THE HASHEMITE UNIVERSITY Faculty of Engineering Biomedical

Engineering Department
GRADUATION PROJECT-(I)
Hemoglobin Detection
STUDENTS

Ahmad Mahdi Ahmad Al-khatib 1736696

Yousef Omar Yousef Ramdan 1737174
ALmutaz Bellah Mahmoud Lafi 1732809
Ameen Munier Ameen Khater 1738337
Hazim Ahmad Yacoub Khawaldeh
1737383

Graduation Project Advisor

Dr. Bassam AL-Naami
 DEPARTMENT OF BIOMEDICAL ENGINEERING
ACKNOWLEDGMENT

Academic Year
2021-2022

ABSTRACT
Table of Contents
Chapter 1

INTRODUCTION

A reduction in the quantity of hemoglobin present in the blood that is below the
levels that are considered normal for a person's age and gender, according to
medical specialists [1, is defined as follows: The reduction in total erythrosit mass
in the blood circulation is described clinically and pathologically, but it is
functionally characterized as a decrease in the oxygen transport capacity of the
blood, resulting in hypoxia in tissues in the surrounding region (or tissue hypoxia).
As a result of a reduction in the number of erythrositis and/or the quantity of
hemoglobin in the blood, the amount of oxygen that can be delivered by blood
reduces as well. Using hemoglobin concertation (hb), hematotic value (Hct), and
the eritrosit number in everyday practice is more common, more expensive, and
more time-consuming than using the less common, more expensive, and more
time-consuming technique of direct measurement of the eritrosit mass in everyday
practice (which is less common, more expensive, and more time-consuming).
Specifically, in the case of the eritrosits, "these metrics are expressed in the
following order: the quantity in g/dl; the percentage of eritrosits in the blood; and
the mm3, and they represent the eritrosits in contrast to the plasm, the liquid in
which they are suspended" (for example). As a result, they are influenced by
changes in the improving plasm compartment without experiencing any changes in
the eritrosits compartment. Measurements of plasm volume (hemodulation) should
be performed after any suitable medication has been administered, or after a certain
period of time has elapsed, in order to confirm the diagnosis of anemia. When
compared to a normal population, it is possible to detect a significant amount of
dispersion in each of the three metrics. A lot of essential parameters, such as age,
gender, race, and altitude of the dwelling area, have an impact on this dispersion.
Among them are the following: Arithmetic average plus/minus two standard
deviation) of the population [2] and 95 percent (arithmetic average plus/minus two
standard deviation) of the population [3] are included in the provided limitations.
There are several different types of anemia that may occur, including Iron
Deficiency [3, hemolitic anemia [4, megaloblastic anemia [5, aplastic anemia [6,7],
and anemia associated with chronic diseases [8]. It is possible to experience
anemia in a variety of ways. It is described as a reduction in the number of red
blood cells in the body that is associated with a low level of iron in the body,
which is one of the factors taken into consideration while reaching this assessment.
It is important to note that this kind of anemia is by far the most prevalent type of
anemia that may develop. Iron is a critical component of hemoglobin, which is a
pigment in the blood that transports oxygen across the body.

Hemoglobin

When you look at the structure of blood, you will see that it is composed of four globin chains, each of which
contains one haem molecule, which is capable of establishing an irreversible connection with oxygen. When oxygen
binds to hemoglobin, its oxygen affinity is changed, resulting in structural changes in the globin chains that surround
and surround the hemoglobin molecule. As a consequence of this "molecular cooperativeness" inside hemoglobin,
which is regulated by a variety of factors such as pH, CO2, and 2,3-diphosphoglycerate, among others, it is feasible
to construct a sigmoidal-shaped oxygen dissociation curve in hemoglobin. When hemoglobin interacts with carbon
dioxide, carbamino molecules are formed as a result of the interaction between the gases. It is the carbamino
chemicals that help to buffer the hydrogen ions that are found inside the red blood cells. This allows carbon dioxide
to be carried more effectively throughout the body. Several theories have been advanced to explain how normal
hemoglobins grow. These theories include changes in the globin chains or the iron atom, as well as changes in the
binding of ligands other than oxygen to the iron atom. It is important for the production and destruction of
hemoglobin. Among males, hemoglobin levels ranged from 13.5 to 18.0 grams per deciliter of blood, while female
hemoglobin levels ranged from 11.5 to 16.0 grams per deciliter of blood. Depending on the species, each erythrocyte
contains around 200–300 million molecules of hemoglobin in each cell [9].

