2) Symptomatic CLL or advanced stage (Rai stage > 0, accelerated disease
progression)
• Chemotherapy
• If CD 20 positive: rituximab
• Targeted therapy with ibrutinib
3) Refractory CLL or early recurrence in fit, young patients:
Allogeneic stem cell transplantation

Prognostic factors
• Advanced age is associated with a poor overall survival rate.

Large granular lymphocytic (LGL) leukaemia

 Is a chronic lymphoproliferative disorder that exhibits an unexplained, chronic

(> 6 months) elevation in large granular lymphocytes (LGLs) in the peripheral
blood.
 It is divided in two main categories:
 T-cell LGL leukemia (T-LGLL).
 Natural-killer (NK)-cell LGL leukemia (NK-LGLL).
Signs and symptoms
 The most common physical finding is moderate splenomegaly.
 B symptoms are seen in a third of cases.
 Recurrent infections due to anaemia and/or neutropenia are seen in almost
half of cases.
 Rheumatoid arthritis is commonly observed in people with T-LGLL
Diagnosis
1) Laboratory findings:
 The requisite lymphocytosis of this disease is typically 2-20x109/L.
 Immunoglobulin derangements including hypergammaglobulinemia, autoantibodies,
and circulating immune complexes are commonly seen.

2) Peripheral blood
The neoplastic lymphocytes seen in this disease are large in size with azurophilic
granules that contains proteins involved in cell lysis such as perforin and granzyme B

3) Bone marrow
The lymphocytic infiltrate is usually interstitial, but a nodular pattern rarely occurs.

4) Immunophenotype
The neoplastic cells of this disease display a mature T-cell immunophenotype, with the
majority of cases showing a;

CD4 CD8
- Ve + Ve

Genetic findings
Clonal rearrangements of the T-cell receptor (TCR) genes are a necessary condition for
the diagnosis of this disease.

Mycosis fungoides
 Mycosis fungoides is an indolent, CD4+ cutaneous T-cell lymphoma that presents on
the skin.
It is characterized by scaly, pruritic, well-demarcated skin plaques and patches.
Clinical features
 Initially, pruritic cutaneous plaques, patches, and brownish nodules develop.
 Subsequently, systemic spread occurs, including lymphadenopathy and
hepatosplenomegaly.
 Sézary syndrome
 A cutaneous T-cell lymphoma with leukemic dissemination of mutated T cells
Epidemiology
 Exact prevalence unknown
 Can be an advanced form of mycosis fungoides or
arise de novo
Clinical features
 Systemic skin lesions
 Erythroderma accompanied by palmar and plantar
hyperkeratosis
 Intense pruritus
 Generalized lymphadenopathy
Diagnostics:
Based on the characteristic triad of pruritic erythroderma,
lymphadenopathy, and atypical T cells (Sézary cells) on peripheral blood smear.

Adult T-cell leukemia/lymphoma

A non-Hodgkin lymphoma associated with infection with the human T-cell
lymphotropic virus I (HTLV-I)

Clinical features:
 Generalized lymphadenopathy
 Hepatosplenomegaly
 Skin lesions: may be similar to those seen in mycosis fungoides
 Lytic bone lesions.
Diagnostics
• Peripheral blood smear: lymphocytes with condensed chromatin and
hyperlobulated nuclei that resemble clover leaves.
• Laboratory tests: hypercalcemia, (↑) LDH
• Immunophenotype: stain positive for CD2, CD4, CD5, CD29, and CD45RO
 Plasma cell tumors (Multiple myeloma)
Plasma cell dyscrasia:

a group of conditions characterized by the abnormal proliferation of the same

type (=monoclonal) of a plasma cell that may also secrete a monoclonal
immunoglobulin and/or immunoglobulin fragment (e.g., light chain).

Plasmacytoma:

An early-stage plasma cell dyscrasia characterized by a single lesion;

affect bones affect soft tissue
solitary plasmacytoma of bone solitary extramedullary plasmacytoma
in rare cases multiple solitary lesions in soft tissue, bone or both (multiple
solitary plasmacytoma

Multiple myeloma:

A malignant plasma cell dyscrasia characterized by uncontrolled proliferation and

the diffuse infiltration of monoclonal plasma cells in the bone marrow.

Classification (Based on immunoglobulin type)

IgG and IgA: typical multiple myeloma; majority of patients
Bence Jones myeloma  Free light chains excreted in urine.
 15–20% of multiple myelomas.
IgD, IgE, and IgM: very rare subtypes of multiple myelomas

Pathophysiology
 Neoplastic proliferation of plasma cells
 Bone marrow infiltration by malignant plasma cells → suppression of
hematopoiesis → leukopenia, thrombocytopenia, and anaemia.
 Cell proliferation → osteolysis → hypercalcemia.
 Overproduction of monoclonal immunoglobulin and/or light chains →
dysproteinemia (a state of pathologically increased synthesis of
immunoglobulins and/or their subunits) → kidney damage (e.g., myeloma cast
nephropathy) and/or paraprotein tissue deposition (may cause amyloidosis)
 Nonfunctioning antibodies → functional antibody deficiency
 ↑ Serum viscosity → hyperviscosity syndrome.
Clinical features
 Often asymptomatic
 Mild fever, night sweats, weakness, and weight loss (B symptoms).
 back bone pain (most common symptom)
 Symptoms of hypercalcemia
 Spontaneous fractures
 Foamy urine (caused by Bence Jones proteinuria).
 Renal insufficiency Increase risk of amyloidosis.
 Suppression of hematopoiesis → leukopenia, thrombocytopenia, and anaemia.
 Increased risk of infection
 Increased risk of petechial bleeding.

Diagnostics
 The following tests are required for patients with suspected multiple myeloma
(MM):
 Serum protein electrophoresis or free light chain assay (best initial test)
 24-hour urine protein electrophoresis
 Bone marrow biopsy (confirmatory test)
 Laboratory tests (CBC and biochemistry) to assess for hypercalcemia, anemia
and renal insufficiency
 Imaging to assess for bone lesions.
Laboratory tests
CBC

 Anemia, thrombocytopenia, leukopenia

Peripheral blood smear:

 rouleaux formation

Biochemistry

 Elevated total protein

 Hypercalcemia
 ↑ Creatinine
Rouleaux; an aggregation of
 Massively increased ESR
erythrocytes with the
 Paraprotein (gamma) gap appearance of a stack of coins
 ↑ β2 microglobulin. on peripheral blood smear.

Electrophoresis and immunofixation

 Serum protein electrophoresis (best initial test): monoclonal gammopathy with M

protein (M spike)
 Urine protein electrophoresis: Bence Jones proteins.
 Monoclonal immunoglobulin light chains produced by neoplastic cells.
 suggestive of plasma cell disorders such as multiple myeloma or Waldenstrom
macroglobulinemia

Urinalysis

 Urine dipstick: negative for protein.

Clusters of plasma cells in multiple

myeloma
Complications of MM:
A) Renal disease (Dysproteinemia-associated kidney disease)

 Myeloma cast nephropathy (myeloma kidney)

 Most common cause of renal injury and renal failure in patients with MM
 Diagnosed by markedly positive urine sulfosalicylic acid test
 Monoclonal immunoglobulin deposition disease (MIDD)
 Light chain deposition disease

B) Hypercalcemia-associated renal damage:

 Leads to hypercalciuria and nephrocalcinosis

Systemic manifestations

 AL amyloidosis:

Light chains can accumulate as amyloids and may lead to restrictive

cardiomyopathy, renal insufficiency, macroglossia, and malabsorption
syndromes.

 Infections
 Immunodeficiency (nonfunctional immunoglobulins) and side effects of
medications

 Secondary plasma cell leukemia

Monoclonal gammopathy of undetermined significance (MGUS)

 Characterized by complete or incomplete monoclonal immunoglobulins (of any
class) detectable in patient serum without accompanying clinical symptoms.

Diagnostics
 Usually, an incidental finding on workup (e.g., protein electrophoresis) for
other conditions such as vasculitis, hypercalcemia, skin rashes, peripheral
neuropathy, increased ESR, and hemolytic anemia
Risk of progression to myeloma
 Determined by the following predictors:
 Presence of non-immunoglobulin G-type M protein.
 M-protein concentration ≥ 1.5 g/dL.
 abnormal serum free light chain (SFLC) ratio

M protein
1. A monoclonal immunoglobulin (paraprotein) that is produced in plasma cell
dyscrasias (e.g., multiple myeloma, Waldenstrom macroglobulinemia,
monoclonal gammopathy of undetermined significance).
 Detected on serum or urine electrophoresis as a spike in the gamma-
globulin zone (monoclonal spike, M spike).
2. A virulence factor of group A streptococci that prevents opsonization by
complement factor C3.

Waldenstrom macroglobulinemia
A type of non-Hodgkin lymphoma associated with abnormal production of
monoclonal IgM antibodies.

Clinical features:
 Peripheral neuropathy
 Impaired platelet function
 Formation of cold agglutinins (IgM) with hyperviscosity syndrome.
 Raynaud phenomenon
 Cerebral venous thrombosis hyperviscosity syndrome

 Impaired vision (e.g., blurry vision) The large size of IgM leads to
increased blood viscosity.
 Retinal haemorrhages, engorged retinal veins
 Headaches
 Myeloproliferative neoplasms (MPNs)
 A group of disorders characterized by a proliferation of normally developed
(non dysplastic) multipotent hematopoietic stem cells from the myeloid cell line.
 According to the WHO classification, the following disorders are
myeloproliferative neoplasms:
Common (classic) Less common
Polycythemia vera Essential thrombocythemia
Primary myelofibrosis Chronic eosinophilic leukemia
Chronic myeloid leukemia Chronic neutrophilic leukemia
Myeloproliferative neoplasms, unclassifiable
 Chronic myeloid leukemia (CML)
 Is a type of myeloproliferative neoplasm involving hematopoietic stem cells
that results in overexpression of cells of myeloid lineage, especially
granulocytes.

Epidemiology
 ♂>♀
 Peak incidence is 50–60 years.

Etiology
 Idiopathic (in most cases)
 Ionizing radiation (e.g., secondary to therapeutic radiation)
 Aromatic hydrocarbons (especially benzene).

Pathophysiology
 Philadelphia chromosome
Reciprocal translocation between chromosome 9 and chromosome 22 →
formation of the Philadelphia chromosome t(9;22) → fusion of the ABL1 gene
(chromosome 9) with the BCR gene (chromosome 22) → formation of the BCR-
ABL gene → encodes a BCR-ABL non-receptor tyrosine kinase with increased
enzyme activity.
 Result: inhibits physiologic apoptosis and increases mitotic rate →
uncontrolled proliferation of functional granulocytes.
Clinical features
1) Chronic phase

 Can persist for up to 10 years and is often subclinical

 When symptomatic, features include:
• B symptoms.
• Splenomegaly: abdominal discomfort in the left upper quadrant
• Lymphadenopathy is not typical in CML.

2) Accelerated phase

 Erythrocytopenia: anemia pleocytosis (or pleiocytosis) is an increased cell count

particularly an increase in white blood cell count, in a
 Neutropenia: infection and fever bodily fluid, such as cerebrospinal fluid.
 Extreme pleocytosis
 Infarctions: splenic and myocardial infarctions, retinal vessel occlusion
 Leukemic priapism.

3) Terminal phase: myelofibrosis

• Extreme splenomegaly : palpable in lower left quadrant or pelvic cavity

Blast crisis
 The blast crisis is the terminal stage of CML.
• Symptoms resemble those of acute leukemia.
• Rapid progression of bone marrow failure → pancytopenia, bone pain
• Severe malaise
 Diagnostics

1) Clinicians should maintain a high index of suspicion if patients present

with the following:

 Severe leukocytosis on routine laboratory testing Splenomegaly

 Constitutional symptoms (e.g., malaise, fatigue) with nonspecific signs of bone
marrow suppression (e.g., anemia, thrombocytopenia)

2) Initial diagnostic workup should include:

CBC: Leukocytosis , Thrombocytosis, Basophilia and eosinophilia.

Peripheral blood smear Blast cells in peripheral blood can indicate the transition to
AP-CML.
Bone marrow aspiration and biopsy: hyperplastic myelopoiesis, elevated granulocytic precursor
cells, especially myelocytes and promyelocytes

3) Diagnostic confirmation:

 Identification of the Philadelphia chromosome and/or the BCR-ABL1 fusion gene.

 Evaluate patients without the Philadelphia chromosome or the BCR-ABL1 fusion gene
for other myeloproliferative disorders.

4) Further laboratory studies

 Leukocyte alkaline phosphatase (LAP):

CML leukemoid reactions

Low LAP High lAP

 Flow cytometry:
Can be used to assess the type and maturity of leukocytes in order to detect progression
to advanced phases of CML.
 Primary myelofibrosis
Bone marrow fibrosis, extramedullary hematopoiesis, and splenomegaly

Pathophysiology:
genetic mutations → hyperplasia of atypical megakaryocytes → ↑ TGF-β → ↑
fibroblast activity → bone marrow obliteration due to fibrosis → displacement of
hematopoietic stem cells → extramedullary hematopoiesis

Clinical features
 Anemia
 Symptomatic splenomegaly
 Thromboembolic events
 Petechial bleeding
 Increased infections
Diagnostics of Primary myelofibrosis
CBC Thrombocytosis, and leukocytosis
↑ Leukocyte alkaline phosphatase, ↑ LDH
Peripheral blood smear: dacrocytes (teardrop cells)
Genetic marker JAK2 mutation
Bone marrow Aspiration Aspiration often fails (dry tap) because of severe marrow fibrosis.

Essential thrombocythemia
 Isolated uncontrolled proliferation of platelets not caused by another condition
(e.g., reactive thrombocytosis, another myeloproliferative neoplasms).

Pathophysiology:
Genetic mutations → activation of the thrombopoietin receptor → proliferation of
platelets

Clinical features
• Increased risk of fetal loss
• Vasomotor symptoms (headache, visual disturbances, acral paresthesias,
ocular migraines)
• Erythromelalgia
• Acute gouty arthritis
• Thromboembolic events
• Petechial bleeding
• Commonly asymptomatic

Erythromelalgia
Diagnostics

 Isolated sustained thrombocytosis (> 450 x 109/L)

 ↑ LDH and ↑ uric acid
 Bone marrow studies: hyperplasia of mature megakaryocytes.
 Polycythemia vera (PV)
 Is a chronic myeloproliferative neoplasm that is characterized by an
erythropoietin-independent, irreversible increase in erythrocyte, granulocyte, and
platelet counts.

Pathophysiology
 The JAK2 (Janus kinase 2) oncogene codes for a non-receptor tyrosine
kinase in hematopoietic progenitor cells.
 JAK2 is essential for the regulation of erythropoiesis,
thrombopoiesis (megakaryopoiesis), and granulopoiesis.
 95% of primary PV patients have a mutation in the JAK2 gene (gain of
function) → ↑ tyrosine kinase activity → erythropoietin-independent
proliferation of the myeloid cell lines → ↑ blood cell mass (erythrocytosis,
thrombocytosis, and granulocytosis) → hyperviscosity and slow blood flow
→ ↑ risk of thrombosis and poor oxygenation.

Clinical features
 Often asymptomatic
 Hyperviscosity syndrome (triad of mucosal bleeding, neurological symptoms, and visual changes)
 Plethora (Plethora)
 Cyanotic lips
 Pruritus; worsens when the skin comes into contact with warm water
 neurological symptoms: dizziness, headache, visual disturbances, tinnitus
 Hypertension
 Splenomegaly
 Peptic ulcer disease
 Symptoms of thrombotic and hemorrhagic complications.

Diagnostics
Increase Hb/Hct, RBCs, Platelets and Leukocytes (> 12,000/μL)
Decrease ESR and EPO

 Arterial O2 saturation: normal

 Description Cause
immature and/or abnormal cells (e.g., Bone marrow infiltration or
Leukoerythroblastic dacrocytes, nucleated red blood cells, fibrosis (myelofibrosis, bone
reaction myelocytes, metamyelocytes, and marrow metastasis).
myeloblasts, giant platelets).
Infections (predominantly
Reactive leukocytosis: cell hyperplasia bacterial, e.g., pertussis)
Leukemoid reaction with proliferation of myeloid or lymphoid ,Severe purulent conditions
elements. (e.g., perforated appendicitis)
,Drugs (e.g., steroids)

Myelodysplastic syndromes (MDS)

 Myelodysplastic syndromes (MDS) are a group of hematological cancers in
which malfunctioning pluripotent stem cells lead to hypercellularity and dysplasia
of the bone marrow.
 This, in turn, leads to cytopenia of one or more cell lines (thrombocytopenia,
erythrocytopenia, leukocytopenia).

Etiology

1) Primary MDS (90% of cases):

 Tends to occur in elderly patients

 Unknown etiology: likely due to spontaneous mutations

2) Secondary MDS (10% of cases):

 caused by exogenous bone marrow damage

 Benzene and other organic solvents
 Radiation damage: therapeutic radiation, radioiodine therapy, ionizing
radiation
 Paroxysmal nocturnal hemoglobinuria

 Treatment-related MDS:
Following cytostatic therapy (alkylating agents, topoisomerase II inhibitors,
azathioprine, etc.)
Clinical features
1) Hepatosplenomegaly
2) Depending on the affected cell line:
Erythrocytopenia; anemia
Leukocytopenia increased susceptibility to bacterial infections, especially of the skin
Thrombocytopenia; petechial bleeding

Diagnostics

 CBC with peripheral smear:

Normocytic or macrocytic anemia (rarely microcytic) of refractory type (refractory
anemia).

 Other possible findings:

 Leukocytopenia and/or thrombocytopenia
 Nucleated RBCs
 Pseudo-Pelger-Huet anomaly:
 Neutrophils with hyposegmented nuclei (usually bilobed)
 Seen in peripheral blood smears of patients undergoing chemotherapy.

 Bone marrow biopsy:

Hypercellular, dysplastic bone marrow with numerous cells of all three cell lines
with blasts, megakaryocytes, etc.

 Ringed sideroblasts:

An immature red blood cell in which abnormal mitochondrial iron deposition

forms a ring around the nucleus.
Seen in bone marrow of patients with heme synthesis disorders.
 Pharmacology

Haematinics:
Haematinics are nutrients required for the formation of blood cells. Iron, vitamin
B12, Erythropoietin and folate are the main haematinics, and deficiencies can
lead to anaemia.

 Pharmacologic treatment of these types of anemia usually involves

replacement of the missing substance.
 An alternative therapy for certain types of anemia and for deficiency in other
types of blood cells is administration of recombinant hematopoietic growth
factors, which stimulate the production of various lineages of blood cells and
regulate blood cell function.
General signs and symptoms of anemia:

 Patients with a 𝑯𝒃 𝒍𝒆𝒔𝒔 𝒕𝒉𝒂𝒏 𝟕𝒈/𝒅𝒍 will have symptoms of tissue hypoxia
(fatigue, headache, dyspnea, pallor, angina, tachycardia, visual impairment,
syncope, lymphadenopathy (enlargement of lymph nodes), hepatic and or splenic
enlargement, bone tenderness, blood loss in feces, neurologic symptoms.
 In addition to general signs and symptoms of anemia, iron deficiency anemia
may cause:
Pica Hunger for ice. dirt, paper, etc.
Koilonychias Upward curvature of the finger and toe nails (spoon nail).
Angular cheilitis Soreness and cracking at the comers of the mouth.

 Anemia leads to decreased O2 carrying capacity of blood and thus O2

availability to tissues (hypoxia).
 A large number of drugs cause toxic effects on blood cells, hemoglobin
production, or erythropoietic organs, which, in turn, may cause anemia.
 Anemia can be at least temporarily corrected by transfusion of whole
blood.
Note that; aspirin is an anti-platelets and
not anticoagulant
 Divalent metal transport {DMT}: carriers Fe+2 into enterocytes at the luminal border.
Transferrin: carriers iron in the blood
Ferroportin (FP): transport iron to plasma at the basolateral border.
Iron:
The total quantity of iron in the body averages 4 to 5 grams, about 65% - 75% of
which is in the form of hemoglobin.
4% myoglobin
1% heme compounds
0.1% combined with the protein transferrin in the blood
Stored for later mainly in the reticuloendothelial system
15% to 30% and liver parenchymal cells, principally in the form of
Or 10-20 ferritin.

