TECHNICAL UNIVERSITY OF MOMBASA

FACULTY OF APPLIED AND HEALTH SCIENCES

DEPARTMENT OF PURE & APPLIED SCIENCES
UNIVERSITY EXAMINATION FOR:
DIPLOMA IN LABORATORY TECHNOLOGY
AAB 2330: BIOMEDICAL TECHNIQUES - HAEMATOLOGY
AND CLINICAL CHEMISTRY
END OF SEMESTER EXAMINATION
SERIES: DEC 2022
TIME: 2HOURS
DATE: Pick Date Select Month Pick Year
Instructions to Candidates
You should have the following for this examination
-Answer Booklet, examination pass and student ID
This paper consists of FIVE questions. Attempt question ONE (Compulsory) and any other TWO questions.
Do not write on the question paper.
SECTION A:
Question ONE

a) Define the following terms: (5 marks)

i. Haematology- Scientific/Biological study of blood,its components and the biochemistry involving
it.

ii. Haemostasis- Self adjusting mechanism that functions to keep a balanced optimum internal
environmentsl state and external environment
iii. Antigens- Any substance that triggers the release of antiges

iv. Secretors- A, B, and O antigens which are widely distributed in tissues and are present in body
secretions.

v. Agglutination clumping of RBCs

b) Outline FOUR functions of plasma proteins. (4 marks)
transport,
storage,
defense
blood clotting,
enzymatic reaction,
inhibition of protessase and maintenance of osmotic pressure

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 c) State FOUR regulatory systems the blood is engaged in. (4 marks)
Sensors that detect changes in temperature
Sensors that detect changes in pH
Sensors that detect changes in oxygen tension
Sensors that detect changes in blood component concentrations
d) Differentiate between Plasma and Serum(4 Marks)
Plasma is fluid portion of blood that contains cellular components consisting of red blood cells,White
blood cells and platelets while Serum is the fluid or Liquid portion of the blood that does not clot when
blood has had the opportunity to clot,
e) State and discuss at least two Romanowsky dyes used in staining of blood samples(5 marks)
Methylene Blue is a cationic stain. In simple terms, it is a blue dye that is charged positively and binds the
negatively charged components of the cells such as the nucleus, DNA or RNA present in the cytoplasm.
These cells usually have a low affinity. It is a popular stain used for a variety of activities, including
bacterial identification and cellular structure in both plant cells and animal cells. Methylene Blue solution
is used to stain the cells,including blood cell. The purpose of staining with methylene blue is to determine
cell mortality. When methylene blue stain is given to a sample, a healthy cell causes the stain to become
colourless.
Methylene blue is used for several other purposes, including as an antidepressant, a treatment for cyanide
poisoning, and a titration indicator in a laboratory. It turns dark blue when it is dissolved in water. It is
utilized in a number of scientific investigations for a variety of procedures. It is an efficient redox indicator
in chemistry because it turns clear when it comes into touch with reducing substances. It is used as a
titration aid in solutions. It’s also employed as a pigment in microbiology to analyze nucleic acid chains. It
can be added to a solution to colour RNA and DNA for visual analysis.
Methylene Blue can be used as a counterstain. For using counterstain you have to decolourize the primary
stain using a decolorizing agent (an acid-alcohol solution)
Hematoxylin and eosin (H&E): Most common staining method using two dyes. Used for staining basic or
acidic structures of the tissue.
Hematoxylin: Basic, positively charged dye. Purple to blue dye: Attracted to acidic, negatively charged
cellular structures such as DNA and RNA in nuclei and on ribosomes in cytoplasm.
Eosin: Acidic, negatively charged dye. Pink to red dye: Attracted to basic, positively charged cellular
structures (many proteins) in cytoplasm
f) State the physical properties of carbohydrates
Steroisomerism - Compound shaving same structural formula but they differ in spatial configuration.
Example: Glucose has two isomers with respect to penultimate carbon atom. They are D- glucose and L-
glucose.
Optical Activity - It is the rotation of plane polarized light forming (+) glucose and (-) glucose. Diastereo
isomers - It the configurational changes with regard to C2, C3, or C4 in glucose.

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 Example: Mannose, galactose.

Annomerism - It is the spatial configuration with respect to the first carbon atom in aldoses and second
carbon atom in ketoses.