Synthesis

A hemoglobin molecule is composed of four polypeptide globin chains. Each contains a haem moiety that has an
organic part (a protoporphyrin ring made up of four pyrrole rings) and a central iron ion in the ferrous state (Fe2þ).
Normal adult hemoglobin molecules (HbA) have a molecular mass of 64,458 Da with a complex quaternary
structure, the function of which has been extensively studied and is described below. Erythrocytes containing
hemoglobin are produced in the bone marrow of the long bones, such as femur and humerus, and flat bones, such as
sternum and ribs. Erythropoiesis is mainly under the control of erythropoietin, which is released from the kidney in
response to cellular hypoxia mediated by hypoxia-inducible transcription factors [10].

Structure and Function

Hemoglobin is a protein found in mammals that has a molecular weight of around 64,500 Da and is made of four
components. Each subunit contains one polypeptide chain and one proton-donating prosthetic group (the haem), in
addition to one proton-donating prosthetic group (the heme group) and one proton-donating prosthetic group (the
heme group), as well as one proton-donating prosthetic group (the heme group) and one proton-donating prosthetic
group (the heme group) (the Hema group). This is due to the fact that one iron atom in the ferrous state is positioned
in the center of a protoporphyrin ring, which is responsible for the ring's crimson hue. Instead of the ferrous iron
atom in free haem being changed to ferric iron via a reaction with oxygen, the ferrous iron atom in hemoglobin is
coupled with oxygen reversibly and remains in the ferrous form regardless of whether the hemoglobin is oxygenated
or deoxygenated (see Figure 1). It is the folding of the polypeptide chains around the haem groups that allows
hemoglobin to accomplish this reversible combination with oxygen. This folding encloses the haem groups in
hydrophobic pockets, which allows them to combine again when they are exposed to oxygen. All four polypeptide
chains analyzed include polypeptide chains that are about the same length as the other three polypeptide chains. The
a-chains are made up of two identical chains that each have 141 amino acids, while the/3-chains are built up of two
further identical chains that each carry 146 amino acids. a-chains are the most common kind of chain seen in
proteins. The a-chains are known as the a-chains due to the fact that they are similar in every way. Many species'
amino acid sequences have been determined by deducing their DNA sequences from their amino acid sequences.
Tertiary structure is defined as the shape of a subunit governed by the geometry of the backbone of each chain in
conjunction with the arrangement of amino acid side chains. A majority of the amino acid residues in both the alpha
and alpha-3 subunits are structured in helices, where the amino groups of the peptide backbone are hydrogen-bonded
to the carboxyl groups of neighboring turns, as can be seen in the illustration.
Helical segments are divided into parts by corners and short, non-helical segments in each subunit, for a total of
eight helical segments per subunit. It is the way in which the four subunits of the tetramer are organized in a
molecule that is known as the QUATERNARY structure of hemoglobin[11].
The structures of hemoglobin with and without oxygen on hemoglobin haem have been identified in sufficient detail
by X-ray analysis to allow many of the atoms, with the exception of hydrogens, to be located within 0.5/ of their real
locations. It has been determined that human hemoglobin and horse hemoglobin from horses are the two species that
have been studied. It was determined via high-resolution research that the changes between the tertiary structures of
the two species are minor and that their quaternary structures are almost similar. Furthermore, a more in-depth
investigation of their quaternary structures at lower resolutions reveals that the two oxygenated forms are
structurally identical to one another in all respects.
Both oxy- and deoxyhemoglobin have quaternary structures that are quite different from one another in a variety of
ways, including their atomic arrangements. The A81 pair in both structures is similar to the a2/32 pair in the first
structure due to the fact that the structure has an orthogonal axis, which is conserved throughout the quaternary
structure change[12]. Essentially, the overall effect of the quaternary structure change between oxy- and
deoxyhemoglobin is to maintain the,-chains in virtually the same positions relative to one another while increasing
the separation of the /3-chains, resulting in an increase in the amount of space available in the central cavity between
them and the haem groups being 6 A further apart from one another. In order to do this, the chains are kept in
approximately the same places relative to one another, but the /3-chains are separated by an increasing amount. The
letter A is used to symbolize the interface between the letters A and B in a sentence[13].

