SIWES TECHNICAL REPORT

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A TECHNICAL REPORT ON STUDENT INDUSTRIAL WORK
EXPERIENCE SCHEME (SIWES)
UNDERTAKEN AT
THE INFECTIOUS DISEASE UNIT (APIN) - JUTH, JOS.
SUBMITTED TO
THE SIWES COORDINATOR
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF NATURAL AND APPLIED SCIENCES
PLATEAU STATE UNIVERSITY, BOKKOS
BY
BENSON, MATTHEW LASSA
PLASU/2013/FNS/0025
COURSE CODE: BCH 399
IN PARTIAL FULFILLMENT OF THE AWARD OF A
BACHELOR OF SCIENCE DEGREE (B.Sc) IN BIOCHEMISTRY
PLATEAU STATE UNIVERSITY, BOKKOS
JANUARY, 2017

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CERTIFICATION
This is to certify that this Technical Report was written by BENSON MATTHEW
LASSA with Matriculation Number PLASU/2013/FNS/0025 under the supervision
of Mr. Larry Barde and Mr. Francis Okonkwo
_____________________________
BENSON MATTHEW LASSA Date
Student
_________________________
Mr. LARRY BARDE Date
Institution-based supervisor
_____________________________
Mr. FRANCIS OKONKWO Date
Departmental SIWES Coordinator
__________________________
Mr. FRANCIS OKONKWO Date
Head of Department
____________________________
Dean, Faculty of Natural Sciences Date
_____________________________
Mr. Emmanuel Nnadi Date
Director of SIWES

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DEDICATION
This is to GOD for His unending GRACE.

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ACKNOWLEDGEMENT
I genuinely appreciate my parents, Mr. and Mrs. Benson Lassa, for their persisting
patience, love and financial support.
I thank Mr. Kumzing Tangtur (APIN) for the inspiration to dig until I get there, for the
exposure through the NACA PrEP Project.
My sincere gratitude to the friends who became family: Ruth Sheku, Chomo Nyam and
Rityilmwa Tungon for proof-reading this report.
I also appreciate my brothers for the tough time they gave which pushed me to work
harder, Joel and Destiny. This is for you guys.

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TABLE OF CONTENTS
Title Page…………………………………………………………i
Certification………………………………………………………...ii
Dedication…………………………………………………………………..iii
Acknowledgement………………………………………………………….iv
Table of Content…………………………………………………………….v
Chapter 1
Brief history of SIWES and Objectives of SIWES……………………1
Brief history of organization………………………………………………….2
Structural organization of INFECTIOUS DIEASE UNIT (APIN), JUTH………….3
Chapter 2
Care and Safety in the Laboratory…………………………………….4
INTRODUCTORY HIV/AIDS ………………………………………5
Chapter 3
SAMPLE RECEPTION UNIT …………………………………………….12
Chapter 4
SAMPLE COLLECTION/SEPARATION UNIT………………………...13
Chapter 5
CD4/CD4% UNIT………………………………………………………….19
Chapter 6
CLINICAL BIOCHEMISTRY UNIT……………………………………26
Chapter 7
HAEMATOLOGY UNIT………………………………………………….36
Chapter 8
MOLECULAR BIOLOGY UNIT……………………………………….47
Experience gained and problems encountered during the period of the SIWES
program…52
Recommendation……………………………………………………...52
References

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CHAPTER 1
BRIEF HISTORY OF S.I.W.E.S.
SIWES was established in 1973 by the Industrial Training Fund (ITF) as one of her
programmes. It was designed to give Nigerian students studying occupationally-related
courses in higher institutions the experience that would supplement their theoretical
learning in order to solve the problem of lack of adequate practical skills preparatory for
employment in industries by Nigerian graduates of tertiary institutions.
The Scheme exposes students to industry based skills necessary for a smooth
transition from the classroom to the world of work. It affords students of tertiary
institutions the opportunity of being familiarized and exposed to the needed experience
in handling machinery and equipment which are usually not available in the educational
institutions.
Participation in SIWES has become a necessary pre-condition for the award of
Diploma and Degree certificates in specific disciplines in most institutions of higher
learning in the country, in accordance with the education policy of government. Usually
there are three modules: The first module is for two months and this is taken by all 200-
level Engineering and Food Technology students in University. This module of industrial
Training is designed to expose the students to engineering and technology operations at
the shop floor level. The second module is for three months. This is for the 300-level
students of Engineering, Food Technology, Geography, Biochemistry, Nursing,
Pharmacy, Geology, Physics, and Library Science. The third module is however for six
months and it is taken by 400-level students of Engineering, Food Technology, Botany,
Microbiology, Industrial Chemistry, Computer Science, Zoology, Agriculture and
Physiotherapy.
SIWES is operated by the ITF, the coordinating agencies (NUC, NCCE, NBTE),
employers of labor and the institutions concerned (universities and
polytechnics).Funded by the Federal Government of Nigeria.
Beneficiaries-Undergraduates students of the following: Agriculture, Engineering,
Technology, Environmental, Science, Education, Medical Science and Pure and Applied
Sciences.
Duration - Four months for polytechnics and Colleges of Education, and six
months for the Universities.
OBJECTIVES OF SIWES
SIWES is a program organized for students of higher institutions to acquire practical
knowledge of their various discipline in a real standard establishment different from the
kind of experience or knowledge gained within the four walls of the classroom or school
laboratory.

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BRIEF HISTORY OF THE ORGANIZATION:
THE INFECTIOUS DISEASE UNIT FORMALLY APIN (AIDS PREVENTION INITIATIVE
NIGERIA) is a non-governmental organization registered with the Nigerian Corporate
Affairs Commission (CAC). APIN has built a strong partnership with the Nigerian
Government, which is memorialized through a memorandum of understanding (MOU)
with the National Planning Commission. APIN is a leading Nigerian organization in the
provision of prevention, care and treatment services to patients with HIV/AIDS
and other diseases of public health significance. Its operating experience in Nigeria
dates back to 2000, when the Harvard School of Public Health (HSPH) initiated HIV/AIDS
research and prevention initiatives in Nigeria, with funding from the Bill & Melinda
Gates Foundation. In 2004, the HSPH project started implementing HIV/AIDS
prevention, care and treatment programs with support from the U.S. President’s
Emergency Plan for AIDS Relief (PEPFAR). These activities culminated in the
establishment of APIN as an independent NGO in 2007, with the aim of building local
capacity and sustainability of HIV/AIDS and other diseases programming in Nigeria.
Presently, APIN operates as a direct implementing partner to CDC having just completed
a 3-year transition from Harvard in line with the US Government’s goal of transitioning
programs to local indigenous organizations. In the four years since its operations as an
implementing partner of the CDC, APIN has managed a cumulative budget of more than
$50m, a reflection of the growing confidence of the USG that APIN has built enough
systems to operate as an independent implementing partner of US funds. In addition to
the US funded activities, APIN is currently soliciting for other funding opportunities to
diversify its funding base in the wider public health arena as part of its response to
current health challenges and its overall sustainability plan.
Apart from its contribution to evidence-based HIV prevention in the country, APIN is
also one of the pioneers in the provision of comprehensive HIV/AIDS care in Nigeria. It
currently supports the Federal Government of Nigeria to provide ARV care and
treatment directly as a USG implementing partner tomore than 74,000 patients in 33
treatment clinics and 75 primary healthcare centers in nine (9) states. These clinics are
equipped with state-of-the-art laboratories with capabilities for diagnosis and
monitoring of HIV including CD4, viral load, DNA PCR and drug resistance monitoring
(DRM). Similar giant strides have been made in prevention, systems strengthening, as
well as in human and infrastructure capacity development efforts.
It has a widespread network of service providers, communities, researchers and persons
living with HIV and AIDS support groups in nine (9) states in Nigeria. APIN has a.
purposive, exemplary and result-oriented leadership, committed and hardworking
human resources and a broad-based support of collaborators in Nigeria and the United
States. It essentially inherited the same dedicated team that has worked on the Harvard
program in the last 6 -10 years.

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STRUCTURAL ORGANISATION OF INFECTIOUS DISEASE UNIT (APIN)-JUTH
In the infectious disease unit, APIN, JUTH, where I underwent my SIWES program, we
have the following sections:
 Sample Reception Unit,
 Clinical Chemistry (Chemical Pathology) Unit,
 Hematology Unit,
 Molecular Biology Unit,
 Sample Separation/Collection Unit; and
 CD4/CD4% Unit

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CHAPTER 2
SAFE WORKING PRACTICES IN A MEDICAL LABORATORY
The following are some of the important points which apply when working with
infectious materials:
1. Never mouth-pipette. Use safe measuring and dispensing devices.
2. Do not eat, drink, smoke, store food, or apply cosmetics in the working area
of the laboratory.
3. Use an aseptic technique when handling specimens.
4. Always wash your hands after handling an infectious material in the
laboratory, when leaving the laboratory and before attending to patients.
Cover any open wound with a water proof dressing.
5. Wear appropriate protective clothing when working in the laboratory. Ensure
it is decontaminated and laundered correctly.
6. Wear protective gloves and when indicated a face mask, for all procedures
involving direct contact with infectious materials.
7. Centrifuge safely to avoid creating aerosols. Know what to do should a
breakage occur when centrifuging.
8. Avoid practices which could result in needle stick injury.
9. Do not use chipped or cracked glassware and always deal with a breakage
immediately and safely.
10. Avoid spillages by using racks to hold containers, work neatly and keep the
bench surface free of any unnecessary materials.
11. Decontaminate working surfaces at the end of each day’s work and following
any spillage of any infectious fluid.
12. Report to the laboratory officer in charge, any spillage or other accident
involving exposure to infectious material.
13. Know how to decontaminate specimens and other infectious materials.
14. Use and control an autoclave correctly.
15. Dispose laboratory waste safely.