SECTION B:

Question TWO
a) Discuss the Biological properties of human blood. (5 marks)

Fluid blood circulates throughout the body by way of the Heart, arteries, veins and capillaries
It transports oxygen, electrolytes, nourishment, hormones,Vitamins and antibodies to tissues and transports
waste products from tissues to the excretory organs.
Blood consists of a fluid portion referred to as plasma that Contains cellular components consisting of red
blood cells,White blood cells and platelets.
When blood has had the opportunity to clot, the fluid or Liquid portion of the blood that does not clot is
called serum.

b) Discuss the functional properties of human blood. (5 marks)

Fluid Connective Tissue: Blood is a fluid plasma connective tissue of 55% plasma and 45% formed
components such as WBCs, RBCs, and platelets. Because these live cells are floating in plasma, blood is a
fluid connective tissue rather than merely fluid.
2. It gives oxygen to the cells: The blood collects oxygen from the lungs and delivers it to various cells
throughout the body. Waste carbon dioxide travels from the bloodstream to the lungs and is expelled.
3. Transports Nutrients and Hormones: Glucose, vitamins, minerals, and proteins are taken into the
bloodstream via the capillaries in the villi lining the intestinal tract. Hormones released by the endocrine
glands are also delivered to other organs by the blood and tissues.
4. Homeostasis: Blood keeps the internal body temperature stable by absorbing and releasing heat.
5. Clotting of the Blood at the Site of Injury: Platelets aid in blood clotting factors at the site of damage.
Platelets and fibrin combine to create a clot at the wound site.

c) Elucidate the composition of human blood. (10 marks)

Blood is a fluid connective tissue with plasma as the fluid portion suspending the following cellular components
Fluid Connective Tissue: Blood is a fluid plasma connective tissue of 55% plasma and 45% formed components
such as WBCs, RBCs, and platelets. Because these live cells are floating in plasma, blood is a fluid connective
tissue rather than merely fluid.

2. It gives oxygen to the cells: The blood collects oxygen from the lungs and delivers it to various cells
throughout the body. Waste carbon dioxide travels from the bloodstream to the lungs and is expelled.

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3. Transports Nutrients and Hormones: Glucose, vitamins, minerals, and proteins are taken into the
bloodstream via the capillaries in the villi lining the intestinal tract. Hormones released by the endocrine glands
are also delivered to other organs by the blood and tissues.

4. Homeostasis: Blood keeps the internal body temperature stable by absorbing and releasing heat.

5. Clotting of the Blood at the Site of Injury: Platelets aid in blood clotting factors at the site of damage.
Platelets and fibrin combine to create a clot at the wound site.

Question THREE
a) State FOUR biological sample used in pathological studies. (4 marks)
Blood
Urine
Saliva
Sperms
Vaginal fluids
b) Discuss the FOUR sampling techniques used in hematological studies (16marks)
Simple Random Sampling
Researchers use two major sampling techniques: probability sampling and nonprobability sampling. With
probability sampling, a researcher can specify the probability of an element’s (participant’s) being included in
the sample. With nonprobability sampling, there is no way of estimating the probability of an element’s being
included in a sample. If the researcher’s interest is in generalizing the findings derived from the sample to the
general population, then probability sampling is far more useful and precise. Unfortunately, it is also much
more difficult and expensive than nonprobability sampling.
Probability sampling is also referred to as random sampling or representative sampling. The word
random describes the procedure used to select elements (participants, cars, test items) from a population.

When random sampling is used, each element in the population has an equal chance of being selected (simple
random sampling) or a known probability of being selected (stratified random sampling). The sample is
referred to as representative because the characteristics of a properly drawn sample represent the parent
population in all way.

Stratified Random Sampling

This procedure known as stratified random sampling is also a form of probability sampling. To
stratify means to classify or to separate people into groups according to some characteristics, such as
position, rank, income, education, sex, or ethnic background. These separate groupings are referred
to as subsets or subgroups. For a stratified random sample, the population is divided into groups or
strata. A random sample is selected from each stratum based upon the percentage that each
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subgroup represents in the population. Stratified random samples are generally more accurate in
representing the population than are simple random samples. They also require more effort, and there
is a practical limit to the number of strata used. Because participants are to be chosen randomly from
each stratum, a complete list of the population within each stratum must be constructed. Stratified
sampling is generally used in two different ways. In one, primary interest is in the representativeness
of the sample for purposes of commenting on the population. In the other, the focus of interest is
comparison between and among the strata.