Hemoglobin (Hb) is widely known as the iron-containing protein in blood that is essential for O2 transport in
mammals. Less widely recognized is that erythrocyte Hb belongs to a large family of Hb proteins with members
distributed across all three domains of life—Bacteria, archaea, and eukaryotes. The three-dimensional structure of
hemoglobin has been studied by the methods of X-ray crystallography. From low-resolution studies, the hemoglobin
molecule was shown to comprise two pairs of identical subunits, the et- and/3-chains[14]. The structure of each
subunit is similar to that of the monomeric respiratory protein myoglobin. The four subunits are arranged
tetrahedrally and the structure is such that the four haem groups, the binding sites for oxygen, are far apart, each in a
separate pocket on the surface of the molecule. The arrangement of the four subunits in the hemoglobin tetramer is
different in deoxygenated and liganded hemoglobin, explaining Haurowitz's observation (1938) that crystals of
deoxyhemoglobin dissolve on exposure to air and that oxyhemoglobin crystal with a different morphology
precipitate from the solution. Electron density maps have now been made at high resolution (- 2.8/~) for horse
methemoglobin (i.e. hemoglobin with the iron atoms in the ferric state with water molecules as haem ligands) and
for horse and human deoxyhemoglobin. With the aid of the amino acid sequences of the a-and /3-polypeptide
chains, which have been determined for both horse and human hemoglobin, atomic models of the structures have
been built from the electron density maps. The accuracy of the latest models is such that from the better-determined
regions of the maps, for example, the polypeptide backbone in helical regions and the internal side chains, the
atoms, except for hydrogens, can be positioned to within 0.5 ~. The surface side chains and some parts of the
structure that are ill-defined in the maps are positioned less well[15].

The connection between Hemoglobin and Iron.

When it comes to producing blood in the human body, a large quantity of iron is necessary.
Blood hemoglobin is an iron-containing protein present in the red blood cells of humans and other animals, as well
as in plant and animal tissues. The transfer of oxygen into the red blood cell and the binding of hemoglobin to
hemoglobin allow for the distribution of oxygen throughout the body, both via the circulation and into the tissues,
thanks to the transfer of oxygen into the red blood cell. Hb is composed of two primary components: heme
molecules, which are iron-containing structures, and globin molecules, which are proteins that surround and protect
the heme molecules. Heme molecules are composed of two major components: heme molecules and globin
molecules. The majority of hemoglobin is made up of heme molecules and globin molecules. Scientists are still
trying to figure out the exact nature of the link between hemoglobin and iron. In the synthesis of hemoglobin, iron is
a critical component, and it may be found in abundance in the natural world. Red blood cells are replaced with new
ones about every six months. Throughout the process, the iron from the old red blood cells is reused and recycled.
Despite the fact that we recycle, our bodies continue to need iron on a daily basis, which is provided by the iron in
our meals[16].
Hemoglobin is responsible for around 70% of the iron found in your red blood cells, according to the World Health
Organization. There are certain proteins in your body that contain a trace amount of iron, which accounts for around
6 percent of the total iron in your body. It is necessary for a variety of cell or tissue activities to be carried out
properly, including respiration and metabolism, that these molecules be present. The presence of iron is also
required for the maintenance of a healthy immune system that is both efficient and reliable.
It is a kind of iron-storing protein that contributes to about 25% of the total amount of iron in the human body,
according to the World Health Organization. Because of the capacity of the circulatory system to carry this protein
throughout the body, it is possible to find it in all of the cells, tissues, and organs of the body. Hemoglobin levels fall
as a consequence of the depletion of the body's iron stores, which is a result of this disorder. The reduction in the
quantity of iron in the body is referred to as iron depletion, and it may result in iron-deficient erythropoiesis in a
very short period of time (red blood cell formation). When iron levels in the human body reach their lowest point,
iron deficiency, commonly known as anemia in medical terminology, occurs. It is the most deadly of the disorders.
It is necessary to invest at least 1.8 mg of iron per day via one's usual diet in order to maintain optimal hemoglobin
levels. Among the iron-dense foods that are presently available on the market are venison, hog, turkey, chicken, and
liver, to name a few. The iron content of vegetables and other comparable goods may vary greatly depending on
their composition. Spinach, jaggery, dates, potatoes, green beans, broccoli, and other similar products can all
provide significant quantities of iron to the body[17].

Iron deficiency (Anaemia)