1) Oral iron:
Drug Volume of drug elemental iron
Ferrous sulfate 325 𝒎𝒈 65 𝒎𝒈
Ferrous gluconate 320 𝒎𝒈 37 𝒎𝒈
Ferrous fumarate 325 𝒎𝒈 106 𝒎𝒈

 Ferric iron binds with transferrin in plasma and transported in other tissues
and stored as ferritin and hemosiderin form.
 This complex bind with receptor on developing red cells in B. marrow, iron
released in the cell, transferrin and transferrin receptor are then recycled,
providing an efficient mechanism for incorporating iron into hemoglobin in
developing cells.

 About 10 - 20% of dietary iron is absorbed, for e.g. a standard diet if

contain 10-15 mg of iron, only 1mg is absorbed.
 Absorption (↑) when iron requirement is (↑) as in pregnancy, menstruation,
growing children.

Storage: Iron storage as Ferritin and hemosiderin form in mucosal cells, liver,
spleen, and bone marrow.
Hemosiderin large particles can be observed microscopically
ferritin So small particles Can be seen in the cell cytoplasm only
with an electron microscope.

Elimination: Minimal amount (about 1 mg/day) are lost in sweat, saliva, and in
exfoliated skin and intestinal mucosal cell.

 hephaestin and ceruloplasmin convert

Hepcidin is the main regulator for stimulation
ferrous to ferric (Fe+2  Fe+3).
or inhibition of releasing of iron from
macrophage.  Ferric to ferrous needs vitamin C and HCL
 Prevents to give anti acids with iron supplement

man woman
An average excretes about 0.6 mg of iron Additional menstrual loss of
each day, mainly into the feces. Additional blood brings long-term iron loss
quantities of iron are lost when bleeding to an average of about 1.3
occurs mg/day.

 Hb has 33% of iron (50 mg in 100 ml of blood).

Daily requirement of iron:
Male 0.5-1 mg
Female 1-2 mg
Children 25 mg

 Iron deficiency anaemia is the only indication for the use of iron.
 The most common cause of iron deficiency anaemia in adults is blood loss.

As iron deficiency anemia develops:

Increased TIBC , ed cell distribution width (RDW) , Free erythrocyte
protoporphyrin FEP
Decreased Ferritin, serum iron , % saturation

 200- 400mg oral elemental iron daily should be given to correct anaemia (25%
absorbed, so 50-100mg iron can be incorporated in Hb).
 Treatment should be continue for 3-6 months, this not only correct the anemia but
will replenish iron stores. (Hb should reach normal level in 1-3 months).
 Failure to respond to oral iron therapy may be due to incorrect diagnosis.

Adverse effects due to oral iron therapy:

1) Owing to the risk of a hypersensitivity reaction, a small test dose of iron dextran
should always be given before full intramuscular or intravenous doses.
3) Large amount of oral iron cause; Necrotizing gastroenteritis, vomiting, nausea,
abdominal pain, bloody diarrhea, dyspnea, metabolic acidosis, coma and death,
black stool (melana) and constipation.
 May be minimized adverse effects by lowering the daily dose or by taking iron
tablets immediately after or with meals.
 2) Parenteral iron:
Drug Rout of adminstration
iron dextran IV or IM
Iron sucrose complex IV or IM
Iron-sodium gluconate IV or IM
Iron-sorbitol-citrate only IM

 Indications:
1. It should be reserved for patients with documented iron deficiency unable to tolerate
or absorb iron (pts. With post gastrectomy, previous small bowel resection,
malabsorption syndrome).
2. Pts. With extensive chronic blood loss who cannot be maintained with oral iron alone.

Iron dextran:
 Combination of ferric hydroxide + dextran
 50 mg elemental iron/ml
 Route of administration: IM, or by IV infusion in 1-2 hours.
 Most adults needs about 1-2 G (20-40ml) iron dextran for iron deficiency
anemia.

Iron-sucrose complex and iron sodium gluconate complex:

Appear to be much less likely than iron dextran to cause hypersensitivity
reactions.

Adverse effects of Parenteral iron:

1) Iron toxicity:

 It is seen in young children who have ingested a no. of iron tablets (more than 10
tablets).
 Adults are able to tolerate large doses of iron.

2) Local pain, tissue staining (brown discoloration of tissues overlying the inj. site),
headache, fever, arthralgia, nausea, vomiting, bronchospasm, urticaria, anaphylaxis,
and death.
Treatment of acute iron toxicity:
1) Whole bowel irrigation should perform.
2) Deferoxamine {iron antidote}; is a potent iron chelating.
3) Supportive therapy for GIT bleeding, metabolic acidosis and shock.

Chronic toxicity “hemochromatosis”:

 When excess iron is deposited in heart, liver, pancreas and other organs cause
organ failure and death.
 It occurs in patients with inherited hemochromatosis (excessive iron
absorption in patients, who receive many red cell transfusions for long period.

Treatment of hemochromatosis:
1) Intermittent phlebotomy, 1 unit of blood removed weekly.
2) Iron chelating agent (deferoxamine IV). Phlebotomy – 350 ml
Transfusion – 500 ml
Deferoxamine:
Is a potent iron chelating; binds iron that has already been absorbed and to
promote its excretion in urine and feces.
It is poorly absorbed when given orally and may increase iron absorption by this
route.
It is given IM or preferably IV.
It is metabolized and excreted in urine (turn urine color orange red).

Adverse effects of Deferoxamine:

1) Hypotension; as a result of Rapid IV administration.
2) Idiosyncratic response such as flushing, erythema, intestinal irritation,
urticarial.
3) Acute respiratory distress syndrome may occur if IV infusion lasting longer
than 24 hours.
4) Neurotoxicity after long term therapy of iron overload condition.

‫في مادة معينة باأللفيوالي سائلة تتصنع بشكل دائم الزمة لمنع ايديما الرئة‬
‫ونقص هذه المادة يؤدي الى هذا المرض‬
Megaloblastic anemias:
are a group of disorders characterized by defective nuclear maturation caused
impaired DNA synthesis, caused usually due to vitamin B12 or folate deficiencies.

Vitamin B12:
 Water soluble with MW 1335 Daltons.
 Produced by micro-organisms and fungi.
 The recommended dietary intake for adult is 2µg/day.

Two important proteins involved in the transport of Vit B12:

Intrinsic factor [IF] Transcobalamins I, II, III [TCII] manly II.
From diet to ileum From ileum to tissues

Vit B12 plays important role in two reactions:

1) Necessary in the synthesis of methionine from homocysteine.

 In this reaction both vit B12 and folic acid are involved.
 B12 acts as a co-enzyme (methyl cobalamin) for methyltransferase.

2) Vit B12 is important in conversion of methylmalonyl CoA to succinyl CoA in

Krebs cycle.

 In this reaction B12 acts as co-enzyme for methylmalonyl CoA

mutase.
 B12 deficiency leads to the production of abnormal fatty acids.
 Role of Vitamin B12:
Impairment of DNA synthesis affects all cells, but because red blood cells must be
produced continuously, deficiency of either vitamin B12 or folic acid usually
manifests first as anemia. In addition, vitamin B12 deficiency can cause neurologic
defects, which may become irreversible if not treated promptly (like; tingling,
stomatitis, toungitis, ulcer, paresthesia).

VITAMIN B12 AND FOLIC ACID PHYSIOLOGIC CONSIDERATIONS:

Vitamin B12 Folate
Sources meat, fish green vegetables, yeast
Daily requirement 2-5 𝝁𝒈 50-100 𝝁𝒈
Body stores 3-5 𝒎𝒈 (liver) 10-12 𝒎𝒈 (liver)
Places of absorption ileum duodenum and jejunum

Clinical Pharmacologic of Vitamin B12:

1) Vitamin BI2 is essential for normal DNA synthesis and fatty acid metabolism.

 A deficiency results in impaired DNA replication, which is most apparent in

tissues that are actively dividing, such as the GI tract and erythroid
precursors.
 The appearance of large macrocytic (megaloblastic) red cells in the blood is
characteristic of this deficiency.
 Vitamin B12 deficiency can also result in irreversible neurologic disorders.
2) Vitamin B12, along with vitamin B6 and folic acid, participates in the
metabolism of homocysteine to cysteine.

 Elevations in homocysteine are associated with accelerated

atherosclerosis.
3) Loss of vitamin B12 from the body is very slow (2 𝝁g/day), and hepatic stores
are sufficient for up to 5 years.

 Vitamin B12 is not synthesized by eukaryotic cells and is normally obtained

from microbial synthesis.
4) Parenteral administration of vitamin B12 is standard because the vast majority
of situations requiring vitamin B12 replacement are due to malabsorption.

 Uncorrectable malabsorption requires life-long treatment.

5) Improvement in hemoglobin concentration is apparent in 7 days and
normalizes {corrective} in 1-2 months.

PAPA  dihydrofolate (inactive)  tetrahydrofolate (active)

Tetrahydrofolate (active) syntheses purines, deoxythymidylate monophosphate (dTMP) and methionine

 Gastritis A = atrophy of antrum.

Gastritis B = atrophy of pylori

Therapeutic uses of vitamin B12:

1) Vitamin B12 is used to treat pernicious anemia.
2) Vitamin B12 is used after partial or total gastrectomy to mitigate the loss or
reduction of intrinsic factor synthesis.
3) Administration of vitamin B12 is used to replace vitamin BI2 deficiency caused
by dysfunction of the distal ileum with defective or absent absorption of the
intrinsic factor-vitamin Bi2 complex.
4) Administration of vitamin B12 is necessary in patients with insufficient dietary
intake of vitamin B12 (occasionally seen in strict vegetarians).

Adverse effects of vitamin BI2 “as a drug”. Hypokalemia cause diastolic

Asystoli and muscle cramps
 Are uncommon, even at large doses.
 Hypokalemia (‫ )تبتلع خاليا الدم الحمراء البوتاسيوم‬and thrombocytosis can occur upon
conversion of severe megaloblastic anemia to normal erythropoiesis with
cyanocobalamin therapy.

Folic acid Pharmacology

 Folic acid is readily absorbed from the gastrointestinal tract. Only modest amounts
are stored in the body, so a decrease in dietary intake is followed by anemia within a
few months.
 Folic acid is converted to tetrahydrofolate by the action of dihy-drofolate reductase
{DHFR}, one important set of reactions involving tetrahydrofolate and dihydrofolate
constitutes, deoxythymidylate monophosphate (dTMP) required for DNA synthesis.

 Rapidly dividing cells are highly sensitive to folic acid deficiency.

 For this reason, anti-folate drugs are useful in the treatment of various
infections and cancers.
 Anemia resulting from folic acid deficiency is readily treated by oral folic acid
supplementation.
 Because maternal folic acid deficiency is associated with increased risk of
neural tube defects in the fetus, folic acid supplementation is recommended
before and during pregnancy.
 Folic acid supplements correct the anemia but not the neurologic deficits of
vitamin B12 deficiency.
 Therefore, vitamin B12 deficiency must be ruled out before one selects folic
acid as the sole therapeutic agent in the treatment of a patient with megaloblastic
anemia.
 Folic acid has no recognized toxicity.

 The use of folic acid alone in the presence of vitamin B12 deficiency may result
in worsening of neurological defects.
Vitamin B12 deficiency Folate deficiency
Vitamin B12 deficiency is treated with  Oral folic acid 5 mg daily for 3 weeks
hydroxycobalamin 1000 𝝁𝒈 (𝟏𝒎𝒈). will treat acute deficiency and 5 mg once
weekly is adequate for maintenance
Five doses 2 or 3 days (or 10 days) therapy.
apart followed by maintenance
therapy of 1000 𝝁𝒈 every 3 months for  Prophylactic folic acid in pregnancy will
life. prevent megaloblastosis in women at risk.

The vitamin may be administered:

orally IM or SC
for dietary deficiencies for pernicious anemia

Treatment of Sickle cell anemias

1) Hydroxyurea:
 Effective in reducing painful episodes by about 50%.
 The necessity of blood transfusions was also shown to be reduced.
 Hydroxyurea increases the production of fetal hemoglobin, which makes red
cells resistant to sickling.

Side effects of hydroxyurea:

 Bone marrow suppression and cutaneous vasculitis.

The goal of sickle cell anemia drugs is to vasodilation, but Hydroxyurea alone cannot
do this, so we must give it a second drug "Pentoxifylline".
 Treatment of Sickle cell anemias “continue”

2) Pentoxifylline:
 Is a synthetic dimethyxanthine structurally similar to caffeine.
 The actions of pentoxifylline include increased erythrocyte flexibility and
decreased blood viscosity.
 It is commonly used to treat intermittent claudication (‫)العرج‬, by improving blood
flow and tissue oxygenation.
 Pentoxifylline appears to inhibit erythrocyte phosphodiesterase, which causes
an increase in erythrocyte cyclic adenosine 5ʹ-monophosphate activity and an
increase in membrane flexibility.

Side effects of Pentoxifylline:

 The primary side effects are GIT symptoms, minimized by administration with
food.

Hematopoieti
c factors

Erythrocyte Granulocyte
platelet
factors factors
factors

Oprelvekin
vitamins Iron Erythropoiesis- Filgrastim Saigramostim
stimulating agents (IL-11) (G-CSF) (GM-CSF)
(B12, B9)
(ESA)
Darbepoetin and
erythropoietin
Hematopoietic growth factors
 Are endogenous glycoproteins that bind to specific receptors on bone marrow
progenitor cells and induce their differentiation and proliferation, thereby increasing
production of erythrocytes and various leukocytes.
 Several growth factors are now available for treating anemia or leukopenia.

 These growth factors are produced by recombinant DNA technology and are
administered parenterally.

1) Erythropoietin and Darbepoetin (erythrocytes):

Erythropoietin (EPO)

 A glycoprotein EPO stimulates stem cells to differentiate into proerythroblasts and

promotes the release of reticulocytes from the marrow and initiation of hemoglobin
formation. EPO, thus, regulates red blood cell proliferation and differentiation in bone
marrow.
 Human erythropoietin (epoetin alfa), produced by recombinant DNA technology, is
effective in the treatment of anemia caused by end-stage renal disease, anemia
associated with human immunodeficiency virus infection, anemia in bone marrow
disorders, anemia of prematurity, and anemias in some cancer patients.
Darbepoetin:

 Is a long-acting version of erythropoietin that differs from erythropoietin by the

addition of two carbohydrate chains. Therefore, darbepoetin has decreased clearance
and has a half-life about three times that of epoetin alfa.
 Due to their delayed onset of action, these agents have no value in acute treatment of
anemia.

 Supplementation with iron may be required to ensure an adequate response.

 The protein is usually administered intravenously in renal dialysis patients, but the
subcutaneous route is preferred for other indications.
 These agents are generally well tolerated, but side effects may include elevation in
blood pressure and arthralgia in some cases; may be due to increases in peripheral
vascular resistance and/or blood viscosity].
 When epoetin alfa is used to target hemoglobin concentrations more than 11 g/dL,
serious cardiovascular events such as thrombosis and severe hypertension & increased
risk of death have been observed.
  The recommendations for all patients receiving epoetin alfa or darbepoetin:
1) Minimum effective dose that does not exceed a hemoglobin level of 12 g/dL.
2) The hemoglobin should not rise by more than 1 g/dL over a 2-week period.
3) If the hemoglobin level exceeds 10 g/dL, doses of epoetin alfa or darbepoetin
should be reduced or treatment should be discontinued.

2) Filgrastim, Pegfilgrastim, and Sargramostim (leukocytes):

Filgrastim sargramostim
Polyethylene glycol (PEG) + Filgrastim = pegfilgrastim

recombinant human granulocyte Recombinant human Granulocyte and macrophage

colony stimulating factor (G-CSF) colony stimulating factor (GM-CSF).

 The endogenous forms of these growth factors are produced by various leukocytes,
fibroblasts, and endothelial cells.
 The addition of a PEG moiety to filgrastim (pegylation) creates pegfilgrastim, whose
molecular size is too large to enable renal clearance, thereby increasing the half-life from
about 3.5 hours for filgrastim to 42 hours for pegfilgrastim.

 Pegfilgrastim is eliminated primarily by neutrophil uptake and metabolism.

 The longer half-life of pegfilgrastim has enabled less-frequent administration for
treating cancer chemotherapy-induced neutropenia.
 Filgrastim, pegfilgrastim, and sargramostim are used primarily to treat neutropenia
associated with cancer chemotherapy and bone marrow transplantation.
Studies indicate that filgrastim may be beneficial in the treatment of aplastic anemia,
hairy cell leukemia, myelodysplasia, drug-induced and congenital agranulocytosis, and
other forms of congenital or acquired neutropenia.
Sargramostim
1) Accelerate myeloid cell recovery in patients who have lymphoma, acute lymphoblastic
leukemia, or Hodgkin disease and patients that are undergoing autologous bone marrow
transplantation or chemotherapy.
2) It has also been used to reduce the incidence of fever and infections in patients with
severe chronic neutropenia.
 Filgrastim and sargramostim are administered subcutaneously or intravenously once
a day for 2 weeks.

Adverse effects:

Gastrointestinal effects, fever, bone pain, myalgia and rash.

Less common effects include pulmonary infiltrates and enlargement of liver or spleen.
 Usually used corticosteroids for thrombocytopenia before platelets growth factors

3) Megakaryocyte Growth Factors: Oprelvekin [IL-11]:

 Stimulates the growth of primitive megakaryocytic progenitors and increases
the number of peripheral platelets.
 IL-11 is used for the treatment of patients who have had a prior episode of
thrombocytopenia after a cycle of cancer chemotherapy.

 In such patients, it reduces the need for platelet transfusions.

 The most common adverse effects of IL-11 are fatigue, headache, dizziness,
and fluid retention.
Thrombopoietin agonist Romiplostim: Eltrombopag:
Rout of administration SC receptor agonist Oral agonist
Chronic idiopathic Chronic idiopathic
Indications thrombocytopenia who thrombocytopenia that is
have failed to respond to refractory to other
conventional treatment. agents.
Limitation of use Unrestricted restricted
Risks ---------------------------------- Hepatotoxicity and
hemorrhage.

Chloramphenicol cause:

Aplastic anemia.

Gray baby syndrome

 Drugs Used In Coagulation Disorders
Hemostasis overview;
Hemostasis: is regulated dynamic process of maintaining fluidity of the blood,
repairing vascular injury, and limiting blood loss while avoiding vessel occlusion
(thrombosis) and inadequate perfusion of vital organs.
 Hemostasis considered as a part of homeostasis.
Blood coagulation: is the conversion of fluid blood to a solid gel (clot), namely
the conversion of soluble fibrinogen into insoluble fibrin.
Clotting factors: present in the plasma as inactive precursors, they are activated
by proteolysis, the addition of the suffix (a) indicates activation of clotting
factors, activation of one factor catalyzes the activation of next factor and so on,
generally there are 12 clotting factors:
1 Factor I Fibrinogen
2 Factor II Prothrombin
3 Factor III Tissue thromboplastin
4 Factor IV Calcium
5 Factor V Proaccelerin
7 Factor VII Proconvertin
8 Factor VIII Antihemophilic factor (AHF)
9 Factor IX Christmas factor, plasma thromboplastin component (PTC)
10 Factor X Stuart-Prower factor
11 Factor XI Plasma thromboplastin antecedent (PTA)
12 Factor XII Hageman factor
13 Factor XIII Fibrin-stabilizing factor

 Note that both warfarin and heparin affect in common pathway (X+ II).
3 stages of hemostasis in order:
1. Vascular spasm. Evaluated by bleeding time
2. Formation of a platelet plug.
3. Blood coagulation.
The first two stages enough to stop bleeding in small vascular injury.
The blood vessel spasm refer to some mechanisms;
 Platelet substances: ADP, TXA2, histamine and serotonin.
 Nervous reflex.
 Vascular smooth muscles tune.

In the past it was evaluated using clotting time

but recently it has been replaced by PT & aPTT.
Mechanisms of Blood Coagulation:

Extrinsic System (In Vivo):

 following tissue damage, the process is initiated by the
activation of tissue thromboplastin (III) which is a cofactor
of VII factor that undergoes proteolysis and becomes VIla,
the formed complex (IIIa+VIIa), activates factors IX and X
in the presence of Ca and phospholipid (PL) which is
released from activated platelets, coagulation is sustained by
further generation of factor X via the complex IXa, VIlla, PL
and IVa, because complex (Illa+VIIa ) is rapidly inactivated
by tissue factor pathway inhibitor and antithrombin III, the
outcome is activated X (Xa).
 Antithrombin III affect both intrinsic and
extrinsic pathway.

Intrinsic System (In Vitro, contact with glass):

it commences when the Xlla adheres to a negatively charged surface, it
catalysis the XI to become XIa that converts IX into IXa which is in the
presence of factors VIlla, PL and IVa activates the X (Xa) at this point the
2 pathways converge.