Convenience Sampling
Convenience sampling is used because it is quick, inexpensive, and convenient. Convenience
samples are useful for certain purposes, and they require very little planning. Researchers simply use
participants who are available at the moment. The procedure is casual and easy, relative to random
sampling. Contrast using any available participants with random sampling, where you must (1) have a
well-defined population, (2) construct a list of members of the population if one is not available, (3)
sample randomly from the list, and (4) contact and use as many individuals from the list as possible.
Convenience sampling requires far less effort. However, such convenience comes with potential
problems, which we will describe. Convenience samples are nonprobability samples. Therefore, it is
not possible to specify the probability of any population element’s being selected for the sample.
Indeed, it is not possible to specify the population from which the sample was drawn.

Quota Sampling
In many large-scale applications of sampling procedures, it is not always possible or desirable to list all
members of the population and randomly select elements from that list. The reasons for using any
alternative procedures include cost, timeliness, and convenience. One alternative procedure is quota
sampling.
This technique is often used by market researchers and those taking political polls. Usually, when
this
technique is used, the population of interest is large and there are no ready-made lists of names
available from which to sample randomly. The Gallup Poll is one of the best known and well
conducted polls to use quota sampling. This poll frequently reports on major public issues and on
presidential elections. The results of the poll are syndicated for a fee that supports it. In this quota
sampling procedure, localities are selected and interviewers are assigned a starting point, a specified
direction, and a goal of trying to meet quotas for subsets (ethnic origins, political affiliations, and so
on) selected from the population. Although some notable exceptions have occurred, predictions of
national elections over the past few years have been relatively accurate—certainly, much more so
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than guesswork.

With the quota sampling procedure, we first decide which subgroups of the population interest us. This, in turn, is
dictated by the nature of the problem being investigated (the question being asked). For issues of national
interest (such as abortion, drug use, or political preference), frequently used subsets are age, race, sex,
socioeconomic level, and religion. The intent is to select a sample whose frequency distribution of characteristics
reflects that of the population of interest. Obviously, it is necessary to know the percentage of individuals making
up each subset of the population if we are to match these percentages in the sample

Question FOUR
a) State FIVE characteristics of lipids (5 marks)
Lipids are relatively insoluble in water.

They are soluble in non-polar solvents, like ether, chloroform, and methanol. Lipids have high energy content
and are metabolized to release calories.
Lipids also act as electrical insulators, they insulate nerve axons.

Fats contain saturated fatty acids; they are solid at room temperatures. Example, animal fats. Plant fats are
unsaturated and are liquid at room temperatures.
Pure fats are colorless, they have extremely bland taste.

The fats are sparingly soluble in water and hence are described are hydrophobic substances. They are freely
soluble in organic solvents like ether, acetone and benzene.
The melting point of fats depends on the length of the chain of the constituent fatty acid and the degree of
unsaturation.
Geometric isomerism, the presence of double bond in the unsaturated fatty acid of the lipid molecule produces
geometric or cis-trans isomerism.
Fats have insulating capacity, they are bad conductors of heat.

Emulsification is the process by which a lipid mass is converted to a number of small lipid droplets. The
process of emulsification happens before the fats can be absorbed by the intestinal walls.
The fats are hydrolyzed by the enzyme lipases to yield fatty acids and glycerol.

The hydrolysis of fats by alkali is called saponification. This reaction results in the formation

of glycerol and salts of fatty acids called soaps.

Hydrolytic rancidity is caused by the growth of microorganisms which secrete enzymes like lipases.
These split fats into glycerol and free fatty acids.
b) Outline the classification of lipids (5 marks)
1. Simple lipids: Esters of fatty acids with various alcohols.

Fats: Esters of fatty acids with glycerol. Oils are fats in the liquid state.

Waxes: Esters of fatty acids with higher molecular weight monohydric alcohols.

2. Complex lipids: Esters of fatty acids containing groups in addition to an alcohol and a fatty acid.
Phospholipids: Lipids containing, in addition to fatty acids and an alcohol, a phosphoric acid residue.
They frequently have nitrogen containing bases and other substituents, eg, in glycerophospholipids the alcohol
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 is glycerol and in sphingophospholipids the alcohol is sphingosine.
Glycolipids (glycosphingolipids): Lipids containing a fatty acid, sphingosine, and carbohydrate.
Other complex lipids: Lipids such as sulfolipids and aminolipids. Lipoproteins may also be placed in this
category.
Precursor and derived lipids: These include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, and
ketone bodies, hydrocarbons, lipid-soluble vitamins and hormones.
Essential fatty acids

Two fatty acids are dietary essentials in humans:

Linoleic acid, which is the precursor of arachidonic acid, the substrate for prostaglandin synthesis.

