COURSE OUTLINE

ANALYSIS OF URINE AND  Safety in the clinical laboratory

 Renal function
OTHER BODY FLUIDS  Introduction to urinalysis
 Physical examination of urine
 Chemical examination of urine
 Microscopic examination of urine
 Quality assurance
 Renal disease
 Urine screening for metabolic disaster
BSMT  Cerebrospinal fluid
 Semen analysis
 Synovial fluid
 Serous
 Amniotic
 Fecal analysis
 Malaria
PROFESSOR

RECOMMENDED BOOK
Analysis of urine and body fluids
By Susan King Strasinger

SCHEDULE: GRADING SYSTEM

Quiz 20%
Recitation 15%
Laboratory 15%
Major exams 50%
Laboratory part
Lecture part
ROOM: Total 100%

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 STANDARD PRECAUTIONS

PRELIM
Proper hand washing and wearing of PPE is a major importance in the
laboratory

IMPORTANCE IN LABORATORY
 HAND WASHING
o Sanitize hands:
 After touching the specimen whether or not gloves are
ANALYSIS OF URINE AND OTHER BODY FLUIDS worn
 B/w patient contact
 B/w task procedures and same patient

 GLOVES
 put the gloves before touching any specimen and non-
intact skin
 remove the gloves from hands after use

 MASK, EYE PROTECTION and FACE SHIELD

 Protects mucus membrane of the eyes, nose, and mouth
during procedures and patient care activities

 WEAR A LABORATORY GOWN

 To protect the skin
 To prevent soiling of clothing during procedures that are
likely to generate splashes or sprays of blood, body fluids,
secretions / excretions

 PATIENT CARE EQUIPMENT

 Reusable equipment should be cleaned and reprocessed
appropriately and single – use items are discarded
properly
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 Documenting regular training in safety standards for
 ENVIRONMENTAL CONTROL employees
 Ensure that the hospital has adequate procedures for the
routine care cleaning and disinfection of environmental TYPES OF SAFETY HAZARDS
surfaces, beds, bedrails, bed side equipment and other BIOLOGICAL HAZARDS
frequently touched surfaces - Most direct contact is through patient specimen
- Chain of infection
 LINEN  Requires a continuous link b/w a source, a method of
 Handel, transport and process linen soiled w/ blood, body transmission and a susceptible host
fluids, secretions and excretions in a manner that - Disposal of biological waste
prevents contamination and transfer of microorganism to
other patients and environment SHARPS
- All sharp objects must be disposed in a puncture –
 OCCUPATIONAL HEALTH AND BLOOD – BORNE PATHOGEN resistant, or leak – proof containers
 Take care to prevent injuries when using needles,
scalpels, and other sharp instruments or devices CHEMICAL HAZARDS
- Chemical spills
SPECIFIC REQUIREMENTS FOR OCCUPATIONAL AND  When contact occurs, the first aid is to flush the area
HEALTH ADMINISTRATION with large amount of water for at least 15 minutes
Requiring all employees to practice standard precautions then se4ak medical attention
Providing laboratory gowns, coats, gloves, and face protection
to employees - Chemical handling
Providing sharp disposals containers and providing recapping  Chemicals should never be mixed together unless
of needles specific instructions are followed
Prohibiting eating, drinking, smoking, and applying cosmetics
in the work area - Chemical labelling
Labelling all bio hazardous materials and containers  Hazardous chemicals should be labelled with a
Providing free immunization for HBV description of their particular hazards such as
Establishing a disinfection protocol for work surfaces poisonous, corrosive, flammable, explosive,
Providing medical follow up teratogenic or carcinogenic

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 - Disposal of chemical FIRE / EXPLOSIVE HAZARDS
 According to the state of federal regulation - Observe general precautions:
 Avoid running
RADIOACTIVE HAZARDS  Watch for wet floors
- Amount of radioactivity present in the clinical laboratory  Keep long hair pulled back
is very small and represents little danger  Avoid dangling jewellery
- Effects of radiation are cumulated related to the amount
of exposure PHYSICAL HAZARDS
- Wet floor falls
ELECTRICAL HAZARDS - Heavy boxes sprains
- Laboratory personnel should continually observe for any - Patients strains
dangerous conditions such as frayed cords and
overloaded circuits
- Equipment that has become wet should be unplugged
and allowed to dry completely before reusing

TYPE OF HAZARDS SOURCE POSSIBLE INJURY

1. Biological - Infectious agents - Bacterial
- Fungal
- Viral
- Parasitic infections
2. Sharps - Needles - Cuts
- Lancets - Punctures
- Broken glass - Blood – borne pathogen exposure
3. Chemical - Preservatives - Exposure to toxic, carcinogen, and causative agents
- Reagents
4. Radioactive - Equipment - Radiation exposure
- Radioisotopes
5. Electrical - Ungrounded or wet equipment - Burns

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 - Frayed cords - Shocks
6. Fire / explosive - Open flames organic chemicals - Burns
- Dismemberment
7. Physical - Wet floors - Falls
- Heavy boxes - Sprains
- Patients - strains
 Iris diaphragm
MICROSCOPY  Controls the angle and amount of light that passes
Set of techniques designed to support and justify the findings through the specimen
of urinalysis
Technique for magnifying microscopic objects that are too  Mirror
small  Provide light source
Compound light microscope
o Have a series of lenses used to magnify small objects  Condenser
 Direct light to pass through specimen
2 MAIN PARTS:
 Mechanical parts
 Essential parts o For support
o Help the viewer to examine or illuminate objects through  Base
lenses  Located at the lowest portion
 Ocular / eye piece  To support the whole unit
 Use to magnify the objects
 Stage
 Objectives  Located at the middle partition
 Magnify the objects  To hold the slides
 Scanner
 Low power objective  Stage clip
 High power objective  Hold the slide fixed on its position
 Oil emersion

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  Revolving nose piece ※ POLARIZING MICROSCOPE
 Controlling knob for the objective - For crystals and lipid content of urine specimen
examination
 Arm - Ability to split two beams of light
 Nose piece with objectives and ocular are attached - Crystals appeared multi-colour objects

 Course and fine adjustment knob CARE OF MICROSCOPE

 Rendering a better viewing or magnification of the  Carry the microscope w/ two hands, supporting the base w/
objective the other
 Always hold the microscope in a vertical position
TYPES OF MICROSCOPE  Clean optical surface w/ a good quality lens tissue and
commercial lens cleaner
 Do not use the 10x and 40x objectives w/ oil
※ BRIGTH FILED MICROSCOPE
 Clean the oil immersion lens after use
- Rendered light is essential in order to view urine
 Always remove the slides w/ Low Power Objective raised
sediments
 Store the microscope w/ Low Power Objective in position and
the stage in the center
※ DARK FIELD MICROSCOPE
- Use special light source and special lens for examining live
microscopic specimen FUNCTIONS OF THE KIDNEYS
- Use dark field condenser  Excretion of metabolic waste of foreign chemicals, drugs and
- Specimen appears bright against black background hormone metabolites
Use to examine microorganism in liquid medium  Regulation of waste and electrolyte balances
 Regulation of acid – base balance
※ PHASE CONTRAST MICROSCOPE  Regulation of arterial balance
- Determined a detailed examination of internal structure in  Regulation of electrolyte production and glucose synthesis
living microorganism of urine sample  Regulation of 1, 25 – dihydroxy vitamin D3 production
(cholesterol)
※ INTERFERENCE CONTAST MICROSCOPE
- Similar to phase contrast
- Provide 3D images of urine sediment

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 RENAL FUNCTION GLUMERULAR FILTRATION
Physiology - GLOMERULUS
- Each kidney contains 1 – 1.5 million of nephrons  Consist of coil of approximately eight capillary lobes
 Located w/in the BOWMAN’S CAPSULE
Kidney  Forms the beginning of renal tubule
- Selectively clear waste products from the blood  Serves as non – selective filter of plasma substances
- Simultaneously maintain body’s essential waste, and
electrolyte balances FACTORS THAT INFLUENCE THE FILTRATION PROCESS
-  Cellular structure of capillary walls and bowman’s capsule
Protein PLASMA FILTRATE MUST PASS THROYGH THREE CELLULAR
- Good indicator of kidney LAYERS:
※ CAPILLARY WALL MEMEBRANE
FUNCTIONS: o Endothelial cell
RENAL BLOOD FLOW
- Immature Hgb 02 body parts ※ BASEMENT MEMBRANE
- Erythropoietin, supply/circulation o Further restriction of large molecules

 RENAL ARTERY ※ VECERAL EPITHELIUM

 Supplies blood to the kidney o Thin membrane that covers the filtration slits by
 Total renal blood flow – 1200 ml/min. intertwining foot process of the podocytes of the
 Total renal plasma flow – 600 – 700 ml/min inner layer of the bowman’s capsule
 Blood enters the capillaries through afferent arteriole,
flows through the glomerulus, then into the efferent  Hydrostatic pressure and oncotic pressure
arteriole o Results from the smaller size of the efferent
 Before returning to the renal vein, blood from the arteriole – this enhances filtration
efferent arteriole enters the peritubular capillaries and o Necessary to overcome the opposition of pressures
the vasa recta and flows slowly through the cortex and from the fluid w/in bowman’s capsule
medulla of the kidney

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 Mechanisms of the renin – angiotensin – aldosterone
system (RAAS) TUBULAR REABSORPTION
o Controls the regulation of blood flow to and w/in the - Cellular mechanism (active and passive transport)
glomerulus
- ACTIVE TRANSPORT
 Responsible for the reabsorption of glucose, amino
acid and salts in the proximal convoluted tubule
 Responsible for the reabsorption of chloride in the
ascending loop of Henle
 Responsible of sodium in the distal convoluted tubule