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HIV & AIDS
Introduction
HIV is a lentivirus (literally meaning “slow virus”; a member of the retrovirus family) that
slowly attacks and destroys the immune system, the body's defense against infection,
leaving an individual vulnerable to a variety of other infections and certain malignancies
that eventually cause death. AIDS is the final stage of HIV infection, during which time
fatal infections and cancers frequently arise.
STRUCTURE AND MOLECULAR FEATURES OF HIV
Virion Structure
HIV-1 virion, according to electron microscopic observation, has a cone shaped core or
capsid which consists of:
a. The major capsid protein p24;
b. The nucleocapsid protein, p7/p9;
c. The diploid single stranded RNA genome; and
d. The three viral enzymes, protease, reverse transcriptase and integrase.
Reverse transcriptase is the hallmark of a retrovirus and is capable of transcribing its
genomic RNA into double stranded DNA. This DNA copy of the retroviral genome is
called a provirus. After integration into the host genome, the provirus serves as a
template for cellular DNA-dependent RNA polymerases to generate new viral RNA
genomes as well as shorter subgenomic messenger RNAs. The unspliced and singly
spliced viral RNAs are translated into the protein components of the viral core and the
envelope proteins and the multispliced viral RNAs into the small accessory/regulatory

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proteins. Surrounding the capsid lies the matrix constituted by myristylated p17 gag
protein, which is located underneath the virion envelope.
The matrix protein is involved in the early stages of the viral replication cycle and plays a
part in the formation and transport of the preintegration DNA complex into the nucleus
of the host cell. The virion envelope consists of a lipid bilayer membrane, derived from
the host cell. Like all retroviruses, an envelope consisting of viral glycoproteins
embedded in a host cell derived lipid bilayer surrounds HIV-1. The virus surface is
constituted by 72 knob containing trimers and tetramers. The envelope glycoproteins
are synthesised as gp160 precursor in the rough endoplasmic reticulum. Aspargine
linked, high mannose sugar chains are added to gp160, which is then assembled into
oligomers. These are then transported to the Golgi apparatus where cellular proteases
cleave gp160 into the external surface (SU) envelope protein or gp120 and
transmembrane (TM) protein or gp41. These proteins are transported to the cell
surface, where part of the central and N-terminal portion of the gp41 is also expressed
on the outside of the virion.
The gp41 glycoprotein has an ectodomain that is largely responsible for trimerisation.
Most of the surface exposed elements of the mature oligomeric envelope glycoprotein
complex are located in gp120. Selected, well-exposed,carbohydrates on the gp120
glycoprotein are modifiedin the Golgi by the addition of complex sugars. The gp120and
gp41 are maintained in the assembled trimer by noncovalent, labile interactions
between the gp41 ectodomain and discontinuous structures composed of N- and C-
terminal gp120 sequences. For entry of the virus in the target host cell, the viral
envelope fuses with the plasma membrane of the cell by a process mediated by the
viral envelope glycoproteins.
GENOME
The size of HIV-1 genome is about 9.8 kb with open reading frames coding for several
viral gene products which are flanked on each end by long terminal repeat (LTR)
sequences.

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The three major genes are gag, pol and env.
 The gag gene codes for the gag precursor protein p55, which is cleaved by viral
protease to generate p24, p17, p9 and p6 gag proteins.
 The pol gene codes for the pol precursor, which is cloven into reverse transcriptase
(RT), protease (PR), and integrase (IN). Protease processes the gag and pol
polyproteins. Integrase is involved in the integration of the proviral DNA,
generated from the viral RNA genome by reverse transcriptase into the host cell
chromosomal DNA.
 The env gene codes for the envelope precursor gp 160, which is cloven into gp120
and gp41. Gene products of other spliced mRNA make up various viral regulatory
and accessory proteins.
 The tat gene codes for the transactivating protein. Tat, which along with certain
cellular proteins, interacts with a region in the RNA loop formed at the 3' LTR
region called Tat responsive element (TAR). Tat is involved in the upregulation
of HIV replication.
 The rev gene produces Rev (regulator of viral protein expression). Rev interacts
with a cis acting RNA loop structure called the Rev responsive element or RRE.
The Rev protein promotes the export from the nucleus of the unspliced or singly
spliced viral RNAs that act, respectively, as genomic RNA/template for the
translation of gag/pol proteins and template for envelope proteins. In the
absence of Rev, no structural proteins are made.
 The nef gene: Another viral gene product, Nef, coded by the nef gene, appears to
have a variety of potential functions, including downregulation of viral
expression. It appears that the Nef mRNA represents the majority of the earliest
mRNA species following integration. However, most studies have indicated a
pleiotropic function of Nef and that it is not always associated with
downregulation of replication. Tat, Rev, and Nef are not incorporated into virion

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particles but are first viral components produced from multiple spliced viral
mRNA. The other accessory viral gene products are Vif, Vpr and Vpu.
 Vif is reported to increase virus infectivity and cellto- cell transmission. It helps in
proviral DNA synthesis and might play a role in virion assembly.
 Vpr helps in virus replication.
 Vpu, whose expression appears to be regulated by Vpr, helps in release of the
virus.
VIRUS LIFE CYCLE
The life cycle of HIV-1 can be considered in two distinct phases . The initial early events
occur within a short time and include viral attachment, entry, reverse transcription,
entry into the nucleus, and integration of the doublestranded DNA (the provirus). The
second phase occurs over the lifetime of the infected cell as viral and cellular proteins
regulate the production of viral proteins and new infectious virions.
Schematic diagram of HIV life cycle
HIV is an RNA virus whose hallmark is the reverse transcription of its genomic RNA to
DNA by the enzyme reverse transcriptase. The replication cycle of HIV begins with the
high-affinity binding of the gp120 protein via a portion of its V1 region near the N
terminus to its receptor on the host cell surface, the CD4 molecule. The CD4 molecule is
a 55 kDa protein found predominantly on a subset of T lymphocytes that are responsible
for helper or inducer function in the immune system. It is also expressed on the surface
of monocytes/ macrophages and dendritic/Langerhans cells. In order for HIV-1 to fuse
to and enter its target cell, it must also bind to one of a group of co-receptors.

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The two major co-receptors for HIV-1 are CCR5 and CXCR4. Both receptors belong to
the family of seventransmembrane- domain G protein-coupled cellular receptors, and
the use of one or the other or both receptors by the virus for entry into the cell is an
important determinant of the cellular tropism of the virus (see below for details).
Following binding, the conformation of the viral envelope changes dramatically, and
fusion with the host cell membrane occurs in a coiled-spring fashion via the newly
exposed gp41 molecule; the HIV genomic RNA is uncoated and internalised into the
target cell. The reverse transcriptase enzyme, which is contained in the infecting virion,
then catalyses the reverse transcription of the genomic RNA into double-stranded DNA.
The DNA translocates to the nucleus, where it is integrated randomly into the host cell
chromosomes through the action of another virally encoded enzyme, integrase. This
provirus may remain transcriptionally inactive (latent), or it may manifest varying levels
of gene expression, up to active production of virus.
Cellular activation plays an important role in the life cycle of HIV and is critical to the
pathogenesis of HIV disease. Following initial binding and internalisation of virions into
the target cell, incompletely reversetranscribed DNA intermediates are labile in
quiescent cells and will not integrate efficiently into the host cell genome unless cellular
activation occurs shortly after infection. Furthermore, some degree of activation of the
host cell is required for the initiation of transcription of the integrated proviral DNA into
either genomic RNA or mRNA. In this regard, activation of HIV expression from the
latent state depends on the interaction of a number of cellular and viral factors.
Following transcription, HIV mRNA is translated into proteins that undergo modification
through glycosylation, myristylation, phosphorylation, and cleavage. The viral particle is
formed by the assembly of HIV proteins, enzymes, and genomic RNA at the plasma
membrane of the cells. Budding of the progeny virion occurs through the host cell
membrane, where the core acquires its external envelope. The virally encoded protease
then catalyses the cleavage of the gag-pol precursor to yield the mature virion. Each
point in the life cycle of HIV is a real or potential target for therapeutic intervention.
MODES OF TRANSMISSION
The transmission of a virus can be greatly influenced by the amount of infectious virus in
a body fluid and the extent of contact with that body fluid. Epidemiological studies
conducted during 1981 and 1982 first indicated that the major routes of transmission of
AIDS were intimate sexual contact and contaminated blood. Moreover, it became
evident that transfusion recipients and haemophiliacs could contract the virus from
blood or blood products and mothers could transfer the causative agent to newborn
infants. These three principal means of transmission—blood, sexual contact and
mother-to-child-have not changed. The other modes of transmission of the virus are:

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(1) by sharing of the needles by the intravenous drug users and (2) by needle stick
injuries.
1. Blood and Blood Products
All blood samples of HIV sero-positive individuals contain circulating infectious virus
whether the individual is asymptomatic or has AIDS. HIV is readily found during acute
(primary) infection. Subsequently, within weeks, the level of free virus detected in the
blood is markedly reduced. The total amount of infectious free virus present in the
blood of asymptomatic individuals averages 100 IP (infectious particles) per ml. In the
years before the screening of blood, HIV present in blood and blood products such as
factors VIII and IX could infect transfusion recipients and haemophiliacs. The potential
risk of infection of transfusion recipients depends on the virus load and appears to be
greatest as an infected individual (as donor) advances to disease. In haemophiliacs, this
transmission could be caused only by free virus and was associated with receipt of many
vials of unheated clotting factors.
2. The Transmission of HIV by Genital Fluids
The transmission of HIV by genital fluids most probably occurs through virus-infected
cells since these can be present in larger numbers than free virus in the body fluids.
Moreover, recent studies suggest that these infected cells transfer HIV to epithelial cells
best when present in seminal fluid, because cell-to-cell contact is increased most
probably via factors in semen. The presence of different levels of infected cells in the
genital fluids probably explains the variations in virus transmission among sexual
partners. The amount of virus in genital fluids is important for sexual transmission.
Generally, 10 to 30 per cent of seminal and vaginal fluid specimens have shown the
presence of free infectious virus and/or virus-infected cells. The finding of HIV in the
bowel mucosa itself provides another reason, besides abrasions, for the high risk of
transmission associated with anogenital contact.
3. Transmission from Mother to Child
Mother to child transmission of HIV includes transmission during pregnancy, during
delivery, and through breastfeeding. HIV-1 is transmitted to the fetus or infant by 13 to
48 per cent of infected mothers. Data from various countries suggest that as many as 15
per cent of babies, breastfed by HIV infected mothers, may become infected through
breastfeeding.
4. Transmission by Needle Stick Injury
The chances of transmission of HIV from infected individual by needle stick injury are
only 0.03–0.3 per cent.

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GROUPS AND SUBTYPES OF HIV
Genetic studies have led to a general classification system for HIV that is primarily based
on the degree of similarity in viral gene sequence. The two major classes of HIV are HIV-
1 and HIV-2. HIV-1 is divided into three groups, known as group M (main group), group
O (outlier group), and group N (new group). Worldwide, HIV-1 group M causes the
majority of HIV infections, and it is further subdivided into subtypes A through K, which
differ in expression of viral genes, virulence, and mechanisms of transmission.
HIV-2 is divided into groups A through E, with subtypes A and B being the most relevant
to human infection. HIV-2, which is found primarily in western Africa, can cause AIDS,
but it does so more slowly than HIV-1. There is some evidence that HIV-2 may have
arisen from a form of SIV that infects African green monkeys.

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CHAPTER 3
SAMPLE RECEPTION UNIT
The receptionist on seat collects samples from patients waiting to be transferred to the
laboratory, put bills on the patient’s cards depending on the kind of tests to be done,
register the patient’s cards and then also register results before they are given out to
patient. Listed below are a few steps to follow when dispatching specimens:
1. Keep a register of all specimens dispatched. Record the name, I.D number, and
health centre of the patient, type of specimen, investigation required, date of
dispatch, and the method of sending the specimen.
2. Check the specimen container is free from cracks, and the cap is leak-proof.
3. Use sufficient packaging material to protect a specimen especially when the
container is a glass tube. When the specimen is fluid use sufficient absorbent
material to absorb it should a leakage or breakage occur.