α-linolenic acid is the precursor for growth and development.

Essential fatty acid deficiency can result in a scaly dermatitis, as well as visual and neurologic abnormalities.
Examples of Lipids

Fatty acids - Oleic acid, Linoleic acid, Palmitoleic acid, Arachidonic acid.

Fats and Oils - Animal fats - Butter, Lard, Human fat, Herring oil.

Plant oils - Coconut oil, Corn, Palm, Peanut, Sunflower oil.

Waxes - Spermacti, Beeswax, Carnauba wax.

Phospholipids - Lecithins, Cephalins, Plasmoalogens, Phosphatidyl inositols, Sphingomyelins.

Glycolipids - Kerasin, Phrenosin, Nervon, Oxynervon.

Steroids - Cholesterol.

Terpenes - Monoterpenes, Sesquiterpenes, Diterpenes, Triterpenes.

Carotenoids - Lycopene, Carotenes, Xanthophylls.

c) Explain the biological roles of lipids (10 marks)

Food material: Lipids provide food, highly rich in calorific value. One gram lipid produces 9.3 kilocalories of
heat.
Food reserve: Lipids provide are insoluble in aqueous solutions and hence can be stored readily in the body as
a food reserve.
Structural component: Lipids are an important constituent of the cell membrane.

Heat insulation: The fats are characterized for their high insulating capacity. Great quantities of fat are
deposited in the subcutaneous layers in aquatic mammals such as whale and in animals living in cold climates.
Fatty acid absorption: Phospholipids play an important role in the absorption and transportation of fatty acids.
Hormone synthesis: The sex hormones, adrenocorticoids, cholic acids and also vitamin D are

all synthesized from cholesterol, a steroidal lipid.

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 Vitamin carriers: Lipids act as carriers of natural fat-soluble vitamins such as vitamin A, D and E.
Blood cholesterol lowering: Chocolates and beef, especially the latter one, were believed to cause many heart
diseases as they are rich in saturated fatty acids, which boost cholesterol levels in blood and clog the arterial
passage.
Antibiotic agent. Squalamine, a steroid from the blood of sharks, has been shown to be an antibiotic and
antifungal agent of

Question FIVE
a) State the alleles found at ABO locus (3 marks)
A allele
B allele
O allele

b) Discuss the ABO blood groups system (17 marks)

ABO Blood Group System

In the ABO blood group system, two types of antigens designated A and B give rise to four blood types:
• Type A individuals have the A antigen.
• Type B individuals have the B antigen.
• Type AB individuals have both A and B antigens.
• Type O individuals have neither A nor B antigens.
The antigens are found in other tissues and biological fluids, including the salivary glands and saliva, pancreas,
kidney, liver, lungs, testes, semen, and amniotic fluid.

Biosynthesis of Antigens

All individuals generate the O antigen, also known as the H antigen. The O antigen is synthesized by
fucosyltransferase, a fucose transferase coded by the FUT genes that adds a fucose on the end of a glycolipid (in
erythrocytes) or glycoprotein (in tissues). An additional monosaccharide is then transferred to the O antigen by a
transferase encoded by the ABO locus.
The specificity of this enzyme determines the ABO blood type:

• The A allele produces the A-transferase that transfers N-acetylgalactosamine to the O antigen and thus
synthesizes the A antigen.
• The B allele produces the B-transferase that transfers galactose to the O antigen and thus synthesizes the B
antigen.
• The O allele has a mutation (small deletion) that eliminates transferase activity and no modification of the O
antigen occurs. As a result, the A and B antigens differ in their terminal sugar molecules.

Subgroups of blood types A and B have been described. The most important are the A1 and A2 antigens. Both A1
and A2 (and A1B and A2B) cells react with anti-A antibodies. A1 cells react more strongly than A2 cells. The
apparent difference between A1 and A2 is that each A1 cell contains more copies of the A antigen than A2 cells.