- PASSIVE TRANSPORT
 Reabsorption of water in all parts of nephron urea in
Proximal convoluted Tubule and ascending loop of
Henle

TUBULAR SECRETION
- Serves two functions
 Elimination of waste products not filtered by the
glomerulus
 Removal of non-filtered substances is in the
Proximal Convoluted Tubules
 Regulation of acid – base balance in the body
 Actual excretion of excess hydrogen ions
 Maintain the normal pH of the blood w/c is 7.4

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INTRODUCTION TO URINALYSIS FACTORS THAT INFLUENCE URINE VOLUME
 Fluid intake
URINE  Fluid loss from nonrenal source
- Ultra filtrate of plasma  Variation in the secretion of anti – diuretic hormone
- Composed of 95% water, 5% solutes  Need to excrete increased amount of dissolved solids
- Consist of urea, creatinine and other organic and inorganic
chemicals dissolved in water SPACIMEN COLLECTION
- Solutes or sediments includes organized and unorganized Clear container. All samples must be collected in clean, sterile
sediments containers. Disposable, wide mouth and flat bottom
- Average daily urine output is 1200ml to 1500ml containers w/ screw caps are usually recommended
Washing of hands
TWO UNIQUE CHARACTHERISTICS OF A URINE SPECIMEN Manner of collection
 Readily available and can be easily collected Label the container. Unlabelled specimen should not be
 Urine contains information about body’s major metabolic accepted in the laboratory. Label should be on the container
functions and not on the lid
Time of collection and examination. Allowable time is not
CONCENTRATION SUBSTANCE MAY BE INFLUENCE more two hours.
Urine preservation. A good urine preservative should be
※ Dietary Intake
bactericidal and should preserve formed elements
※ Physical Activity
※ Body Metabolism
※ Endocrine Functions
※ Body Position

URINE VOLUME
- Depend on the amount of water that the kidney excrete

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CHANGES IN UNPRESERVED URINE
ANALYTE CHANGES CAUSE
Color Darken Oxidation or reduction of metabolite
Clarity / transparency Decreased Bacterial growth and precipitation of amorphous material
Odor Increased Bacterial multiplication causing breakdown of urea to ammonia
pH Increased Breakdown of urea to ammonia by urease – producing bacteria/loss of CO2
Glucose Decreased Glycolysis and bacterial use
Ketones Decreased Volatilization and bacterial metabolism
Bilirubin Decreased Exposure to light/photo oxidation to biliverdin
Urobilinogen Decreased Oxidation to urobilin
Nitrate Increased Multiplication of nitrate – reducing bacteria
RBC and WBC cells and cast Decreased Disintegration in dilute alkaline urine
Bacteria Increased Multiplication
Trichomonas Decreased Loss of motility, death

URINE PRESERVATIVES
PRESERVATIVE ADVANTAGE DISADVANTAGE
Refrigeration Does not interfere w/ chemical test Increased SG., precipitate amorphous urates and phosphate
Thymol Preserves glucose and sediments well Interfere w/ acid precipitation test for protein
Decease bacteria
Boric acid Preserves protein and formed elements well May precipitate crystals
Increased bacteria
Formalin Excellent sediment preservative Interferes w/ chemical test for glucose, blood, and leukocytes
Toluene Does not interfere with routine tests Floats on surface of specimens
Sodium Fluoride Prevents glycolysis Inhibits reagent strip tests for glucose, blood, and leukocytes
Phenol Inhibits reagent strip tests for glucose, blood, Causes an odor change
and leukocytes
Commercial Preservative Tablets Convenient when refrigeration not possible May contain one or more of the preservatives
Saccomanno fixative Preserves cellular elements

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TYPES OF URINE SPECIMENT - For bacterial cultures and routine urinalysis
Random specimen
- Most commonly received specimen Suprapubic aspiration
- Useful for routine screening test - Introduction of a needle through the abdomen into the
bladder
First morning specimen - Bacterial culture and cytological examination
- Concentrated
- Ideal screening specimen 3 glass collection
- Determine prostatic infection
Fasting specimen - 1st container – first urine specimen
- Second morning - 2nd container – midstream collected urine sample
- 2nd voided specimen after a period of fasting - 3rd container
- Recommended for glucose monitoring - Contain the remaining urine after the prostate gland
has been massaged
2 hours post prandial
- Use primarily for monitoring insulin therapy in persons w/ Pediatric specimen
diabetes mellitus - Pediatric urine collector
- Correlate w/ blood glucose tests

Glucose tolerance specimen

- Urine is tested for glucose and ketones

24 – hour (or timed) specimen

- Creatinine, protein, urea
- Ideal for quantitative determination of nitrogenous and
non – nitrogenous compounds of urine

Catheterized
- For bacterial cultures

Midstream clean (catch specimen)

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PHYSICAL EXAMINATION OF URINE - 2 additional pigment: uroerythrine and urobolin
Color
- Straw to black Pink pigment orange brown color
- Normal color is yellow due to urochrome

LABORATORY CORRELATION OF URINE COLOR

COLOR CAUSE CLINICAL / LABORATORY CORRELATION
Colorless Recent fluid consumption Commonly observed with random specimens
Pale/light yellow Polyuria or diabetes insipidus Increased 24-hour volume
Diabetes mellitus Elevated specific gravity and positive glucose test result
Dark yellow/amber Concentrated specimen First morning specimen, After strenuous exercise or in first morning specimen
Caused by dehydration, Less fluid intake
Orange Bilirubin Positive to foam test, positive chemical test for bilirubin
Acriflavine Possible green fluorescence, negative bile test
Phenazopyridine, rifampicin Positive orange foam and thick orange pigment
Yellow – green Bilirubin oxidised to biliverdin Positive colored foam
Yellow – brown
Green Pseudomonas infection Positive urine culture
Blue – green Amitriptyline Antidepressant
Methocarbamol Muscle relaxant, may be green-brown
Indican Bacterial infections (D. aeruginosa)
Pink RBCs Cloudy urine test results for blood and RBC’s visible microscopically
Red Hgb Clear urine with positive chemical test results for blood; intravascular hemolysis
Myoglobin Clear urine with positive chemical test results for blood; muscle damage
Porphyrins Negative chemical test results for blood
Rifampin Tuberculosis medication
Brown RBC’s oxidize to methemoglobin Seen in acidic urine after standing; positive chemical test result for blood
Brown and pink color Due to uroerythrin that attaches to as
when stored in refrigerator

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Black Methemoglobin Denatured hemoglobin
Homogentisic acid Seen in alkaline urine after standing
Phenol derivatives Interfere with copper reduction tests
Metronidazole Interfere with copper reduction tests
Melanuria Genetic disorder of phenylalanine and tyrosine metabolism
Alkap-tonuria
o Nonsquamous epithelial cells
CLARITY o Abnormal crystals
- Refers to transparency or turbidity o Lymph fluid
Clear o Lipids
Hazy
Cloudy SPECIFIC GRAVITY
Turbid - Measures of the density of the dissolved chemicals in the
Milky specimen
- Screen the renal concentrating ability of the kidneys
CAUSE OF URINE TURBIDITY
Non – pathologic URONOMETER
o Squamous epithelial cells - Consist of a weighted float attached to a scale that has been
o Mucus calibrated in terms of urine specific gravity
o Amorphous phosphates, carbonates, urates - Not recommended by the national committee for clinical
o Semen, spermatozoa laboratory standards
o Fecal contamination - Large volume of urine is needed
o Radiographic contrast media - Temperature should be corrected (20oC)
o Talcum powder
o Vaginal creams
REFRACTOMETER
Pathologic - Concentration of dissolve particles determines the angle of
o RBCs w/c the light passes through a solution
o WBCs - When using a refractometer, a drop of urine is placed on the
o Bacteria prism, instrument is focused on a good light source, and the
o Yeast reading is taken directly from the specific gravity scale.

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HARMONIC OSCILLATION DENSITOMETRY ODOR
 Based on the principle that the frequency of a sound wave - Freshly voided urine has a faint aromatic odor
entering a solutions will change in proportions to the density ODOR CAUSE
of the solution. Aromatic Normal
Foul, ammonia –like Bacterial decomposition, Urinary Tract
Clinical correlation Infection
 Isothenuric – SG. Is 1.000 Fruity, sweet Ketones
 Hypostenuric – SG. Below 1.010 Maple syrup Maple Syrup Urine Disease
 Hyperstenuric – SG. Above 1.010 Mousy Phenylketonuria
Rancid Tyrosinemia
pH Sweaty feet Isovaleric acidemia
- Reflection of the ability of the kidney to maintain thin normal Cabage Methionine malabsorption
hydrogen ion concentration in plasma and extracellular fluid Bleach Contamination
- 6.5 – 7.0 neutral
Acidic
CHEMICAL TESTING
o A high diet in meat and protein
REAGENT STRIP
o Prolonged diarrhea starvation (E.coli)
Store w/ desiccant in an opaque, tightly closed containers
o During sleep (decrease pulmonary ventilation increase
Store below 30oC; do not freeze
in respiratory acidosis)
Do not expose to volatile fumes
o 6.0 pH
Do not use past the expiration date
Do not use if chemical pads become discoloured
Alkaline
Remove strips immediately prior to use
o UTI (Urinary Tract Infection)
o Renal tubular acidosis
o Metabolic alkalosis (vomiting) CONFIRMATORY TESTING
o Vegetarian - Test using different reagents/methodologies to detect the
o 7.0 pH same substances as detected by the reagent strips
pH
o based in double indicator principle
o methylred
o bromthymol blue