Intrinsic pathway Extrinsic pathway

aPTT PT

Formation of Thrombin and Fibrin:

The Xa in the presence of Va, PL and Ca catalyzes the conversion
of prothrombin into IIa which cleaves fibrinogen into insoluble
fibrin (la), in the presence of Ca, XIII convents into XIIIa that
stabilizes fibrin, thrombin also promotes aggregation of
thrombocytes, cell proliferation and contraction of smooth muscle,
on the other hand it exerts anticoagulant effect by activating the
protein C pathway.
 Regulation of Coagulation and Fibrinolysis:
There are 2 systems that antagonize coagulation;

Fibrin Inhibition: Fibrinolysis:

Is accomplished by the
Represented by 𝜶𝟏-antiprotease, 𝜶𝟐-macroglobulin,
conversion of plasminogen
𝜶𝟐-antiplasmin and antithrombin in addition to
(inactive protein) into plasmin
proteins C and S that art activated via
(active enzyme) which lysis
thrombomoduline, these proteins inactivate factors
fibrin, and thus preventing
II, IX, X, XI and XII.
thrombosis.

Notes:
Active plasmin degrade fibrin, fibrinogen.
Some drugs and substances activate plasminogen into plasmin:

Extrinsic: t-PA, urokinase.

Intrinsic: Factor XIIa, High-molecular-weight kininogen (HMWK), kallikrein.
Exogenous: Streptokinase.

Drugs used in clotting disorders

Anticlotting drugs Drugs that facilitate clotting
Anticoagulants. Replacement factors.
Thrombolytics. Vitamin K (1972).
Antiplatelet drugs. Antiplasmin drugs.

Direct thrombin inhibitor (Dabigatran)

Anticoagulants Indirect thrombin inhibitor (heparin, warfarin)
Direct factor Xa inhibitors (Rivaroxaban).
t-PA derivatives.
Thrombolytics
Streptokinase.
Aspirin.
Antiplatelet drugs Glycoprotein llb/llla inhibitors.
ADP inhibitors (clopidogrel).
PDE/adenosine uptake inhibitors.

Direct thrombin inhibitor; reacts with thrombin directly.

Indirect thrombin inhibitor; react with antithrombin III or other inhibitors.

Rivaroxaban inhibit the activity of Stuart factor (factor Xa) and thrombin
Drugs Affecting Clot Formation and Resolution:
Interfere with the clotting cascade and thrombin
Anticoagulants formation. Heparin, Warfarin
 Prevent new thrombus to form.
Antiplatelets Alter the formation of the platelet plug Aspirin, Clopidogrel
{Disaggregation of platelets}.
Thrombolytic Break down the thrombus that has been formed by Alteplase,
drugs stimulating the plasmin system. Urokinase,
Strentokinase

Drugs Used In Coagulation Disorders:

Drugs used in Acute Myocardial Infraction, Deep Vein Thrombosis,
thrombosis: Pulmonary Embolism and acute ischemic syndrome.
Drugs used in Vasculitis, hemophilia and vitamin K deficiencies
bleeding disorders: diseases.
ANTICOAGULANTS:
Substances that prevent clotting, according to their actions on thrombosis they could be divided into 2
categories:

I. Indirect Thrombin Inhibitors.

II. Direct Thrombin Inhibitors.

Pharmacology of Indirect Thrombin Inhibitors:

 They are so called because their anticoagulant effect is exerted by interaction with antithrombin III
(2, 9, 10, 11, and 12), the prototype drug of this group is heparin it is so called because it was firstly
extracted from the liver.

Mechanism of action:

 Heparin acts indirectly by binding to antithrombin III, causing a rapid anticoagulant effect, in vivo
and vitro, effect occurs within minutes after I.V injection, in the absence of heparin, antithrombin III
interacts very slowly with thrombin and factor Xa. Heparin molecules bind antithrombin III inducing a
conformational change that accelerates its rate of action about 1000-fold.

 Heparin serves as a true catalyst, allowing antithrombin III to rapidly combine with and inhibit
circulating thrombin and factor Xa, antithrombin III inhibits II, IX, X, XI, and XII

Chemistry of Heparin:

 Heparin is a large sulfated polysaccharide polymer obtained from animal sources.

Type High molecular weight heparin Low molecular weight heparin

(unfractionated heparin) {UFH}
MW 15,000-20,000 2000-6000
T 1/2 short (0.5 - 1 hour) about 5 to 6 hours
Duration Short Long
Activity Both vivo and vitro (mainly in vitro) Both vivo and vitro (mainly in vivo).
inhibition especially thrombin (II) and Xa only Xa and has less effect on II
Side effects Frequent does Less frequent
Clinical Used in machines More common clinically
Monitoring aPTT Not necessary

 Agents of LMWHsHs: enoxaprin, daltepaiin and tinzaparin.

Low molecular weight heparin:

Equal efficacy. Given SC every 12-24hr.

Increased bioavailability after Is cleared unchanged by kidney (do not use in renal
S.C injection. failure!)
Laboratory monitoring is unnecessary, since they do not
Given once or twice daily prolong the aPTT, the plasma level and pharmacokinetics
are predictable. So can be used in outpatient therapy.
Less frequent hemorrhage. Less likely to cause thrombocytopenia.
Pharmacokinetics of Heparin:

Absorption:

 Heparin is poorly absorbed from the gut, because of its charged and large molecules, so it
must be given parenterally, either by deep S/C (effects appear in 1-2 hours after injection) or
I.V.
 I.M injection is contraindicated, because of the formation of hematoma.
 Orally contraindicated also.

Distribution and metabolism:

Heparin rapidly bind to plasma proteins inactivated in the liver and excreted in the urine;
diseases of liver and kidney prolong its t 1/2.

Placental barrier:

Heparin doesn't cross placental barrier, so it could be given safely in pregnancy.

Therapeutic Uses:
a. Deep vein thrombosis and pulmonary embolism: decreases the incidence of recurrent
thromboembolism.
b. Prophylaxis of postoperative venous thrombosis in patients undergoing elective surgery
(hip replacement) and those in the acute phase of MI.
C. Pregnant women with thromboembolism or with prosthetic heart valves.
d. The drug is also used in extracorporeal devices (dialysis machines) to prevent thrombosis.
Heparin Toxicity:
Bleeding; is the major side effect of heparin.
Heparin is monitored by two ways;
1) Anti-Xa units (0.2-0.7 units).
2) Activated partial thromboblastin time (aPTT): defined as the time required for plasma to
clot in the presence of kaolin (activator of XII), cephalin (substitute PL) and Ca, normally

Normal PTT In heparin therapeutic

25 to 35 seconds 1.5-2.5 normal (~ 𝟗𝟎 𝒔)
Treatment of Heparin Toxicity:
Protamine Sulfate: (antidote of heparin) is a highly basic positively peptide that combines
with negatively charged heparin, forming a complex devoid of anticoagulant activity.
For every 100 units of heparin remaining in the patient, administer 1mg; the infusion should
not exceed 50 mg in 10-minute period, because protamine can exert anticoagulant effect.

For example: In heparin toxicity, every 100 mg of protamine sulfate is given. If the original dose was
1000 mg of heparin, we do not give 10 mg of protamine, but we measure the concentration of the drug
in the plasma when toxicity occurs, and suppose that it is 600 mg, we give + mg of protamine.

Indirect Thrombin Inhibitors- Pharmacokinetics:

 Fondaparinux:

UFH LMWH Fondaparinux

IV or S.C S.C S.C
Rout of Initiated as IV bolus.
administration  Then followed by once or twice once daily.
lower doses or daily
continuous infusion
The dose is titrated
Dose using activated Weight-based dosing
adjustment partial
thromboplastin time
(aPTT)
 Anti-Xa  Factor Xa levels.
units.
Monitoring aPTT  Factor Xa  Usually not
levels. needed.
 Usually not
needed

Protamine Protamine sulfate

Antidote Protamine sulfate sulfate partially Doesn’t NOT reverse
the action

The monitoring of LMWH is usually not needed except:

 Renal impaired.
 Pregnant.
 Obese patients.
 Pharmacology of direct Thrombin Inhibitors: {hirudin and lepirudin}
Mechanism of action:

Exert their anticoagulant effect by directly binding to the active site of thrombin (inhibition)
in contrast to indirect thrombin inhibitors as LMWHs which act through binding (activation)to
antithrombin.

Clinical Use:

 hirudin is a specific irreversible thrombin inhibitor administered parenterally for patients

with heparin-induced thrombocytopenia and monitored by aPTT, it has a short t 1/2 (1 hour),
contraindicated in patients with renal disease.

Oral direct thrombin inhibitors (Dabigatran) and oral direct Xa

inhibitors:

 Offer significant advantages over warfarin.

 It appears that the oral anti-Xa drugs and oral direct thrombin inhibitors are poised to
challenge warfarin’s dominance in the prevention and therapy of thrombotic disease.

D. Direct Oral Factor Xa inhibitors:

1. Chemistry and pharmacokinetics:

Oral Xa inhibitors, including the small molecules rivaroxaban, apixaban, and
edoxaban, (note suffix -ban).
 Have a rapid onset of action and shorter half-lives than warfarin.
 These drugs are given as fixed oral doses and do not require monitoring.
 They undergo cytochrome P450-dependent and cytochrome P450-independent
elimination.

2. Mechanism and effects:

These small molecules directly bind to and inhibit free factor Xa and factor Xa
bound in the clotting complex.
3. Clinical use:
Venous thromboembolism following hip or knee surgery.
Rivaroxaban Prevention of stroke in patients with atrial fibrillation, without
valvular heart disease.
Apixaban Prevention of embolic stroke in patients with nonvalvular atrial
fibrillation.

4. Toxicity:
 Like other anticoagulants, the factor Xa inhibitors can cause bleeding.

 No reversal agents exist.

Pharmacology of Oral Anticoagulants: Warfarin (the prototype drug) and dicumarol:

Pharmacokinetics:
 Warfarin is well absorbed, following oral administration has a high
bioavailability, 99 % of warfarin bind to plasma albumin, which prevents its
diffusion into the CSF, urine and breast milk, drugs that have a greater affinity for
albumin binding site, such as sulfonamides, can displace the anticoagulant and
lead to a transient elevated activity.
 It has a low volume of distribution, but a long t1/2 36 hours, metabolized in the
liver into inactive metabolites that are excreted in the urine and stool.
 Warfarin does cross the PBB.

Mechanism of action:
 Normally vitamin K is involved in the synthesis of active prothrombin and other
clotting factors (VII, IX and X), this appears in conversion of glutamic acid to y-
carboxyglutamic acid in the presence of O2, CO2 and active form of vitamin K
(KH2) (hydroquinone).
 The above reaction requires reactivation of vitamin K, that occurs by oxidation
of vitamin K epoxide (KO) into its active form via vitamin K epoxide reductase
enzyme (VKER).
 VKER is inhibited by warfarin, the result is inactive biologically
(noncoagulatory) proteins, in addition to this action warfarin activates
physiologic anticoagulants especially C and S proteins.

Sources of Vitamin K:
K1 K2 K3
phytonadione menaquinone Menadione
green leafy vegetables gut flora synthetic
Fat soluble Fat soluble water soluble

 K3 is no longer used medically because of its ability to produce hemolytic

anemia and lacks coagulatory property.

Properties of Warfarin:
1) It is active only in Vivo.
2) Onset of anticoagulant effect is only after 8 to 12 hours; maintenance dose is
achieved in 24 hours, even if it is given I.V (rare).
3) The anticoagulant effect persists for a few days, after the drug has been
stopped.

Clinical Indications for warfarin:

 Oral anticoagulants are used for the treatment of chronic forms of thrombosis
rather than acute which is treated by heparin, it is also utilized for the prophylaxis
of thrombosis.

Monitoring of Warfarin therapy:

International Normalized Ratio (INR): the ratio of patient’s PT to PT of plasma
obtained from healthy subject on no medication.
INR for a healthy person INR for warfarin therapy
𝟎. 𝟗 − 𝟏. 𝟑 𝟐. 𝟓 − 𝟑. 𝟓

Prothrombin time (PT): is the time required for plasma to clot in the presence of
exogenous thromboplastin, the normal PT is 11-15 seconds.
high INR (ex; 5) Low INR (ex; 0.5)
high chance of bleeding high chance of having a clot
 Adverse Effects of warfarin:
a. Bleeding disorder:
Is the most common side effect, especially hemorrhage, of the bowel or the brain,
which is reversed by administration of vitamin K1 and fresh plasma.
 However, reversal following administration of vitamin K takes
approximately 24 hours.
b. Fetus toxicity:
In addition to bleeding there is a high risk of abnormal bone formation, because
warfarin prevents the formation of y-carboxyglutamic acid, which found in the
bone, so it should be avoided in pregnancy.
c. Hepatitis and necrosis of soft tissues are rare symptoms.
Bilirubin may displace warfarin, causing Kernicterus

Because it inhibit prostaglandin, which protect gastric mucosa.

 Platelet AGGREGATION INHIBITORS:
 Platelet aggregation inhibitors decrease the
formation or the action of chemical signals that
promote platelet aggregation.
 The last step in this response to vascular
trauma depends on a family of membrane GP
receptors that-after activation-can bind adhesive
proteins, such as fibrinogen, von Willebrand
factor, and fibronectin.
 The most important of these is the GP Ilb/IIIa
receptor that ultimately regulates platelet
interaction and thrombus formation.
 Thus, platelet activation agents, such as TXA2, ADP, thrombin, serotonin, and
collagen, all promote the conformational change necessary for the GP Ilb/IIIa
receptor to bind ligands, particularly fibrinogen.
 Fibrinogen simultaneously binds to GP Ilb/IIIa receptors on two separate
platelets, resulting in platelet cross-linking and aggregation.

VWF functions;
1) By GP Ilb/IIIa forms bridges between platelets and subendthelium.
2) Carrier of VIII clotting factor.

Clinical Uses:
These agents are beneficial in the prevention and treatment of occlusive
cerebrovascular, peripheral vascular disease {PVD} and cardiovascular diseases,
in the maintenance of vascular grafts and arterial patency, and as adjuncts to
thrombin inhibitors or thrombolytic therapy in myocardial infarction.
Prostaglandin Thromboxane A2
Vasodilation. Vasoconstriction.
Inhibit platelet aggregation. Activate platelet aggregation.
 Antiplatelet drugs:

I) Cyclooxygenase/TX2 synthase inhibitors: Aspirin.

II) Adenosine di phosphate (ADP) receptor inhibitors (P2Y12);
Irreversible Reversible
Clopidogrel,Ticlopidine Prasugrel, Ticagrelor, cangrelor

III) Phosphodiesterase inhibitors:

Dipyridamole Cilostazol
PDE-3 + PDE-5 PDE-3

IV) Glycoprotein Ilb/IIIa inhibitors: Abciximab, Eptifibatide, Tirofiban, Defibrotide.

A. Aspirin:
 Is the only irreversible COX inhibitor that prevents TXA2 synthesis, the
inhibitory effect is rapid, apparently occurring in the portal circulation.
 Aspirin is frequently used in combination with other drugs having anticlotting
properties for example, heparin or clopidogrel.
 NSAIDs, such as ibuprofen, inhibit COX-1 by transiently competing at the
catalytic site.
 Ibuprofen, if taken concomitantly (‫ )في نفس الوقت‬with, or 2 hours prior to
aspirin, can antagonize the platelet inhibition by aspirin.
 Therefore, aspirin should be taken at least 30 minutes before ibuprofen or at
least 8 hours after ibuprofen.

B.Ticlopidin eand clopidogrel:

 These drugs irreversibly inhibit the binding of ADP to its receptors on platelets
and, thus, inhibit the activation of the GP Ilb/IIIa receptors required for platelets to
bind to fibrinogen and to each other.

C. Abciximab:
 is a monoclonal antibody, that binds to GPIlb/IIIa receptors, the antibody
blocks the binding of fibrinogen and von Willebrand factor; consequently,
aggregation does not occur, another member of this group is eptifibatide.
E. Dipyridamole:
 A coronary vasodilator is employed prophylactically for angina pectoris.
 It is usually given in combination with aspirin or warfarin; it is ineffective when
used alone.
 Dipyridamole increases intracellular levels of cAMP by inhibiting cyclic
nucleotide phosphodiesterase, resulting in decreased thromboxane A2 synthesis.
 It may potentiate the effect of prostacyclin to antagonize platelet stickiness
and, therefore, decrease platelet adhesion to thrombogenic surfaces.
 Dipyridamole is effective for inhibiting embolization from prosthetic heart
valves.

Thrombolytic Agents

Mechanism of Action:
Activate plasminogen to plasmin, which in turn breaks down fibrin threads in a
clot to dissolve a formed clot.

Indications:
Acute MI, severe pulmonary emboli, DVT and to clear occluded venous catheters.

Contraindications:
 Allergy.
 Any condition that worsens through dissolution of clots.
 Pregnancy and lactation.
 Alteplase and Reteplase bind directly with fibrin and lysis it.
 Not activate plasmin.
 Streptokinase and Urokinase activate plasmin directly.

 The major application of the thrombolytic agents is as an alternative to

percutaneous coronary angioplasty in the emergency treatment of coronary
artery thrombosis.
 Under ideal conditions (ie, treatment within 6 h), these agents can promptly
recanalize the occluded coronary vessel.
 Very prompt use (ie, within 3 h of the first symptoms) of t-PA in patients with
ischemic stroke is associated with a significantly better clinical outcome.

Adverse effects of Thrombolytics:

1) Cardiac arrhythmias.

2) Hypotension.

3) Bleeding:
 Is the most important hazard and has about the same frequency with all the
thrombolytic drugs.
 Cerebral hemorrhage is the most serious manifestation.
 Bleeding of GI, genitourinary, respiratory and retroperitoneal also occur.

4) Hypersensitivity reactions:
Rash, flushing (‫)توهج‬, bronchospasm, and anaphylactic reaction, specifically for
Streptokinase.
Streptokinase; a bacterial protein, can evoke the production of antibodies that
cause it to lose its effectiveness or induce severe allergic reactions on
subsequent therapy.
 Patients who have had streptococcal infections may have preformed
antibodies to the drug.
 Bleeding Disorders Treated With Clotting Factors

1) Hemophilia:
Genetic lack of clotting factors that leaves the patient susceptible to excessive
bleeding from any injury.

2) Liver disease:
Clotting factors and proteins needed for clotting are not produced.

3) Bone marrow disorders:

Platelets are not formed in sufficient quantity to be effective.

Drugs used in bleeding disorders

 Vitamin K.
 Plasma fractions.
 Fibrinolytic inhibitors aminocaproic acid.
 Serine protease inhibitors aprotinin.

HEMOSTATIC DRUGS
1. Vitamin K:
 Is a fat-soluble vitamin, which requires bile salts for absorption form the gut.

Pharmacokinetics:
tablets ampules
5mg 50 mg
The onset of action is delayed for 6 hrs and accomplished by 24 hrs.
 Clinical Indications of vitamin K:
1. Warfarin toxicity: infusion should be done slowly, because it can cause
dyspnea, chest and back pain leading even to death.
2. Vitamin K deficiency: as supplemental therapy either due to disorders of liver,
intestine diseases, or in hospitalized patients in ICU because of poor diet,
parenteral nutrition, recent surgery, multiple antibiotic therapy and uremia.
3. Vitamin K is currently administered to all newborns to prevent hemorrhage
disease of vitamin K deficiency, which is especially common in premature
infants.

2. Plasma Fractions:
A) Plasma factors:
Freeze concentrates of plasma containing prothrombin, factors IX and X and VII
are commonly available for treating deficiencies of these factors e.g. hemophilia
B is treated by IX factor.

B) Desmopressin Acetate:
 Increases activity of VIII factor and used to treat hemophilia A (deficiency of VIII
factor or von-Willebrand disease.
 Desmopressin acetate can be used in preparation for minor surgery such as
tooth extraction without any requirement for infusion of clotting factors if the
patient has a documented adequate response.

c. Cryoprecipitate:
Plasma protein fraction obtained by whole blood; used to treat deficiencies of
fibrinogen such as that occurs in DICS and liver disease, VIII deficiency and von-
Willbrand disease if desmoprosin is not indicated.

Von-Willebrand disease is a disorder which is characterized by spontaneous bleeding

from mucous membrane, excessive bleeding from wounds, menorrhagia and a
prolonged bleeding time (normally 2-7 minutes) in the presence of normal count of
thrombocytes, (140,000-450, 000) because vWF stabilizes factor VIII by binding to it,
deficiency of vWF is associated with a secondary decrease in VIII.
3. Pharmacology of Fibrinolytic Inhibitors:
 Aminocaproic acid and its analog tranexamic acid.