Worldwide, anemia and iron deficiency are problems that affect individuals of all ages. In addition to affecting
persons of all ages, they are severe public health challenges that are often encountered in ordinary clinical practice.
Irrespective of age, iron deficiency and anemia are major public health problems that affect people of all ages and
have an influence on people of all ages, and they are frequent medical diseases that doctors face regularly in their
offices[18]. Although the prevalence of iron deficiency anemia has decreased in recent years in many parts of the
world, iron-deficiency anemia remains the most common cause of anemia worldwide, and it has a significant impact
on the lives of young children and premenopausal women in both low- and high-income countries. Future
breakthroughs in the diagnosis and treatment of this disease are quite likely, and there is little question about that.
Iron is needed by the body for a multitude of functions for it to function effectively, including respiration, energy
generation, DNA synthesis, and cell proliferation. Iron is also required by the body for the production of red blood
cells. A number of mechanisms have been discovered that enable the human body to save iron. When red blood cells
are ruptured, these processes include the ability to recycle the metal and the development of a strategy for holding
metal in the absence of an excretory mechanism. Despite this, since high quantities of iron may be detrimental, its
absorption is limited to 1–2 mg daily, and a significant percentage of the iron needed daily (about 25% of total daily
needs) is given by macrophages, which phagocytose and recycle old erythrocytes as well as the iron. In order to
maintain proper iron levels in the body, hepcidin is a hormone that controls total body iron levels. It aids in the
prevention of both iron deficiency and excess in the body. Deficiency in iron reserves is defined as a decline in iron
reserves that occurs before the development of overt iron deficiency anemia or that persists without advancing to
anemia in the absence of other factors[19]. A more severe condition in which low iron levels are accompanied by
anemia, as well as the presence of microcytic hypochromic red blood cells, is hemochromatosis. It is defined by the
presence of hypochromic red blood cells in the blood (microcytic hypochromic red blood cells). Extrapolating from
iron-restricted erythropoiesis, it is feasible to conclude that the transport of iron to erythroid precursors is hampered,
regardless of how well-stocked the iron stores are with the metal in question. 3,4 The presence of chronic
inflammation, such as that produced by autoimmune disorders, malignancies, and viral diseases, as well as that
induced by chronic renal disease, among other circumstances, may result in normal or even increased iron stores as a
result of iron sequestration. Having iron deficiency and anemia is common in the elderly and those suffering from
chronic illnesses, and it may be detected in people suffering from chronic renal disease as well. The presence of an
iron deficiency or elevated hepcidin levels in the blood may still be used to detect a considerable percentage of the
anemia that is frequent in elderly persons. The following conditions are present in iron-poor erythropoiesis, which
occurs when there is an insufficient mobilization of iron from the body's iron reserves: Learn more about iron
deficiency anemia and other related issues by reviewing the Glossary of Terms and Phrases. Recent breakthroughs
in our understanding of systemic iron homeostasis, which are outlined in the following section, the origins,
pathophysiological aspects, and treatment options for iron deficiency and accompanying anemia in adults will be
discussed in detail in this review. If readers wish to learn more about the look, symptoms, and diagnosis of iron-
deficiency anemia by laboratory testing, they are referred to further resources. They are also pointed in the direction
of other sites that provide knowledge on challenges that are unique to children or expectant moms, for example[20].

Causes of Iron-Deficiency (Anemia)

Anemia is most often caused by malnutrition, starvation, and hunger in developing countries, especially in children
and pregnant women. Anemia is most often caused by poverty, malnutrition, and hunger in the world's poorest
countries, according to the WHO. The phytates present in grains sequester iron in a compound that is not readily
absorbed by the body, lowering iron bioavailability. According to the WHO, chronic blood loss is caused by
hookworm infections and schistosomiasis, both of which induce persistent bleeding. Hookworm is a common
ailment in Africa. People in affluent countries have long followed a strict vegan or vegetarian diet. Anemia is one of
the most common health issues connected with a strict vegan or vegetarian diet. Also, high menstrual losses might
cause malabsorption and chronic blood loss. Men and older people with persistent blood loss from the digestive
tract, including occult blood, may need a gastrointestinal biopsy to rule out malignancy. gastrointestinal blood loss,
particularly in the small intestine, and its causes. Video-capsule endoscopy is becoming increasingly widespread and
may be utilized to establish where gastrointestinal blood loss occurs, especially in the small intestine, and what
causes it when standard testing for iron deficiency anemia in individuals with gastrointestinal blood loss comes back
negative. Regular blood donors are at risk for iron deficiency, and their iron levels should be monitored to ensure
they are not deficient. Intravascular hemolysis causes anemia by excreting iron in the urine. A lack of iron
exacerbates the condition (e.g., in paroxysmal nocturnal hemoglobinuria). Anemia may be caused by blood loss,
hemolysis, and moderate inflammation in endurance athletes. Iron absorption impairment is a common but
underappreciated cause of anemia in women using NSAIDs and anticoagulants (anticoagulants). NSAIDs and
anticoagulants may induce blood loss as a side effect, in addition to their other undesirable effects. It is considered
normal for many causes of iron shortage to present at the same time. Anemia is caused by a lack of iron mixed with
intestinal worm infestations in developing countries. Anemia is especially harmful to children under five. The results
of real-time polymerase chain reaction assays on feces revealed that the degree of iron deficiency is linked to the gut
burden of the hookworm Ancylostoma duodenale (hookworm). Long-term schistosomiasis causes anemia, which is
caused by the body's inflammatory processes[21]. Chronic schistosomiasis often causes anemia. Women with
hypermenorrhea may have iron malabsorption and other issues as a consequence of their sickness. Iron deficiency
anemia may occur in people with end-stage renal disease due to dialysis blood loss, inadequate hepcidin clearance,
inflammation, or the use of certain drugs (e.g., proton-pump inhibitors and anticoagulants). Anemia is increasingly
frequent with age, particularly among the elderly. It has been associated with iron deficiency, inflammatory diseases,
low erythropoietin levels, and malignancy. Increasing subclinical inflammation, higher hepcidin levels, and
impaired iron absorption in obese patients are all likely to lead to mild iron deficiency. According to several studies,
early-stage congestive heart failure patients had higher blood levels of hepcidin than later-stage patients. Iron
deficiency is thought to be caused by poor iron absorption and persistent inflammation in people with congestive
heart failure[22].