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 o clinical significant  Color: tallow
 treatment of UTI o Early detection of liver disease
 precipitation / identification of crystals o Degradation production of Hemoglobin
 determination of unsatisfactory specimen o Clinical significance
 Hepatitis
Protein  Cirrhosis
o Cause of turbidity  Biliary absorption
 Crystals, casts o Didzo reaction
o Most indicative parameter of a renal disease o Positive result = pink - violet
(proteinuria) o Fouchet test = (+) green – blue
o Icto test = (+) blue - purple
Ketones
o 3 intermediated products of fat metabolism Urobilinogen
 Acetone 2% o Bile pigment that results from the degredation of
 Acetoacetic acid 20% Hemoglobin
 β – hydroxybotyrate 78% o Produces in the intestine from the reduction of
o Rothera’s tests (acute) bilirubin by intestinal bacteria
o Gerhands test and lindemas test (diacetic acid) o Clinical significance
o Han’s method  Ehrlich tests
 Betahydroxybutyric acid  Liver disorder
 Haemolytic disorder
Blood
o Cause of turbidity Nitrite
o Pseudo peroxidase principle o Rapid screening test for UTI
 Change in chemical pads that represents our o Clinical significance
blood  Pyelonephotis
o Berodine test = (+) green blue colored complex  Cystisis
o Gemochromogen = (+) pink color  Antibiotic therapy evolution
o Turbididty
Bilirubin  Bacteria
o Foam’s test

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 Leukocyte
o Leukocyte esterase

Specific gravity
Glucose
o Monitoring of diabetes mellitus
o Hyperglycemia
o Glycosuria

CAUSE OF PROTENURIA
※ Pre – renal proteinuria
o Bence jones proteinuria
 Multiple myeloma

※ Renal proteinuria
o Time renal disorder
 Glomerulus tubules

※ Post – renal proteinuria

o Not the true renal disease
o Other organs near the kidneys
o Protein in the urine as it passes through the structures
of lower urinary tract such as uretra, ureter
o Clinical significance
 UTI
 Vaginal secretions
 Prostatic fluid

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 MICROSCOPIC EXAMINATION OF URINE

MIDTERMS
Specimen preparation
o Should be examined while fresh or adequately
preserved

Specimen volume
ANALYSIS OF URINE AND OTHER BODY FLUIDS o 10 ml to 15ml

Centrifugation
o 5 minutes at a relative centrifugation force of 1,200
rpm

Sediment preparation
o Decant (0.5 – 1ml)

Volume of sediment examination

o 20ul

Examination of sediments
o 10 fields both in Low Power Objective and High Power
Objective

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SEDIMENT STAIN
STAIN ACTION FUNCTION
1. Sternheimer – malbin (crystal violet Delineate structure and contrasting colors of Identifies WBC, epithelial cells and casts
and saffranin O) the nucleus and cytoplasm
2. Toluidine blue (metachromatoc Enhances nuclear details Differentiates WBC and renal tubular epithelial cells
stain)
3. 2% acetic acid Lyses RBC and enhances nuclei of WBC Distinguishes RBCs from WBCs, yeast, oil droplets
and crystals
4. Lipid stains: oilred O and sudan III Stains triglycerides and neutral fats orange red Identifies free fat droplets and lipid containing cells
and casts
5. Gram stain Differentiate gram (+) and gram (-) bacteria Identifies bacterial casts
6. Hansel stain Methylene blue Identifies urinary eosinophils
Eosin Y stain
Eosinpphilic granules
7. Prussian blue stain Stains structures containing iron Identifies yellow brown granules of hemosiderin in
cells and casts

URINARY SEDIMENTS ARE CLASSIFIED INTO: URINE SEDIMENTS CONSTITUENTS

ORGANIZED SEDIMENTS UNORGANIZED SEDIMENTS Red Blood Cells
o Includes o Flat droplets o Appear as smooth, non – nucleated, biconcave disks
 epithelial cells o Mucous thread o Measures approximately 7 mm in diameter
 leukocytes o Crystals o Must be identified using high per objective
 erythrocytes o Artefacts o Reported as the average number seen in 10 high power
 bacteria fields
 parasite
 semen
 casts

o HYPERSTEURIC

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  Concentrated urine
 Cells shrink due to loss of water
 May appear crenated or irregularly shaped

o HYPOSTEURIC
 Dilute urine
 Cells absorb water, swell, and lyse rapidly, releasing o Monocelural cells
their Hgb and living only the cell membrane  Lymphocytes, monocytes, macrophages, and
 Appear as large empty cell called “ghost cells” histocytes may be present in small numbers
o Frequently confused w/ yeast cells, oil droplets, and air  Usually fewer than five leukocytes per high power field
bubbles found in normal urine
 Pyuria
White Blood Cells o Increase in urinary WBCs indicates the presence of
o Larger than the RBCs, size of 12 um an infection or inflammation in the genitourinary
system

Epithelial cells
o Squamous epithelial cells
 Largest cells found in urine sediment
 Contain abundant, irregular cytoplasm and a
o Neutrophils
prominent nuclear about the size of an RBC
 Predominant WBC found in urine sediment
 Reported in terms of rare, few, moderate, and many
 When exposed to hypotonic urine absorb water and
ether in low power or high power magnification base
swell
on laboratory protocol
 Brownian movement of the granules produces a
 Originate from the linings of vagina and female urethra
sparkling appearance referred to as “glitter cells”
and the lower portion of male urethra
 Midstream clean – catch technique contain less
o Eosinophils
squamous contamination
 Primarily associated w/ drug – induced intestinal
 Variation of the squamous epithelial cells is clue cells
nephritis
 Clue cells are indicative of vaginal infection by
 Preferred stain is HANSEL however; wright stain can
Gardeneralla vaginalis
also be used.

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  Absorbed lipids present in glomerular filtrate

o Transitional epithelial cells

 Urothelial cells
 Smaller than squamous cells and appear in several Bacteria
forms (spherical, polyhedral, caudate) o Not normally present in urine
 Has the ability to absorb large amount of water o May produce (+) positive nitrate, pH above 8.0
 Originate from the lining of renal pelvis, ureters, o In the form of cocci or bacilli
bladders and upper portion of male urethra o Reported as few, moderate, or many per low power
 Transitional cells w/ abdominal morphology maybe objective
indicative of malignancy or viral infection o Significant for UTI

Mucous
o Protein material produced by glands and epithelial cells
o Appear as thread – like structures
o Renal tubular epithelial cells o No clinical significance
 Vary depending on the area of the renal tubules from o Reported as few, moderate, many per low power field
w/c they originate o Tamm – horsefall protein is its major constituents
 Cells from proximal convoluted tubules have coarsely
granulated cytoplasm
 Cells from collecting ducts are finely granulated

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Yeast CASTS
o Appear as small, retractile oval structure w/ buds or o Formed w/in the lumen of the distal convoluted tubules and
mycelia collecting ducts
o Reported as few, moderate, many per high power field o Their shaped is representative of the tubular lumen
o Primarily candida albicans o Parallel sides and some rounded ends and may contain
o May be seen in urine of diabetic patient, immune additional elements present in the filtrate
compromised patients, and women w/ vaginal miniliasis o Reported as the average number per 10 low power fields
o Major constituents is TAMM – HORSFALL protein
 Glycoprotein excreted by RTE cells
of the distal convoluted tubules and
upper collecting ducts
o Cylindriuria (presence of urinary casts)

Parasites TYPES OF CASTS:

o T. vaginalis (trophozoite) Hyaline Cast
 Pear – shaped flagellate w/ undulating membrane
o Sexually transmitted
o S. hematobium
o E. vermicularis
o Frequently seen casts
o Consist almost entirely of Tamm – Hors Fall protein
Spermatozoa
o Presence of 0 – 2 per Low Power Field is considered
o Urine is toxic to sperm
normal
o Tapered oval head with long, thin tail
o Strenuous exercise, dehydration, heat exposure and
emotional stress causes increase of hyaline cast
o Pathologically, in acute glomerulonephritis, chronic
renal disease, pyelonephritis, and congestive heart
failure may all cause in the increase of this cast.
o It appears colorless in unstained sediment
o Have a refractive index similar to urine
o Sternheimer – Malbin Stain produces pink color in
hyaline cast
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Red Blood Cells Casts Bacterial Casts
o Contains bacilli both within and bound to the protein
matrix
o Seen in pyelonephritis
o Identification can be difficult because packed casts
packed with bacteria can resemble granular casts
o Shows bleeding within the nephron
o Confirmation of bacterial casts is best made by
o RBC casts are primarily associated with damage to the
performing a Gram stain on the dried or
glomerulus (glomerulonephritis) that allows passage of
cytocentrifuged sediment
the cells through the glomerular membrane
o Can are easily detected under low power by their
Epithelial Casts
orange-red color
o They are more fragile than other casts and may exist as
fragments or have a more irregular shape as the result
of tightly packed cells adhering to the protein matrix
o Represent the presence of advanced tubular
White Blood Cell Casts destruction, producing urinary stasis along with
disruption of the tubular linings
o Associated with heavy metal and chemical or drug-
induced toxicity, viral infections, and allograft
rejection.
o Signifies infection or inflammation within the nephron o Cells visible are smaller, cuboidal and columnar shaped
o Most frequently associated with pyelonephritis cells
o Primary marker for distinguishing pyelonephritis
(upper UTI) from lower UTIs. Mixed Cellular Casts
o They are also present in nonbacterial inflammations o Most frequently encountered include RBC and WBC
such as acute interstitial nephritis casts in glomerulonephritis
o Most frequently composed of neutrophils; therefore, o WBC and RTE cell casts, or WBC and bacterial casts in
they may appear granular unless disintegration has pyelonephritis
occurred, multi-lobed nuclei will be present. o When mixed casts are present, there should also be
homogenous casts of at least one of the cell types, and
they will be the primary diagnostic marker