Molecular Basis of ABO system

A single gene, ABO, on chromosome 9, encodes A- and B- transferases. The ABO gene (approximately 20
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kilobases) is organized into seven exons. Most of its coding regions are located in exons 6 and 7 of the ABO locus
including the domain responsible for catalytic activity. The products of the A and B alleles differ by four amino
acid substitutions. However, amino acid residues at positions 266 and 268 are the most important in determining
whether a gene product is an A-transferase or B-transferase. The A1 allele and A2 allele differ in a single
nucleotide deletion before the translation stop codon. The resulting A2 reading-frame shift abolishes the stop
codon, yielding a product with an extra 21-amino acid residue at the C-terminus. Subgroups of blood types O have
also been reported. The sequence of the O1 allele has a deletion of a single nucleotide at exon 6. This nucleotide
deletion leads to a reading-frame shift generating a truncated protein, which lacks the catalytic domain. While the
O1y allele also has a single nucleotide deletion, it differs from O1 by nine nucleotides within the coding sequence.
O1 and O1y have identical phenotypes. In addition, an O2 allele is inactivated by a substitution mutation at
glycine (position 268) by arginine. Additionally, a few dozen other rare O alleles that yield inactive products have
been documented.

Secretors

A, B, and O antigens are widely distributed in tissues and are present in body secretions. They are known as
secretors. The O antigen is the substrate for the A- and B- transferase because the A- and B- transferase can only
utilize a fucosylated substrate. The O antigen is synthesized by fucosylation of the terminal galactosyl residue
catalyzed by the fucosyltransferase, which is encoded by FUT genes. However, non-secretors have O antigens on
erythrocytes synthesized by FUT1, and thus have A or B antigens in blood. Homozygous FUT1 mutations
produce very rare erythrocyte O-deficient phenotypes in which the erythrocytes express no O and thus no A or B,
regardless of ABO genotype. Individuals with such phenotypes can be either secretors or non-secretors. The non-
secretors who also have no O (or A or B) in their secretions are known as Bombay (Oh) phenotypes.

Inheritance of A and B Antigens

A and B alleles are dominant. For AO and BO heterozygotes, the corresponding transferase synthesizes the A or B
antigen. A and B alleles are co-dominant in AB heterozygotes because both transferase activities are expressed.
The OO homozygote produces neither transferase activity and therefore lacks both antigens. The inheritance of A
and B alleles obeys Mendelian principles. Blood Group Typing and Protein Profiling, an individual with type B
blood may have inherited a B allele from each parent or a B allele from one parent and an O allele from the other;
thus, an individual whose phenotype is B may have the BB (homozygous) or BO (heterozygous) genotype.
Conversely, if the blood types of the parents are known, the possible genotypes of their children can be
determined. When both parents are type B (heterozygous), they may produce children with genotype BB (B
antigens from both parents), BO (B antigen from one parent, O from the other heterozygous parent), or OO (O
antigens from parents who are both heterozygous). Thus, blood group typing can be used for paternity testing.

Absorption–Elution Assay

The absorption–elution assay is highly sensitive and can be used for testing dried bloodstains. It indirectly detects
the presence of antigens. It is imperative to note that successful agglutination reactions usually require intact cells
and is very difficult to carry out because blood cells lyse when they are dry. In this method, the lysed cells
containing antigen are immobilized on a solid phase and at low temperatures, the antigen is allowed to bind to its
corresponding antibody: anti-A antibodies, anti-B antibodies, or anti-O lectins. (The anti-O lectin is isolated from
plants reacts strongly with the O antigen present in type O blood, but has some cross-reaction with the A antigen).
The excess unbound antibodies are removed by washing and the bound antibodies are then eluted at higher
temperatures (antigen–antibody binding can be affected by temperature). The eluted antibodies can then be
identified by an agglutination assay using A, B, and O indicator cells (freshly prepared red blood cells of each
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blood group). The blood stains containing A antigen can bind to anti-A antibodies. The eluted anti-A antibody can
form agglutination with A cells. Likewise, for type B blood, the eluted anti-B antibody can form agglutination
with B cells; for type AB blood, the eluted antibodies can form agglutination with both A and B cells; and with
type O blood, the eluted antibodies can form agglutination with O cells.

Absorption – the stain is mixed with serum/lectin containing antibodies and incubated for antibody to be removed
by absorption/neutralization. The resultant fluid is tested for antibody activity.
Agglutination – clumping of RBCs
Agglutinogens – antigens on RBCs

Blood group Antigens

Antibodies
O - Anti- A & Anti- B
A A Anti- B
B B Anti- A
AB A&B -

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