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CHAPTER 4
SAMPLE COLLECTION/SEPARATION UNIT
Blood collection
Blood samples are collected for measurement of blood lipids and glucose. For
lipid measurements, serum should be used in preference to plasma to avoid the diluting
effect of anticoagulants, which results in about 3% difference in concentrations.
Nevertheless, some countries may have special reasons to use plasma, e.g. to retain
comparability with the earlier surveys. For glucose measurement, plasma is used and
glycated hemoglobin measurement requires whole blood.
 Equipment
Equipment for sample drawing
For sample drawing the following equipment is needed:
 needles (preferably vacutainer needles), size 20G to 22G
 tubes
 vacutainer holder
 tourniquet
 disinfection swabs
 micropore tape
 dental rolls
 adhesive dressing
 rubber gloves
 pillow or other support
 needle disposal box
Chemically clean evacuated tubes with appropriately reduced pressure are used in
sample drawing. If plasma specimens are taken, EDTA is used as anticoagulant. Tubes
with liquid EDTA reduce the risk of hemolysis that sometimes occurs with tubes using
EDTA in powder form. For glucose determination the fluoride tubes are needed. Plastic
vacuum tubes are preferred to glass tubes. Plastic vacuum gel tubes are most
convenient.
 Sample drawing and handling
Fasting before the sample collection
If fasting glucose, lipoprotein fractions and fasting triglycerides are to be measured the
samples are collected after a fasting period. For glucose alone, fasting of four hours is

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sufficient. For triglyceride measurement, the fasting period should be minimally 10
hours and maximally 14 hours (too long fasting causes major changes in energy
metabolism with implications for blood triglycerides). This implies that the survey
timetable is planned so that fasting samples are taken in the morning. When the
primary lipids of interest are serum total cholesterol and high density lipoprotein
cholesterol, blood can be taken at any time of the day with the patient non-fasting. In
the case of drawing non-fasting samples it is recommended that blood sample drawing
is spread throughout the day.
Position of patient
The position of the patient can influence the cholesterol values. Standardization of the
position is necessary. It is recommended that all blood samples should be drawn in a
sitting position and that the patient remain in sitting position for 15 minutes prior to
blood collection. This waiting period will allow equilibration of the concentrations of
blood components. Preferably, blood is not collected from the arm that is used for
blood pressure measurement, i.e. blood should usually be drawn from the left arm.
Use of tourniquet
Prolonged venous occlusion can cause changes in concentrations of blood constituents.
Therefore, the use of a tourniquet should be minimized. If a tourniquet is used to search
for a vein, it should be released before withdrawal of blood begins. In any case, the use
of a tourniquet should be limited to less than one minute.
Sample drawing procedure
Blood samples are taken from the vein in the antecubital fossa. Before blood collection,
the patient is asked to remove tight clothes that might constrict the upper arm.
The phlebotomist (person collecting the blood) sets the tourniquet around the upper
arm of the patient, searches the proper vein by inspecting and palpating and then
sterilizes the injection site. The vein can be anchored by placing the thumb about two
centimeters below the vein and pulling gently to make the skin a little taut. After that,
the needle, beveled upward, is pushed smoothly and quickly into the vein, to minimize
the possibility of hemolysis as a result of vascular damage. Immediately after the
insertion, the tourniquet is released to minimize the effect of hemoconcentration.
The order in which the various tubes are filled is determined by the risk of
contamination and coagulation. It is recommended that the order below is followed:
I. tubes for serum,
II. citrate filled tubes,
III. gel tubes,
IV. heparin filled tubes,

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V. EDTA filled tubes,
VI. Fluoride filled tubes.
Another consideration that might affect the order of tube filling is the priority of the
assay for which the tubes are needed, in case insufficient blood flow cuts the sampling
short.
If there are any problems with blood flow during blood taking (e.g. collapsing vein), the
procedure is discontinued and an attempt is made on the other arm. If that also fails, no
further attempts are made and the blood collection for this particular patient is
recorded as "failed".
Depending on the scheduled analyses the following tubes need to be filled:
Type of analysis Specimen type Type of tube
Lipids Serum 10 ml plain vacutainer, preferably with gel
Plasma glucose Plasma
4 ml tube filled with glycolytic inhibitors
potassium oxalate and sodium fluoride
Full blood count whole blood 3 ml tube with anticoagulant K2EDTA
If vacuum tubes are used, the tube is placed into the adapter. When taking several
tubes the next tube is changed immediately after the previous one is filled. In case there
is suspicion that not enough blood will be obtained to fill all the tubes, they are filled in
the order of priority of the assay for which they are needed. To assure proper mixing
tubes pre-filled with EDTA, gel or fluoride is inverted about 8 times towards the stopper
while the next tube is filling up (It may simplify the manual of operations to prescribe
inverting all tubes, since it does not harm plain tubes).

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Before the patient leaves the examination site and before the rack is moved anywhere,
all the tubes are labeled with the patient identification number and name.
Clotting
After the identification of the tubes, the timer is started. The blood samples are allowed
to clot at 15-24 °C. If vacuum gel tubes are used, the temperature is at least 20°C
(optimum 20-22°C), because the gel viscosity changes in colder temperature. The
clotting time is minimally 30 minutes and maximally one hour.
Storage and transfer of whole blood samples
Samples are refrigerated to 4°C immediately after collection. They can also be shipped
in refrigerated packaging at 4°C. At that temperature they are stable for 7 days. If it is
anticipated that analysis can not occur within 7 days, samples are frozen immediately at
-70°C (-20°C is not sufficient).
SAMPLE SEPARATION
TEST: Separation of plasma from blood samples
AIM: In-vitro separation plasma cells from blood samples collected in 10 mls EDTA
vacutainer tubes into three cryovials each bearing patients ID, project no, (JU-05-0251)
number of weeks, date, and time of collection.
MATERIALS: Transfer /disposable pipettes, biohazard bags, tablemats, sample racks,
polyphorus rack, 2mls cryovials, gloves, centrifuge, 70% alcohol, arch files, rim of
papers,10% diluted bleach solution,
PRINCIPLE:
When blood is collected in EDTA vacutainer tubes and centrifuge at the speed of 2500
RPM for five minutes, the components of blood having their different densities separate
into layers, which consists of plasma, lymphocytes, and red blood cells. The plasma is
extracted into two cryovials and store in freezers to be used for HIV viral load assays.
PROCEDURES
1. Collect blood in 10 mls EDTA vacutainer tube arranged in sample racks and mix
gently.
2. Label 3 cryovials each with the Pepfar ID, type of specimen, sample date, and
arrange in polyphorus racks.
3. Centrifuge the blood samples in the EDTA tube at 2500 revolutions per minute
for 5 minutes in the centrifuge at room temperature.
4. Set the bio-safety cabinet in preparation for dispensing of samples: switch on the
safety cabinet, place the tablemat on the floor of the cabinet; set the biohazard
bags and the transfer pipettes to begin work.

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5. Carefully check the patients JU-number on the tube with the labels on the
cryovials to be sure of correspondence before dispensing.
6. Gently aspirate the top layer of plasma using the transfer pipette to within 2-3
mm above the Buffy coat.
7. Transfer 1ml of the plasma into each three properly labeled cryovials and close
securely.
8. Dispose of the transfer pipettes after dispensing of each patient’s plasma.
9. Catalogue the patients’ number into a logbook and enter into the computer
system for backup of information.
10. Rack the cryovials into the cryobox according to the catalogue number.
11. Aliquot and store the three vials at -800
C.
For mother specimen, two cryovials are separated, banked, and stored at -800c
awaiting viral genotyping and sequencing. For babies specimen, 2 cryovials i.e.
one with plasma for viral load assay and the other with whole blood for DNA
PCR.
Centrifuging
When samples for glucose measurements are taken, the samples are centrifuged no
later than 20-35 minutes after the sample is drawn.

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For serum samples, blood is centrifuged within one hour after blood collection.
For plasma samples, blood is cooled and centrifuged as soon as possible and separated
immediately after centrifuging. Stoppers are not left opened during the centrifuging.
The centrifuge is not allowed to be cold and blood specimens are centrifuged at a
temperature 15-24°C. For serum preparation blood is spun for 10 minutes at 1500 g. For
plasma, the conditions are 15 minutes at 2000g to 3000g.

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CHAPTER 5
CD4/CD4% UNIT
CLUSTER OF DIFFERENTIATION MOLECULES (CDs)
The involvement of lymphocytes with the immune response and various diseases
can be assessed by enumeration of specific cells bearing particular membrane proteins
called cluster of differentiation molecules (CDs). CDs are functional cell surface proteins
or receptors that can be measured in situ and from peripheral blood, biopsy samples, or
other body fluids. They often are used as a nomenclature system to differentiate
between leukocyte subpopulations.
CDs have both biological and diagnostic significance. In normal individuals the
concentration of these molecules in serum is very low. When one’s immune system is
activated in response to disease, the concentration of these molecules usually rises and
fluctuates. Monitoring CD levels may help in the management of the disease. For
example, circulating levels of CD54 are directly related to the progress and prognosis of
the skin cancer metastatic melanoma. CD23 is a B-cell growth factor, and elevated levels
are associated with chronic lymphocytic leukemia. CD35 is a potent agent for the
suppression of complement-dependent tissue injury in autoimmune inflammatory
disease. Elevated levels of CD8 have been found in childhood lymphoid malignancies
and in HIV-infected individuals. It has been established that the CD4 molecule is a cell
surface receptor for HIV-1, and considerable research is currently being done on the role
of both cell-bound and soluble CD4 in AIDS.
CD4 T Lymphocytes
The CD4 T lymphocytes; a subpopulation of the lymphocytes also known as T helper
cells, are coordinators of the body's immune response, e.g., providing help to B cells in
the production of antibody, as well as in augmenting cellular immune response to
antigens.
The “CD” or cluster of differentiation is a protein expressed on the surface of the
cells of the hematopoetic system. The expression of these proteins is used in
lymphocyte nomenclature. Over 300 CD molecules have been reported so far. These
proteins are often associated with the specific function of the cells. Cells with different
functions express different CD molecules (for e.g.: CD3+ cells are total T lymphocytes,
CD4+ cells are T helper cells, CD8+ cells are cytotoxic T lymphocytes and CD19+ are B
lymphocytes).
CD4 T lymphocytes occupy the central position in regulating immune functions.
CD4 T lymphocytes are the primary targets of HIV. The relentless destruction of CD4 T
lymphocytes by HIV, either directly or indirectly, results in the loss of HIV-specific
immune response, recall antibody response and, finally, non-specific immune response
in the AIDS stage.