22
 o Example:
 In glomerulonephritis, the predominant casts
will be RBC
 in pyelonephritis, the predominant casts will be
WBC

Granular Casts URINARY CRYSTALS

Frequently found in the urine are rarely of clinical significance.
The primary reason for the identification of urinary crystals is
to detect the presence of the relatively few abnormal types
This may represent disorders such as liver disease, inborn
errors of metabolism, or renal damage caused by
o Coarsely and finely granular cast crystallization of iatrogenic compounds within the tubules.
o Seen after strenuous exercises Usually reported as rare, few, moderate, or many per High
o In disease state, granules may represent disintegration of Power Field
cellular casts and protein aggregates filtered by the Precipitation of urine solutes
glomerulus Crystal formation takes place at low temperature with
o Easily visualized under low power objective, final concentrated high specific gravity specimen
identification at High Power Objective Valuable aid in the identification of crystals is the pH of the
specimen because this determines the type of chemicals
Waxy Casts precipitated
o Granular casts remain in the tubules for an extended Organic and iatrogenic compounds crystallize more easily in
period an acidic pH whereas inorganic salts are less soluble in neutral
o Granules disintegrated, cast matrix develops a waxy and alkaline solutions.
appearance
o Structures becomes more rigid, end of the cast may NORMAL CRYSTALS IN ACIDIC URINE
appear jagged/ broken  Amorphous Urates
o Diameter becomes broader o Color: Brick dust or yellow brown
o Representative of extreme urine stasis, indicating chronic o Frequently encountered in specimens that have been
renal failure. refrigerated and produce a very characteristic pink
sediment

23
 o Accumulation of the pigment, uroerythrin, on the
surface of the granules is the cause of the pink color  Sodium Urates
o Found in acidic urine with a pH greater than 5.5, o Needle-shaped
whereas uric acid crystals can appear when the pH is o Seen in synovial fluid during episodes of gout
lower o Do appear in the urine

 Calcium Oxalate
 Uric Acid Crystals o Most common form: dihydrate that is easily recognized
as a colorless octahedral envelope or as two pyramids
o Seen in a variety of shapes, including rhombic, four- joined at their bases
sided flat plates (whetstones), wedges, and rosettes o Shape: Monohydrate calcium oxalate crystals are oval,
o Usually appear yellow brown, but may be colorless and or dumbbell shaped
have a six-sided shape, similar to cystine crystals o Birefringent under polarized light
o Highly birefringent under polarized light, which aids in o Associated with foods high in oxalic acid, such as
distinguishing them from cystine crystals tomatoes, asparagus and ascorbic acid
o Associated with increased levels of purines and nucleic o Primary pathologic significance form in cases of
acids are seen in patients with leukemia who are ethylene glycol (antifreeze) poisoning
receiving chemotherapy, in patients with Lesch-Nyhan
syndrome and in patients with gout.

24
 NORMAL CRYSTALS IN ALKALINE URINE Calcium Phosphate Crystals
PHOSPHATE o may appear as colorless, flat rectangular plates or thin
- Majority of the crystals found in alkaline urine prisms often in rosette formations.
o The rosette forms may be confused with sulfonamide
Amorphous Phosphate Crystals crystals when the urine pH is in the neutral range.
Granular in appearance, similar to amorphous urates o Calcium phosphate crystals dissolve in dilute acetic acid
and sulfonamides do not.
o Common constituent of renal calculi.

Triple Phosphate Crystals (ammonium magnesium phosphate) Calcium Carbonate Crystals

o easily identified by their prism shape that frequently
o mall and colorless, with dumbbell or spherical shapes.
resembles a “coffin lid”.
o As they disintegrate, the crystals may develop a feathery
o They may occur in clumps that resemble amorphous
material.
appearance.
o birefringent under polarized microscope. o they can be distinguished by the formation of gas after the
o often seen in highly alkaline urine associated with the addition of acetic acid.
presence of urea-splitting bacteria o Also a birefringent which differentiate from bacteria.
o Have no clinical significance.

25
 Ammonium Biurate Crystals o Thick cystine polarizing crystals is made using cyanide-
o exhibit the characteristic yellow-brown color of the urate nitroprusside test.
crystals
o frequently described as “thorny apples” because of their
appearance as spicule-covered spheres.
o resemble other urates in that they dissolve at 60_C and
convert to uric acid crystals when glacial acetic acid is
added.
 Cholesterol Crystals
o Rarely seen unless specimens have been refrigerated,
because the lipids remain in droplet form.
o Resembles a rectangular plate with a notch in one or more
corners.
o Associated with disorders producing lipiduria, such as
nephrotic syndrome
o Seen in conjunction with fatty casts and oval fat bodies.
ACETIC ALKALINE o Highly birefringent with polarized light.
 Amorphous urates - amorphous phosphate
 Calcium oxalate - Calcium carbonate  Sulfonamide crystals
 Ammonium urates - ammonium biurate o Finding of this crystal in the urine of patients being treated
for UTIs are common.
ABNORMAL URINARY CRYSTALS IN ACIDIC URINE o Inadequate patient hydration
 Cystine Crystals o Still the primary cause of sulfonamide crystallization.
o found in the urine of persons who inherit a metabolic o Variety of crystal shapes and colors which are most
disorder that prevents reabsorption of cystine by the renal frequently encountered include needles, rhombic,
tubules (cystinuria). whetstones, sheaves of wheat, and rosette with colors
o Appears colorless, hexagonal plates and may be thick or ranging from colorless to yellow – brown.
thin. o Diazo reaction for confirmation.
o May be seen in the presences of ammonia.
o May be difficult to differentiate from color less uric acid
crystals

26
  Leucine
o Yellow-brown spheres that demonstrate concentric circles
and radial striations.
o Seen less frequently than tyrosine crystals.
o Should be accompanied by tyrosine crystals when present.

 Ampicillin crystals
o Appear as colorless needles that tent to form bundles
following refrigeration.

 Bilirubin
o Present in hepatic disorders producing large amount of
bilirubin in the urine.
o Appears as clumped needles or granules with the
characteristic yellow color of bilirubin.
o Positive chemical test result for bilirubin would be
CRYSTALS ASSOCIATED WITH LIVER DISORDERS expected.
 Tyrosine o Disorders that produce renal tubular damage, such as viral
o Appear as fine colorless to yellow needles that frequently hepatitis, bilirubin crystals may be found incorporated into
form clumps or rosettes. the matrix of casts.
o Usually seen in conjunction with leucine crystals in
specimens with positive chemical test results for bilirubin.
o May be encountered in inherited disorders of amino acid
metabolism.

27
 URINARY SEDIMENT ARTIFACTS
Starch granules Diaper fiber
 Contamination may occur when cornstarch is the  May initially mistaken for cast
powder used in powdered gloves.  Usually much longer and more refractile
 Highly refractile spheres, usually with a dimpled center  Examination under polarized light can frequently
 Resemble a fat droplet when polarized, producing a differentiate b/w fibers and cast.
Maltase cross formation.
 Occasionally confused as RBCs.
 Differentiation b/w starch and pathologic elements
may include chemical test for blood or protein and the
presence of oval fat bodies or fatty cast.

Hair
 May initially mistaken for cast
 Usually much longer and more refractile
 Examination under polarized light can frequently
differentiate b/w fibers and cast.
Oil droplets
 Highly refractile QUALITY ASSURANCE AND MANAGEMENT IN
 May resemble RBCs to inexperience laboratory
personnel. THE URINALYSIS LABORATORY
 Contamination by immersion oil or lotions and creams
 Maybe seen with fecal contamination QUALITY ASSURANCE
 The overall process of guaranteeing quality patient care
 Continual monitoring of the entire process test ordering and
Air bubbles specimen collection through reporting and interpreting
 Highly refractile results.
 May resemble RBCs to inexperience laboratory
personnel.
 Occur when the specimen is placed under a cover slip.