Figure 1: - Prevalence of anemia in the middle east by age and gender.

Diagnosis of iron deficiency

The process of identifying whether or not a medical disease exists is referred to as clinical diagnosis. Among the
most essential clinical issues to address when measuring iron status in patients are screening for the iron deficit in
otherwise healthy individuals and diagnosing iron deficiency in patients with overt anemia[23]. Low-cost screening
strategies, such as the use of serum ferritin and hemoglobin levels in combination with other laboratory tests to
identify people at risk for specific diseases, have been demonstrated to be beneficial. A patient's blood may be tested
for the iron deficit if both results are within the normal range. If both findings are normal, it is possible to rule out
the existence of an iron deficiency in the patient's bloodstream. A low serum ferritin level in the presence of a
normal hemoglobin level indicates that the patient's iron reserves have been exhausted. Additional tests such as
erythrocyte protoporphyrin, MCV, or serum receptor may be required to determine how severe his or her iron
shortage has become. Low ferritin and hemoglobin levels in the blood indicate a serious sickness that needs prompt
medical intervention. As soon as feasible, efforts should be made to replenish the iron stores that have been depleted
in the body. When anemia is present in the context of normal or increasing serum ferritin levels, an additional
investigation should be carried out to rule out the potential that there are other possible reasons for anemia that have
not yet been found[24].

Table 1: - The causes of iron deficiency with examples on each cause.

Blood loss due to Iron deficiency

Normally, the quantity of iron in human blood is between 0.4 and 0.5 milligrams per milliliter of blood. A negative
iron balance may be a consequence of blood losses due to physiological, pathological, or iatrogenic reasons. Several
studies have shown that premenopausal women's iron storage is influenced more by the quantity of menstrual blood
they lose than they are by their dietary iron intake. Most women have excessive menstrual bleeding, and in referral
areas, ID affects about half of those who suffer from the disorder, according to the National Institute of Health. It is
possible that an underlying bleeding issue is present in up to 20% of women who have significant menstrual
bleeding that is exacerbated by the IDA (most often, von Willebrand disease).
It is recommended that in future investigations, those with hemoglobin levels lower than the figures below (the
values are shown in g/100 ml of venous blood of individuals living at sea level) should be considered to have
anemia, according to the results[25].

-children aged 6 months to 6 years: 11

-children aged 6–14 years: 12
-adult males: 13 -adult females, non-pregnant: 12
-adult females, pregnant: 11”
Table 2: The mean and lower standard deviation (~2 SD) of normal hemoglobin concentrations (g/l) in a Caucasian
population

Interaction in the testing Kit

Iron storage in bone marrow may be predicted using the serum ferritin concentration, as previously stated, but only
when there is no acute-phase reaction, which is very unusual in patients (10-12). The present analysis did not reveal
any additional important relationships between ESR, fibrinogen, and ferritin concentrations, except a statistically
significant correlation between the three variables. According to our results, the association between CRP and
ferritin concentrations 160 ug/L did not reach statistical significance in the current investigation, in contrast to the
findings of Witte et al (4-6).
In accordance with the nomogram suggested by Witte et al. (ferritin concentrations 160 g/L vs ESR, n = 30), we
would misdiagnose six iron-deficient patients and two patients with trace iron reserves (as indicated by bone marrow
iron stain) if we followed their suggestions.
The inclusion of patients with ferritin concentrations of more than 160 ug/L in nomograms did not enhance the
usefulness of the nomograms provided by Witte and colleagues, as previously reported (4-6). Despite the fact that
both investigations discovered statistically significant connections between ESR and CRP and ferritin (P 0.001), the
nomograms were unable to discriminate between people who had no iron stores, trace iron reserves or positive iron
reserves in their bone marrow[26].
 Table 3. Unadjusted, and age-adjusted correlation between ferritin and serum anemia parameters in women
BMI; body mass index, Hb; hemoglobin, RBC; red blood count, Fe; iron, Htc; hematocrit, TIBC; total iron-binding
capacity, MCV; mean corpuscular volume, MCHC; Mean corpuscular hemoglobin concentration, RDW; red
distribution width.