Mechanism of action:
Inhibit competitively fibrinolysis (conversion of plasminogen into plasmin or
fibrinolysin) thus preventing lysis of fibrin.

Pharmacokinetics:
The drugs are rapidly absorbed orally, and cleared from the body via the kidneys,
administered IV as loading dose of 5 g, over 30 minutes to avoid hypotension.

Clinical Uses:
They are used as adjunctive therapy (‫ )كعالج مساعد‬in hempophilia, bleeding form
fibrinolytics, (streptokinase, urokinase and alteplase), prophylaxis for rebleeding
from intracranial aneurysms, post GIT bleeding, hemorrhage of the urinary bladder,
secondary to radiation- and drug-induced cystitis and postprostatoectomy.

Adverse Effects:
Intravascular thrombosis, hypotension, diarrhea, myopathy, nasal stuffiness and
abdominal discomfort.

Contraindications:
DICS and genitourinary bleeding from the upper tract because of high incidence of
excessive clotting.

4. SERINE PROTEASE INHIBITORS: APROTININ

 Aprotinin is a competitive inhibitor of trypsin, chymotrypsin, plasmin and
kallikrein, leading to inhibition of XII factor.
 It also inhibits plasmin-streptokinase complex in patients who have received
the thrombolytic agent.
 Aprotinin was shown to reduce bleeding—by as much as 50%—especially for
open heart procedures and liver transplantation.

Adverse effects:
Cardiac arrhythmias, MI, CHF, hypotension, increased risk of renal failure, heart
attack, and stroke.
 Antineoplastic Drugs:
Cancer:
 Is a term used for diseases in which abnormal cells divide without control and
are able to invade other tissues by metastasis.
Nomenclature;
 In general benign tumor attaching with the suffix (- oma).
Epithelial Mesenchymal
Adenoma Lipoma
Papilloma Fibroma

 In general malignant tumor + sarcoma or carcinoma:

Epithelial Mesenchymal
Carcinoma Sarcoma
Adenocarcinoma, squamous cell carcinoma Liposarcoma , ostosarcoma

 Categorized based on the functions/locations of the cells from which they

originate:
Skin or in tissues that line or cover internal organs. E.g.,
Carcinoma Epithelial cells.
 80-90% reported cancer cases are carcinomas.
Sarcoma Bone, cartilage, fat, muscle, blood vessels, or other
connective supportive tissue.
White blood cells and their precursor cells such as the
Leukemia bone marrow cells, causes large numbers of abnormal
blood cells to be produced and enter the blood.
Lymphoma Cells of the immune system that affects lymphatic
system.
Myeloma B-cells that produce antibodies- spreads through
lymphatic system.
CNS cancers Cancers that begin in the tissues of the brain and spinal
cord.
Benign vs malignant tumor:
Benign tumors Malignant tumors
Not cancerous. Cancerous.
They can often be removed, and, in Cells in these tumors can invade
most cases, they do not come back. nearby tissues and spread to other
parts of the body.
Cells in benign tumors do not spread The spread of cancer from one part of
to other parts of the body. the body to another is called
metastasis.

THE SEVEN WARNING SIGNS OF CANCER (C-A-U-T-I-O-N):

C Change in bowel or bladder habits
A A sore that does not heal
U Unusual bleeding or discharge
T Thickening or lump in the breast, testicles, or elsewhere
I lndigestion or difficulty swallowing
O Obvious change in the size, color, shape, or thickness of a wart, mole, or mouth sore
N Nagging (‫ )مزعج‬cough or hoarseness )‫(بحة بالصوت‬

Four characteristics that distinguish them from normal cells:

 Uncontrolled proliferation.
 High differentiation and loss of function.
 Invasiveness.
 Metastasis.

There are two main categories of relevant genetic change:

 The activation of proto-oncogenes to oncogenes.
 The inactivation of tumour suppressor genes.
1. The activation of proto-oncogenes to oncogenes:
 A genes that normally control cell division, apoptosis and differentiation which
can be converted to oncogenes that induce malignant change by viral or
carcinogen action.

2. The inactivation of tumor suppressor genes:

 Normal cells contain genes that suppress malignant change- termed tumor
suppressor genes (anti-oncogenes).
 Mutations of these genes are involved in many different cancers.
 The loss of function of tumor suppressor genes can be the critical event in
carcinogenesis.

Cancer Therapeutic Modalities (classical):

Surgery  1/3 of patients without metastasis Respond to

surgery and radiation.
 If diagnosed at early stage, close to 50% cancer
Radiation could be cured.
 50% patients will undergo chemotherapy, to remove
Chemotherapy micrometastasis.
 However, chemotherapy is able to cure only about
10-15% of all cancer patients.

Chemotherapy drug; classification based on the mechanism of action:

Antimetabolites: Drugs that interfere with the formation of key biomolecules

including nucleotides, the building blocks of DNA.
Genotoxic Drugs: Drugs that alkylate or intercalate the DNA causing the loss
of its function.
Plant-derived These agents prevent proper cell division by interfering with
inhibitors of mitosis: the cytoskeletal components that enable the cell to divide.
Other Chemotherapy These agents inhibit cell division by mechanisms that are not
Agents: covered in the categories listed above.

 Liquid cancers are not treated with surgery, but with chemotherapy, unlike solid cancers,
which require surgery before chemotherapy.
 The most common organs in which cancer has spread, in order:
 Liver  lung  brain  bone
Anticancer drugs; {A-A-A-C-M-T-V}
A – A – A:

A A A
ALKYLATING AGENTS ANTIMETABOLITES ANTIBIOTICS
1) Usually start with {c}. Usually end with: Usually end with;
2) May end with:  ate.  mycin.
 mide.  purine.  bicin.
 fan.  bine.
 lan  Xed.
 amine Remember; Mitoxantrone
 ine Remember; 5-Fluorouracil
 tin

C – M – T – V:
CAMPOTHECIN ANALOGUES MISCELLANEOUS TAXANES VINCA ALKALOIDS
Ends with: No uniform rule Ends with: start with:
tecan taxel Vin

Drugs affecting hormones;

Steroid hormones Glucocorticoids,
Estrogens, Progestins.
Selective estrogen receptor modulator (SERM) Tamoxifen, Toremifene
“end with - fen”
Selective estrogen receptor degrader (SERD) Fulvestrant
Aromatase inhibitors “end with zole” Letrozole, Anastrozole
Antiandrogens Flutamide, Bicalutamide
5-𝜶 reductase inhibitors “end with ride” Finasteride, Dutasteride
GnRH analogues “ends with elin” Nafarelin, Triotorelin

SERM: Inhibit breast estrogen and stimulate uterus estrogen.

SERD: Degrade the estrogen receptors.
Aromatase: Convert testosterone into estrogen.
5-𝜶 reductase: Convert hydrotestosteron into testosterone.
GnRH: Gonadotrophin inhibitor.
Cell cycle:

1) Interphase;

 G1 phase: organelles growth.

 S phase: DNA duplicating.
 G2 phase: prepare for mitosis.
 G0 phase: resting phase.

2) Mitotic phase:

 Prophase.
 Metaphase.
 Anaphase.
 Telophase.
After completion of mitosis, the resulting daughter cells have two options:
 They can either enter G1& repeat the cycle.
 They can go into G0 and not participate in the cell cycle.
Growth fraction
The ratio of proliferating cells to cells in G0.
large percentage of proliferating cells & few cells in G0 mostly of cells in G0
high growth fraction low growth fraction

Cancer Cells Susceptibility AND growth Cycle

 Rapidly dividing cells are generally more sensitive to chemotherapy, whereas
slowly proliferating cells are less sensitive.
 In general, non-dividing cells (those in the G0 phase) usually survive (‫)تنجو من‬
the toxic effects of many of these agents.
 Ideal anti-cancer should eradicate cancer cells without harming normal cells.
 Normal cells with high rate of division are also affected causing many side
effects:
Hair follicles: Hair loss (alopecia).
Decrease in immunity & increased
Lymphatic system:
susceptibility to infection.
Bone marrow: Aplastic anemia.
Mucous membrane of the stomach Nausea & vomiting.

People being treated with chemotherapy are given filgrastim and pegfilgrastim.
According to their actions on the cell cycle, antitumor agents are divided into:
Cell Cycle-Specific (CCS) Agents: Cell- Cycle Non-Specific (CCNS) Agents:
effective for high growth-fraction effective for both low growth fraction
tumors, (only against replicating cells), tumor (solid) as well as for high growth
Affect specific phase in cycling cells can kill both Go and cycling cells
antimetablities, antitumor antibiotics Alkylating agents, antitumor antibiotics
(bleomycin), vinca alkoloids, (dactinomycin and mitomycin)
etoposide and taxanes. anthracyclines, camptothecins and
platinum analogs.

Principles of Cancer Chemotherapy:

Goal of treatment:
 Curative which requires the eradication of every neoplastic cell.
 Palliation of cancer symptoms.
 Improved quality of life increased time to tumor progression.

Chemotherapy is used in three main clinical settings:

 The primary treatment in patients who present with solid
Primary induction advanced cancer and many hematologic.
chemotherapy:  No alternative treatments exists.
 Used before radiation.
 The administration of therapeutic agents before a main
treatment.
Neoadjuvant  Used before surgery.
chemotherapy:  Neoadjuvant therapy aims to reduce the size or extent of
the cancer before using radical treatment intervention.
 Neoadjuvant hormone therapy prior to radical
radiotherapy for adenocarcinoma of the prostate.
 Refers to use of chemotherapy as adjuvant to local
Adjuvant treatment such as surgery or radiation therapy.
chemotherapy:  The goal is to reduce the risk of local and systemic
recurrence.
Effects of various treatments on the cancer cell burden in a hypothetical patient:
 There is no chance of diagnosing cancer unless the number of affected cells is
more than 109 = 1g mass. 106 = 1 mg mass
 Clinical symptoms usually first appear at this stage.
 With the development of cancer cells may reach (1012) and = 1 km mass.
 At this stage, the development of cancer is very rapid and may lead to
death.
CURATIVE CHEMOTHERAPY:
solid tumors disseminated cancers
testicular carcinoma leukemia
 Combination-drug chemotherapy
 Tumor burden is initially reduced by reduces the chance of drug resistance.
surgery and/or radiation.  Each drug is chosen to have a
 Treatment of occult micrometastases different cellular site of action or
is continued after clinical signs of different cell-cycle specificity.
cancer have disappeared.  Each drug is chosen to have a
different organ toxicity.

PALLIATIVE CHEMOTHERAPY:
 Initial remissions are transient, with symptoms recurring between treatments.
 Survival is extended, but the patient eventually dies of the disease.
Principles of Cancer Chemotherapy:
 Tumor susceptibility and the growth cycle:

Rapidly dividing cells are generally more sensitive to anticancer drugs where as
non-proliferating cells (those in phase Go) usually survive the toxic effects of
these agents.

 Tumor growth rate:

 The growth rate of tumor initially is rapid, but decreases as the tumor size
increases due to inadequate vascularization.
 Reducing the tumor burden through surgery or radiation, increases their
susceptibility to anticancer drugs.

 Combinations of the drugs:

Combination therapy is more effective than monotherapy in most cancers, the

advantages include anticancer drugs provide maximal cell kill and may slow or
prevent subsequent development of cellular drug resistance.
The following principles are important for selecting appropriate anti-tumor drugs
to use in combination chemotherapy:
a. Each drug should be active when used alone against the particular cancer.
b. The drugs should have different mechanisms of action.
c. Cross-resistance between drugs should be minimal.
d. The drugs should have different toxic effects.

 Log Kill Phenomenon:

 Destruction of cancer cells by chemotherapeutic agents follows first-order

kinetics (that is, a given dose of drug destroys a constant fraction of cells).
 The term "log kill" is used to describe this phenomenon.
 For example, a diagnosis of leukemia is generally made when there are about
109 (total) leukemic cells.
 Consequently, if treatment leads to a 99.999% kill, then 0.001% (105) of 109
cells (or 104 cells would remain).
 This is defined as a 5-log kill.
 At this point (5-log kill), the patient appears asymptomatic; that is, the patient
is in remission.
 For most bacterial infections, a five-log (100,000- fold) reduction in the number
of microorganisms results in a cure, because the host’s immune system can
destroy the remaining bacterial cells. However, in treating cancer, because tumor
cells are not as readily eliminated, additional treatment is required to totally
eradicate the leukemic cell population.
 For this reason, most cancer treatment begins with debulking (‫ )اﻟﺗﺧﻔﯾف‬by
surgery and/or radiation in order to initially reduce the neoplastic cell
burden before chemotherapy, immunotherapy, or a combination of these
treatment modalities.

 Pulse therapy:

 Involves intermittent treatment with very high doses of anticancers that are too
toxic to be used continuously.
 This method is used successfully in therapy of acute leukemias and testicular
carcinomas.

Rescue therapy:

 Toxic effects of antitumor drug can be alleviated (‫ )تخفيفه‬by this method.

 high doses of methotrexate may be given for 36-48 hrs and terminated before
severe toxicity occurs to cells of the GIT and bone marrow, Leucovorin which is
accumulated more readily by normal than tumor cells, is then administered, this
results in rescue of normal cells because leucovorin bypasses the
dihydropholate reductase step in folic acid.
 Other examples include the use of Mensa that traps acrolin, released from
cyclophosphamide, hence reducing the incidence of hemorrhagic cystitis.
 Dexrazoxane inhibits free radicals and affords protection of cardiomyocytes
against anthracydines (Doxorubcin).
Methotrexate - leucovorin dihydropholate reductase step in folic acid
Mensa – acrolin (cyclophosphamide) Reducing the incidence of hemorrhagic cystitis.
Dexrazoxane - Doxorubcin affords protection of cardiomyocytes
Resistance to Cytotoxic Drugs:
 Inherited resistance:

Some neoplastic cells are inherently resistant to most anticancer drugs, (e.g.
malignant melanoma renal and brain cancers absence of response on the first
exposure), properly due to mutation of p 53 suppressor (decreased its activity),
gene resulting in resistance to radiation therapy and to a wide range of anticancer
drugs.

 Acquired resistance:

Develops in response to exposure to a given anticancer agent, (mutation) after

prolonged administration of suboptimal drug doses Resistance could be
minimized by short term intensive intermittent therapy with combinations of
drugs.

Alkylating Agents {first A}:

 The first anticancer agents developed.
 Chemically related to mustard gas used in WW1.
 Are more effective in treating slow-growing tumors because they are cell-cycle
nonspecific.
 Alkylating agent induced damage to cancer cells accumulates even during
non-active portions of the cell cycle.
 All alkylating agents are toxic to hematologic cells, therefore
myelosuppression is a predictable side effect.
 The drugs used to treat CNS cancers are used because they cross the BBB not
because they are better agents
 binds with guanine DNA intrastrand.
Mechlorethamine Melphalan, Busulfan
Lomustine Carmustine
Dacarbazine Cisplatin
Temozolomide Carboplatin, oxaliplatin
Ifosfamide Chlorambucil
Cyclophosphamide
Two example in alkylating agents:
Chlorambucil Cyclophosphamide
Phase: non specific non specfic
Chronic Lymphocytic breast, testicular and other
Indications: Leukemia (CLL), solid tumors, leukemia,
Ovarian Carcinoma. lymphoma, neuroblastoma and
immunosuppression
hemorrhagic cystitis, BM
Bone marrow suppression, suppression, cardiotoxicity
Side Effects: drug is carcinogenic and may  Administer Mensa to
be teratogenic. prevent hemorrhagic
cystitis (binds to the
toxic metabolite).
Metabolized to phosphoramide
Crosslinks DNA by binding to
MOA: mustard which is a DNA
both strands.
alkylating agent.
decreased cellular uptake and decreased cellular uptake and
Resistance: increased repair of drug increased repair of drug
induced DNA damage induced DNA damage

Antimetabolites {second A}:

General MOA: Blocking the synthesis of DNA and/or RNA

Folate antagonists: End (ate / xed) Methotrexate, pemetrexed, raltitrexed

Pyrimidine Antagonist: End with (bine) Cytarabine, fluorouracil, gemcitabine.
Purine Antagonists: End (bine / purine) Fludarabine, mercaptopurine, pentostatin.

 Antimetabolites are structurally related to normal compounds that exist within

the cell (analogs).
 They generally interfere with the availability of normal purine or pyrimidine
nucleotide precursors, either by:
 Inhibiting their synthesis.
 Competing with them in DNA or RNA synthesis.

 Their maximal cytotoxic effects are in S-phase and are, therefore, cell cycle
specific.
Methotrexate (MTX)
 MTX is structurally related to folic acid (vitamin B9) and act as antagonist of it.
 It inhibits mammalian dihydrofolate reductase (DHFR).
 The inhibition of DHFR can only be reversed by:
 1000-fold excess of the natural substrate, dihydrofolate (FH2).
 By administration of leucovorin, which bypasses the blocked enzyme and
replenishes the folate pool {rescue therapy}.

Mercaptopurine (6-MP) and 6-thioguanine (6-TG):

 Purine Antagonists:

M.O;
6-MP and 6-TG are activated by hypoxantheine-
guanine phosphororibosyltransferases (HGPRTases)
to form 6- mercaptopurine-ribose phosphate (6-MPRP)
and other toxic metabolites (like; TIMP).
 (6-MPRP) and other toxic metabolites (like; TIMP);
inhibit several enzymes of de novo purine nucleoside
synthesis.

Pharmacokinetics (6-MP and 6-TG):

 Rout of administration: low oral bioavailability
 Distribution: distributed widely except for the CSF
6-MP: Is metabolized in the liver to 6- methylmercaptopurine, catalyzed by
xanthine oxidase.
Allopurinol (purine analog), can lead to accumulation of 6-MP (toxicity), because
allopurinol inhibits xanthine oxidase, so it is important to reduce the dose of 6-
MP in patients receiving allopurinol.
6-TG not interact with allopurinol, which can be used in full doses with
allopurinol, excretion mainly occurs via the kidneys.
Note that: Allopurinol (common used in cancer patients to reduce hyperurecemia
following tumor lysis)
 5- Fluorouracil
Mechanism of action:
 5-Fluorouracil is a pyrimidine antagonist that needs
to be converted to 5-fluoro-2ʹ-deoxyuridine-5ʹ-
monophosphate, (5-FdUMP), which inhibits thymidylate
synthetase and thus the production of dTMP and DNA.
 Inside our body there is not enzyme to lysis 5-FU, but
exist in cancer cells.

Resistance:
 Is usually due to decreased conversion to F-UMP.

Pharmacologic properties:
 5-Fluorouracil is administered IV; it is also used topically to treat skin cancers.

Vinca alkaloids
VinBlastine and VinCristine (oncovan)
VinBlastine VinCristine
Uses ABVD MOPP
A Adriamycin (doxorubicin) M Mechlorethamine
State B Bleomycin O Vincristine (Oncovin),
V Vinblastine P Procarbazine
D Dacarbazine P Prednisone
 Hodgkin’s disease.  Childhood leukemias.
INDICATION  Lymphomas Breast carcinoma.
 Childhood tumors-Wilm’s tumor.
(Neuroblastoma).
 Testicular tumors
 Hodgkin’s disease.
 Bone marrow suppression.  Peripheral neuritis.
TOXICITY Anorexia.  Paresthesia.
 Nausea, vomiting. & Diarrhea.  Muscle weakness.
 Alopecia
VII. ADJUNCT AGENTS:

Amifostine (Ethyol):
Is a cytoprotective agent that is dephosphorylated to active-free thiol, which then acts
as a scavenger of free radicals.
It is used to:
1) Reduce the incidence of neutropenia-related fever.
2) Reduce infection induced by alkylating agents and platinum-containing agents (e.g.,
cisplatin.)
3) Reduce renal toxicity associated with platinum-based drug therapy.
4) Reduce xerostoma (sensation of dry mouth) in patients undergoing irradiation of
head and neck regions.
Characteristics of “Important” Anticancer Drugs
1) Alkylating agents: non-specific cell cycle
Drugs Mechanism Used Adverse effect
Non-Hodgkin’s, 1) BMS.
Cyclophosphamide attacks guanine ovarian, breast CA, 2) Mucositis.
N7— dysfunctional neuroblastoma 3) Hemorrhagic cystitis.
DNA 4) hepatotoxicity (high
dose)
Testicular. 1) Nausea, vomiting
Ovarian. (use ondansetron)
Cisplatin cross links DNA Bladder. 2) Nephrotoxicity
strands lung CA. (use amifostine)
3) Neurotoxicity
(use deafness)
1) BMS.
Procarbazine 2) Pulmonary toxicity,
Hodgkin’s (MOPP) 3) hemolysis.
4) Neurotoxicity.
5) Leukemogenic.