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CD4 T Lymphocytes and HIV
Within hours of exposure to HIV, CD4 T lymphocytes are found to be infected showing
active viral replication. The infected CD4 cells release virions by budding through the cell
membrane or by lysis of the infected cells. The released virus particles then infect
uninfected CD4 T lymphocytes. CD4 T lymphocytes also serve as important reservoirs of
HIV: a small proportion of these cells carry HIV provirus integrated in the host DNA
without active virus multiplication.
During the primary HIV infection, the number of CD4 T lymphocytes in the
bloodstream decreases by 20% to 40%. HIV brings about the lysis of HIV infected cells as
well as bystander uninfected cells using various mechanisms such as lysis of the cells
infected with HIV. Billions of CD4 T lymphocytes may be destroyed every day, eventually
overwhelming the immune system's regenerative capacity. In acute HIV-1 infection, in
addition to the decline in CD4 T lymphocytes counts, qualitative impairments of CD4 T
lymphocytes function are detected. The impairment of HIV-1-specific CD4 T
lymphocytes function occurs very early in acute infection. Following acute primary HIV
infection, one may remain free of HIV-related illnesses, often for years, despite ongoing
replication of HIV in the lymphoid organs and relentless destruction of the immune
system. However, during the period, the immune system remains sufficiently competent
to provide immune surveillance and to prevent most infections. Although the decrease
in the total number of T lymphocytes marks the decrease in immune competence,
sometimes the quantitative loss of CD4 T lymphocytes may not be matched by the
qualitative functions. A number of assays such as cytokine induction, antigen-induced
proliferation, measurement of activation markers etc can assess the functions of
lymphocytes. However, the total CD4 T lymphocytes number still remains the most
robust marker of immune competence.
The progressive loss of CD4 T lymphocytes eventually results in the loss of an
ability to mount desirable immune response to any pathogen and vulnerability to
opportunistic pathogens characteristic of AIDS. The estimation of peripheral CD4 T

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lymphocytes counts is relied upon for taking a decision on initiation of ART. The
estimation of peripheral CD4 T lymphocytes counts has also been used as a tool for
monitoring disease progression and the effectiveness of antiretroviral treatment (ART).
The changes in the CD4 T lymphocytes counts are important indicators of the response
to ART. HIV plasma virus load is a sensitive indicator of the progression of HIV disease.
However, due to the relatively high cost of virus load estimation, the CD4 T lymphocytes
count remains the most important key indicator for initiation and monitoring of ART and
a measure of the effectiveness of the treatment in clinical trial evaluations.
CD4 Count
A CD4 count is a lab test that measures the number of CD4 T lymphocytes (CD4 cells) in
a sample of your blood. In people with HIV, it is the most important laboratory indicator
of how well the immune system is working and the strongest predictor of HIV
progression.
CD4 count is also used to help HIV care provider decide when to start antiretroviral
therapy (ART).
 The CD4 count of an uninfected adult/adolescent who is generally in good health
ranges from 500 cells/mm3
to 1,600 cells/mm3
.
 A very low CD4 count (less than 200 cells/mm3
) is one of the ways to determine
whether a person is living with HIV.
PRINCIPLE OF CD4+/CD4% COUNT:
The principle of the test is an immunoflorescent technique based on an antigen-
antibody complex formation. The antibody is labeled with a flurochrome dye.
PRINCIPLE OF FLOW CYTOMETRY:
Flow cytometry refers to a technology that simultaneously measures and analyzes
multiple physical and chemical characteristics of single cells or other biological particles,

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as they flow in a fluid stream past optical and/or electronic sensors. It provides
information about their relative size, relative granularity or internal structure, and
fluorescence in several spectral regions emitted by fluorochrome labeled probes which
bind specifically and stoichiometrically to cellular constituents such as protein antigen
and nucleic acids. Individual cells stained with fluorescent labels or absorption dyes are
suspended in physiological solution and introduced under a slight pressure through a
flow chamber into the centre of a stream of cell-free sheath fluid. The light scattered by
the individual particle and the fluorescence emitted by the cells is used for analysis and
sorting of the cells based on the fluorescent antibody directed against a specific surface.
This combination of scattered and fluorescence light is picked up by the detectors in the
flow cytometer. These detectors then produce electronic signals that are proportional
to the optical signals received. The visible light undergoes deflection based on the size
and internal structures of the cell. FSC (Forward Scatter) correlates with the cell volume.
SSC (Side Scatter) depends on the inner complexity of the particle (i.e. shape of the
nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
The fluorescence emitted by the cell depends upon the florescence tagged specific
monoclonal antibodies against the cell surface markers. The data collected on each cell
or events are stored in the computer. This data is then processed and analyzed to
provide information about cell populations within the sample.
Flow cytometric methodologies (METHOD OF CD4 COUNTING)
Immunofluroscence analysis by flow cytometry is the gold standard for CD4 T
lymphocytes measurements and also the method of choice if a large throughput of
samples is required. The flow cytometric assays work on the principle of scattering of
light due to different sizes, granularity of the cells passing thorough the laser beam, and
also by the fluorescence emitted by the cells after staining with the specific monoclonal
antibodies to cell surface markers that are tagged with different fluorescence dyes. The
population of interest can be thus identified and gated for further analysis within the
population of interest. The monoclonal antibodies specifically bind different surface

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receptors like CD4 for T helper cells. Relative percentages of the cells expressing the
specific receptor (eg. CD4) on its surface are obtained from the flow cytometer and the
absolute counts can be calculated with the help of absolute lymphocyte count obtained
from haematology analyzer as detailed below.
Absolute CD4 T lymphocytes count determination by flow cytometer may be performed
using dual- and single-platform methods.
Dual-platform approach
The dual-platform approach estimates absolute CD4 T lymphocytes counts by a
mathematical formula using two independent parameters; CD4 T lymphocytes
percentage obtained by flow cytometer and total (WBC) and differential lymphocyte
counts estimated by a haematology analyzer or a haemocytometer. The lymphocyte
population can be gated (identified) in a flow cytometer using the suitable surface
markers for leucocyte subpopulations and the percentage of CD4 T lymphocytes
amongst the total lymphocyte percentages i.e. gated population can be obtained. An
absolute CD4 T lymphocytes count is then derived using a mathematical formula:
(%CD4+ T-cells x the absolute lymphocyte count).
Because the percentage of CD4 T lymphocytes is obtained from the reference
lymphocyte populations, the purity of the lymphocyte gate is most essential. Hence, for
the sample with a high proportion of lymphocytes, the percentage of CD4 T
lymphocytes can be easily derived from a homogeneous gate that includes forward
scatter (FSC, size of the cell populations) and right angle side scatter (SSC, granularity of
the cell populations) patterns. However, when the sample has a high proportion of non-
lymphocytes (monocytes, basophils and immature red blood cells), this traditional
FSC/SSC lymphocyte gate tends to be unreliable as nonlymphocytes have been shown to
contaminate the gates, then this morphological gating remains questionable. Hence,
this gating strategy is now considered as unacceptable.
Single-platform approach
The single-platform approach enables absolute CD4 T lymphocytes counts to be derived
directly without the need for a haematological analyzer. This can be assessed either by
counting CD4 T lymphocytes populations in a precisely determined blood volume or by
using the known numbers of fluorescent microbeads admixed to a known volume of
CD4-stained blood.
It is required to pipette small amounts of reagents, e.g.: 10 μl to 25μl and hence the
pipetting technique is very crucial for reliable use of the single platform approach.

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Partec CyFlow Counter (volumetric system)
CyFlow is another desktop single-platform technology made by Partec,
Germany. It is a volumetric software controlled absolute count system equipped with
either a single 532 nm green solid-state laser used for one fluorescence parameter or
two lasers with a mercury arc lamp applicable for 2 or 3-color analyses. Data acquisition
and analysis are performed in real time with FlowMax software. It can be used as a
mobile system that can run on car batteries. It works on simple no lyse and no wash
protocol. However, the robustness and reproducibility is under evaluation.
3-Step Protocol for CD4 counting and CD4% enumerating with a
volumetric absolute counting FCM
1. take 20 µl whole blood from the patient into a sample tube
2. CD4: add 20µl of the monoclonal antibody (CD4 mAb PE) and incubate for 15
minutes at room temperature in the dark
2. CD4%: add 10µl of antibody (CD4 mAb PE) and 10µl antibody (CD45 mAb DY647)
and incubate for 15 minutes at room temperature in the dark
3. CD4: add 800µl of the no lyse buffer
3. CD4%: add 400µl buffer 1. Directly prior measurement add 400µl buffer 2
INTERFERENCES OF CD4 COUNTING
a.CLOT: blood clot can block the flow cuvette depending on the size of the clot.

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b. HAEMOLYSIS: the destruction of red blood cells will result to high background
noise thereby interfering with the actual count of the CD4 cells.
c.LIPAEMIA: excess of fat in the blood can mask antigen site and change the reaction
environment.
RESULT REPORTING/BIOLOGICAL REFERENCES INTERVALS
In reporting CD4 counts, absolute CD4 T-lymphocytes count is expressed in cells/µl or
cells/mm3
{example, 500cells/ µl} while CD4 T-lymphocyte percentage value is
expressed in percentage {example, 20%}.
Biological Reference Values;
 Absolute CD4 count = 365 cells/µl – 1571cells/ µl
 CD4% count = 15% - 50%

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CHAPTER 6
CLINICAL CHEMISTRY
CLINICAL BIOCHEMISTRY (also known as clinical chemistry or chemical
pathology) is the laboratory service absolutely essential for medical practice or branch
of laboratory medicine in which chemical and biochemical methods are applied to the
study of disease.
The results of the biochemical investigations carried out in a clinical chemistry
laboratory will help the clinicians to determine the diseases (diagnosis) and for follow-
up of the treatment/recovery from the illness (prognosis).
The use of biochemical tests:
Biochemical investigations are involved in every branch of clinical medicine.
The results of biochemical tests may be of use in:
1. Diagnosis and in the monitoring of treatment.
2. Screening for disease or in assessing the prognosis.
3. Research into the biochemical basis of disease
4. Clinical trials of new drugs
Biochemical investigations hold the key for the diagnosis and prognosis of diabetes
mellitus, jaundice, myocardial infarction, gout, pancreatitis, rickets, cancers, acid-base
imbalance etc. Successful medical practice is unimaginable without the service of clinical
biochemistry laboratory.
In the clinical chemistry laboratory, the following tests are assayed:
1. liver function,
2. kidney function (or renal function),
3. electrolytes,