28
 INCLUDED ARE THE FOLLOWING: o Do not assume any information about the specimen or
 Procedure manuals patient
 Pre analytical factors o Do not re-label an incorrectly labelled specimen
 Analytical factors o Do not discard the specimen until the examination is
 Post analytical factors complete
o Leave the specimen exactly as you receive it
o Identify problems on specimen requisition with date, time,
PROCEDURE MANUAL and initials or signatures
Contains all the procedures performed in the urinalysis o Make person responsible for specimen collection
section. participate in the solution of the problem
o Report all mislabeled specimen
THE FOLLOWING ARE INCLUDED:
Principle or purpose of the test ANALYTICAL FACTORS
Patient preparation Processes that directly affect the testing of the specimen
Specimen type and method of collection  Quality control
Reagent, standard and control  Materials, procedures, techniques that monitor the
Instrumentation, calibration, and maintenance protocol and accuracy, precision and reliability of a laboratory tests.
schedules  Performed to ensure that acceptable standards are met
Procedures during the process of patient testing.
Normal values  Performed at scheduled time
Limitations of the test
References  Internal quality control
o Used to verify accuracy and precision
PREANALYTICAL FACTORS o Levy Jennings chart
Test request
o Complete data of the patient  External quality control
o Testing of unknown sample received from outside
Patient preparation agency
o Proper instruction to the patient or relative of the patient o Provides unbiased validation of the patient test results

Specimen collection

29
  Reagent o Standardized reporting format
 All reagents and reagent strips must be properly labelled o With reference ranges
with the following o Written procedures should be available for reporting of
o Date of preparation or opening critical values
o Purchase date Interpretation of result
o Expiration date
o Appropriate safety information SUMMARY OF QUALITY ASSURANCE ERRORS
Pre analytical
 Instrumentation and equipment
 Patient misidentification
 Instrumentation
 Wrong test ordered
o Refractometers
 Insufficient urine volume
o Automated reagent strip readers
 Incorrect urine specimen type collected
o Automated microscopy instruments
 Delayed transport of urine to the laboratory
 Incorrect storage or preservation of urine
 Equipment
o Refrigerators
o Centrifuge Analytical
o Microscopes  Sample misidentification
o Water baths  Erroneous instrument calibration
 Reagent deterioration
 Testing procedures  Poor testing technique
 Detailed, concise testing instruction written in a step by  Instrument malfunction
step manner  Interfering substance present
 Misinterpretation of quality control data
 Personnel and facilities
Post analytical
POST ANALYTICAL FACTORS  Patient misidentification
Process that affect the reporting of results and correct  Poor handwriting
interpretation of data.  Failure to send report
 Failure to call critical values
Reporting of result  Poor quality of instrument printer
 Inability to identify interfering substances

30
 RENAL DISEAS o Attachment of this autoantibody to the basement
membrane, followed by complement activation, produces
the capillary destruction.
RENAL DISEASE
 Disorders throughout the body can affect renal function  Chronic glomerulonephritis
 Often classifies as being glomerular, tubular, interstitial, or o progression to chronic glomerulonephritis and end-stage
vascular, based on the area of the kidney primarily affected. renal disease may occur.
o Gradually worsening symptoms include fatigue, anemia,
GLOMERULAR DISORDER hypertension, edema, and oliguria.
 Acute poststreptococcal glomerulonephritis o Examination of the urine reveals hematuria, proteinuria,
o sudden onset of symptoms consistent with damage to the glucosuria as a result of tubular dysfunction, and many
glomerular membrane. varieties of casts, including broad casts.
o may include fever; edema, most noticeably around the o A markedly decreased glomerular filtration rate is present
eyes; fatigue; hypertension; oliguria; and hematuria. in conjunction with increased BUN and creatinine levels
o Symptoms usually occur in children and young adults and electrolyte imbalance.
following respiratory infections caused by certain strains
of group A streptococcus that contain M protein in the cell  Nephrotic syndrome
wall. o marked by massive proteinuria (greater than 3.5 g/d),
low levels of serum albumin, high levels of serum
 Rapid progressive (centric) glomerulonephritis lipids, and pronounced edema.
o has a much poorer prognosis, often terminating in renal o Increased permeability of the glomerular membrane is
failure. attributed to damage to the membrane and changes in
o Symptoms are initiated by deposition of immune the electrical charges in the basal lamina and
complexes in the glomerulus, often as a complication of podocytes, producing a less tightly connected barrier.
another form of glomerulonephritis or an immune
systemic disorder such as systemic lupus erythematosus
(SLE).

 Goodpasture syndrome
o Morphologic changes to the glomeruli resembling those in
Rapidly Progressing Glomerular Nephritis Summary of Laboratory Testing in Glomerular Disorders
o An autoimmune disorder termed

31
DISORDERS URINALYSIS TEST OTHER SIGNIFICANT o Ascending bacterial infection of the bladder
TEST o Clinical Course: Acute onset of urinary frequency and
Acute Macroscopic hematuria  Antistreptolysin O titer
burning resolved with antibiotics
glomerulonephritis Proteinuria  Anti–group A streptococcal
RBC casts enzymes
Granular casts, hyaline  Acute pyelonephritis
WBCs o Infection of the renal tubules and interstitium related to
Rapidly progressive Macroscopic hematuria  BUN interference of urine flow to the bladder, reflux of urine
glomerulonephritis Proteinuria  Creatinine
from the bladder, and untreated cystitis
RBC casts  Antiglomerular basement
membrane antibody
Goodpasture Macroscopic hematuria Antiglomerular basement  Chronic pyelonephritis
syndrome Proteinuria membrane antibody o Recurrent infection of the renal tubules and interstitium
RBC casts caused by structural abnormalities affecting the flow of
Chronic Hematuria Serum compliment levels urine
glomerulonephritis Proteinuria
Glucosuria
Cellular and granular casts LABORATORY RESULTS IN TUBULAR DISEASE
Waxy and broad casts Disorders Primary urinalysis result Other significant
Nephrotic Heavy proteinuria Serum albumin test
syndrome Microscopic hematuria Cholesterol Acute tubular necrosis Microscopic hematuria Hemoglobin
Renal tubular cells Triglycerides Proteinuria Hematocrit
Oval fat bodies RTE cells Cardiac enzymes
Fat droplets RTE cell casts
Fatty and waxy casts  Hyaline, granular, waxy, casts
Cystitis Leukocyturia Urine culture
TUBULAR DISORDERS Bacteriuria
Microscopic hematuria
 Acute tubular necrosis Mild proteinuria
o Damage to the renal tubular cells caused by ischemia or Increased pH
toxic agents Acute pyelonephritis Leukocyturia Urine culture
o Decrease blood flow by causing lack of oxygen Bacteriuria Blood cultures
presentation to the tubules or the presence of toxic WBC casts
substances in the urinary filtrate. Bacterial casts
Microscopic hematuria
Proteinuria
 Cystisis Chronic pyelonephritis Leukocyturia Urine culture

32
 Bacteriuria Blood cultures
WBC casts BUN Tyrosyluria
Bacterial casts Creatinine
Microscopic hematuria o Most frequently seen is a transitory tyrosinemia in
Creatinine
Proteinuria clearance premature infants
o caused by underdevelopment of the liver function
required to produce the enzymes necessary to complete
URINE SCREENING FOR METABOLIC the tyrosine metabolism.
o This condition seldom results in permanent damage
DISORDERS o may be confused with PKU when urinary screening tests
URINE SCREENING FOR METABOLIC DISORDERS are performed
 many of the abnormal results obtained in the routine o ferric chloride test produces a green color.
urinalysis are related to metabolic rather than renal disease. o Nitroso – Napthol (+) result to orange red color

AMINO ACID DISORDERS Alkaptonuria

Phenylketonuria o The name alkaptonuria was derived from the observation
o estimated to occur in 1 of every 10,000 to 20,000 births that urine from patients with this condition darkened after
o if undetected, results in severe mental retardation. becoming alkaline from standing at room temperature.
o caused by failure to inherit the gene to produce the o the term “alkali lover,” or alkaptonuria, was adopted.
enzyme phenylalanine hydroxylase. o Without this enzyme, the phenylalanine-tyrosine pathway
o Once discovered, dietary changes are made cannot proceed to completion, and homogentisic acid
o eliminate phenylalanine, a major constituent of milk, from accumulates in the blood, tissues, and urine.
the infant’s diet can prevent the excessive buildup of o Later, brown pigment becomes deposited in the body
serum phenylalanine and can thereby avoid damage to the tissues (particularly noticeable in the ears).
child’s mental capabilities. o Deposits in the cartilage eventually lead to arthritis.
o Urine testing can be used as a follow-up procedure in o A high percentage of persons with alkaptonuria develop
questionable diagnostic cases liver and cardiac disorders.
o a screening test to ensure proper dietary control in o HOMOGENTISIC ACID REACTS
previously diagnosed cases  screening tests for metabolic disorders including the
o a means of monitoring the dietary intake of pregnant ferric chloride test, in which a transient deep blue color
women known to lack phenylalanine hydroxylase is produced in the tube test.
o ferric chloride test, (+) blue green color  Benedicts test (+) – yellow precipitate

33
  Addition of alkali to freshly voided urine and to CYSTINE DISORDERS
observe for darkening of the color  Cystinuria
 Silver nitrate and ammonium hydroxide (+) black urine o marked by elevated amounts of the amino acid cystine in
the urine.
Melanuria o The presence of increased urinary cystine is not due to a
o produces a darkening of urine defect in the metabolism of cystine but, rather, to the
o reacts with ferric chloride, sodium nitroprusside inability of the renal tubules to reabsorb cystine filtered by
(nitroferricyanide) and Ehrlich reagent the glomerulus.
o In the ferric chloride tube test, a gray or black precipitate o laboratory screening determinations are based on the
forms in the presence of melanin and is easily observation of cystine crystals in the sediment of
differentiated from the reactions produced by other amino concentrated or first morning specimens.
acid products. o A chemical screening test for urinary cystine can be
o A red color is produced by the reaction of melanin and performed using cyanide-nitroprusside produces a red-
sodium nitroprusside. purple color in a specimen that contains excess cystine.
 Cystinosis
Maple Syrup Urine Disorder
o The incomplete metabolism of cystine results in crystalline
o MSUD is also included in newborn screening profiles using
deposits of cystine in many areas of the body, including
MS/MS
the cornea, bone marrow, lymph nodes, and internal
o MSUD is caused by an IEM, inherited as an autosomal
organs.
recessive trait.
o The amino acids involved are leucine, isoleucine, and
 Homocystinuria
valine.
o Silver nitroprusside test (+) red purple
o urine specimen that produces a strong odor resembling
maple syrup that is caused by the rapid accumulation of
keto acids in the urine. PORPHYRINE DISORDERS
o urine screening test most frequently performed for keto Porphyrins
acids is the 2,4-dinitrophenylhydrazine (DNPH) reaction o the intermediate compounds in the production of heme.
w/c produce a yellow or white precipitate
Porphyrias
o Disorders of porphyrin metabolism
o can be inherited or acquired from erythrocytic and hepatic
malfunctions or exposure to toxic agents.