2) Antimetabolite agents (cell cycle specific):

Drugs Mechanism Used Adverse effect
1) Leukemia, 1) BMS.
Methotrexate inhibits DHF 2) Lymphomas. 2) mucositis,
reductase (S 3) Breast CA. 3) crystalluria.
phase) 4) Psoriatic arthritis leucovorin (folinic
and rheumatoid acid) rescue
Purine 1) ALL. 1) BMS.
antimetabolite (S 2) immunosuppression 2) Hepatotoxicity
6-Mercaptopurine phase) bioactivated (jaundice, necrosis).
by HGPR  (Azathioprine forms 3) GI distress
transferase 6-MP).
Pyrimidine 1) Breast, ovarian. 1) BMS.
antimetabolite 2) head, and neck CA 2) GI irritation.
5-Fluorouracil bioactivated to 3) topical for basal cell 3) Alopecia.
inhibit thymidyiate CA and keratoses
synthetase
4) Antibiotic agents (cell cycle specific):
Drugs Mechanism Used Adverse effect
BMS — delayed CHF
(dexrazoxane is an
1) Hodgkin’s (ABVD). iron-chelating agent
Intercalated, forms 2) Breast. preventing the
free radicals, 3) Endometrial. formation of free
Doxorubicin
inhibits 4) Lung. radicals; it is not a free
topoisomerase 5) ovarian CA. radical “trapper”),
2) alopecia,
3) vesicant,
4) radiation “recall”
Complexes with Fe 1) Hodgkin’s. 1) Pneumonitis.
and O2  DNA 2) Testicular. 2) Pulmonary fibrosis.
Bleomycin strand scission (G2 3) Head, neck. 3) mucocutaneous.
phase) 4) skin CA. 4) Alopecia.
5) hypersensitivity

5) Vinca alkaloids (cell cycle specific):

Drugs Mechanism Used Adverse effect
1) Hodgkin’s (ABVD).
Vincristine and (↓) Microtubular 2) Testicular CA. BMS, GI, alopecia
polymerization  3) Kaposi’s.
spindle poisons 1) Hodgkin’s (MOPP).
(M phase) Neurotoxicity
Vinblastine 2) Leukemias.
3) Wilms.

M phase Vinca alkaloids, pacilitaxel

G2 phase Bleomycin
S phase 6-Mercaptopurine, Methotrexate, hydroxyurea

Neurotoxicity Procarbazine, Vinblastine, Cisplatin

Pulmonary fibrosis Bleomycin
Nephrotoxicity Cisplatin
Hepatic, (↑) jaundice 6-Mercaptopurine, Cyclophosphamide
TOXICITY OF ANTICANCER DRUGS:
 Rapidly proliferating cells such as the bone marrow, GI tract mucosa, hair
follicles, and gonads are most sensitive to cytotoxic drugs.
 Most often bone marrow suppression (BMS) is dose-limiting.

 Anticancer drug dosage is usually carefully titrated to avoid excessive

neutropenia (granulocytes <500/dl,) and thrombocytopenia (platelets
<20,000/dL); colony stimulating factors, erythropoietin, and thrombopoietin
can be supportive  ↑ infections and need for antibiotics.

Other Dose-Limiting or “Distinctive” Toxicities

Toxicity Drug(s)
Renal Cisplatin, methotrexate.
Liver 6-MP, busulfan, cyclophosphamide
Pulmonary 6-MP, busulfan, cyclophosphamide.
Cardiac Doxorubicin, daunorubicin.
Neurologic Vincristine,* cisplatin, paditaxel.
Immunosuppressive Cyclophosphamide, cytarabine, dactinomycin, methotrexate.
Other Cyclophosphamide Hemorrhagic cystitis).
Procarbazine Leukemia
asparaginase Pancreatitis
 Immunosuppressants Pharmacology
 The Immune system is designed to protect the host from harmful foreign
molecules.
 However, in certain instances its powerful destructive mechanisms do more
harm than good, e.g. hypersensitivity reactions, autoimmune disorders, and
rejection reactions to transplanted tissues.
 Drugs that suppress immune mechanisms play an important role in treating
these conditions.

IMMUNE MECHANISMS:
Cell –mediated immunity Humoral (antibody –mediated
immunity).
T- lymphocytes B- lymphocytes

Drugs that modulate immune function:

Immunosuppressant:
Corticosteroids prednisone
Immunophilin ligands cyclosporine, tacrolimus, sirolimus
Mycophenolate mofetil Mycophenolate mofetil
Cytotoxic drugs azathioprine
Immunoglobulin based agents etanercept

Immune potentiators
Aldesleukin Interferons

Cell Mediated Immunity:

 Involves ingestion and digestion of antigen by antigen-presenting cells.
 Activated TH cells secretes IL-2, which stimulates TH1 & TH2.
 TH1 produce TNF-β and IFN-γ which activate:
NK cells Cytotoxic T cells Macrophages
kill tumor & virus, infected cells kill bacteria
IFN-γ TNF-β and IFN-γ
Humoral Immunity
B-lymphocytes bind to antigen and are induced by interleukins (IL-4 & IL- 5)
produced by TH2 which in turn causes B-cells proliferation & differentiation into:
 Memory B cells.
 Antibody secreting plasma cells.
 Antibodies produced by plasma cells bind to antigens on the surface of
pathogens and trigger the precipitation of viruses and the destruction of bacteria
by phagocytic cells or lysis by the complement system.

Cytokines:
Soluble, antigen-nonspecific signaling proteins
that bind to cell surface receptors on a variety of
cells.
Cytokines include;
Interleukins.
Interferons (IFNs).
Tumor Necrosis Factors (TNFs).
Transforming Growth Factors (TGFs).
Colony-stimulating factors (CSFs).

Classification of Immunosuppressant drugs:

An agent that can suppress or prevent the immune response.
Inhibitors of cytokine (IL-2) Calcineurin inhibitors: Cyclosporine and
Tacrolimus (FK506).
Sirolimus (rapamycin).
Inhibitors of cytokine gene Corticosteroids
expression:
(Antimetabolites): Myclophenolate Mofetil
Cytotoxic agents: • Leflunomide • Azathioprine • Methotrexate.
Alkylating agents: Cyclophosphamide.
 antilymphocyte globulins (ALG).
Immunosuppressive  antithymocyte globulins (ATG).
antibodies that block T cell  Rho (D) immunoglobulin.
surface molecules  Muromonab-CD3.
 Basiliximab and Daclizumab.
 Cyclosporine (CSA); Calcineurin inhibitors:
Cyclosporine is a fungal polypeptide composed of 11 amino acids.
Calcineurin; a phosphatase necessary for dephosphorylation of transcription
factor Nuclear Factor of Activated T cells (NFATc) required for interleukins
synthesis (IL-2).

Mechanism of action for Cyclosporine:

1. Acts by blocking activation of T cells by inhibiting interleukin-2 production.

2. Decreases proliferation and differentiation of T cells.
3. Cyclosporine- immunophilin complex inhibits calcineurin,
4. Suppresses cell-mediated immunity.

Kinetics for Cyclosporine:

 Effective orally and I/V.

Uses of Cyclosporine:

 Organ transplantation (kidney, liver, heart) either alone or with other

immunosuppressive agents (Corticosteroids).
 Autoimmune disorders endogenous uveitis (‫)التهاب القزحية‬, rheumatoid arthritis,
active Crohn’s disease, psoriasis, nephrotic syndrome, severe corticosteroid-
dependent asthma.
 Graft-versus-host disease after stem cell transplants.

Adverse Effects (Dose-dependent):

 Therapeutic monitoring is essential.

 Nephrotoxicity (increased by NSAIDs and aminoglycosides).
 Liver dysfunction.
 Hyper (glycaemia, tension and kalemia).
 Viral infections (Herpes - cytomegalovirus).
 Lymphoma, Hirsutism, Neurotoxicity (tremor) Gum hyperplasia and
anaphylaxis after I.V.
 Tacrolimus (TAC); Calcineurin inhibitors:
 Chemically not related to cyclosporine, both drugs have similar mechanism of
action.
 The internal receptor for tacrolimus is immunophilin ( FK-binding protein, FK-
BP).
 Tacrolimus FKBP complex inhibits calcineurin.

Kinetics for Cyclosporine:

 Effective orally and I/V.

Indications:

Organ and stem cell transplantation, prevention of rejection of liver and kidney
transplants and atopic dermatitis and psoriasis (topically).

Toxic effects:

 Anaphylaxis.
 GIT disturbances.
 NO hirsutism or gum hyperplasia.
Drug interactions as cyclosporine.
 Hyper (glycaemia, tension and kalemia).
 Nephrotoxicity (more than CsA).
 Neurotoxicity (more than CsA).

What are the differences between CsA and TAC?

 TAC is more preferred than CsA due to:
 TAC is 10-100 times more potent than CsA in inhibiting immune responses.
 TAC has decreased episodes of rejection.
 TAC is combined with lower doses of glucocorticoids.
 TAC is more nephrotoxic and neurotoxic.
 Sirolimus (SRL - (Rapamycin) ):
 Macrolide antibiotic.
 It is not a calcineurin inhibitor, but inhibits the response of T cells to IL-2 and
thereby blocks activation of T- & B cells.
 Sirolimus inhibits the response of T cells to IL-2 and thereby blocks
activation of T- & B cells.
 SRL blocks the progression of activated T cells from G1 to S phase of cell
cycle (Antiproliferative action).
 Inhibits B cell proliferation & immunoglobulin production.

Inhibitors of cytokine gene expression (Corticosteroids):

Prednisone, Prednisolone, Methylprednisolone and Dexamethasone have both
anti-inflammatory action and immunosuppressant effects.

Anti-inflammatory action:

 Induce lipocortin-1 synthesis, which binds to cell membranes preventing the

phospholipase A2.
 This leads to diminished eicosanoid production and cyclooxygenase
expression, decrease production of inflammatory mediators as
prostaglandins, leukotrienes, histamine, and bradykinin.

Immunosuppressant action:

 Suppress both T&B cells by decrease production of cytokines IL-1, IL-2,

interferon, reducing both B cell clone expansion and antibody synthesis
respectively.

Kinetics Orally parenterally topically and by inhalation (asthma).

Dynamics Anti-inflammatory and immunosuppressant.
 Solid organ allografts & haematopoietic stem cell
transplantation.
Indications  Autoimmune diseases as refractory rheumatoid arthritis,
systemic lupus erythematosus, asthma or chronic rejection of solid
organ allografts..
Adverse Adrenal suppression, Osteoporosis, Hypercholesterolemia, Hyperglycemia,
Effects Hypertension, Cataract and Infection
 Azathioporine
CHEMISTRY:
 Is a derivative of mercaptopurine, a prodrug which is cleaved to 6-
mercaptopurine then to 6-mercaptopurine nucleotide, thioinosine monophosphate
(nucleotide analog, TIMP).
 Inhibits de novo synthesis of purines required for lymphocytes proliferation.
 Prevents clonal expansion of both B and T lymphocytes.

Pharmacokinetics:
 Orally or intravenously.
 Widely distributed but does not cross BBB.
 Metabolized in the liver to thiouric acid (inactive metabolite) by xanthine
oxidase and excreted primarily in urine.

Drug Interactions:
 Co-administration of allopurinol with azathioprine may lead to toxicity due to
inhibition of xanthine oxidase by allopurinol.

USES:
 Acute glomerulonephritis, Systemic lupus erythematosus, Rheumatoid
arthritis, Crohn’ s disease and autoimmune hemolytic anemia.

Adverse Effects:
 Bone marrow depression: leukopenia, thrombocytopenia,
 GIT toxicity, Hepatic dysfunction and Increased risk of infections.
 MYCOPHENOLATE MOFETIL
Chemistry:
 Is a semisynthetic derivative of mycophenolic acid from fungus source.

Metabolism:
 Is a prodrug, hydrolyzed to mycophenolic acid, it is extensively bound to
plasma protein, metabolized in the liver by glucuronidation and excreted in urine.

Mechanism of action:
 Inhibits de novo synthesis of purines.
 Mycophenolic acid is a potent inhibitor of inosine monophosphate (IMP),
crucial for purine synthesis which leads to deprivation of proliferating T and B
cells of nucleic acids.

CLINICAL USES:
 In solid organ transplantation.
 Hematopoietic stem cell transplant patients.
 Combined with tacrolimus as prophylaxis to prevent graft versus host
disease.
 In autoimmune disorders: Rheumatoid arthritis, & dermatologic disorders.

Sulfapyridine Anti-bacterial
Peptic ulcer and chron
5-ASA Anti-inflammatory
disease
 Antibodies:
 Sometimes used as a quick and potent immunosuppressive therapy to prevent
the acute rejection reactions.
Polyclonal antibodies: Monoclonal antibodies:
Antilymphocyte globulins (ALG). Basiliximab.
Antilymphocyte globulins (ALG). Daclizumab.

Polyclonal antibodies:
Bind to the surface of circulating T lymphocytes, which are phagocytosed in the
liver and spleen giving lymphopenia and impaired T-cell responses & cellular
immunity.

Kinetics:
 Given i.m. or slowly infused intravenously.
 Half-life extends from 3-9 days.

Adverse Effects:
 Antigenicity.
 Anaphylactic and serum sickness reactions (Fever, Chills, Flu-like syndrome).
 Leukopenia, thrombocytopenia.
 Risk of viral infection.

Monoclonal antibodies:
 Are IL-2 receptor antagonists, Bind to CD25, Block IL-2 stimulated T cells
replication & T-cell response system.

Given I.V.
Drug Basiliximab Daclizumab
Potential More potential Less potential
Half life 7 days 20 days

Glycoprotein Ilb/IIIa inhibitors: Abciximab

Rho (D) immunoglobulin:
 Is a human IgG preparation that contains antibodies against red cell Rho (D)
antigens.
 Maternal antibodies to Rh-positive cells are not produced in subsequent
pregnancies, and hemolytic disease of the neonate is averted (‫)بتم تجنبها‬.

Infliximab
Is targeted against TNF-α, a proinflammatory cytokine, and thereby decreases
formation of interleukins and adhesion molecules involved in leukocyte
activation.
 Infliximab induces remissions in treatment-resistant Crohn’s disease.
 In combination with methotrexate, infliximab improves symptoms in patients
with rheumatoid arthritis.
 It also is effective in the treatment of ulcerative colitis, ankylosing spondylitis,
and psoriatic arthritis.
 Infusion reactions and an increased rate of infection may occur.

Etanercept:
 Is not a true Mab, binds with high affinity to TNF-α.
 Etanercept is used in arthritis, psoriasis, and ankylosing spondylitis, and it is
being investigated in other inflammatory diseases.
 1. Salmonella
 Gram negative bacilli, Facultative anaerobic.
 Belong to Enterobacteriaceae (Gammaproteobacteria).
 Motile by peritrichous flagella (distributed over the entire cell).
 Tolerate stomach acidic pH.
 Live in intestines of all vertebrates.
 Not part of human normal microbiome {Potential pathogen}.
 Encapsulated bacteria.
 Contain endotoxin.
 Contain O antigen.
 Contains pili which encoded by plasmids.
 Caused osteomyelitis (for sickle cell disease patients).
 Positive for hydrogen sulfate (H2S)

Classification of Salmonella:

Antigenic structure and Virulence factors:

1) O antigen {somatic antigen}:

 Antigenic variation.
 Thermostable.
 2) H antigen:

A. Phase I H antigens:
Responsible for immunological identity expressed by some serotypes.
B. Phase II antigens:
Non-specific antigens, found in many other serotypes.

3) Vi (virulence) = capsule:

 Superficial of the somatic antigen.

 Found only in serotypes S.Paratyphi C, S.Dublin, and S.Typhi.
 Heat sensitive.

4) Salmonella Pathogenisity

A. Salmonella Pathogenisity Islands (SPI 1):

Encodes for cell invasion, intracellular survival and the production of Vi antigens
capsule.
B. Salmonella Pathogenicity Island 2:
Encodes genes essential for intracellular replication

Pathogenesis of Salmonella Typhi:

Ingestion  Mesenteric lymph nodes  Blood circulation (macrophage) 
Reltculoendolhelial system (spleen  Liver  Gallbladder {associated with
gallstone})  Peryer’s patches of small intestine.

Typical Enteric fever stages:

First week Bacteremia
Splenomegaly.
Second Liver necrosis.
week Involvement of RES.
Ulceration of the peyer’s patches.
Third week Intestinal bleeding.
Intestinal ulceration.
Chronic Chronic carrier.
stage Involvement of other organs.
Transmission of S. Typhi S. Paratyphi:
 Humans are the only source of these bacteria.
 Acquired through consumption of water or food contaminated by feces of an
acutely infected or convalescent person or a chronic, asymptomatic carrier.
 Risk is high in - low and middle- income countries.
 Infection may occur due to travelling to endemic areas.

The Diagnosis of Enteric fever: {Widal test}

The most accurate bone marrow 90%
The second accurate blood cultures 80% – 90%
The third accurate feces cultures 50% – 80%
The least accurate urine culture 10% – 25%

 Infection depends on the dose, as the concentration of Salmonella must be

≥ 105 for infection to occur.

Enteric fever treatment & prevention

Treatments:
1) Compensate the lost electrolytes; Fluid and electrolyte replacement in cases of
diarrhea.
2) Antibiotic treatment:
 Chloramphenicol, Trimethoprim and sulphamethoxazole (co-trimoxazole),
Ampicillin.

 Fluoroquinolones

 Cephalosporins (e.g: ceftriaxone and cefaperazone) in cases of resistance

to ciprofloxacin
Note: Antibiotics may prolong excretion of salmonellae in the faeces.

Prevention:
1) Vaccination: live attenuated mutant strain of Salmonella Typhi, Vi capsular
polysaccharidevaccine.
2) Improve hygiene and sanitation.
 2. Brucella
Aerobic, gram- negative coccobacilli.
Belong alphaprotobacteria.
Facultative intracellular.
Noncapsulated.
Non motile.
Cause zoonotic infection called brucellosis (malta fever, undulent fever).
In animal hosts, the Brucella lives as an intracellular parasite in organs such as the
uterus and placenta.
It has been proposed that there is only B. melitensis based on DNA-DNA
hybridization.
 Some studies depend that B. melitensis is the only species, but there are
many species, the most important of which are: B. abortus and B. suis,
where the most dangerous is B. melitensis.

Antigenic structure and Virulence factors:

A) Antigenic structure:
 Two antigens part (epitopes) of LPS are recognized: A&M.

 The A&M ratio varies from one brucella to another.

 B.melitensis has the highest concentration of M antigen and cause
of serious infections.
B) Virulence factors:

Inhibits:
Phagosomal fusion and lysosomal destruction → allows
Lipopolysaccharide intracellular survival.
(LPS) Complement deposition (opsonization).
Endotoxin:
Less toxic than in other species of bacteria.
Weak immune system inducer.
Superoxide Provides defense against oxidative burst
dismutase and activity → allows intracellular survival.
catalase:
Type IV secretion Multi-protein complex.
system: Allows Brucella to reach and replicate within a cell’s
endoplasmic reticulum.

Brucella melitensis Goat, sheep and camels.

B. abortus the consumption of raw milk
B. suis Domestic swine populations
Pathogenesis and clinical manifestations
 Brucella approved that caused apportion of animals.
 How does Brucella bypass stomach acid and bile salt?
It secretes the enzyme urease, which breaks down the amino
stomach acid acids and highlights the amine group, which is very basic,
raising the pH to neutralize the stomach acid.
bile salt It secretes the enzyme cholylglycine hydrolase which performs
the hydrolysis of bile salt.
The Brucella into the peyer's patches and then into the blood.

Transmission of Brucella
Ingestion Spoil milk
Direct contact Touching the wool )‫ (صوف‬of an infected
animal.
Inhalation Dust from wool or other dried material
of infected animals.
Accidental inoculation Laboratory workers who handle
culture of the organism.
Transplantation and blood transfusion Very rare.
Diagnosis of Brucella:
1) Culture:

 Definitive.
 Done in the first week.
 Shows positive within 3-7 days.
 Longer incubation (up to 6 weeks) may be needed.
 Bone marrow is the gold standard (most sensitive).
 Requires special culture techniques.

2) Serum tube agglutination test (SAT):

 Measures antibodies against a Brucella antigen (smooth lipopolysaccharide).