Chapter 2
Optical systems of hemoglobin and iron deficiency detection

Diagnostic markers such as oxygen saturation, hemoglobin (Hb) concentration, and pulse rate must be monitored in
order to identify the physiological status of a patient. It is necessary to first identify the hemoglobin content of an
individual in order to examine that individual's or another's physiological state or the physiological status of another
person. It is possible to identify and monitor anemia (a low hemoglobin level) and polycythemia vera (a high
hemoglobin level) using the information supplied, allowing for early intervention in both cases. The usage of this
approach may also be used to identify impending post-operative bleeding, as well as the presence of autologous
retransfusions, which both have the potential to be life-threatening occurrences. A variety of approaches are being
researched for the detection of hemoglobin concentrations, and these methods are being further investigated. The
collection and evaluation of blood samples is one example of how this may be performed. Due to the time lag
between blood collection and analysis, it is impossible to provide real-time patient monitoring in an emergency
situation, which is the negative side of the coin. Fortunately, there are some solutions. Online patient monitoring
with little risk of infection is made possible by non-invasive technology, which also allows for real-time data
monitoring, allowing for rapid clinical response to the information collected. The great majority of total blood
absorption in the visible and near-infrared wavelength ranges is accounted for by blood plasma, which is mostly
composed of water and hence absorbs the most light. In these wavelength ranges, the most light is absorbed by
blood plasma, which has the highest absorption capacity. Another issue to consider is the pace at which hemoglobin
derivatives are absorbed into the body. The technique of measuring the transmission or reflection of light through
tissue has been used to detect pulsatile fluctuations in blood volume inside a tissue for many years and is well-
established. When it comes to the medical world, photoplethysmography is the word used to describe this process
(PPG). The hemoglobin content, oxygen saturation, and pulse may all be measured with the use of a newly invented
optical sensor device that measures each of these parameters using three different wavelengths at the same time. A
non-invasive multi-spectral measurement technique is based on the transmission of practically monochromatic light,
generated by light-emitting diodes (LEDs) with wavelengths ranging from 600nm to 1400nm and transmitted
through a small patch of skin on the finger, which is used as a non-invasive and non-invasive multi-spectral
measurement technique because it is non-invasive. Since the sensor produced in this inquiry was totally incorporated
into a wearable finger clip and is capable of full-fledged wireless communication, the usage of a small board-sized
wireless-enabled CPU was necessary[27].

Figure 2. Prototype Hemoglobin Sensor System

The dispersion of the reaction reagent crystals is critical to the accuracy of the detection process. We discovered that
the distribution of reaction Reagent crystals, as well as the size of the crystals, had a substantial impact on the
detection sensitivity of reaction Reagent in our experiments. A confirmation of this was obtained in a laboratory. It
has been found experimentally that when the reaction membrane for hemoglobin and potassium ferricyanide is
disposed of using the fast evaporation technique, the reaction will proceed more quickly than usual. Additional
enhancements to the project's authenticity and sensitivity have been made. It was feasible to investigate the
performance measurement of the optic hemoglobin biosensor with the help of the detecting system, and the findings
were found to be satisfactory. As shown in Fig. 1, it is possible to generate the calibration curve for the biosensor
that is utilized for the detection of hemoglobin concentration. Because the relationship between the reflectance value
and hemoglobin concentration is linear, as indicated in the graph, it is possible to demonstrate that a relationship
exists between the two variables. Additionally, the biosensor may be used to detect hemoglobin at concentrations
ranging from 5.0 to 16.0 grams per deciliter (g/dL), with a correlation coefficient (r) of 0.991 between the two
measurements (see Fig. 5). At room temperature, the findings of the experiment may be acquired in 60 seconds if
the experiment is carried out properly. Due to its focus on the requirements of the medical sector, this breakthrough
biosensor has the potential to meet the growing need for on-the-spot hemoglobin testing in the field of medicine,
among other applications. It was really a computer that was used to simulate and record the requisite empirical
equations based on the data collected during preliminary calibration. The data collected during preliminary
calibration was input into the computer, which then recorded the required empirical equations. Respondents'
responses, as measured in the field, were then utilized to determine the hemoglobin content of their blood using
empirical formulae developed in the laboratory and derived from the responses recorded in the field[28].
 Figure 3. Calibration curve of the biosensor for the determination of hemoglobin in the range of 5.0-16.0 g/dL.