34
 two screening tests for porphyrinuria use the Ehrlich reaction
and fluorescence under ultraviolet light in the 550- to 600-nm
range.
Ehrlich reaction
o can be used only for the detection of ALA and
porphobilinogen.
Watson-Schwartz test for differentiation between the
presence of urobilinogen and porphobilinogen

MUCOPOLYSACCARIDE DISORDER
PURINE DISORDERS
 known as Lesch - Nyhan disease that is inherited as a sex-
linked recessive result in massive excretion of urinary uric acid
crystals.

CARBOHYDRATE DISORDER
 increased urinary sugar (melituria) is most frequently due to
an inherited disorder.
 Presence of galactosuria, indicating the inability to properly
metabolize galactose to glucose.

35
 CEREBROSPINAL FLUID

FINALS
CEREBROSPINAL FLUID
 One of the major fluids in the body
 Flows through the subarachnoid space between the arachnoid
matter and the pia matter.

FUNCTIONS:
 provides a physiologic system to supply nutrients to the
nervous tissue
ANALYSIS OF URIN AND OTHER BODY FLUIDS  remove metabolic wastes
 produce a mechanical barrier to cushion the brain and spinal
cord against trauma.

VOLUME:
Adult – 140 ml to 170 ml
Neonate – 10ml to 60ml

SPECIMEN COLLECTION AND HANDLING

 collected by lumbar puncture between the third, fourth, and
fifth lumbar vertebrae
 collected in three sterile tubes
 test to be performed on STAT basis
 if not possible
o chemistry and serology tubes are frozen
o microbiology at room temperature
o cell count to be placed inside the refrigerator

36
CLINICAL SIGNIFICANCE OF CSF APPEARANCE DIFFERENTIAL COUNT ON CSF SPECIMEN
APPERANCE CAUSE MAJOR SIGNIFICANCE  valuable diagnostic aid
Crystal clear Normal  performed on a stained smear
Hazy, WBCs Meningitis  percentage of mononuclear and polynuclear present
turbid, RBCs Hemorrhage  lymphocytes and monocytes
Traumatic tap o cells found in normal CSF
milky, Microorganisms Meningitis  Adult
cloudy Protein Disorder that affect blood – o Predominance of lymphocytes to monocytes
brain barrier  Monocytes
Bloody RBCs Hemorrhage o Prevalence to children
Traumatic tap  Pleocytosis
Xanthochromic Hemoglobin Hemorrhage o Presence of increased numbers of normal cells
Lysed cells CELLS SEEN IN CEREBROSPINAL FLUID
TYPE OF CELL CLINICAL SIGNIFICANCE MICROSCOPIC FINDINGS
Bilirubin RBC degradation
Carotene Increased serum levels Lymphocytes Normal All stages of development
Viral, tubercular, fungal meningitis may be found
Protein Disorders affecting blood-brain Bacterial meningitis Granules may be less
Neutrophils
barrier Early cases of viral, tubercular, and prominent than
Melanin Meningeal melanosarcoma fungal meningitis in blood
Cerebral hemorrhage
Monocytes Normal Found mixed with
CELL COUNT Viral, tubercular, fungal meningitis lymphocytes
 Red blood cell count Macrophages RBCs in spinal fluid May contain phagocytized
o Usually determined only when traumatic tap has occurred RBCs
appearing as empty
vacuoles or ghost
 White blood cell count cells,
o Lysis of RBC prior to WBC count using acetic acid Blast forms Acute leukemia Lymphoblasts,
o Neubauer counting chamber myeloblasts,
monoblasts
o Counted in four corner squares and the center square on
Ependymal, Diagnostic procedures Appears in cluster
both sides of hemocytometer choroidal, and (Pneumoencephalography)
Normal adult CSF count spindle-shaped cells
0 WBC/ ul – 5 WBC/ ul Malignant cells Metastatic carcinomas Seen in clusters with
200 WBCs or 400 RBCs appear clear Primary central nervous system fusing of cell borders
carcinoma and nuclei

37
CHENISTRY TEST o TRICLOROACETIC ACID
 Protein determination  It will precipitate the albumin and globulin equally

PROTEIN FORMATION DYE – BINDING TECHNIQUE

 Albumin o Advantage of using a smaller size and less interference
o Compromises the majority of CSF protein from external sources
 Pre -albumin o Use coomassie brilliant blue g 250
 Alpha globulin  Bind to variety of protein
o Include haptoglobin and ceruloplasmin o Beer’s law
 Transferrin  The concentration of the protein present will
o Major beta globulin present determine the amount of blue color procedure.
 IgG
 IgA ELECTROPHORESIS
Detection of oligoclonal bands representing inflammation
within the Central Nervous System
CLINICAL SIGNIFICANCE
Presence of two or more oligoclonal bands in the
 ELEVATED TOTAL PROTEINS
cerebrospinal fluid that are not present in the serum can be
o Damage to the blood brain barrier (meningitis and
valuable tool in the diagnosis of multiple of multiple sclerosis,
hemorrhage)
encephalitis, neurosyphilis, neoplastic disorders
o Production of Ig within the Central Nervous System
OLIGOCLONAL BANDS
o Decreased clearance of normal protein from the fluid
o Located in the gamma region of the protein
o Degeneration of neutral tissue
electrophoresis
 LOW VALUES
GLUCOSE
o Fluids is leaking from the Central Nervous System
※ NORMAL VALUE: 60% - 70% of plasma glucose
※ Blood glucose is drawn about two hours prior to spinal tap to
METHODOLOGY allow time for equilibration between the blood and the fluid.
PRINCIPLES OF TURBIDITY
※ Specimens should be tested immediately to avoid glycolysis
o SSA
※ Elevated CSF glucose result in elevated plasma glucose
 Combination of sodium sulfate
※ Low CSF glucose results to meningitis, bacterial meningitis,
tubercular meningitis

38
 ※ Normal CSF glucose results to viral meningitis. Neisseria meningitidis (gram (-) cocci)

CEREBROSPINAL FLUID LACTATE AMNIOTIC FLUID

 Meningitis cause
Amniotic fluid
GLUTAMIN Present in amnion
Produced in CNS by the brain cells from ammonia and α – keto Provide protective cushion for the fetus and allow movement
glutarate Amniotic fluid increases throughout the pregnancy
Serves as to remove the toxic metabolic waste product
Fetal urine after the first trimester
ammonia from CNS
Amniocentesis
NORMAL VALUE: 8mg/dl – 18mg/dl
o Needle aspiration into the amniotic sac
Elevated levels associated with liver disorder
Increased synthesis of glutamine is caused by the excess
ammonia in the CNS TYPES OF AMNIOCENTESIS
Determination of CNS glutamine provides an indirect test for ※ Transabdominal
the presence of excess ammonia in the CSF ※ Vaginal
Frequently requested for patients in coma with unknown
origin AMNIOCENTESIS
75% of children with Reye’s Syndrome have elevated CSF ※ Performed after the 14th week of gestation
glutamine levels
 Maximum of 30 ml in sterile syringe
MICROBIOLOGY TEST  First two to three ml collected is discarded
 Identification of the causative agent of meningitis  For bilirubin analysis (HDN) must always be protected from
 24 hours in cases of bacterial meningitis to six weeks for light
tubercular meningitis  NORMAL AMNIOTIC FLUID: colorless
 Method used for primary diagnosis in CSF is Gram Stain, acid –  TURBID, SLIGHT IMODERATE: cells
fast stain, India ink preparation and latex agglutination test  BLOOD – STRAKED: traumatic tap
 For blood culture Abdominal trauma
 Streptococcus pneumoniae (gram (+) cocci)
Haematophilus influenzae (peumorphic gram (-) rods)
Escherichia coli (gram (-) rods)

39
KHEIHAUER – BESKE TEST
 For fetal hemoglobin o SPHINGOMYELIN
 Lipid that is produced at a constant rate after about 26
※ YELLOW: presence of bilirubin weeks of gestation
※ DARK GREEN: presence of meconium
※ DARK RED BROWN: associated with fetal death o LECITHIN – SPHINGOMYELIN RATIO
 Determine the fetal lung maturity
 Prior to 35 weeks of gestation lecithin – sphingomyelin
TESTS FOR FETAL DISEASE
ratio
 Hemolytic disease of the newborn (HDN)
 Usually less than 1.6 because large amount of
o Evaluates severity of fetal anemia due to HDN
lecithin is not yet produce at this time.
o Measurement od amniotic fluid bilirubin performed by
 lecithin – sphingomyelin ratio reaches 2.0 or higher a
spectrophotometric analysis
preterm delivery is relatively safe.
 Neural tube defects
 Foam stability index
o Increase levels of AFP in both maternal circulation and
o Measures the individual lung – surface lipid concentration
amniotic fluid
o Foam or shake tests
o AFP (alpha – feto – protein)
o Amniotic fluid is mixed with 95% ethanol, shaken for 15
 Major protein produced by the fetal liver during early
seconds, allowed to sit undisturbed for 15 minutes
gestation (prior to 18 weeks)
o POSITIVE RESULT
 Presence of continuous line of bubbles around the
TESTS FOR TETAL MATURITY outside edge.
 Fatal lung maturity
o Compared to lecithin – sphingomyelin ratio
HUMAN CHROMIC GONADOTROPHIN HORMONE
Quality determined to detect whether the female patient is
o LECITHIN
pregnant or not
 Primary component of surfactant that make up for
Hormone produce by the placenta after implantation
alveolar lining and account for alveolar stability
It can support pregnancy by allowing the production of
 Produce at relatively low and constant rate until the
progesterone, which help to prepare the lining of the uterus
35th week of gestation
for implantation.
 Stability of fetal lung alveoli.