 Evolution titers (fourfold increase in the titer between acute and convalescent
serum obtained ≥ 2 weeks apart).
 Provides a definitive diagnosis.
 Limited by the time required.
 Positive titers providing a presumptive diagnosis:
 1:160 outside endemic regions.
 1:320 within endemic regions.

3) Enzyme-linked immunosorbent assay (ELISA):

 Comparable sensitivity and specificity to SAT.

 Tests for immunoglobulins against cytoplasmic
proteins.

4) Rose Bengal agglutination

 Can be used as a screening tool.

 Rapid.

5) Polymerase chain reaction ((PCR).

 Detects Brucella DNA.

 Can provide a presumptive diagnosis.
The prevention & treatment of Brucellosis:
The treatment:
1) Most cases of brucellosis are mild and require no treatment.
2) Combinations of antibacterial drugs: doxycycline and rifampin
or streptomycin for several weeks.
3) Vaccination:
 An attenuated vaccine for animals exists.
 Vaccine usually is not given to human because the vaccine sometimes
causes brucellosis.

The prevention:
Pasteurization of dairy products.
Immunization of uninfected domesticated animals.
Proper disinfection of urea and removal of reproductive tissues.
Slaughter {‫ }ذبح‬of infected animals.
 Plasmodium:
 Obligate intracellular parasite belong
apicomplexan group.
The Apicomplexa
 Are not motile in their mature forms
 Non motile.
and are obligate intracellular
 Contain enzymes that penetrate the host’s parasites.
tissues at the apical area.  They’re characterized by formation
of oocyst.
 Life cycle involves transmission between
 Cantains apical complex, which
several hosts.
contains proteins facilate cell
 Cause malaria, affects 10% of the world’s membrane pore “rhoptries and
population. micronemes”

 Vector borne disease by mosquito. They might be intracellular parasite:

Inside the red blood cells  Plasmodium.

Life cycle of parasites: Inside the white blood cells.

Inside the tissues.
1) Definitive host: The host that harbors the
sexually reproducing stage of a parasite.

2) Intermediate: The host in which the parasite undergoes asexual reproduction.

In plasmodium
Definitive host Mosquito
Intermediate host Human

 Plasmodium species:

Species Fever cycle (Hr) Clinical condition

Plasmodium falciparum 36 – 48 Malignant tertian malaria
Plasmodium malariae 72 Quartan malaria
Plasmodium ovale 36 – 48 Benign tertian malaria
Plasmodium vivax 36 – 48
Plasmodium knowlesi 24 Quotidian (‫ )كل يوم‬malaria

P. falciparum levels of parasitaemia may reach up to 30% or more of circulating

erythrocytes.
 Lower levels (<5%) are found with other species.
 More details about plasmodium species:
Species Disease / other details Infection site
Plasmodium  The most dangerous type. infects mature and
falciparum  Caused malignant, sever hemolytic. immature RBCs.
Plasmodium  Symptoms of the resulting diseases
ovale are milder than Plasmodium falciparum.
 Form hypnozoites, a dormant hepatic Attack only
phase (relapse phenomenon). reticulocytes.
Plasmodium
 In addition caused benign not
vivax
malignant.
Plasmodium  The same symptoms and diseases,
knowlesi but the difference is due to the different
Plasmodium life cycle / fever cycle. Attacks only
malariae senescent cells.

Plasmodium: Life cycle:

Three prominent stages:
1) Liver phase. 2) Erythrocytic cycle. 3) Sporogonic phase.
A) Infected mosquito bites human;
sporozoites migrate through
bloodstream to liver of human.
B) Sporozoites undergo schizgony in
liver cell; trophozoites called merozoites
are product.
C) Merozoites released into blood
stream from liver.
D) merozoites developed into ring stage
in RBC.
E) Ring stage grow and divides,
producing more merozoites.
F) merozoites are released when RBC ruptures; some develop into male and
female gametocytes or may infect new red blood cells.
G) Another mosquito bites infected human and ingests gametocytes.
h) In mosquito's digestive tract, gametocytes unite to form zygote.
I) Resulting sporozoites migrate to salivary glands of mosquito.
 Differential characteristics of Plasmodium Species
 Parasites alter the shape, size, lipid content and osmotic properties of RBCs,
resulting in the formation of vesicles that can be stained with Giemsa stain.

Erythrocytes
P. Falciparum P. Vivax P. Ovale
Schuffner dots - + +
Maurer dots + - -

Parasites
P. Falciparum P. Vivax P. Ovale
All sexual stages seen - + +
Band forms - - -
Double infections + - -
Double chromatin dots + - -
Banana shaped gametocytes + - -

 Note that (-) does not mean the absence of the property exclusively, but the (+)
confirms its existence very clearly; For example, Banana shaped gametocytes are
present at (P. Vivax and P. Ovale) but are more visible in (P. Falciparum).

Especial for “P. falciparum“

Plasmodium: Epidemiology
 Depends on climatic factors.
 Usually In tropical and subtropical areas.
P. falciparum Predominant (80% ) in tropical areas like Africa
P. vivax is Tolerant of lower ambient temperatures like the Mediterranean sea.

 Rare in very high altitudes; during colder seasons, in deserts.

 Affect all ages, high prevalence among children.
 Repeated infections or exposure results in relative immunity and less severe
disease.
 They are not protection antibody forming.
 Although it continuously changes antigens, exposure to a second infection
has less severe symptoms.
 Visitors to endemic areas are more severely affected.
 Transmission by contaminated blood transfusions or needle sharing can rarely
occur.

Plasmodium pathophysiology
 People with blood diseases such as sickle cell anemia, thalassemia or
hemoglobin C often tend to be less likely to get malaria because RBCs do not live
long.
 There are many virulence factors of Plasmodium in the attack, and they are as
follows:

Parasite Factors Host Factors Geographic and

Social Factors
Drug resistance Immunity Access of treatment
Multiplication rate proinflammatory Culture / economic
Cytokines factors
Invasion pathway Genetics (anemia) Political stability
Cytoadherence Age; no cerebral Transmission
malaria in infant intensity
Rosetting pregnancy
Antigenic polymorphism
Antigenic variation (PIEMP)
Malaria toxin “hemozoin”
 Virulence factors:
1) MicroRNAs (miRNAs):

 Small non-coding RNAs released into blood circulation.

 It is not coded for protein production, but Supports survival and
maintenance of the biological functions of the body.
 Biomarkers of specific organ dysfunction in malaria patients.
 The presence of miRNA indicates that the organ is inactive.
 Present in both humans, parasites and insects.
 The presence of (miRNA) in the human body or the insect supports the
growth of Plasmodium.

2) Circumsporozoite protein (CS):

 Outer protein coat of the sporozoites.

 Inhibits protein synthesis in infected hepatocytes.
 Enhance immune evasion.

3) Erythrocyte membrane protein 1 (PfEMP1):

 Expressed by asexual stages of P. falciparum.

 For the pathogenicity of severe malaria.
 Binding by CD36 “scavenger receptor”.

4) Complement receptor 1 (CR1):

 Receptor for the P. falciparum merozoite protein PfRh4.

 Enhance rosette (cluster) formation leading to blockage of blood flow in
brain capillaries.
 Central nervous system effect  cerebral malaria. Cerebral malaria  90% co-
exist with pneumonia.

5) Duffy blood group:

 Blood group antigen  {Fya and Fyb}.

 receptor for a family of proinflammatory cytokines termed chemokines
 Receptor for Plasmodium vivax or P. Vivax merozoites ligand.
 Duffy blood group absent in Africa  absent of P. Vivax
 6) ICAM1:

 Cytoadherence of Plasmodium falciparum–infected erythrocytes to host

endothelium.
 Found on endothelial surface.

7) HLA-polymorphism:

 Carriers of HLA Class I Bw53 and HLA class II DRB1*1302-DQB1*0501

protected against severe malaria.

8) RBC sialoglycoproteins (e.g glycoprotein A):

 P falciparum receptor site.

9) Glucose-6-phosphate dehydrogenase:

 Used to synthesize parasitic DNA.

10) Gerbich blood group: minor sialoproteins GPC and GPD:

 Maintains erythrocyte shape and membrane stability.

 Receptor for Plasmodium falciparum erythrocyte-binding antigen 140
(EBA140 or BAEBL).
 Mediates invasion into human erythrocytes.

The complex (anti-antigen) may accumulate on the kidney, causing transient

Glomerulonephritis

11) Malaria toxin “hemozoin”

 Plasmodium, which is inside the RBC, performs anaerobic metabolism,

where it increases the proportion of ATP and degradation hemoglobin,
producing the hemozoin  may cause hemolysis.
 P. falciparum caused acidosis as a result of metabolism.

Fever cycle:

After the RBCs burst, the plasmodium is released into the blood.

It is recognized by the macrophage and secretes IL-1, TNF-𝜶 , which is then activated by the hypothalamus; which raises
the body temperature.
Available Malaria Diagnostic Tests:
1) Clinical:

 Anemia.
 Increase liver enzyme expression, reticulocytes and jaundice.
 Black water fever.
 Dark urine.
 Thrombocytopenia  as a result of Rosetting formations; platelets life
span decrease.

2) Microscopy; “thick and thin blood films”

Thin blood smear: Allows us to determine the type of parasitic species

present, by studying the parasitic form (dots).
Thick blood smear: Allows us to determine the intensity of infection.

3) RTDs:

 We use it when the shape of the parasite is clear and easy to identify.
 Rapid diagnosis {serological}; 2-lines and contain lactose filter paper.
 When the parasite-laden blood binds to the antibody, the first line is
formed.
 The fluid completes the movement until the formation of the second line.

4) Highly sensitive RDTs.

5) Molecular test.
6) Ultra-sensitive molecular.

Indication:
Clinical Microscopy and RTDs HS-RTDs and US molecular
molecular
Symptoms 100 – 200 𝒑𝒂𝒓𝒂𝒔𝒊𝒕𝒆𝒔 / 𝝁𝒍 2–10 <1

 More sensitive diagnostic tools are required with cases of lowdensity

infections.
Treatment:
 Antimicrobial drugs:
 Quinolines.
 Phenanthrene methanols , Qinghaosu (Artemisinin), Quinones.
 Folate Antagonists (Trimethoprim, pyrimethamine, proguanil).

Management:

1) Draining wetlands and removing standing water.’

2) Insect repellents (‫)طارد الحشرات‬.

3) Mosquito netting and protective clothing.

 Especially when travel into endemic area.

4) Vaccination:
 Do not give lifelong immunity.

Clinical outcome of plasmodium infection:

 Asymptomatic infection.
 Fever (symptomatic).
 Sever malaria (metabolic acidosis, severe anemia, cerebral malaria).
 Death.

Fever cycle: releasing parasites into blood.

Incubation period: The period it takes for a microbe to adapt to the environment
and then multiply begins.
Hemolysis occurs as a result of:
 Following replication of merozoites.
 Hemozoites “parasitic toxin”
 Babesiosis
 Piroplasm (‫ )مثل االجاص‬parasite belong apicomplexan.
 Intracellular parasite infects RBCs only.
 Zoonotic disease of humans.
 Caused by the hemoprotozoan.
 For infection to occur, the parasite must attach to the skin for 24-36 hours.

Three Host in life cycle:

Definitive host Ixodes scapularis tick
Intermediate host Rodent mainly white-footed mice.
Accidental host Human
 Naturally, infected Ixodes bite and infect one of the white-footed mice, then
the Ixodes again infect some mice, absorbing the damaged RBC and biting the
human being, so they are called the accidental host.

 There is no study that proves that mice that have acquired the infection
transmit the infection to humans.
 The infective part is the sporozoites but quickly turns into merozoites in
the mouse.

Three main species:

Babesia microti North America and
B.ducani New England
Babesia divergens in Europe

Life cycle:
 The same life cycle of
Plasmodium, but the difference is
that it does not go through the liver
stage.
Babesia: Transmission
1) Transfusion of packed red cells (blood - plasma).
2) Rarely transplacental.
3) Reported in immunocompetent and immunocompromised individuals.

Immunocompetence is the ability of the body to produce a normal immune response following
exposure to an antigen.
 Immunocompetence is the opposite of immunodeficiency or immuno-incompetent
or immuno-compromised.

Babesiosis: Clinical Manifestations

1) Asymptomatic to severe illness:

 Presence of associated tick-borne illnesses such as ehrlichiosis,
anaplasmosis, and borreliosis.
 The presence of risk factors:
 Old age.
 Asplenia / hyposplenism.
 HIV-infected individuals.
 Malignancy.
 Patients on immunosuppressive agents:
 Rituximab and on tumor.
 Necrosis factor-alpha inhibitors like infliximab and etanercept.

2) Incubation period:
 Depends on the route of transmission:

tick bite 1–4 weeks

transfusion of contaminated blood Up to 6 weeks

3) Signs & symptoms: The same as Plasmodium, but no cerebral malaria.

Highly chills, sweats, headache, anorexia, vomiting,
nonspecific diarrhea, abdominal pain, joint pain, dark-colored urine-------ETC.
Signs: Fever, pharyngeal erythema, hepatomegaly, splenomegaly, jaundice,
glomerulonephritis and retinal hemorrhages.

4) Blood parameters& chemistry:

 Hemolytic anemia with elevated reticulocytes count.
 Thrombocytopenia and normal to slight decreased WBC.
 Elevated liver enzymes, BUN, Creatinine, proteinurea.

Kidney function enzymes

 Babesiosis: Diagnosis
1) Microscopy:
 Giemsa stain of blood films: Can be mistaken for
malaria.
 Look for intraerythrocytic ring forms;
 In the Babesia maltese crosses (merozoites in
tetrads).

 Extracellular forms (parasites);

 Banana shape in plasmodium.

2) Molecular tests:
 Highly sensitive and specific.
 Useful for low parasitemia.
 Confirm infection.
 Determine species.

3) Serologic tests:
 Indirect florescence antibody “IFA” and ELISA.
 In endemic areas support or confirm the diagnosis of human babesiosis.
 Useful in screening blood donors for Babesia infectns.

Treatment and management:

Almost the same as Plasmodium;

 Qinine and clindamycin, alternatives atovaquone and azithromycin.
 Avoidance of areas known to be tick infected.
 Using appropriate insecticides.
 Wearing appropriate clothing.
 Performing daily tick inspections if one ventures into wooded areas where
ticks live.
 Yersinia pestis
 Gram negative facultative anaerobe coccobacilli.
Bipolar staining: the ends of
 They do not form spores or capsules, but Y. the bacilli stain more intensely
pestis produces a capsule-like envelope. than the middle.

 Non motile at room temperature.

 Retain staining at the ends of the cells
(bipolar staining).
 Member of Enterobacteriaceae
(Gammaproteobacteria).

 Catalase positive, but oxidase-negative.

 Evade free radical inside macrophage.
 Contains pPCP1, PMT1 and pCD1 plasmids
carry virulence factors.
 In addition, outer membrane carry other
virulence factors.
 Vector borne disease.

Virulence and pathogenicity:

1) Plasminogen activator protease: promotes adherence to basement membranes,
prevent opsonization and phagocytic migration, and facilitates metastatic spread.

2) Type III secretion system (TTSS):

 Yersinia outer proteins (YOPs): prevent phagocytic killing of the bacteria.

 Inhibit production of cytokines reducing the inflammatory immune response to
infection.

3) Adhesin (YadA): attachment, complement inhibitor.

4) Lipopolysaccharide endotoxin: not recognized by TLRs, trigger inflammation,

fever, and blood clotting.

 Phagocytes not start or incomplete if start.

5) F1 protein for capsule.

Yersinia pestis: pathogenesis
 Plague is divided into three main types — bubonic, septicemic and pneumonic —
depending on which part of your body is involved.

1) Bubonic plague
 It's named after the swollen lymph nodes (buboes) that typically develop in the first week after
you become infected.
Possible bubonic plague signs and symptoms may include:

 Sudden onset of fever and chills.

 Headache.
 Enlargement of lymph node.
 Gastritis and arthritis.

2) Septicemic plague
 Septicemic plague occurs when plague bacteria multiply in your bloodstream.
Possible Septicemic plague signs and symptoms may include:
 Nausea, vomiting, diarrhea, and abdominal pain.
 Disseminated intravascular coagulation (DIC).
 Gangrene in the limbs may turn black.
 Organs malfunction (lung, spleen).
 Death if not treated.

3) Pneumonic plague
 Pneumonic plague affects the lungs. It's the least common variety of plague but the most
dangerous.

 Signs and symptoms can begin within a few hours after infection, and may include:

 Fever.
 Headache.
 Weakness.
 Cough.
 Hemoptysis.
 Dyspnea.
 Chest pain.
 Yersinia pestis life cycle:

 First stage: sylvatic cycle

 Lifespan spent cycling between wild animals and vectors {flea}.
 In this case, the infection is not transmitted from the animal to humans.
 The flea feeds on the blood of an infected animal, and it takes infection from it to bite
humans later.

 Second stage: urban cycle

 City rate congregate and infect the flea.
 The main source of infection is the flea, and sometimes it is transmitted from mice
directly to humans, but it is rare.
 The only way to transmit infection with pneumonic plague is through coughing.
 Plaque: Diagnosis
Method Part to diagnosis
Culture: bubo aspirate, blood or sputum
Wrigh’s, Giemsa, wayson and Gram stain. bubo aspirate, sputum
Molecular tests: PCR for FI (capsule) antigen in bubo aspirate.
Immunoflourescence: Antibody applied to bubo aspirate or sputum.

Method Sensitivity Specify Time

Culture. Highly sensitive highly specific 2-3 days
Wrigh’s, Giemsa, wayson moderate Moderate Rapid within
and Gram stain {bi polar}. sensitive specific minutes
Molecular tests: highly sensitive Highly specific Rapid within hours
Immunoflourescence: moderate highly specific Rapid within
sensitive minutes

 We put the mucus on a slide with an antibody made against the antigen Yersinia, and
upon interaction, the complex is stained with fluorescence.

Plaque: Treatment & management

Treatment Protective measures Prevention
Reduces the risk of death Isolation of people with Rodent control
(11%). pneumonic plague
Pneumonic or septicemic: Avoidance of
antibiotics must be started Limiting access to their endemic areas
within 24 hours after rooms
symptoms appear
Wear a mask, eye protection, Eradication of
Streptomycin or gentamicin a gown, and gloves fleas on
domestic pets
Doxycycline, levofloxacin, Chemoprophylaxis with
and ciprofloxacin. doxycycline or ciprofloxacin
 Rickettsia
 Obligate intracellular.
 Needs NAD for metabolic function.
 Gram negative coccobacilli, aerobic.
 Gram-negative only in classification and when dyeing, we do not dye it
with a gram stain, but it is more correct to dye it with a dye
immunoflorousence.
 Life cycle involves both an arthropod vector and a vertebrate host.
 Arthropod vectors: ticks, lice, fleas, or mites.
 Mainly infect endothelial cells.
 Rickettsiaceae comprises of one pathogenic genera.
 Some studies clime that Rickettsiaceae comprises two genus;
Rickettsia and Orientia, but they differ in surface proteins.
 Two groups based on clinical manifestation:
 Spotted fever group (SFG).
 Typhus group.
 All rickettsia diseases include fever and rash.

Transmission of Rickettsiae
Nymph Adult
Rarely to infect human Main human infection

1. The fertilization and laying )‫ (تبيض‬of the female occurs.

2. Bacteria (Rickettsiae) are kept inside the eggs.
3. The eggs hatch and the bacteria are transferred to the larvae.
4. Now the larvae go one of two ways:
 Turn into Nymph.
 Somehow it was passed on to a rodent and then again swallowed up by the
Nymph.
5. Either the nymph transmits the infection to an animal or it becomes an adult;
when it becomes an adult, it is transferred to humans (main pathway).
 ‫العدوى ليست حيوانية اي انها‬
‫ال يمكن ان تنتقل من الحيوان‬
nymph ‫المصاب فقط من ال‬

Rickettsia Virulence Factors

1) Outer membrane protein:

 Adherence.
 Induced phagocytosis.
2) Phospholipase C:

 Escape phagocytosis.
3) Recruitment of actin:

 During replication within the cytoplasm, it secretes substances that recruit

actin for transport from one cell to another.
Spotted Fever Disease: Pathophysiology
Spread Necrotic eschar (‫ )مكان العضة‬creation by local spread into Vascular
endothelium tropism (‫( )تتنقل‬vasculitis).
Result 1) Increased vascular permeability:
 Hypovolemia.
 Hypotension.

2) Focal areas of endothelial proliferation  perivascular

infiltration: Leading to thrombosis and leakage of red blood cells
into the surrounding tissues.
Complications 1) Vascular lesions occur throughout the body.
2) Systemic manifestations: neurologic manifestations
(encephalitis), pulmonary and renal failure, cardiac abnormalities.