Figure 4.

Biosensor for Hemoglobin Optics for Hemoglobin Optics Hemoglobin Optics Biosensor for Hemoglobin Optics As
shown in Figure 5, this is a mock-up of an experimental assembly of the ocular hemoglobin biosensor for the
purpose of testing. One or more of the following components make up an electrochemical biosensor: a plastic
substrate, a transparent film, a reaction membrane, and an electrical connection, among other things. Comparing the
response membrane that was chosen to the other alternatives, it was found to be very absorbent in comparison.
Preparing the reaction membrane prior to application consisted of pouring the reaction reagent on top of the
membrane, which was then immediately transported to a dry box at 37 degrees Celsius. It took us 10 minutes of
reaction time to get crystals of the reaction reagent that were equally spread across the reaction vessel. In order to
get the reaction reagent, it was essential to dilute a phosphate-buffered solution containing 11.0 g/L saponin and
11.0 g/L potassium ferricyanide in a phosphate-buffered solution of acetic acid, which was previously prepared. The
following steps were taken to create the reaction reagent: It is shown in Fig. 2 that the optic hemoglobin biosensor is
formed of a response membrane of sufficient size that is directly fastened to a plastic substrate by means of a bond,
in addition to a transparent film that has been coated on the sensor's surface[28].
 Figure 5. Schematic of the optic hemoglobin biosensor.

Measurement of Red Blood Cells (RBCs) Parameter

Highly consistent measurement is made when determining the hemoglobin content and other features of red blood
cells, as well as when
evaluating other aspects of red blood cells. Manufacturers provide a broad variety of quality control techniques and
processes to facilities that use particle counting and sizing equipment, as well as specific technical help to ensure
that the technology is properly implemented. In contrast, the use of such technologies may be constrained by
practical considerations, especially during field research in remote places where it may be difficult to get blood
samples to a laboratory on the same day that they are collected. The latter is particularly true in the case of research
conducted in underdeveloped nations, which is a major source of funding. The use of instruments such as the
Hemocue (Hemocue AB, ngelhlam, Sweden), which can be found here (Hemocue AB), is thus critical to achieving
success (in Swedish). However, when measuring the hemoglobin concentrations of venous blood samples, it is
feasible to achieve findings that are quite constant. When measuring the hemoglobin concentrations of capillary
blood samples, however, it is possible to obtain results that are substantially varied. According to the researchers,
this discrepancy in findings might be due to insufficient capillary blood collecting procedures. A new guidebook
released by Helen Keller International delves into the use of techniques such as hemocue and capillary sample
collection, both of which are becoming more popular in a range of therapeutic contexts. Blood collection techniques
such as hemo cueing and collecting capillary samples are increasingly being used in a wide range of therapeutic
contexts. Another excellent source of information is the International Nutritional Anemia Consultative
Organization's guideline on "Measurements of Iron Status," which was provided by this organization[29].

The significance of hemoglobin as a screening indication for iron deficiency

According to the majority of experts on anemia, dietary iron insufficiency is responsible for around one-half of all
cases of anemia, and "up to a prevalence of iron deficiency anemia of 40%, the prevalence of iron deficiency would
be nearly 2.5 times that of anemia.". The prevalence rates for each of these ratios, as previously discussed, appear to
vary significantly depending on a variety of factors, including the age and gender of the individuals being studied,
their location within the world, and the prevalence rates of other causes in the general population, among others. The
consequence is that the frequency of anemia in a community may only provide a very poor estimate of the predicted
prevalence of iron-deficiency deficiency anemia in the general population (based on the frequency of anemia in the
community). With no globally recognized diagnostic standards for iron deficiency, investigating the diversity in
correlations between prevalence rates of iron deficiency and anemia in diverse populations is difficult. In addition,
there are no internationally accepted diagnostic criteria for anemia. Despite the fact that the approach has only been
employed in a limited amount of research, it is obvious that the use of bone marrow iron is too intrusive to be used
in field investigations on a consistent basis. Despite the fact that iron indicators have been studied in a variety of
different combinations, the cutoffs for iron insufficiency vary from one indication to another depending on which
indicator is used[30].
There is a lack of standardization between different commercial kits for measuring the concentration of transferrin
receptors in some developing countries; the threshold for zinc protoporphyrin can range between 40 and 70 mol/mol
haem depending on whether or not the cells have been washed prior to the assay, and there is a lack of
standardization between different commercial kits for measuring the concentration of transferrin receptor in some
developing countries. For the most part, the history of therapeutic treatments has relied on monitoring changes in
hemoglobin concentration in response to a specific treatment program in order to determine the efficacy and
effectiveness of the therapeutic interventions. Even though its sensitivity decreases when the incidence of anemia
decreases, it remains a useful diagnostic tool when employed for this reason despite its lower sensitivity. If there is
still a high prevalence of anemia at the end of the intervention, it may be difficult to assess if the intervention was
suboptimal in terms of effectiveness or efficiency (even if the baseline values have been significantly reduced). This
is owing to the likelihood that residual anemia is unrelated to iron deficiency, making it difficult to determine
whether or not the intervention was unsuccessful in this case (despite the fact that the baseline values have been
significantly reduced). A recent study found that the prevalence of anemia among newborns and young children was
the most reliable epidemiological predictor of iron status in this age group, despite the limitations mentioned above.
Compared to this, the other indicators that are now available have had a terrible result[30].