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4. bone profile,
5. lipid profile; and
6. carbohydrate/diabetic tests.
Renal Function Tests (RFT):
RFT is any clinical and laboratory procedure designed to evaluate various aspects of
renal (kidney) capacity and efficiency and to aid in the diagnosis of kidney disorders.
Such tests can be divided into several categories, which include:
(1) concentration and dilution tests, whereby the specific gravity of urine is
determined at regular time intervals following water restriction or large water
intake, to measure the capacity of the kidneys to conserve water,
(2) clearance tests, which give an estimate of the filtration rate of the glomeruli, the
principal filtering structures of the kidneys, and overall renal blood flow,
1. Albumin (ALB).
2. Creatinine (CREj).
3. Urea.
4. Uric Acid.
Albumin (ALB)
Location: Synthesized in the Liver.
Function:
 Carries ions and molecules (e.g. Bili & Ca).
 Maintains oncotic pressure.
 Regulates plasma volume.
Disease associated: Increased: In Dehydration.
Decreased: In Oedema, Nephrotic syndrome and Burns
Note: Low levels also affect binding site availability and can give falsely low levels of
total Calcium (Ca).
Creatinine (CREj)

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Type: Organic Compound.
Location: Muscle tissues and then released into blood.
Function: Plasma Creatinine is mostly derived from breakdown of Creatinine and then
excreted by kidney into urine. Therefore, it uses to assess the kidney function.
Disease associated: Increased in Kidney diseases.
Note: Creatinine is generally used for the measurement of Glomerulur Filtration Rate
(GFR) by estimating the Creatinine from the plasma and urine.
Urea
Type: Organic Compound of breakdown of amino acids in the Liver.
Location: Plasma and Urine.
Function: Urea is used as a marker of renal function and as an indication of reduced
blood flow. (Congestive Cardiac Failure)
Disease associated:
Increased in:
1. Renal Failure.
2. High Protein Diet (Increased catabolism due to Starvation or Tissue damage may
cause moderate elevation).
3. Dehydration.
4. Heart Failure.
Decreased in:
1. Advanced Liver Disease.
2. Hemodialysis.
Uric Acid
Type: Organic Molecule of end products of purine (Adenine and Guanine) catabolism.
Location: Purines can be synthesized in the body or can be ingested from foodstuff.
Function: Man is therefore prone to clinical gout and renal damage, whereas other
mammals.
Disease associated:
Increased in:
1. Gout in which hyperuricaemia may be genetically determined or could be
secondary to increased turnover of nucleic acids (Malignant tissue, tissue
damage).
2. Uremia due to kidney disease.
3. Diabetic Acidosis.
4. Reduced excretion may be due to renal glomerular dysfunction or acidosis.

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Liver Function Tests (LFT):
LFT is any laboratory procedure that measures and assesses various aspects of liver
function. Because of the diversity of liver function and the varied and complicated
metabolic processes that may be affected by disease states, more than 100 tests have
been devised to test liver function. These reactions bear upon the metabolism of
proteins, fats, carbohydrates, bile, and the detoxification and clearance of drugs and
toxic chemicals performed by the liver.
1. Alkaline phosphatase (ALP).
2. Alanine Aminotransferase (ALT).
3. Aspartate Aminotransferase (AST).
4. Total Bilirubin (BILT2).
5. Total protein (TP).
6. Albumin (ALB2).
Alkaline phosphatase (ALP)
Type: Enzyme.
Location: Mainly in Liver and Bone.
Function: Hydrolyses phosphate at high pH in vitro.
Disease associated: Increased: In Liver, Bone and Malignant diseases.
Note: ALP increases during bone growth and in pregnant women.
Alanine Aminotransferase (ALT)
Type: Enzyme.
Location: Mainly in Liver.
Function: Catalyzes the transformation of amino acids from ketoglutrate to Alanine to
form Pyruvate.
Disease associated: Increased: In Liver diseases.
Method of Alanine Aminotransferase (ALT):
 Pyruvate is reduced to lactate in association with oxidation of NADH (Reduced
form) to NAD+
(Oxidized form). The later reaction is catalyzed by LDH.
 (NAD: Nicotinamide-Adenine Dinucleotide).
 Oxidation-Reduction Reaction: it is a chemical reaction whereby electrons are
removed (Oxidation) from atoms of the substance being oxidized and transfer to
those being reduced (Reduction).
Aspartate Aminotransferase (AST)
Type: Enzyme.

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Location: Liver, Heart and Skeletal Muscles
Function: Catalyzes the transformation of amino acids from ketoglutrate to Aspartate to
form Oxaloacetate.
Disease associated: Increased: In Heart and Liver diseases
Total Bilirubin (BILT2)
Type: Organic Compound (conjugated and unconjugated forms).
Location: Liver.
Function: Bilirubin is a Bile pigment formed from the breakdown of Heme group of
hemeprotiens (e.g. hemoglobin).
Disease associated: Increased: In Acute hemolysis and Liver diseases. Increase levels of
Bilirubin may cause Jaundice.
Bilirubin (Conjugated or Direct)
Type: Conjugated Bilirubin is a Water soluble form of Bilirubin.
Location: Conjugation with Glucuronic acid takes place in the Liver by Uridyl-
Diphosphate Glucuronyl Transferase (UDGT) and passes into the Bile system.
Function: Conjugated Bilirubin is broken down in the gut o form stercoblinogen and
accounts for the normal color of faeces.
Disease associated: Increased in Choletasis (Obstruction of Bile Flow).
 Bilirubin Fractions
1. Unconjugated "Indirect"; (Alpha-Bili).
2. Monoconjugated; (Beta-Bili).
3. Diconjugated "Direct"; Gamma-Bili).
4. Protein; (Sigma-Bili).
Total protein (TP)
Type: The sum of all proteins and globulins.
Location: Most proteins are synthesized in the Liver and released into biological fluids.
Function: It measures the sum of circulating proteins: Albumin, Alpha-1, Alpha-2, Beta
and Gamma Globulins. Albumin must be measured at the same time to distinguish any
increase or decrease in fractions
Disease associated:
Increased in: Multiple Myeloma and Immunoglobulin Disorders.
Decreased in: Liver and Kidney Diseases.
Albumin (ALB2)
Type: Protein.
Location: Synthesized in the Liver.

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Function:
1. Carries ions and molecules (e.g. Bili & Ca).
2. Maintains oncotic pressure.
3. Regulates plasma volume.
Disease associated: Increased: In Dehydration.
Decreased: In Oedema, Nephrotic syndrome and Burns.
Note: Low levels also affect binding site availability and can give falsely low levels of
total Calcium (Ca).
Electrolytes (E,U,C):
1. Bicarbonate (HCO3-
& CO2).
2. Chloride (CL-
).
3. Potassium (K+
).
4. Sodium (Na+
).
Bicarbonate (HCO3-
& CO2)
Type: HCO3-
: is the second largest organic anion in plasma. CO2: is an organic gas.
Location: CO2 transports from tissue to the lungs in the form of HCO3-
in the plasma.
Function: HCO3-
regulates acid-base balance and CO2 is the end products of metabolism
of foodstuffs.
Disease associated: Increase of HCO3-
with decrease of pH is screen in respiratory
acidosis. Increases of both are seen in metabolic alkalosis.
Decrease of HCO3-
with increase of pH is seen in respiratory alkalosis. Decreases of both
are seen in metabolic acidosis.
Chloride (Cl-
)
Type: Anion Ion.
Location: Extracellular Fluid (ECF).
Function: Maintain electrical neutrality of ECF.
Disease associated:
Increased in:
1. Hyperventilation (Excess breathing).
2. Drugs (NH3CL or KCL).
3. Dehydration.
Decreased in:
1. Hypoventilation (Inadequate removal of CO2 from the blood by lungs).
2. Vomiting.
3. Diarrhea.
4. Diabetic ketoacidosis (Ketone bodies in blood displace of both HCO3-
& Cl-
)

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Note: When (HCO3-
) ions increase; (Cl-
) ions decrease, and when (Na+
) ions increase; (Cl-
)
ions increase.
Potassium (K+
)
Type: Cation Ion.
Location: Intracellular Fluid (ICF) inside cells.
Function: It helps in nerve impulse transmission, contractility of cardiac and skeletal
muscles.
A pump mechanism in the cell membrane transfers Na from the inside cell to outside in
exchange for K.
Disease associated:
Increased in:
1. Kidney failure.
2. Urinary obstruction.
3. Tissue damage (Crush injuries and massive hemolysis).
4. Addison's disease.
Decreased in:
1. Diarrhea.
2. Vomiting.
3. Diuretic medications.
4. Cushing's Syndrome where hypokalemia in the rule.
Note:
1. K and H ions are secreted into distal tubule in exchange for Na ions
2. If (K) is high, the heart stops beating in Diastole (The fully relaxed state).
3. If (K) is low, the heart stops beating in Systole (The fully contracted state).
Sodium (Na+
)
Type: Cation Ion.
Location: Extracellular Fluid (ECF) and regulated by aldosterone.
Function:
1. Maintain water balance and osmotic pressure.
2. Maintain the pH of plasma.
Disease associated:
Increased in (= increase of body Sodium or decrease of body water in the
following):
1. Dehydration due to sweating.
2. Diabetes insipidus (Either lack of ADH or nephrogenic).
3. Increase of glucose and urea in urine which contribute to osmolilaity of urine and
increase water excretion.

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4. Excess of Na solution by IV (e.g. NaCl or NaHCO).
Decreased in:
1. Diuretic medications in CHF, RD and HPT.
2. Kidney diseases.
3. Congestive Heart Failure.
4. Hepatic Cirrhosis.
Bone Profile/Mineral Tests:
1. Calcium (Ca2+
).
2. Phosphorous (PHOS).
Calcium (Ca2+
)
Type: Inorganic Cation Element.
Location: 99% in Bone and 1% in Plasma.
Function: Ca is essential for the normal function of heart, nerves impulses, muscle
contractility and blood coagulation.
Disease associated: Increased in: Hyperparathyroidism, Carcinoma metastatic to bone
and multiple myeloma.
Decreased in: Hypoparathyroidism, Vitamin D deficiency, Malabsorption, Kidney disease
and Muscle Tetany.
Note: Bone releases Ca to prevent hypokalemia or to control high level with assisting of
the kidney and parathyroid gland.
Phosphorous (PHOS)
Type: Inorganic Element.
Location: Bone.
Function:
1. Give the bone its rigidity as calcium-phosphate salts.
2. Part of ATP structure.
3. Part of phospholipids structure.
4. Part of DNA and RNA structures.
Disease associated:
Increased in:
1. Renal diseases with secondary low calcium.
2. Hypoparathyroidism.
3. Hyperthyroidism.
Decreased in:
1. Hyperparathyroidism.
2. Malabsorption that may leads osteomalacia (Soft bones; Low Ca & PHOS levels).