1) Spotted Fever Group:

A) Rocky Mountain spotted fever:
 Highest incidence: 60 - 69 years, highest Death: in children < 10 years.
 April and September.
 Fever, headache, rash, toxicity, mental confusion, and myalgia.
 Rash in palms and soles.
 Muscle tenderness: in gastrocnemius region.
 Complications: DIC, thrombocytopenia, encephalitis, vascular collapse, renal, heart
failure.
 B) Rickettsialpox:
 Self-limiting disease.
 Papulovesicle )‫ (حبة على راسها سائل‬and develops into black eschar in 3 to 5
days.
 A diffuse rash distributed randomly in the body.
 No rash in palms or soles.

2) Typhus group
Epidemic Louse Borne Typhus Fever Endemic (Murine) Typhus
 Headache, malaise, and myalgia. Maculopapular (‫ )حب صغير مفروش‬rash not
 Rash begins on trunk not extremities. petechial; starts on trunk and spreads to
extremities.

Spotted Fever Disease: Diagnosis

1) Clinical picture:
Important to begin treatment.

2) Serologic tests:
 Indirect fluorescent antibody (IFA):
 Sensitive and specific.
 Available in reference laboratories.
 Immunofluorescence or immunoenzyme methods:
Examination of skin lesions.
 Weil felix test (agglutination test):
 Cross reacting antibodies.
 Somatic antigens of non-motile Proteus.
 Not reliable: low sensitivity and specificity.

3) Culture:
 Cell cultures.
 Difficult and hazardous.
Spotted Fever Disease Management & Treatment

Risk Factors for fatal infection:

 Age.
 Glucose-6-phosphate dehydrogenase deficiency.
 G6PD; is a main source of energy in RBCs (NADP+  NADH).
 Delayed treatment.

Antibiotics:
 Highly effective if given during the first week of illness.
 Doxycycline for both children and adults.
 Tetracyclines & Sulfonamides are contraindicated.
 Affects on teeth color and late child growth.

Prevention:
Avoidance of tick contact.
Frequent tick removal in tick infested areas.
Vaccines are not licensed.
 Trypanosomiasis
Hemoflagellates:
Site of diagnosis Intracellular Extracellular
The presence of flagella Absent Present

Classification:
There are main two species; Trypanosoma cruzi or brucei.

Type T. Cruzi T. brucei

Site American Trypanosoma African Trypanosoma
Transmitted by kissing bug Tse Tse fly.
Main disease Chagas Sleeping sickness
Virulence factors Pore-forming protein: Escape Variable surface
phagosome. glycoprotein (VSG) coa:
Penetrin: Promote adhesion. invade immunity.

 Trypanosoma brucei can be subdivide into 3 species:

species T.brucei rhodesiense T.brucei gambiense T.brucei brucei
Harmful for Human Human Animal
Epidemiology West African East African ------------------------

African Trypanosomiasis: Life cycle:

1) The infected tsetse bug bites humans and injects the infective form (metacyclic
trypomastigotes) from it salivary glands.
2) The parasite divides by binary fission to become trypomastigotes.
3) It goes into the blood or the lymph (and returns from the lymph to the blood in
the nocturnal time).
4) A new tsetse insect feeds on the blood of the infected in the evening, taking
the form of trypomastigotes.
5) The trypomastigotes begin binary fission within the Gut of the tsetse and
become procyclic.
6) They complete fission to become epimastigotes.
7) The epimastigotes go to the salivary gland of the insect to transform into
metacyclic trypomastigotes and the loop repeats.
American trypanosomiasis: Life Cycle
1) During feeding, the next insect defecates on human skin, where its excrement
contains the infectious form "trypomastigotes".
2) The person feels itching and scratches his skin. The infected stool from the
injection site enters his body.
3) When entering the body, the parasite chooses one of two paths:
Intracellular (mesenchymal tissues) Extracellular
Amistigotes Trypomastigotes

4) In the same way, a new insect feeds on the blood of the patient in the nocturnal
time to take the parasite and infect another human.
 Note the life cycle inside the Gut of the insect:
Trypomastigotes  epimastigotes  trypomastigotes.

Type T. Cruzi T. brucei

Tike Kissing bug Tse Tse
Infective form trypomastigotes metacyclic trypomastigotes
Site of Infective form Stool Salivary glands
Diagnosis Tissue biopsy; amistigotes trypomastigotes
PBS; trypomastigotes

In hemoflagellate parasites, there is a structure called kinetoplast;

 It is present in the mitochondria and contains cyclic DNA for the production of
proteins and is not related to movement.
Types trypomastigotes epimastigotes Amistigotes
Kinetoplast : nucleus Posterolateral Centrally Near the nucleus
Undulated membrane Present Present ------------------------
Motility Motile Motile Non-motile
Site Extracellular Extracellular Intracellular
Transmission of Trypanosoma:
T. Cruzi T. brucei
Human  human Oral – fecal rout
Cattle  human Sexually, blood transfusion and transplant organs
Tse Tse  human Congenital or transplacental
Vector transmission

African Trypanosomiasis: Pathology and Pathogenesis

 Incubation periods (recurrent):
T.brucei rhodesiense T.brucei gambiense
Weeks to months Months to years

American Trypanosomiasis: Pathology and Pathogenesis {chagas}

Phases Acute phase Chronic phase

Harmful for For children For adult
Major signs Romana’s sign. Cardiomyopathy
Inoculation chagoma. Gastrointestinal Chagas
Maybe both of the above.
Diagnosis +Ve for: -Ve for smear.
Smear, culture or PCR +Ve for PCR
70 – 80 % of chronic Chagas is asymptomatic or indeterminate throughout life.
Trypanosomiasis: Immune Response

1) T.brucei:

 T-cell independent:

 IgM against surface glycoproteins.

 Parasitic destruction by lysis and opsonization.
 Production of polyclonal nonspecific heterophile antibodies: antibodies to
DNA, and rheumatoid factor.

 Formation of circulating Immune complexes:

 Immune complexes with invariant nuclear and cytoplasmic antigens.

 Responsible for anemia and vasculitis as a result of deposition.

2) T. Cruzi

 Antibodies against T.cruzi cross-react with host tissue.

Trypanosoma: Diagnosis, Treatment &Prevention

Diagnosis: Treatment: Prevention:

1) Agents that penetrate 1) Patient isolation
1) Microscopy: the blood–brain barrier and treatment.
“Suramin, pentamidine, 2) Using insecticides.
2) Animal inoculation: or eflornithine” 3) Fly control.
 Serologic tests.
 Molecular tests. 2) Use of drugs that may
 Xenodiagnosis (chagas). reduce severity of acute
 Culture (N, N, N). chagas disease
(Allopurinol).

Type T. Cruzi T. brucei

Diagnosis Tissue biopsy; amistigotes trypomastigotes
PBS; trypomastigotes
 Lymphatic Filariasis
Nematodes (cylindrical).
Thread like worms.
Pseudocoelomate round worms.
Male and female are together inside host.
Vectors and intermediate host:

Anopheles Culex Aedes and Mansonia mosquitoes

Africa America the Pacific and in Asia

 Carry endosymbiotic (‫ )تكافلية‬bacteria Wolbachia in their gut.

 Wolbachia is very important in Filariasis life cycle.

Filariasis three species: Timory in Indonesia

 Wuchereria bancrofti.
 Brugia malayi.
 Brugia timori. The same in everything

Wuchereria bancrofti Brugia malayi and Brugia timori

No nucleus Two nucleus in the tail.

 Wuchereria is transmitted from insects directly to humans. There is no

intermediate animal.
In general, the filariasis of all kinds settles in the lymph, but by research, we
examine the blood, not the lymph, especially at nocturnal (9pm – 2am); because the
parasite moves from the lymph to the blood during that period, and the activity of
the vector is also great during this period.

Life cycle:
Mosquito salivary gland L3 Filariasis larvae
Human lymph L4 Male + Female
Human blood L1 Microfilaria
Mosquito thorax L2 Rhabditiform larvae
Life cycle events:
1) The insect injects from its salivary gland the infective form “Filariasis larvae”.
2) During 9-14 days it turns into a "L4".
 After 6-12 months, the meeting takes place here and produces the male and
the female.

3) It produces a membrane and turns into a "microfilaria" during its passage into
the blood.
4) The insect picks up the infective form and enters the gut and then passes into
the thorax to become "Rhabditiform larvae".
5) It goes back to the salivary gland and belongs to type L3.

Filarial: Pathology and pathogenesis

Depend on the vitality of the parasite:
 Live filarial worms.
 Dead filarial worms by antibiotic.
Live filarial worms:
 One of the most important signs that the lymph nodes are enlargement,
especially in the groin area.
 Cause vasodilatation  Lymphangioactasia.
 Lymphangioactasia + spread  lymph occlusion  lymph vessel dysfunction.
 Secondary infection from; staphylococcus and streptococcus.

Dead filarial worms: Risk factors:

 Release of Wolbachia antigens  secondary infection. Age.
 The rupture of lymph vessels  chyluria. Antifilarial drugs.
Metabolic factors.
Both types lead to Elephantiaisis.

Clinical Aspects:
 Acute manifestations:

 Filarial fever.
 Lymphadenitis, lymphedema, lymphangitis, epididymoorchitis.

 Chronic manifestations:

 Hydrocele,chyluria,elephantiasis.

Occult filariaisis:

 Due to hypersensitivity reaction to microfilarial antigens; Microfilariae are

not found in blood.
 Classical features of lymphatic filariasis are absent.

 Tropical pulmonary eosinophilia:

Massive enlargement of the lymph nodes and spleen in children or chronic

cough.
Nocturnal bronchospasm, and pulmonary infiltrates in adults.
Intense eosinophilia, elevated levels of IgE, high titers of filarial antibodies.
Lymphatic Filarial: Diagnosis
 The most specific test is for IgG4.

Lymphatic Filariasis: Treatment and Prevention

Treatment:
Diethylcarbamazine(DEC): Albendazole:
Eliminates microfilariae from the blood. Affects both microfilariae and adult worms.

 Antihistamines and corticosteroids: for allergy.

 Doxycycline; kills endosymbiotic Wolbachia bacteria.
 Secondary bacterial infection treatment.
 Pressure bandage or plastic surgery for elephantiasis.

Prevention:
 Addition of DEC to salt.
 Mosquito control.
 Visceral Leishmaniasis:
 Hemoflagellates.
 Obligate intracellular parasites inside phagocytes.

 Life cycle involve two hosts:

Definitive host Intermediate host
Mammalian female sand fly

Infective stage: in macrophages

promastigote Amastigote

Visceral Leishmania vs cutaneous Leishmania

Visceral Leishmania cutaneous Leishmania
You can get out of the macrophage It stays inside the neutrophil and you
and injure many organs such as the can't get out of it.
marrow and reticuloendothelial cells.

Types trypomastigotes epimastigotes Amistigotes promastigote

Kinetoplast : nucleus Posterolateral Centrally Near the nucleus Present
Undulated membrane Yes Yes -------------------- No
Motility Motile Motile Non-motile ----------------
Site Extracellular Extracellular Intracellular extracellular

Visceral Leishmaniasis: Virulence Factors

Protect against hydrolytic enzymes of Disseminate through the bloodstream
macrophage phagolysosome: to visceral organs:
 Lipophosphoglycan (LPG).  Resistance to microbicidal
 Membrane bound acid properties of normal serum.
phosphatase.  Ability to survive 37°C.

Bone marrow aspirate showing intracellular and extracellular

Leishman Donovan bodies, Giemsa stain, 1000X.
Epidemiology:
L. donovani L. infantum L. chagas
In tropics and subtropics: Central and South
adults Children / infant America, central and southern Asia, Africa,
Europe, and the Middle East.

 High incidence in patients with HIV infection.

Pathogenesis:
 Symptoms are not visible; but within (3-12) months, it swells and becomes
reactivated, causing pigmentation.
 Enlargement of liver, spleen, lymph nodes, bone marrow, small intestine, skin
pigmentation.
 Double quotidian fever: twice/day, reappear at irregular intervals.
 Diarrhea, malabsorption, weight loss.
 Anemia, tachycardia, thrombocytopenia.
 Leukocyte count < 𝟒𝟎𝟎𝟎/ 𝐦𝐦𝟑; agranulocytosis.
 Elevated IgG, NOT PROTECTIVE.
 Glomerulonephritis due immune complexes.
 After the treatment, a macule is formed on the skin and then transformed into a
pigmented nodule to become its name; Post-Kala azar dermal leishmaniasis (PKDL).

Diagnosis:
Aspirate: bone marrow, liver, spleen, or lymph nodes.
Culture (N, N, N media).
Serological tests: DAT, ELISA, IFAT, rK39Ag test.

Visceral Leishmaniasis: Management &Treatment:

Insect control  Use of insecticides
measures
Elimination of  Treating human cases.
mammalian  Destroying infective dogs.
reservoirs
 Miltefosine: Less toxic and safe.
Treatment  Pentavalent antimonial drugs: lower percent of fatal cases.
 Pentamidine, amphotericin B, or liposomal amphotericin B: use for resistant
cases, may be toxic
 Borrelia
 Belong Spirochaetaceae family.
 Slender, rod shaped, with multiple (7-20) axial flagella.
 Microaerophilic; can live in small amount of oxygen, but not in high
concentration.
 Stain by Giemsa, Wright stains.
 Can be cultured artificially, but need special growth requirements;
Nacetylglucosamine, fatty acids, amino acids, nucleic acids.
 Contain multiple circular and linear plasmids.
Species Diseases Vector
Borrelia burgdorferi: Cause Lyme disease tick.
B recurrentis: Epidemic relapsing fever louse
B hermsii: Endemic relapsing fever tick

Virulence Factors:
 Variable outer membrane proteins: due to recombination
between linear plasmids.
 Use of manganese in place of iron in enzymes and electron
transport chain: Circumventing body’s natural defense
mechanisms -the lack of free iron in human tissues and fluids.
 Use of SodA (Superoxide dismutase): metalloenzyme to
degrade ROS.
 Not contain lipid A, but contains endotoxin similar lipoprotein.
OspA OspC
Bind to fibronectin (enhance
Depend on temperature, colonization) and serum factor H
pH), for binding to ticks (provide complement resistance,
midgut, low in human. protect against phagocytosis and
antibody).
Of course, OspA is little in humans, but in the case of arthralgia it is high.
Serum factor H is important in activating the alternative complement pathway, and
Borrelia inhibits it so that the classical pathway is activated.
Relapsing fever: Epidemiology
1) Endemic (tick-borne): Borrelia hermsii;
 North America.
Reservoir: Rodents, rabbits, birds, lizards.
accidental host: Human
Infected tick Live years, trans-ovarial passage.

Trans-ovarian passage; the bacteria build up inside the

 Infection occur at night, by bite.
insect's eggs, and they pass them on to the host in the
 Associated with Wooded areas. form of larvea or nymph, both of which cause infection.

2) Epidemic (louse-borne): Borrelia recurrentis;

 East and Central Africa and, Peruvian Andes.
Reservoir and host: Human
Infected louse: Live 2 months and No trans-ovarial passage.
Infection: Crushing of louse by scratching.

 Increase risk: Over crouding, low hygiene, poverty.

 Bacteria multiply in lice hemolymph not feeding parts or excrement.
 Neither eggs nor intermediate mammals, where when the skin is rubbed in
the place of the louse, transmission of infection occurs (excrement).

Relapsing Fever: Pathogenesis, Immunity& Manifestations

 After short appearance in blood it’s Sequestered in internal organs.
 Antibody production due to altered OMPs  fever.
 The symptoms appear after 7 days (Incubation period) of infection.
Relapsing cycles:
with tick borne (endemic) with louse borne( epidemic)
one – two per week three – four per week

 The louse born is more severe than tick.

 Massive spirochetemia develops, high fever (for 1 week), rigors, severe
headache, muscle pains, and weakness.
 In louse born: myocarditis, cerebral hemorrhage, and hepatic failure, death
(rate40%).
Relapsing Fever: Diagnosis, Treatment, Prevention:

Diagnosis:
 Microscopy: Giemsa or Wright staining of blood smears.
 Spirochaetaceae shown in PBS.
 Culture and serology: in reference laboratories.

Treatment:
 Doxycycline or tetracycline (louse-borne) and alternatives; erythromycin
and ceftriaxone.
 Risk of jarisch-Herxheimer; 24 hrs following treatment in high level of
bacteremia.
 After treatment with antibiotics, Borrelia wall may rupture, accompanied by
endotoxin shock caused by the release of an endotoxin similar to
lipoprotein, and this results in several problems, including; Kidney failure,
heart failure, rash, headache and arthralgia.

Prevention:
 Insecticide treatment.
 Rodent control.
 Improve hygiene.

Lyme disease; Borrelia burgdorferi:

 Endemic: in regions of United States, Canada, and temperate Europe and Asia.
 Risk of tick bite in wooded habitat.
Reservoir: White-footed mice.
Vector: Ixodes ticks.
Human infection By tick nymph.
Animal infection Nymph or larvae
life cycle Completed within 2 years.
  No deer, no disease 
Life cycle of lyme disease:
 The tick must be on the deer to complete its life and without the presence of
the deer the life cycle is not complete.
1) When it is on the deer, it meets and lays eggs, where it feeds on the deer's
blood and does not cause infection to the deer, but it goes down to the ground
and lays its eggs in the ground.
2) In spring:
 The eggs hatch to become larvae.
 During this period, it can infect animals.
3) In winter:
It turns into nymph and either infects animals or humans until spring comes.
4) Either the insect develops or it goes on the deer.

Borrelia burgdorferi: Pathogenesis& Immunity

Pathogenesis:
Osps of B burgdorferi: Facilitate persistence of infection by other bacteria.
Use of Host proteases: Facilitate dissemination.
Peptidoglycan and outer Induce inflammation.
membrane components:
Immune modulation: inhibition of mononuclear and NK function, lymphocyte
proliferation, cytokine
Chronic disease: Autoimmune arthritis due to humoral response to OspA.

Immunity:
Humoral; IgG develop after weeks or months after primary
infection.
Activation of classical complement.
No protective long lasting immunity.
Lyme disease: Manifestations, Diagnosis, Treatment & Prevention

Manifestation:
 Bull's eye ring: annular lesion.
 Erythema migrans.
 Fluctuating arthritis: may become chronic.
 Fever, fatigue, myalgia, headache, joint pains, and mild neck
stiffness, neurological or cardiac abnormalities.

Diagnosis:
 Typical clinical findings.
 Culture: need special media and experience.
PCR: B burgdorferi specific DNA.
EIA (screening Ab) confirmed by immunoblot (detect specific Ag): in later stages.
Immunoblot
1) There are special proteins secreted by Borrelia, we put them on the
electrophoresis device, and they separate and move from negative to positive,
forming a band.
2) We put the used gel on a cellulose filter paper.
3) We combine the filter paper with an antibody from the patient and another
marked antibody, where when the two antibodies interact together, the result
appears, and the first one alone (from the patient) does not give a result.

Treatment& Prevention:
 Doxycycline and b-lactams (e.g amoxicillin, cefuroxime) and alternatives:
macrolides (azithromycin, clarithromycin).
 No treatment for chronic cases.
 Prevention: protective clothing, nymph removal, insect repellents, Prophylactic
doxycycline in endemic areas.
 Francisella tularensis
 Family: Francisellaceae.
 Tiny, Gram negative coccobacilli.
 Facultative anaerobe , encapsulated.
 Cause: Tularemia (Rabbit fever).
 Common in North America.
 Morphologically similar Brucella.
 Grow on Chocolate agar and Cysteine–glucose blood agar.
 Zoonotic & arthropod born infection;
 Skin bite, cut wound, inhalation, ingestion, Human-human (pneumonic
tolaremia).
 Infective dose <100.
Vectors in animals Ticks and deer flies.
Reservoir: Ticks (transovarial)

Pathogenesis:
 LPS not recognized by TLRs.
 Escapes phagosome to macrophage cytoplasm.
 Lesion at the site of infection, becomes ulcerated and early bacteremia may
occur.
 Multiply within hepatocytes, kidney, alveolar epithelial cells and others.
 It infects reticuloendothelial organs, forming granulomas.

Immunity:
 T - cell–dependent: (either CD4+ or CD8+ cell) is the main resistant mechanism.
 Natural acquired long lasting immunity is also developed (protective antibodies).
Life cycle:
A) During phagocytic ingestion of bacteria: the
bacteria interact with special components in the
phagocyte "early endocyst, late endocyst" that help it
to multiply inside the phagocyte and do not interact
with the lysosome.
B) It is released from the phagosome and replicated in
the cytosol.
 There are two possible occurrences of infection:
1) Infecting more phagocytes after initial dead.
2) Invasion of various other cells and especially
(reticuloendothelial organs).