40
 Glycoprotein composed of 237 amino acids with molecular  Synoviocytes
mass of 25.7 kDa. o Cells lining the synovial fluid
 Arthritis
TYPES OF PREGNANCY TEST  Arthrocentesis
 ENZYME IMMUNO ASSAY o Collection by needle aspiration
o Quick and accurate test that can be used on site and in the
laboratory NORMAL SYNOVIAL FLUID VALUE
o A test that uses antibodies and color changes to identify a Volume <3.5mL
substance. Color Pale yellow
Clarity Clear
Viscosity Able to form a string 4 – 6 cm long
Erythrocyte count <2000 cells/uL
Leukocyte count <2000 cells/uL
Neutrophils <20% of the differential count
Lymphocytes <15% of the differential count
Monocytes and macrophage 65% of the differential count
Crystals None
Glucose <10 mg/dl lower than the blood glucose
 AGGIUTINATION Lactate <250 mg/dl
o Inhibition based assay Total protein <3 g/dl
Uric acid Equal to blood volume
 SANSWICH ELISA
APPERANCE AND VISCOSITY
Clear and pale yellow
o normal
SYNOVIAL FLUID Deeper yellow
o Presence of inflammation
SYNOVIAL FLUID Greenish tinge
 Referred to as JOINT FLUID o Bacterial infection
 Viscous liquid in the cavities of the movable joints – Turbidity
diarthroses o Presence of White Blood Cells
 Ultrafiltrate of the plasma across the synovial membrane Milky

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 o Crystals are present

VISCOSITY
 Polymerization of hyaluronic acid
 Essential for the proper joint lubrication

MEASUREMENT OF DEGREE OF HYALURONATE DIFFERENTIAL COUNT

POLYMERIZATION  Should be performed on cytocentrifuge preparation or on
 Ability of the fluid to form a string (4 – 6 cm) thinly smeared slides
 Rope or mucin clot test = 2 – 5% acetic acid, normal synovial  Fluids should be incubated with hyaluronidase prior to slide
fluid forms a solid clot surrounded by clear fluid preparation.
 Mucin clot test is reported as:  The primary cells seen in normal synovial fluids are:
o Solid clot o Monocytes
o Soft clot o Macrocytes
o No clot o Synovial tissue cells
o Friable clot  Neutrophils should be less than 25%
 Lymphocyte should be less than 15%
CELL COUNT  Eosinophils
※ Total leucocyte count  Reiter cells
※ Red Blood Cell counts  RA cells
※ Count should be done or performed as soon as possible
※ Very viscous fluid may need to be pretreated by adding one CELLS AND INCLUSION SEEN IN SYNOVIAL FLUID
drop of 0.05% hyaluronidase in phosphate buffer per milliliter Cells/Inclusion Description Significance
of fluid and incubating of 370C for 5 minutes Neutrophils PMN Leukocytes Bacterial sepsis
※ Neubauer counting chamber is used for manual counting on Lymphocytes MN leukocytes Crystal included inflammation
thoroughly mixed specimen like in Cerebrospinal Fluid Macrophage Normal
counting (monocyte) Viral infection
Synovial lining cells Similar to macrophage, but may normal
※ Dilution using Normal Saline Solution be vacuolated
LE cells Neutrophils containing Lupus erythrmatosus
characteristics ingested round
body

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Reiter cells Vacuolated macrophage w/ Reiter’s syndrome o Differentiate inflammation and sytic arthritis
ingested neutrophils Nonspecific inflammation
RA cells (ragocytes) Neutrophils with dark cytoplasmic Immunologic inflammation
o Lactate level >250 mg/dL (septic arthritis)
granules containing immune  Total protein
complex o Hemorrhagic and inflammatory disorders
Cartilage cells Large, multinucleated cells osteoarthritis
Rice bodies Shows collagen & fiber Tuberculosis, sepsis &  Uric acid
rheumatoid arthritis
Fat droplets Retractile intracellular & Traumatic injury
extracellular globules MICROBIOLOGY TEST
Hemosiderin Inclusions w/in clusters of synovial Pigmented villonudular  Gram stain
cells synovitis
 Culture
o Chocolate agar
CRYSTAL IDENTIFICATION o Staphylococcus
- Important diagnostic test for the elevation of arthritis o Streptococcus
- Crystal formation results in an acute, painful inflammation o Hemophilus species
- Primary crystal seen in synovial fluid o Neisseria gonorrhea
o Monosodium urate (uric acid)
 found in gout SEROLOGY TESTING
o Calcium pyrophosphate  Association of immune system in the inflammation process
 Seen with pseudo gout  Performed on serum
o Hydroxy apatite  Synovial fluid analysis
 osteoarthritis o Serves as confirmatory measure in cases that are
o Cholesterol difficult to diagnose.
 extracellular  Rheumatoid arthritis and lupus erythematosus
o Corticosteroid  Arthritis is a frequent complication of lyme disease (borrelia
 Injection burgdorferi)

CHWEMISTRY TEST
 Glucose
o Normal synovial fluid glucose should not be more than
10 mg/dL lower than the blood value
 Synovial fluid lactate levels

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SEROUS FLUIDS SPECIMEN COLLECTION
Collected by needle aspiration
Thoracentesis
SEROUS FLUID
Paracentesis
Close cavities of the body are each lined by two membranes
EDTA tube – for cell and differential count
referred to as serous membrane
Heparinized tube
Fluid between the membranes
o Chemical
Provides lubrication
o Serological
Normally, only small amount is present
o Microbial
Ultrafiltrate of plasma
o Cytologic analysis
Its production and reabsorption are subjected to hydrostatic
and colloidal pressures
LABORATORY PROCEDURE
Appearance
EFFUSION
Cell count and differential count
 Increase of fluid between membranes
Chemistry tests
Microbiology
 TRANSUDATE
Cytology
o Systemic disorder that disrupts the balance in the
Differentiation between exudate and transudate
regulation of fluid filtration and reabsorption

 EXUDATE PLEURAL FLUID

o Directly involve the membranes of a particular cavity ※ Obtained from the pleural cavity
Transudate Exudate ※ Transudate or exudate
Appearance Clear Cloudy ※ Additional tests
Fluid: serum protein <0.5 >0.05 o Pleural fluid cholesterol
ratio  Greater than 60mg/dl
White Blood Cell count <1000/uL >1000/uL o Pleural fluid to serum cholesterol ratio
Spontaneous clotting NO YES  Greater than 0.3
Pleural fluid <60mg/uL >60mg/uL o Fluid to serum total bilirubin ratio
cholesterol  0.6 or greater
Pleural fluid: bilirubin <0.06 >0.6
ratio
CORRELATION OF PLEURAL FLUID APPEARANCE & DISEASE
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APPEARANCE DISEASE o Associated with pancreatitis
Clear, Normal o Esophageal rupture and malignancy
pale yellow  Triglycerides
Turbid, white Microbial infection o Chylous effusion
Bloody Hemothorax
Hemorrhagic effusion MICROBIOLOGY
milky Chylous material from thoracis duck leakage ※ Associated with pleural effusion
Pseudocylous material from chronic inflammation o S. aureus
o Enterobacteriacea
o Anaerobes
HEMATOLOGY TEST o M. Tuberculosis
Differential count
CELL SIGNIFICANT TESTS
Neutrophil Pneumonia, pancreatitis, pulmonary infection
Gram stain
Lymphocyte Tuberculosis, viral infection, autoimmune disorder,
Culture
malignancy
Mesothelial cells Normal and reactive forms have no clinical
Acid fast stain
significance
Plasma cells Tuberculosis PERICARDIAL FLUID
Malignant cells Metastatic carcinoma Primarily result of damage in the permeability of the
eosinophil Allergic and parasitic infection membranes due to
Associated with trauma (presence of air or blood in o Infection
the pleura cavity) o Malignancy
o Trauma
CHEMISTRY TEST o Metabolic disorder
 Glucose
o Fluid values should be compared with plasma values APPERANCE
o Values less than 60mg/dl is considered decreased  Clear and pale yellow
 pH  Turbid
o lower than 7.3 (chest tube drainage)  Blood streak
o lower than 6.0 – indicates esophageal rupture  Milky
 Amylase  Grossly bloody

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 SEMEN
LABORARTORY TESTS
PHYSIOLOGY
※ Transudate or exudate
 Composed of four fractions contributed individually by
※ Hematology test
o Testes and epididymis 5%
※ Cytologic examination o Seminal vessels 60%
※ Bacterial cultures and gram stain o Prostate 20% - 30%
o Bulbourethral gland 5%
PERITONEAL FLUID
Ascites PROCESSES TAKING PLACE IN VARIOUS PARTS OF MALE
Fluid in the peritoneal cavity GENITAL ORGAN
Clear and pale yellow  Epididymis
Turbid o Sperm maturation and storage
May appear green
Blood streak  Seminiferous tubules
Chylous o Spermatogenesis