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Note:
1. When the level of Ca is high, the PHOS level is low and vice versa.
2. After the meal, PHOS level decrease because it moves into the cell along with
glucose.
Lipid Profile Tests:
1. Cholesterol (CHOL).
2. Triglycerides (TRIG).
3. High Density Lipoprotein (HDL).
4. Low Density Lipoprotein (LDL).
Cholesterol (CHOL)
Type: Cyclic Aliphatic Organic Hydrocarbons
Location: Adrenal gland, Liver and Gallstone.
Function:
1. Precursor of steroid hormone.
2. Part of lipoproteins structure (LDH).
3. Part of cell membrane.
Disease associated: Increased in: Atherosclerosis, Hypothyroidism, Nephrosis Diabetes
Mellitus, Obstruction of Bile flow.
Decreased in: During Starvation and in Hyperthyroidism.
Note: 2/3 of cholesterol is esterified with fatty acids (In the Liver). Routine assays
measure the total cholesterol level (i.e. Ester and free cholesterol).
Triglycerides (TRIG)
Type: Triglycerides consists of glycerol esterified with 3 fatty acids that may reaches the
Liver from fat sources or synthesizes from glucose.
Location: Adipose Tissues.
Function: In case of Starvation or Hypoglycemia; Triglycerides release from adipose
tissues to give energy.
Disease associated:
Increased in:
1. Diabetes Mellitus.
2. Nephrosis.
3. Liver Destruction.
4. Lipid metabolism disorders.

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High Density Lipoprotein (HDL)
Type: Macromolecules that contain of a combination of 50% proteins, 28%
phospholipids and 20% cholesterol and apoprotien A-I & A-II.
Location: HDL is a catabolic product of chylomicrons and VLDL. It is synthesized in the
Liver and intestine.
Function: HDL removes cholesterol from peripheral cells to the Liver. This stats by the
activation of lecithin- cholesterol
acyltransferase (LCAT) by apoprotiens of HDL LCAT.
Disease associated:
Increased in:
1. Protective against atherosclerosis and coronary heart diseases.
2. Physical activity.
Decreased in:
1. Atherosclerosis and coronary heart diseases.
2. Smoking.
3. Obesity.
Carbohydrate/Diabetic Tests:
Glucose
Type: The simplest organic form of Carbohydrate.
Location: In all Cells.
Function:
1. The major source of energy.
2. At low levels of blood glucose, the Liver synthesizes it from glycogen, adrenalin,
cortisol, glucagons, thyroxin and growth hormone and raise blood glucose levels.
Disease associated:
Increased in:
1. Diabetes Melitus.
2. Cushing's Syndrome (Excess of cortisol which produces more glucose via
gluconegenesis).
3. Acromegaly (Excess of growth hormone).
4. Hyperadrenalinism (Excess of adrenalin causes the conversion of glycogen to
glucose in the Liver).
Decreased in:
1. Glycogen storage disease (Inability of the Liver to convert glycogen back to
glucose).
2. Galctosemia (Increase of galactose in the blood due to deficiency of the enzyme
Galactose-1-Phosphate
3. Liver diseases.

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CHAPTER 7
HAEMATOLOGY UNIT
Hematology refers to the study of the numbers and morphology of the cellular
elements of the blood—the RBCs (erythrocytes), WBCs (leukocytes), and platelets
(thrombocytes)—and the use of these results in the diagnosis and monitoring of disease
(infections).
WHITE BLOOD CELLS (LEUKOCYTES)
White blood cells (leukocytes), unlike red cells, are nucleated and independently motile.
Highly differentiated for their specialized functions, they do not undergo cell division
(mitosis) in the bloodstream, but some retain the capability of mitosis. As a group they
are involved in the body's defense mechanisms and reparative activity. The number of
white cells in normal blood ranges between 4,500 and 11,000 per cubic millimetre.
Fluctuations occur during the day; lower values are obtained during rest and higher
values during exercise. Intense physical exertion may cause the count to exceed 20,000
per cubic millimetre. Most of the white cells are outside the circulation, and the few in
the bloodstream are in transit from one site to another. As living cells, their survival
depends on their continuous production of energy. The chemical pathways utilized are
more complex than those of the red cells and are similar to those of other tissue cells.
White cells, containing a nucleus and able to produce ribonucleic acid (RNA), can
synthesize protein. They comprise three classes of cells, each unique as to structure and
function, that are designated granulocytes, monocytes, and lymphocytes.
PLATELETS (THROMBOCYTES)
The blood platelets are the smallest cells of the blood, averaging about two to four
micrometres in diameter. Although much more numerous (150,000 to 400,000 per cubic
millimetre) than the white cells, they occupy a much smaller fraction of the volume of
the blood because of their relatively minute size. Like the red cells, they lack a nucleus
and are incapable of cell division (mitosis), but they have a more complex metabolism
and internal structure than have the red cells. When seen in fresh blood they appear
spheroid, but they have a tendency to extrude hairlike filaments from their membranes.
They adhere to each other but not to red cells and white cells. Tiny granules within
platelets contain substances important for the clot-promoting activity of platelets.
RED BLOOD CELLS (ERYTHROCYTES)
The red blood cells are highly specialized, well adapted for their primary function of
transporting oxygen from the lungs to all of the body tissues. Red cells are
approximately 7.8 micrometres in diameter and have the form of biconcave disks, a
shape that provides a large surface-to-volume ratio. When fresh blood is examined with
the microscope, red cells appear to be yellow-green disks with pale centres containing
no visible internal structures. When blood is centrifuged to cause the cells to settle, the

PLSU/2013/FNS/0025
volume of packed red cells (hematocrit value) ranges between 42 and 54 percent of
total volume in men and between 37 and 47 percent in women; values are somewhat
lower in children. Normal red blood cells are fairly uniform in volume, so that the
hematocrit value is determined largely by the number of red cells per unit of blood. The
normal red cell count ranges between four million and six million per cubic millimetre.
Hemoglobin
About 95 percent of the dry weight of the red blood cell consists of hemoglobin, the
substance necessary for oxygen transport. Hemoglobin is a protein; a molecule contains
four polypeptide chains (a tetramer), each chain consisting of more than 140 amino
acids. To each chain is attached a chemical structure known as a heme group. Heme is
composed of a ringlike organic compound known as a porphyrin, to which an iron atom
is attached. It is the iron atom that reversibly binds oxygen as the blood travels between
the lungs and the tissues. There are four iron atoms in each molecule of hemoglobin,
which, accordingly, can bind four atoms of oxygen. The complex porphyrin and protein
structure provides the proper environment for the iron atom so that it binds and
releases oxygen appropriately under physiological conditions. The affinity of hemoglobin
for oxygen is so great that at the oxygen pressure in the lungs about 95 percent of the
hemoglobin is saturated with oxygen. As the oxygen tension falls, as it does in the
tissues, oxygen dissociates from hemoglobin and is available to move by diffusion
through the red cell membrane and the plasma to sites where it is used. The proportion
of hemoglobin saturated with oxygen is not directly proportional to the oxygen
pressure. As the oxygen pressure declines, hemoglobin gives up its oxygen with
disproportionate rapidity, so that the major fraction of the oxygen can be released with
a relatively small drop in oxygen tension. The affinity of hemoglobin for oxygen is
primarily determined by the structure of hemoglobin, but it is also influenced by other
conditions within the red cell, in particular the pH and certain organic phosphate
compounds produced during the chemical breakdown of glucose, especially 2,3-
diphosphoglycerate.
In this laboratory, the following tests are carried out
 Blood Group Test
 Genotype Test
 Full Blood Count
 Malaria Rapid Test
BLOOD GROUP TEST
Analysis
A needle is inserted into the vein and blood into a tube. During the procedure, the
elastic band used is reserved to restore circulation. Once the blood has been collected,
the needle is removed and a band aid or gauze is applied.
Procedures

PLSU/2013/FNS/0025
 Venous blood is collected into EDTA sample bottle
 Antiserum A, B, and D were placed on the white tile separately in three spots
 Three separate drops of blood were dropped unto each of the spots
 Each spot was then mixed together with the tip of a clean glass slide or an inverted
rubber pipette
 The tile was rocked for three minutes to view agglutination
Results
Blood type (or blood group) is determined, in part, by the ABO blood group antigens
present on red blood cells.
A blood type (also called a blood group) is a classification of blood based on the
presence or absence of inherited antigenic substances on the surface of red blood cells
(RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids,
depending on the blood group system. Some of these antigens are also present on the
surface of
Red blood cell compatibility
• Blood group AB individuals have both A and B antigens on the surface of their RBCs,
and their blood serum does not contain any antibodies against either A or B antigen.
Therefore, an individual with type AB blood can receive blood from any group (with AB
being preferable), but can donate blood only to another type AB individual.
• Blood group A individuals have the A antigen on the surface of their RBCs, and blood
serum containing IgM antibodies against the B antigen. Therefore, a group A individual
can receive blood only from individuals of groups A or O (with A being preferable), and
can donate blood to individuals with type A or AB.

PLSU/2013/FNS/0025
• Blood group B individuals have the B antigen on the surface of their RBCs, and blood
serum containing IgM antibodies against the A antigen. Therefore, a group B individual
can receive blood only from individuals of groups B or O (with B being preferable), and
can donate blood to individuals with type B or AB.
•Blood group O (or blood group zero in some countries) individuals do not have either A
or B antigens on the surface of their RBCs, but their blood serum contains IgM anti-A
antibodies and anti-B antibodies against the A and B blood group antigens. Therefore, a
group O individual can receive blood only from a group O individual, but can donate
blood to individuals of any ABO blood group (i.e. A, B, O or AB). If anyone needs a blood
transfusion in a dire emergency, and if the time taken to process the recipient's blood
would cause a detrimental delay, O Negative blood can be issued.
GENOTYPE TEST
Principle
Different types of haemoglobin (Hb) have different migration rate because Hb s are
proteins. They can be made to run to either anode or cathode depending on the charge
carried by the Hb. The buffer used determines the charge carried by the Haemoglobin
(Hb). Here alkaline cellulose acetate is used in an alkaline buffer at pH 8.4, this tends to
make the Hb to migrate to the anode where there is negative charge.
Procedure
Step 1: Preparation of Haemolysate
The anticoagulant blood was centrifuge at 2500rpm for five minutes. The supernatant
plasma was discarded and packed cells were washed with large volume of saline three
times. After the final washing the red cells were lysed by adding equal volume of
distilled water, one quarter (1/4) volume of toluene and one drop of 3% potassium
cyanide and was mixed properly.
Step 2: Electrophoresis
The buffer was poured into the electrophoresis chamber. Two wicks were socked in the
buffer and positioned such that they made contact with the buffer. The cellulose
acetate paper was re-socked in the buffer and left to soak for 20minutes. Excess buffer
was removed by keeping the plate between absorbent papers.
Using an applicator stick 0.5ml of the haemolysate samples test and control were
applied approximately 3cm away from the cathode. The cellulose acetate membrane
plate was immediately placed in the electrophoretic chamber (tank). The chamber was
connected to the power supply and the plate was electrophoreses for 20 minutes at
approximately 350. After 20 minutes, the power supply was disconnected and the result
was read.