Tularemia: Clinical Manifestations

 Most common, acute onset of fever, chills, malaise.
Ulcero-glandular  Local papule become necrotic.
 Swollen and painful regional lymph nodes.
Typhoidal tularemia  Infective dose: > 108.
 Abdominal manifestations, prolonged fever {not relapsed}.
Pneumonic tularemia  Directly from infected human.
 Spread from different organs.
Oculo-glandular  Painful purulent conjunctivitis.

Tularemia: Diagnosis, Treatment &Prevention:

Diagnosis: Treatment & Prevention
1) Culture:  Gentamicin or streptomycin.
 Require special media, risk of  Doxycycline or ciprofloxacin (no
laboratory infection. relapses seen).
 Use of rubber gloves, eye protection
2) Serology: when handling infected wild mammals.
 Direct immunoflourescence (in  Ticks removal.
reference labs).  Live attenuated vaccine: for
 Agglutination test: positive (1:40; in laboratory workers and those who
first week, 1:320; in 3-4 week). cannot avoid contact with infected
animals.
 Single high antibody titers
considered diagnostic.
 Bartonella
 Family: Bartonellaceae, related to the genus Brucella.
 Gram-negative, coccobacilli.
 Stain very poorly using Gram stain.
 Facultative intracellular in RBCs.
 Infect erythrocytes and endothelial cells.
 Arthropod born.
 3 most relevant medically species are:
B. henselae B. quintana B. bacilliformis
Cat scratch disease(CSD) Trench fever Oroya fever(Carrion’s disease)
Flea Louse Sand fly

Life cycle:
 Any of the three has the same mechanism, as well as affecting humans and
animals with the same mechanism.
 The bartonella must pass by 2 niches; vascular endothelium and RBCs.
1) The insect injects Bartonella into the skin.
2) Macrophage or dendritic cells received it.
3) The macrophage transports the bartonella
to vascular endothelium then it released into
the peripheral circulation.
4) In the peripheral circulation the RBCs
invade and multiply within them.
5) After doubling, take one of two ways:
a) RBC lysis  hemolysis.
b) Remain in the RBCs.

Transmission:
B. henselae B. quintana B. bacilliformis
Mainly zoonotic infection from Human body louse. Infected sand flies.
cat, but may directly from tick.
Epidemiology:
B. henselae B. quintana B. bacilliformis
Cat scratch Trench fever Oroya fever(Carrion’s
disease(CSD) disease)
Common in children Found only in Peru (endemic), Populations prone to
and adolescents, Ecuador, and Colombia, infestations with lice,
except for associated with a history of such as the homeless.
immunocompromised recent travel to these regions.
adults).

By B. henselae and B. Quintana.

Host risk factors:
 Production of vascular endothelial factor,
 Immunocompromised:
causing vascular proliferation
 Chemotherapy.
(angiogenesis).
 Organ transplant.
 AIDS→ bacillary angiomatosis.  Multiplication within immature capillaries,
 Homelessness. swollen endothelium shields,
 Poor hygiene. Bartonella from both innate and adaptive
immune responses.
 Seen mainly in face.

Cat Scratch disease (CSD):

It often ends on its own, but in some children it may develop into secondary
symptoms: fever, febrile lymphadenopathy (axilla and neck), CNS involvement,
hepatosplenomegaly, endocarditis and so on.

Trench fever:
 It is often associated with poor hygiene areas and homeless.
 It is normal to be in the form of a fever and ends within 4-5 days, but in some
people relapses (in 4-5 days) occur and may last for weeks.
 Other complications: Bacteremia and endocarditis, headache, loin pain,
lumbago, knee and ankle pain, sever shin pain.
Oroya fever (Carrion’s disease):
 Acute hemolytic anemia.
 Nodular, highly vascular skin lesions called “verruga
peruana lesions”.
 It is very similar to bacillary angiomatosis, but it is in the
lower extremities and not in the face.
 Giemsa-stained blood smear showing parasitism of all
erythrocytes with B. bacilliformis.

Bartonella: Diagnosis ,Treatment &Prevention

Laboratory tests:
Serologic Demonstrate seroconversion.
testing  IFA the most accurate for B henselae.
Microscopy Warthin-Starry silver stain.
 The confirmatory stain for B henselae.
Molecular(PCR) Amplify ribosomal RNA gene fragments from tissue samples.
Culture Special agar medium to detect Bartonella quintana & B.
henselae.

Treatment &Prevention:
Reduce lymph node enlargement: Azithromycin or erythromycin
treat bacteremia in immunocompromised: Erythromycin or doxycycline
in Bartonella endocarditis: Valve replacement
 Ehrlichia & Anapalsma
 Anaplasmataceae family.
 Obligate intracellular in WBCs.
 Small gram negative.
 Lack LPS and peptidoglycan.
Type Ehrlichia chaffeensis Anapalsma phagocytophilum
Vector Tick Tick
Reservoir Deer Rodent
Obligate in Monocytes Neutrophil
Associated diseases Human Monocytic Human Granulocytic
Ehrlichiosis (HME) Anaplasmosis (HGA)

Life cycle of both:

1) The infectious form is called "dense core
cell", as it has uh receptors on neutrophils and
monocytes.
2) Endocytosis of dense core  phagocytosis.
 These microbes are resistant to
phagosome fusion and ROS.
3) Inside the phagocytes it become vegetative
cells called “reticulocyte”.
4) Reticulocyte differentiate into morula (can be
seen under microscope).

Epidemiology:
HME southeastern and lower Midwestern United States
HGA Northern states, Asia and Europe.

 Note that in the United States specifically, at the beginning we say that it is
Borrelia, and through the blood smear we can distinguish between it and
anaplasma;
Borrelia anaplasma
Soiralcheates Morula
Signs and Symptoms:
 Manifestations may be mild.
 Findings are clinically similar to Rocky Mountain spotted fever (RMSF), but
rashes are less commonly seen.
RMSF symptoms:
Rash in palms and soles.
Complications: DIC, thrombocytopenia, encephalitis, vascular collapse, renal,
heart failure.

 May be life threatening: depending on the patient’s age and general health.
 Flulike: fever, chills, nausea, muscle aches, and headache.
 Leukopenia, thrombocytopenia.
 Diarrhea, malaise.
 Ehrlichiosis or anaplasmosis are considered in any case of unexplained acute
fever in patients exposed to ticks in endemic areas.

Diagnosis, Treatment &Prevention:

Serology:
IFA or If the initial titer of antibodies 1:64 or fourfold or greater rise in antibody;
we can diagnostic patients with clinical pictures.
Microscopy:
Observation of intracytoplasmic inclusions (morulae).
PCR:
Test of Ehrlichia DNA.
Laboratory findings:
Low leukocyte count, thrombocytopenia, anemia, and impaired liver and renal
function.
Treatment: Doxycycline.
Prevention: Avoid wooded areas and tick bites.
 Herpesviridae (Cytomegalovirus, herpes simplex, and EpsteinBarr (EBV))
Characteristics of the previous three viruses:
 DNA viruses “double strands”
 Enveloped viruses.
 Include latency phase.
Common Kaposi sarcoma
name CMV EBV associated herpesvirus
(KSHV),
Designation HHV-5 HHV-4 HHV-8
 Close contact.  Direct contact.
 Sexual transmission.  Saliva.
 Saliva.
Transmission  Congenital.  Blood transfusion.
 Kissing.
 Body fluids.  Organ Transplant.
 Blood transfusion.
 Organ Transplant.
 B lymphocytes.
 T lymphocytes.
Primary  B-cell.  Peripheral blood
infection site  B lymphocytes.  Oral epithelium Mononuclear cell.
 Monocytes.  Oral epithelium.
 Monocytes.
 Neutrophils.
Latency site  Vascular endothelial  B lymphocytes  B lymphocytes.
cells

1) Heterophile negative 1) (Primary infection).

mononucleosis. Infectious mononucleosis.

2) Severe congenital 2) Tumors including: Tumors including:

infection.
A)) B-cell tumors: 1. Kaposi
 Burkitt lymphoma. sarcoma.
Diseases 3) Infections in
 Nasopharyngeal
immunocompromised
carcinoma. 2. Some B-cell
(gastroenteritis, retinitis,
lymphomas.
pneumonia). B)) some T-cell tumors.

Herpes type 𝜷-herpesviridae 𝜸-herpesviridae 𝜸-herpesviridae

Epstein Barr virus (EBV)

 𝜸-herpesviridae, two strains (types 1and 2).
 Enveloped, double-stranded DNA virus.
 Infects more than 95% of the world's population.
Most viruses depend on the host, but EBV does not
depend on the host because there is a tugment region
between the envelope and capsid.
 The tugmen contain proteins for translation,
replication and transcription to build its structure and
capsid.

Life cycle & Latency:

Latency Target cells Disease

Latency type III Naïve B cells lymphoblastic lymphoma (LBL)
Nasopharengyal carcinoma (NPC).
Latency type II Germinal center resiratory tract carcinoma.
Latency type I Dividing B memory cells Germinal center B cells lymphoma
Latency type 0 Quesient B memory cells

1) When EBV reaches the body, it invades the naive B cells.

2) When the cells are infected, they start dividing and this is accompanied by the
latency type III.
 During proleferation some antigens are displayed on the naïve cell surface:
 EBV nuclear antigens (EBNAs).
 latent membrane protein (LMPs1,2).
 EBV-encoded small RNAs (EBERs).
 microRNAs.
3) The naïve B cells are activated and go to the germinal center for differentiation
and this is accompanied by a Latency type II.
 naïve B Cells display almost the same antigens as the third type.
4) They differentiate into memory cells and exit to the peripheral circulation
accompanied by Latency type I.
5) Memory cells begin to divide, and this is accompanied by the division of the
virus.
 The cell cannot tolerate the viral load and it explodes by lytic cycle and
this is accompanied by the Latency type 0.
 Latency Target cells Antigens expressed
 EBV nuclear antigens (EBNAs).
Latency Naïve B cells  latent membrane protein
type III (LMPs1,2).
 EBV-encoded small RNAs (EBERs).
 microRNAs.
 EBER1/2 RNA.
Latency Germinal center,  EBNA-1.
follicles  LMP-1 (type IIa).
type II
 EBNA-2 (type IIb).
 EBER1/2 RNA.
Latency Dividing B memory  EBNA-1.
cells  LMP-1 (type IIa).
type I
 EBNA-2 (type IIb).
 BART RNA

How does EBV get into B cells?

The virus binds to the {CD21 {CR2}& CD23} , C3d blocked.

 During the activation of the naïve B cells, monoclonal IgM (against the virus)
and also heterophile polyclonal IgM are secreted.
 This heterophil IgM react with RBCs of some animals like; horse, sheep,
bovine and the agglutination is occur.
 Trypanosoma and leishmania secrete heterophil IgM so nonspecific.
Manifestations of EBV:
Lymphoproliferative Syndrome:

 Incidence 1- 2% after renal transplantations and 5- 9% after heart– lung

transplantations.
 Greatest in patients with primary EBV.

Burkitt Lymphoma:

 Enlargement of the jaw especially in the children.

 Endemic Burkitt lymphoma (BL) in Africa.
 The risk is greatest, where there is a high incidence of malaria.
Diagnosis of Burkitt lymphoma:
 In Burkitt Lymphoma the level of IgA against {EBV-VCA} is increased.
 Screening high IgA antibody to VCA and early EBV antigens.

Other EBV-associated lymphomas:

 Hodgkin lymphoma: present in Reed-Sternberg cells (multinucleated cells of B-

cell origin).
 Nasopharyngeal Carcinoma: endemic in southern China.
 Posttransplant lymphoproliferative disorders (PTLD).
In AIDS patients: associated with hairy leukoplakia of the tongue, interstitial
lymphocytic pneumonia (appear with CMV also).

Treatment:
Supportive.
Corticosteroids: in Laryngeal obstruction as a result of lymphadenopathy.
Acyclovir: in Hairy leukoplakia in patients with AIDS.
Infections mononucleosis:
At the beginning of the disease there are no symptoms, but it develops in some
people (increases with AIDS) to mononucleosis disease.
Mononucleosis disease called also: glandular disease, kissing disease or mono.

Mononucleosis symptoms:
 Fever.
 Malaise.
 Lymphadenopathy.
 Sole throat.
 Complication: ↑ liver enzymes, CNS, hepatosplenomegaly.

EBV-Immunity& Diagnosis:
1) Heterophile IgM antibodies are produced during an active EBV infection, not
specific.
 The titer is not important, it is important to be positive.
2) Finding atypical lymphocytes and the heterophile antibody test are insensitive
for acute EBV infection, not specific.
3) IgM antibody to the EBV VCA antigen is useful in the acute phase.
 VCA IgG and EBNA antibodies arise after the acute phase of illness.
4) Monospot test:
 Serological test.
 Take a blood from horse, sheep, or bovine and immerge
it with patient serum (IgM); if positive the agglutination
occur.
5) Downy cell test;
 Infected T cells.
 Large nucleus, large cytoplasm and irregular shape In
general.
 Neither heterophile IgM nor downy cells determine EBV.
IgM increased in some cases:
In children; IgM before 4 years remain high and if EBV infect them the IgM titer
remain positive to almost 4 weeks.
In adult the IgM titer remain positive for more than 4 years then replaced by late
IgG.

In CBC of EBV:
 Monocytosis.
 Lymphocytosis.
 ↑ Bilirubin.
 ↑ Liver enzymes.

CMV
 Beta herpesviridae.
 Cause neonatal infection (one of TORCH infection).
 TORCH stand for; Toxoplasma – rubella – CMV – herpes type VII.
 With the above infections the IgM will be increase, increase risk of abortion
and congenital transmitted.

 Cause cytopathic effects: Owl’s eye cells (nuclear inclusions) and cytomegaly.
 In general, we do not search for the type of virus according to changing the
shape of the cell because it does not change it much, but in CMV virus, cell
shape changes and it is called (megalo) because it enlarge the cell.

Three main genes:

 Immediate early (IE).
 Early (E) gene.
 Late gene (L).
Viral replication:

 The virus enters in the form of a nucleocapsid and in the cytoplasm leaves the
capsid and releases the tegument proteins, which include: PP71 and PP65.
PP65 PP71
contributes to viral Degradation of death domain-associated protein (Daxx)
replication and induce activation of the IE genes.

Proteins in IE (pIE): Promote the expression of early and late genes.

Early proteins (PE): Transcription factors and polymerases involved in viral
DNA replication, transcription, and protein synthesis.
Late proteins (PL): Structural proteins synthesized after viral DNA
replication.

 After the construction of the capsid and the DNA, they are assembled by the
nucleus and then go to the Golgi apparatus for the manufacture of envelope and
then exit from the cell.

Epidemiology:

 Virus shedding in body fluids (saliva like) remain up to years.

 Day care centers may transfer infection to children from asymptomatic
patients.
 Infants may excrete virus for up to 5 years after birth following congenital and
perinatal infections.

Pathogenesis
Latency occur in the B cells, but may infect other type of the cells mainly
pluripotent stem cells (CD34) or monocytes.
 Pluripotent stem cells (CD34) problem  leukemia, pancytopenia.
 The immune response may cause pneumonia.
Immunity:
In immunocompetent Primary infection occur, reactivation is subclinical.
In immunocompromised Primary infection and reactivation are symptomatic.
In allograft recipient Infected monocytes differentiate to macrophages once react
with activated T lymphocytes producing new CMV.

Primary infection usually asymptomatic, and secondary is severe symptoms.

 The inhibition of monocytes caused secondary infection.

Manifestations:
 Asymptomatic
 Mononuleosis like syndrome in young adults.
 Negative heterophile IgM.
 Congenital defects (1st trimester):
 Deafness (‫)الصمم‬, psychmotor mental retardation, hepatosplenomegaly,
jaundice, anemia, thrombocytopenia, DIC, pancytopenia, low birth weight,
microcephaly, and chorioretinitis (‫)التهاب المشيمية والشبكية‬.
 In immunocompromised:
Interstitial pneumonia (following BM transplant), chorioretinitis, gastroenteritis,
neurologic disorders, and CMV retinitis (in AIDS patients).
 Also EPV caused interstitial pneumonia.
 Most study bind post-transplant organs infection with MCV.

CMV: Diagnosis
Culture: Detects cytopathic effect (owel’s eye).
PCR: Using plasma or leukocytes, high sensitive.
Biopsy: Demonstrate inclusions in tissues, best for CMV and
gastrointestinal CMV diagnosis.
Serogonversion: IgM, in non-immucompromised patients with primary infection.
 Because in AIDS patients IgM already high.
CMV: Treatment and Prevention
Treatment:
Ganciclovir: inhibit viral replication
Immune globulin: for CMV pneumonia in bone marrow transplant recipients
Foscarnet: inhibits the CMV polymerase
Cidofovir: limited to ganciclovir resistant infections in immunosuppressed
patients.
Valganciclovir

Herpes virus 8
 𝛾- Herpesviridae.
 Known as KS-associated herpesvirus (KSHV).
 Associated with primary effusion lymphoma (PEL) (100%) and multicentric
Castleman disease (MCD) with 50% of AIDS-related cases.
 Endemic in Africa, some parts of Italy, Greece, Spain and Brazil.
 The prevalence is more than 40% in adults.
 Higher rates are seen in cohorts (‫ )مجموعة‬of men who have sex with men (MSM)
in the developed world and in association with HIV infection.

Transmission:

In endemic areas Transferred by close contact with infected saliva.

In non-endemic areas Sexual transmission occur.
Accidental transmission Infected blood products, organ transplantation.
Pathogenesis:
 Infects oral epithelium.
In Latency:
 Present in B-cell s (spindle cells) (tumor cell of endothelial origin).
 B -cells and spindle cells also express lytic antigens

Manifestations:
Classic KS: Endemic KS:
strength Indolent Aggressive
Site of infection On lower extremities. on extremities, oral cavity
and torso
Epidemiology Mediterranean Ashkenazi jews. In central Africa.

Iatrogenic KS: In posttransplant patients.

Epidemic of AIDS associated: most aggressive, in mouth, torso, and internal
organs.

Diagnosis & Treatment:

 Diagnosis:
Indirect Immunofluorescence: Sensitivity 70% - 90%.
PCR from peripheral blood: False negative seropositive in patients without KS.

 Treatment:
 Foscarnet and ganciclovir: inhibit lytic replication.
 No treatment for latently infected cells or vaccine is available.
 Parvovirus B19
Belong Parvoviridae family.
Naked virus.
The smallest virus “20nm”.
ssDNA linear.

Pathogenesis:
1) This virus invades immature nuclear red blood
cells.
2) The virus binds to the human P blood group
antigen.
3) There is a co receptor integrin to help it to invade
the nucleated red blood cell, which are not present
in the rest of the cells that express P antigen.

Life cycle:
1) Attachment and endocytosis.
2) Uncoating.
3) Host machine utilization:
Host DNA polymerase DNA replication
Host RNA polymerase DNA transcription

4) Assembly takes place inside the nucleus and out by the

lysis cycle.

Main B19 viral protein

 Nonstructural protein NS-1.
 VP1 and VP2: capsid proteins
Parvovirus B19: Clinical Consequences:
1) Aplastic crisis: in patients with chronic hemolytic anemias.
2) Persistent anemia with reticulocytopenia: in immunocompromised patients.
3) Persistent bone marrow failure and an acute hemophagocytic syndrome.
4) Autoimmune neutropenia, thrombocytopenia, and hemolytic anemia.

Disease associated with B19 virus:

1) Erythema Infectiosum (5th disease):
 Acute disease.
Host Transmission
Children, young adults Respiratory, blood &blood products , trans placental

Clinical features:

 Fever, malaise, headache, myalgia, itching.

 Rash appears on the face, giving a “slapped-cheek” appearance.
 Lymphadenopathy or splenomegaly.
 Mild leukopenia and anemia.
 Arthritis or vasculitis.
 Hydrops fetalis or still birth.

Diagnosis & treatment:

 Elevated IgM in late acute phase.

 Immunocompromised patients may require IV immunoglobulins.

2) Roseola Infantum (exanthem subitum):

 Acute disease.

Host Transmission
Infants-children Droplet infection (e.g., saliva)

Clinical features:

 High fever.
 May be with generalized convulsions (seizures).
 Leukopenia.
 Macular rash.

Diagnosis & treatment:

 Diagnosis is based on the characteristic history and physical

examination.
 Self-limiting no treatment is needed.