LABORATORY TEST  Seminal vessels

 Hematology test o Produce majority of the fluid
 Chemistry test o Fluid contains fructose
o Glucose o It does not become motile until exposed to the SV fluid
o Amylase
o Alkaline  Prostate gland
o Phosphate o Produce acidic fluid, 30% of the semen volume
o BUN o Contains phosphatase, citric acid, zinc and proteolytic
o Creatinine enzymes

 Microbiology test  Bulbourethral glands

o Gram stain
o Bacterial culture

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SPECIMEN COLLECTION 1.0 No forward progression
※ Collected following a period of sexual abstinence of at least 3 0 No movement
days and not longer than 5 days
※ Prolong abstinence tend to have a higher volume and SPERM MORPHOLOGY
decrease motility  Evaluated with respect to both head and tail appearance
※ Warm sterile glass/plastic container  Form a thinly, stained slide
※ Should be collected in a room provided by the laboratory  Air dried slides are stable for 24 hours
within one hour  At least 200 sperm should be evaluated
※ Fresh specimen is clotted and should liquefy within 30-60
minutes.
※ Specimen awaiting should be kept at 370C
※ All semen specimen is potential reservoir for HIV and hepatitis
virus

PARAMITERS FOR SEMEN ANALYSIS

Appearance
Morphology NORMAL SPERM
Volume - Oval
pH - Shaped head approximately 5um long and 3um wide
motility - Flagella tail approximately 45um long
Sperm count/concentration
Viscosity
o Refers to the consistency of the fluid
o Increased viscosity and incomplete liquefaction will
impede sperm motility

SPERM MOTILITY GRADING

GRADE CRITERIA
4.0 Rapid, straight – line motility
3.0 Slower speed, some lateral movement
2.0 Slow forward progression, noticeable lateral movement

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SPERM COUNT EXAMPLE
1. Using a 1:20 dilution, an average of 60 sperm are counted in
the 5 RBC counting squares. Calculate the sperm
concentration/ml and the total sperm count in a specimen
with a volume of 4ml.

60 sperms x 1,000,000 = 60,000,000 sperm/ml

60, 000,000 x 4ml = 240,000,000 sperm/ejaculate

ADDITIONAL TESTING
SPERM CONCENTRATION Sperm visibility
- NORMAL VALUE: 20 – 160 million o This is suspected when a specimen has a normal sperm
- Neubauer counting chamber concentration with markedly decreased motility
- Dilute the specimen (1:20) o Elevated by mixing the specimen with an eosin –
- Diluting fluid contains sodium bicarbonate (NaHCO3) and negrosin stain, prepare a smear and counting the
formalin number of dead sperm cells in 100 sperm
- Sperms are counted in four corners and center square of large o Living cells are not infiltrated by the dye
center square o Dead cells stain red against the purple background
- Only fully developed sperms should be counted o Normal viability requires 75% living cells.

CALCULATION OF SPERM CONCENTRATION Seminal fluid fructose

 Dilution used o Resorcinol test
 Size and number of squares counted o Check for the presence of fructose
 Sperm concentration/ml o Normal quantitative level of fructose is equal to or
 Number of sperms counted can be multiplied by 1,000,000 greater than β umol/ejaculation
 Total sperm count
o Multiply the number of sperms by the specimen Anti-sperm antibodies
volume o Can be present in both male and female
o Detected in semen, cervical mucosa or serum
o Considered a possible cause of infertility

48
 o Male anti-sperm antibodies are more frequently SPECIMEN FUNCTION TEST
encountered  HAMSTER EGG PENETRATION
o Blood testes barrier separates sperm from the male o Sperm are incubated with species nonspecific hamster
immune system. When this is disrupted, the antigens eggs and penetration is observed microscopically
on the sperm produce an immune response that
damages the sperm  CERVICAL MUCUS PENETRATION
o Observation od sperm penetration ability of partner’s
Microbial and chemical testing midcycle cervical mucus.
o Presence of more than 1 million leukocytes per ml
indicates infection, frequently the prostate  HYPO – OSMOTIC SWELLING
o Routine aerobic and anaerobic culture and tests for o Sperm exposed to low sodium concentrations are
chlamydia trachomatis, myocoplasm hominis, and evaluated for integrity and sperm visibility
ureaplasma urealyticum.
 INVITRO A CHROMOSOME REACTION
CHEMICAL TESTING o Evaluation of the acrosome to produce enzymes
Determination of the levels of acid phosphatase, zinc, etc. essential for own penetration

Post vasectomy semen analysis SPERM FUNCTION TESTS

o Detect the presence or absence of spermatozoa  Commonly performed in specialized andrology laboratories
 More sophisticated semen analysis
TWO TESTS FREQUENTLY USED:  Assess the characteristics of the sperm and the functional
MAR ability
o Mix agglutination reaction
o Used to detect the presence of IgA antibodies

IMMUNOBEAD TEST
FECAL ANALYSIS
o A more specific procedure to detect the presence of FECAL ANALYSIS
IgG, IgM, and IgA Antibodies.  End product of body metabolism
 Produce valuable diagnostic information
 Macroscopic, microscopic, chemical

49
PHYSIOLOGY  Intestinal constipation
- Normal fecal specimen contains bacteria cellulose, f) Mucus or blood steaked
gastrointestinal secretions, bile pigments, water  Colitis
- Small intestine is the primary site for the final breakdown and  Dysentery
reabsorption of compounds  Malignancy
- Large intestine is capable of absorbing approximately 3000ml  Constipation
of water g) Green
 Oral antibiotics
SPECIMEN COLLECTION  Green vegetarian
 Instruction
 Random specimen MICROSCOPIC
 Time specimen Leukocytes
o Three days collection o Ulcerative colitis
o Bacterial dysentery
MACROSCOPIC SCREENING o Invasive bacterial pathogen such as salmonella,
Color shigella, E. coli
Appearance o May be examined as wet preparation or stained
a) Black o Wright’s or Gram stain or methylene blue
 Upper Gastrointestinal tract bleeding o Lactoferrin latex agglutination
 Ion therapy  Fecal leukocyte
 Bismuth
b) Red Undigested muscle fibers
 Lower Gastrointestinal tract bleeding o Monitor patients with pancreatic insufficiency (cystic
 Beets fibrosis)
 Rifampicin
c) Pale yellow, white or gray PROCEDURE
 Bile duct obstruction a) Emulsify a small amount of stool in 2 drops of 10% eosin in
 Barium sulfate alcohol
d) Bulky or frothy b) Coverslip and stand for 3 minutes
 Pancreatic disorder c) Examine under high power objective for 5 minutes
e) Ribbon – like d) Count the number of undigested fibers

50
FECAL FATS KINDS OF BLOOD FILM
 Steatorrhea Thick
 Malabsorption disorders o Used to search for malaria parasite
 Lipid in microscopic exam o Consist of many layers of Red Blood Cells and White
o Sudan III, Sudan IV, oil red O Blood Cells
o During staining, dehemoglobinization occurs.
CHEMICAL TESTING OF FECES Thin
 OCCULT BLOOD o Used to confirm malaria parasite
o Bleeding in excess of 25ml/150g of stool is o Giemsa stain
considered pathologic  The recommended stain to be used
o Mass screening tests for occult blood
o Guaiac reagent LIFE CYCLE
 The sexual phase of malaria life cycle takes place in the
 APT TEST (FECAL HEMOGLOBIN) stomach of the mosquito
o Neonates pink color indicates presence of fatal  Soon after the female anopheles’ mosquito has ingested blood
blood from infected person, the male gametocytes each produce 4 –
8 flagella gametocytes
 When the cysts rupture, they release sporozoites which
eventually enter the salivary glands
 After species of mosquito and ambient temperature but which
MALARIA is usually between 7 and 14 days, the anopheline mosquito
can transmit malaria.
MALARIA
 Caused by a small living organism called parasite STAGES OF MALARIA
 Transmitted from person to person through female anopheles ※ Trophozoite stage
mosquito bite o Most commonly, often called as the ring stage,
 Symptoms although it sometimes takes the form of an incomplete
 Most reliable and the gold standard for the diagnosis is the ring
microscopic examination of the patient stained blood film by o A growing stage, where in the parasite within the red
trained microscopist.
cells may vary in size from small to quite large. Pigment
appears as the parasite grows.

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※ Schizont stage
o Stage where malaria parasite starts to reproduce
o Reproduction is referred as asexual because the
parasite is neither male nor female but reproduces
itself by simple division
Trophozoite stage schizont stage

※ Gametocyte stage c) PLASMODIUM MALARIAE

o Sexual parasite that become either male or female in o Less common species
preparation for the next stage, which takes place in the o One that occurs throughout much of the world
stomach of the female anopheline mosquito

MALARIA SPECIES
a) PLASMODIUM FALCIFARUM
o The commonest species in the hotter parts of the
world and responsible for much sickness and even
death Trophozoite stage schizont stage

d) PLASMODIUM OVALE
o Relatively rare species
o Reported from time to time in many counties,
especially in Africa.

Trophozoite stage gametocyte e) PLASMODIUM KNOWLESI

o New malaria species
b) PLASMODIUM VIVAX
o Commonest species in the cooler parts of the tropics
o Largest malaria parasite found in humans.
o Cause of much illness

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