Fluorescence spectral classification of iron deficiency anemia and thalassemia

Thalassemia (Thal), sickle cell anemia (SCA), and iron deficiency anemia (IDA) are the three most prevalent blood
illnesses in the world, with thalassemia (Thal) being the most frequent in poor nations such as India and the
Democratic Republic of the Congo. Despite the fact that a complete blood count (CBC) is a well-established
diagnostic technique for them, there is much misunderstanding in the general community when it comes to
differentiating between Thal and IDA blood samples when utilizing a full blood count. For the objective of correctly
distinguishing between the two types of anemia mentioned above, we have developed a novel spectral approach that
is unique. For the development of this technique, the identification and measurement of a specific collection of
fluorescent metabolites identified in the blood samples of patients with Thal and IDA served as the foundation for its
development[31].

In order to diagnose anemia, the clinician will send blood samples to the laboratory for complete blood count (CBC)
analysis. RBC indices [less than 10 g/dl] and ferritin levels are both important in anemia diagnosis, and the next
battery of tests would be hemoglobin electrophoresis and/or high-performance liquid chromatography (HPLC) to
identify blood disorders such as Thal, IDA, sickle cell anemia (SCA), among others. In a variety of situations, these
procedures, which are both time-consuming and costly, are used.

A new technique called spectral diagnosis, which involves the detection and quantification of a specific set of
fluorescent biomolecules has been shown to be effective in the diagnosis of cancers such as liver cancer, cervical
cancer, pancreatic cancer, and other types of cancer, with sensitivity and specificity in the range of 90 percent in
some cases. Spectral diagnostic is a method for detecting and quantifying cancer cells that makes use of fluorescent
proteins to do so. Thal anemia and sickle cell anemia are two prominent hereditary blood illnesses that are produced
by the same gene mutation. Thal anemia and sickle cell anemia are caused by two different gene mutations.

Hb levels in the IDA samples were found to vary from 6.2 to 8.2 g/dl, whereas those in the Thal samples were found
to range from 4.2 to 7.4 g/dl, according to the findings. In spite of the fact that the hemoglobin level in the IDA
participants was about 1.3 times higher than that in the Thal patients, there was a significant overlap between the
hemoglobin levels in the Thal and IDA populations.
When it was found that porphyrin was the fluorescent component of RBCs, it was determined that the intensity of
the fluorescence was roughly equal to the number of RBCs present in the bloodstream.

The role of spectrum and wavelengths played in the detection of iron

deficiency.

To test non-invasively red blood cell zinc protoporphyrin, we gently touch the red vermilion of the subject's lower
lip and slowly glide the probe over the surface. The ‘blood absorption index' detects suitable spots for measurement
of the red blood cell zinc protoporphyrin and indicates them to the examiner by lighting an LED indicator linked to
the optical fiber probe. Following are places on the lower lip where mucosal tissue features (such as hemoglobin,
light scattering coefficient, blood vessel size, and epithelial thickness) allow quantification of red blood cell zinc
protoporphyrin. The blood absorption index specifies measurement locations but is not used in the computation.
Instead, dual-wavelength excitation and spectral fitting are utilized to quantify zinc protoporphyrin at a suitable
location. Following these findings, we show how the blood absorption index may identify acceptable tissue areas for
quantitative measurements[32]

A non-invasive optical method for diagnosing iron deficiency has been provided. To make haemoglobin, iron must
be added to protoporphyrin IX[32].
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Appendices
Table1: -Causes of iron deficiency

Table 2: -The mean and lower standard deviation (~2 SD) of normal hemoglobin concentrations (g/l) in a Caucasian
population

Figure 1: - Calibration curve of the biosensor for the determination of hemoglobin in the range of 5.0-16.0 g/dL.