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FULL BLOOD COUNT
A blood count is a routine haematology test. It can be done as part of a general
investigation of an acute illness, for regular monitoring of HIV infection, and to check for
drug side-effects. To do this test, a small sample of blood is taken from a vein in the arm
(venesection or venepuncture).
A full blood count (FBC), also known as a complete blood count (CBC) can:
 Detect whether someone is anaemic. Anaemia is a shortage of haemoglobin, the
substance that allows red blood cells to transport oxygen to the body's tissues.
Normal haemoglobin levels in HIV-negative people are 12 to 16 grams per decilitre
for women and 13.5 to 18 for men, but are often a little lower in people with HIV.
Anaemia can contribute to the symptoms of fatigue and breathlessness. It is more
common among people with HIV than in the general population and may be caused
by HIV itself, opportunistic infections or certain antiretroviral drugs (e.g.
zidovudine/AZT).
 Measure the haematocrit, which is the percentage of red blood cells in the total
blood volume. It shows the oxygen-carrying capacity of the blood and tells whether
the blood is too thick or too thin. The average range is 40 to 54 percent.
 Measure the number and different types of white cells in the blood. The total number
of white blood cells in an average healthy adult is 4000 to 11,000. If there are
insufficient numbers of white cells, especially of neutrophils (neutropenia), then the
risk of bacterial and fungal infections is higher. However, this only applies to
neutrophil levels below 500. People with HIV often have slightly lower levels of
neutrophils than normal and this is not significant. Neutropenia can be a side-effect
of treatment of AZT, ganciclovir or cancer chemotherapy. It can be treated by
reducing or stopping the dose of these drugs, or with G-CSF, which increases the
number of neutrophils in the blood by stimulating production in the bone marrow.
Eosinophils are white cells involved in allergic-type reactions. Their number can be
elevated during certain parasitic infections. A normal amount is 0 to 8% of the total
white blood cell count.
 Measure the level of platelets. These are small cells essential for normal blood
clotting. A shortage of platelets (thrombocytopenia) may result in easy bruising and
bleeding. An excess of platelets can make the blood flow too stodgy. An average
count is 150 to 440, although people with HIV often have fewer than average. As long
as the count is not too low, the condition does not need treatment. If it gets so low
that bleeding occurs, treatment with intravenous gammaglobulin or steroids may be
required.

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Cell Type Relative
Reference
Range
Term for
Increased
Increase
Few Potential
causes
Term for
Decreased
Decrease Few
potential
causes
Neutrophil 40-80% Neutrophilia Bacterial
infection,
arthritis, trauma,
vasculitis,
surgery, AML,
CML
Neutropenia Often
associated
with
chemotherapy
Lymphocyte 25-35% Lymphocytosis Viral infection,
ALL, CLL,
Multiple
Myeloma, TB,
Crohn’s Disease
Lymphopenia or
Lymphocytopenia
Following a
recent
infection,
Immune
suppression
drugs, HIV
infection,
acute stress
Monocyte 2-10% Monocytosis Bacterial
infection,
malaria,
sarcoidosis,
ulcerative colitis,
neoplasms
Monocytopenia Acute
infections,
stress,
glucocorticoids
, aplastic
anemia
Eosinophil 0-5% Eosinophilia Allergic reaction,
helminth
infection, fungal
infections,
Eczema,
Eosinophilic
leukemia
Not applicable
Basophil 0-1% Basophilia Allergic
reactions,
Helminth
infections,IDA,
AML, CML,
Myelodysplasia
Not applicable

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Haematological features of HIV infection
 Lymphopenia—CD4 lymphopenia may be masked by CD8 lymphocytosis in stage
2; improved by antiviral therapy.
 Neutropenia—marrow suppression by virus or therapy; splenic sequestration.
 Normochromic/normocytic anaemia due to suppression of marrow by virus or
therapy. Microangiopathic haemolysis associated with TTP.
 Thrombocytopenia—suppression of marrow by virus or therapy or shortened
survival due to immune destruction (may respond to antiviral therapy), infection,
TTP or splenic sequestration.
 Bone marrow suppression—direct HIV effect or complication of antiretroviral
therapy, ganciclovir, trimethoprim or amphotericin B therapy.
 Bone marrow infiltration—by NHL, Hodgkin’s disease, granulomas due to M.
tuberculosis and atypical mycobacteria or disseminated fungal disease.
The Full Blood Count Using Mindray BC 5380:
The MINDRAY Bc5380 provides a precise and comprehensive FBC. It provides
measurements for haemoglobin, red cell count, red cell indices (size and volume),
platelet count and Leucocyte count with full differential (neutrophils, lymphocytes,
monocytes, eosinophils and basophils). A Nucleated red cell count is under evaluation
and will be added to the repertoire shortly. Additionally, some calculated parameters
are provided on the FBC report which are often used to assess iron and thalassaemia
status

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Reagents
 M-53D Diluent
It provides a stable environment for counting and sizing blood cells.
 M-53LEO (I) Lyse
It breaks down red blood cell walls and cooperates with the M-53LEO (II) lyse to 4-
differentiate WBCs.
 M-53LEO (II) Lyse
It cooperates with the M-53LEO (I) lyse to 4-differentiate WBCs, and dyes Eosinophils.
 M-53LH Lyse
It breaks down red blood cell walls and converts hemoglobin to a hemoglobin complex
to determine the HGB. It 2-differentiates WBCs to Basophils and other WBCs, and
determines WBC amount.
 M-53 Cleanser
It is a cleaning solution formulated to prime and cleans the fluidic channels and tubing
and gets rid of blood albumin and sediment.
 M-53P Probe Cleanser
It is used to clean the analyzer regularly.
PROCEDURE:
1. Entering of work manifest into the programming computer.
2. Arrangement and sorting of the sample according to the manifest and loading
them into the analysis racks of the mindray autoanalyser.
3. Begin or start run and recording of results.
RANGE OF RESULTS
Parameter Male Female
Haemoglobin g/L 135 - 180 115 - 160
WBC x109
/L 4.00 - 11.00 4.00 - 11.00
Platelets x109
/L 150 - 400 150 - 400
MCV fL 78 - 100 78 - 100
PCV 0.40 - 0.52 0.37 - 0.47
RBC x1012
/L 4.5 - 6.5 3.8 - 5.8
MCH pg 27.0 - 32.0 27.0 - 32.0
MCHC g/L 310 - 370 310 - 370
RDW 11.5 - 15.0 11.5 - 15.0
Neutrophils 2.0 - 7.5 2.0 - 7.5
Lymphocytes 1.0 - 4.5 1.0 - 4.5
Monocytes 0.2 - 0.8 0.2 - 0.8
Eosinophils 0.04 - 0.40 0.04 - 0.40
Basophils < 0.1 < 0.1

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MALARIA RAPID TEST
Procedure to malaria rapid testing;
1. Open the alcohol swab. Grasp the 4th finger on the patient’s left hand.
Clean the finger with the alcohol swab. Allow the finger to dry before
pricking
2. Open the lancet. Prick patient’s finger to get a drop of blood.
3. Discard the lancet in the Sharps Box immediately after pricking finger. Do
not set the lancet down before discarding it.
4. Use the capillary tube to collect the drop of blood.
5. Use the capillary tube to put the drop of blood into the square hole
marked “A.”
6. Discard the capillary tube in the Sharps Box.
7. Add buffer into the round hole marked “B.”
8. Wait 15 minutes after adding buffer after which you read the result.
ERYTHROCYTE SEDIMENTATION RATE (ESR):
The erythrocyte sedimentation rate (ESR) is a common hematological test for
nonspecific detection of inflammation that may be caused by infection, some cancers
and certain autoimmune diseases. It can be defined as the rate at which Red Blood Cells
(RBCs) sediment in a period of one hour.
PRINCIPLE OF ESR
When anticoagulated blood is allowed to stand in a narrow vertical glass tube,
undisturbed for a period of time, the RBCs – under the influence of gravity- settle out
from the plasma. The rate at which they settle is measured as the number of millimeters
of clear plasma present at the top of the column after one hour(mm/hr). This
mechanism involves three stages:

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Stage of aggregation: It is the initial stage in which piling up of RBCs takes place. The
phenomenon is known as Rouleaux formation. It occurs in the first 10-15 minutes.
Stage of sedimentation: It is the stage of actual falling of RBCs in which sedimentation
occurs at constant rate. This occurs in 30-40 minutes out of 1 hour, depending upon the
length of the tube used.
Stage of packing: This is the final stage and is also known as stationary phase. In this,
there is a slower rate of falling during which packing of sedimented RBCs in column
occurs due to overcrowding. It occurs in final 10 minutes in 1 hour.
METHOS OF ESR DETERMINATION
There are two main methods to determine ESR :
Wintrobe’s method
Westergren’s method
Each method produces slightly different results. Mosely and Bull (1991) concluded that
Wintrobe’s method is more sensitive when the ESR is low, whereas, when the ESR is
high, the Westergren’s method is preferably an indication of patient’s clinical state.
WESTERGREN’S METHOD
It is better method than Wintrobe’s method. The reading obtain is magnified as the
column is lengtheir. The Westregren tube is open at both ends. It is 30 cm in
length and 2.5 mm in diameter. The lower 20 cm are marked with 0 at the top and 200
at the buttom. It contains about 2 ml of blood.
REQUIREMENTS:
Anticoagulated blood, Westergren tube, Westergren stand, Rubber bulb (sucker)
PROCEDURE:
a) Mix the anticoagulated blood thoroughly.
b) Draw the blood into the tube upto 0 mark with the help of rubber bulb.
c) Wipe out blood from bottom of the tube with cotton.
d) Set the tube upright in stand. Make sure the pipette fits snugly to eliminate
possible leakage and that the pipette is in vertical position.
e) Leave the tube undisturbed for 1 hour.
f) At the end of 1 hour, read the result.

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NORMAL VALUE:
For males : 0-10mm/hr
For females : 0-15 mm/hr
Clinical Significance of ESR
The erythrocyte sedimentation rate (ESR) is a non-specific test. It is raised in a wide
range of infectious, inflammatory, degenerative, and malignant conditions associated
with changes in plasma proteins, particularly increases in fibrinogen, immunoglobulins,
and C-reactive protein. The ESR is also affected by many other factors
including anaemia, pregnancy, haemoglobinopathies, haemoconcentration and
treatment with anti-inflammatory drugs.
Causes of a significantly raised ESR :
 All types of anemias except sickle cell anemia
 Acute and chronic inflammatory conditions and infections including: HIV disease,
Tuberculosis, African trypanosomiasis (rises rapidly)
 Drugs, including oral contraceptives
Causes of Reduced ESR:
 Polycythaemia
 Newborn infants
 Dehydration

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CHAPTER 8
MOLECULAR BIOLOGY UNIT
Molecular biology is a branch of biology that deals with molecular basis of
biological activity. This field overlaps with other areas of biology and chemistry,
particularly genetics and biochemistry. Molecular biology briefly concern itself with
understanding the Interaction between the system of a cell, including the interacting
between the different type of DNA, RNA and protein biosynthesis as oven as learning
how these interaction are regulated.
Polymerase Chain Reaction (PCR):
The polymerase chain reaction (PCR) is a laboratory technique for DNA replication that
allows a “target” DNA sequence to be selectively amplified. PCR can use the smallest
sample of the DNA to be cloned and amplify it to millions of copies in just a few hours.
Discovered in 1985 by Kerry Mullis, PCR has become both and essential and routine tool
in most biological laboratories.
Principle of PCR
The PCR involves the primer mediated enzymatic amplification of DNA. PCR is based on
using the ability of DNA polymerase to synthesize new strand of DNA complementary to
the offered template strand. Primer is needed because DNA polymerase can add a
nucleotide only onto a preexisting 3′-OH group to add the first nucleotide. DNA
polymerase then elongates its 3 end by adding more nucleotides to generate an
extended region of double stranded DNA.
Components of PCR
The PCR reaction requires the following components:
DNA Template: The double stranded DNA (dsDNA) of interest, separated from the
sample.
DNA Polymerase: Usually a thermostable Taq polymerase that does not rapidly
denature at high temperatures (98°), and can function at a temperature optimum of
about 70°C.
Oligonucleotide primers: Short pieces of single stranded DNA (often 20-30 base pairs)
which are complementary to the 3’ ends of the sense and anti-sense strands of the
target sequence.
Deoxynucleotide triphosphates: Single units of the bases A, T, G, and C (dATP, dTTP,
dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA
synthesis.
Buffer system: Includes magnesium and potassium to provide the optimal conditions for
DNA denaturation and renaturation; also important for polymerase activity, stability and
fidelity.
Procedure of PCR
All the PCR components are mixed together and are taken through series of 3 major
cyclic reactions conducted in an automated, self-contained thermocycler machine.
Denaturation:
This step involves heating the reaction mixture to 94°C for 15-30 seconds. During this,

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the double stranded DNA is denatured to single strands due to breakage in weak
hydrogen bonds.
Annealing:
The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds. This allows
the primers to bind (anneal) to their complementary sequence in the template DNA.
Elongation:
Also known at extension, this step usually occurs at 72-80°C (most commonly 72°C). In
this step, the polymerase enzyme sequentially adds bases to the 3′ each primer,
extending the DNA sequence in the 5′ to 3′ direction. Under optimal conditions, DNA
polymerase will add about 1,000 bp/minute.
Types of PCR
In addition to the amplification of a target DNA sequence by the typical PCR procedures
already described, several specialised types of PCR have been developed for specific
applications.
 Real-time PCR
 Quantitative real time PCR (Q-RT PCR)
 Reverse Transcriptase PCR (RT-PCR)
 Single-cell PCR
 Touch down PCR, etc

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Medical Applications of PCR:
 Genetic testing for presence of genetic disease mutations. Eg:
hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism
 Detection of disease causing genes in suspected parents who act as carriers.
 Study of alteration to oncogenes may help in customization of therapy
 Can also be used as part of a sensitive test for tissue typing, vital to organ
transplantation
 Genotyping of embryo
 Helps to monitor the gene in gene therapy
Infectious disease Applications:
a) Analyzing clinical specimens for the presence of infectious agents, including HIV,
hepatitis, malaria, tuberulosis etc.
b) Detection of new virulent subtypes of organism that is responsible for epidemics.
In APIN-JUTH, 3 assays are carried out in the molecular biological units:
 HIV viral load
 HEPATITIS viral load
 EID (EARLY INFACT DIAGNOSIS)
HIV/HEPATITIS VIRAL LOAD ASSAY
The HIV Viral load is the number of copies of the human immunodeficiency virus
in your blood and other parts of your body. The HIV Viral load test helps monitor your
disease, guide HIV therapy, and predict how your disease may progress. Keeping your
viral load low can reduce complications of HIV disease and extend your life.
There are several methods of measuring HIV Viral load: PCR (Polymerase Chain
Reaction), bDNA(branched-chain DNA),NASBA(nucleic acid sequence based
amplification).
In APIN-JUTH, PCR method is used, it involves using an enzyme to multiply the HIV
RNA in the blood sample. (RNA is the part of HIV that knows how to make copies of
virus).this makes it easier to measure the amount of HIV RNA in the blood sample. Using
COBAS® Ampliprep/ COBAS Taqman®HIV-1 Test Analyzer

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Principle
The COBAS® Ampliprep/COBAS® Taqman® Hiv-1 test v2.0 is a nucleic acid
quantification of Hiv-1 RNA in human plasma. The procedure is based on three major
processes;
 Specimen preparation: to isolate Hiv-1 RNA.
 Reverse transcription of target RNA to generate complementary DNA (cDNA).
 Simultaneous PCR amplification and of target cDNA and detection of cleaved dual-
labeled oligonucleotide probes specific to the target.
Procedure
 Perform start up procedures such as inspection, lo on and daily maintenance
 Remove reagents from storage and load immediately (insert A for cassette 1 and
insert B-E for cassette 2-4 respectively)
 Load the corresponding consumables
 Remove sample from storage and prepare worklist
 Attach barcode clips to sample rack and arrange sample tube
 Create order and print worklist
 Vortex controls and samples for 3 seconds
 Pipette controls and samples into the tubes respectively (1100µl)
 Check instrument status and start run
 Remove prepared samples and load the instrument
 When run is completed, review and accept results
 Remove instrument waste
Detection
Detection of PCR product in a COBAS® TaqMan® Test utilizes real-time PCR
technology. The use of dual-labeled fluorescent probes allows for real-time detection of
PCR product accumulation by monitoring of the emission intensity of fluorescent
reporter dyes released during the amplification process. The amplification of HIV-1
target and HIV-1 IC are measured independently at different wavelengths. This process
is repeated for a designated number of cycles each cycle effectively increasing the
emission intensity of the individual reporter dyes, permitting independent identification
of HIV-1 target and HIV-1 IC.
HIV Viral Load Results/ Interpretation
 All HIV-1 OD values <0.20 are not calculated and they are reported as HIV-RNA not
detected (<400copies/ml)
 All HIV-1 OD values >0.20 are calculated but because the copy number is above the
linear range of the assay, it is reported as HIV-1 RNA Not determined
 All HIV-1 OD values =0.20 are calculated and they are reported as HIV-1 RNA
Detected (>400copies/ml).
EARLY INFANT HIV-1 DIAGNOSIS (EID) 18MONTHS
Aim: Qualitative in-vitro test for the detection of HIV-1 DNA in human whole blood for
early infant diagnosis.

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Qualitative Dried Blood Spot (DBS) DNA Polymerase Chain Reaction Assay Using Roche
Amplicor® HIV-1 DNA Test
Procedure for EID (DBS)
 Collect dried blood spot sample (DBS) using appropriate clinical procedure
 Excise punches from Whatmam 903 filter by manual cutting, excise 12mm circular
punch that include the optically most dense dried blood spot area, while cutting follow
the circular path of the paper
 Transfer the excised blood spot into input S-tubes of 22
 Add 1100µl of SPEX (buffer) into each tube containing the excised blood spot
 Vortex 1 vial of the CTM (-) C thoroughly seconds and transfer 100µl into S-tubes and
place in position 1
 Vortex 1 vial HIV- IL (+) C for 20seconds, transfer 1000µl to S-tubes and place at position
2
 Incubate the 24 input S-tubes containing the sample & control in an Eppendorf
Thermomixer at 560
C and 1000rpm for 10 minutes
 Attach a bar code label chip to each 5k 24 rack position while a sample input 5-tubes is
to be placed, attach specific barcode for the control also.
 After incubation transfer the input S-tubes from thermomixer 150 rack to the 5k 24
rack, and immediately insert it into COBAS® Ampliprep / COBAS Taqman®
Early Infant Diagnosis Results/ Interpretation
HIV-1 RESULT IC RESULT INTERPRETATION
<0.2 >0.2 HIV-1 DNA not detected. The sample is presumptive negative for HIV-1
DNA.
<0.2 <0.2 Inhibitory sample; The HIV-1 DNA if present would not be detectable. A new
sample must be collected and processed.
>0.8 Any HIV-1 DNA detected. Sample is positive for the presence of HIV-1 DNA.
>0.2, <0.8 ANY Equivocal, results are indeterminate/inconclusive

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EXPERIENCE GAINED
 I learnt a lot of the practical aspects involved in medical biochemistry and also
biochemistry in general.
 I got to know about and learnt the use of the laboratory equipment.
 I learnt to obey all laboratory rules for my safety and that of the patients.
 I learnt to relate properly with other co-workers and supervisors.
PROBLEMS ENCOUNTERED
 In most cases, safety rules are not taken into consideration and the necessary
safety gadgets and equipment even if in place, not every student knows what to
use for the required emergency.
 It is suggested that some form of allowance should be given to the students by the
employers as a form of encouragement and to assist in their cost of living, basically
feeding, transportation and accommodation especially in areas far from the
students’ neighborhood.
RECOMMENDATIONS
 I propose that more time should be given to the students of BIOCHEMISTRY after
graduation to gain more laboratory knowledge through INTERNSHIP.
 I recommend that government should provide placements for students
undergoing SIWES in the several fields of Nigerian Economy.
 I recommend that more preference should be given to the power sector so as to
provide adequate light to various Medical laboratories in the country.

PLSU/2013/FNS/0025
REFERENCES
 http://laboratoryinfo.com/polymerase-chain-reaction-pcr/
 http://laboratoryinfo.com/common-blood-collection-tubes-their-additives-and-
laboratory-uses/
 Encyclopedia Britannica student and home edition/fbc/clinical_chemistry
 http://www.thl.fi/publications/ehrm/product2/part_iii5.htm
 Textbook of Medical Biochemistry Eighth Edition/Biochemistry of AIDS
 Laboratory Guidelines for enumerating CD4 T Lymphocytes in the context of
HIV/AIDS
 King Saud University Collage of Applied Medical Sciences Department of Clinical
Laboratory Sciences/Practical Biochemistry CLS 432
 Oxford Handbook of Clinical Haematology, Second edition
 Coast Provincial General Hospital Antiretroviral Therapy (Art) Programme/
Standard Operating Procedures For Laboratory Services
 https://www.cdc.gov/malaria/index.html
 https://www.aids.gov/hiv-aids-basics/just-diagnosed-with-hiv-aids/understand-
your-test-results/viral-load/index.html

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