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DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCES BAYERO UNIVERSITY, KANO STUDENTS INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES) REPORT BY MAHMUD ABDULLAHI GINYA SCI/08/BCH/000313 A PRE-REQUISITE FOR THE AWARD OF A GRADE IN SIWES SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF BACHELOR OF SCIENCE IN BIOCHEMISTRY SUPERVISED BY
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Department of Biochemistry

Oct 26, 2014

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Page 1: Department of Biochemistry

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF SCIENCES

BAYERO UNIVERSITY, KANO

STUDENTS INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

REPORT

BY

MAHMUD ABDULLAHI GINYA

SCI/08/BCH/000313

A PRE-REQUISITE FOR THE AWARD OF A GRADE IN SIWES SUBMITTED IN

PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

BACHELOR OF SCIENCE IN BIOCHEMISTRY

SUPERVISED BY

MALL. A.A MUHKTAR

Page 2: Department of Biochemistry

CERTIFICATION

I hereby certify that the Students’ Industrial Work Experience Scheme (SIWES) report

submitted to the Department of Biochemistry, Bayero University Kano, as a requirement for

the award of degree of Bachelor of Science in Biochemistry, has been composed by myself

and has not been presented or accepted in any previous application for a degree. The work, of

which this is a record, has been carried out by myself unless otherwise stated and where the

work is mine, it reflects personal views and values. All sources of information have been

acknowledged by means of references including those of the Internet.

…………………………………… ………………………………….

Mahmud Abdullahi Ginya Signature/Date

…………………………………… …………………………………..

Mall. A.A Muhktar Signature/Date

…………………………………… …………………………………..

Mall. Nasir Abdullahi Signature/Date

SIWES Coordinator

Page 3: Department of Biochemistry

DEDICATION

Page 4: Department of Biochemistry

ACKNOWLEDGEMENTS

Page 5: Department of Biochemistry

TABLE OF CONTENTS

TITLE PAGE…………………………………………………………………………………i

CERTIFICATION....................................................................................................................ii

DEDICATION.........................................................................................................................iii

ACKNOWLEDGEMENT........................................................................................................iv

LIST OF FIGURES...................................................................................................................v

GLOSSARY.............................................................................................................................vi

PREAMBLE

The Industrial Training Fund (ITF)...........................................................................................

Functions of the ITF ...........................................................................................................

The Students Industrial Work Experience Scheme (SIWES)...................................................

Objective of SIWES..................................................................................................................

Importance of SIWES................................................................................................................

CHAPTER ONE

Place of Industrial Training........................................................................................................

Brief History of the Organization..............................................................................................

CHAPTER TWO

EQUIPMENT, REAGENTS AND PREPARATION.........................................................

1.1 Equipments........................................................................................................

1.1.1 Ethylene Diamine Tetracetic Acid (EDTA) Bottles.........................................

1.1.2 Genotype Electrophoresis Machine...................................................................

1.1.3 Reflotron Machine.............................................................................................

1.1.4 Accu-check……………………………………………………………………

1.1.5 Centrifuge Machine……………………………………………………………

1.1.6 Autoclave……………………………………………………………………...

1.1.7 Incubator………………………………………………………………………

1.1.8 Thermostatic Drying Oven……………………………………………………

1.1.9 FACS Count Machine (CD4 Machine)……………………………………….

1.2 Reagents and their Preparation………………………………………………..

Page 6: Department of Biochemistry

CHAPTER THREE

METHODS/ANALYSIS OF ROUTINE WORK DONE IN THE VARIOUS SECTION

OF THE LABORATORY

2.1 Clinical Chemistry Section…………………………………………………...

2.2 Haematology Section………………………………………………………...

2.3 Medical Microbiology Section……………………………………………….

2.4 Immunology Section…………………………………………………………

Page 7: Department of Biochemistry

PREAMBLE

THE INDUSTRIAL TRAINING FUND (ITF)

The Industrial Training Fund was established by Decree No. 47 of 8th October, 1971, with

the aim of “promoting and encouraging the acquisition of skills in industry and commerce

with a view to generating a pool of indigenous manpower sufficient to meet the needs of the

economy”(ITF, 2010). This was the first of the three Manpower Training and Development

Agencies created by the Fedhjjeral Military Government during the Second National

Development Plan period (1970 – 1974).

After the establishment of agency, ITF rose to its challenges by organizing programs that

could among other things, expose students of Nigerian Universities and Polytechnics to

practical experience in their course of study and provide a means through which students

could meet as well as interact with industrial personnel.

Based on the foregoing, the student work experience programme which basically prepares

students for the world of work has become an innovative phenomenon in the process of

manpower development and training in Nigeria, hence, the introduction of the Students

Industrial Work Experience Scheme (SIWES) into the educational training system

FUNCTIONS OF ITF

Key functions of ITF include:

Encouraging greater involvement of employers, particularly small employers, in the

organization and development of training programmes and facilities including the

establishment of Group Training Schemes and Centres in certain areas of economic

activity;

Building of training facilities of its own, in identified areas of national need;

Organizing research and studies into training as a support to other activities of the Fund;

Establishing uniform Nation al Vocational Apprenticeship Training Scheme in the

country;

Page 8: Department of Biochemistry

Seeking to harmonize ITF’s non-formal training programmes with the curricula of

formal educational institutions;

Bearing a proportion of the direct cost of on-the-job and off-the-job training of Nigerian

employees.

(ITF, 2010)

STUDENTS INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

The Students Industrial Work Experience Scheme (SIWES) is a Skill Training Programme

designed to prepare and expose students of Nigerian University to the industrial work

situation they are likely to encounter after graduation. The need for the establishment of the

scheme aroused when there was a growing concern among industrialists that graduates of

institutions of higher learning lacked adequate practical background required for employment

in industries. Therefore, the employers were of the opinion that the theoretical education in

higher institutions was not responsive to the needs of employers of labour.

As mentioned above, SIWES programme was designed to complement classroom teaching in

the course of studies and to acquaint students with the skills needed in the industries after

graduation. It is an effort to bridge the gap existing between theory and practice of

engineering and technology, science, Agriculture, Medical, Management and other

professional educational programs in Nigerian institutions. The scheme is funded by the

Federal Government of Nigeria and jointly coordinated by the National Universities

Commission (NUC) and Industrial Training Fund (ITF).

OBJECTIVE OF SIWES

To provide students with an opportunity to apply their knowledge in real work situation,

thereby bridging the gap between theory and practice.

 To expose students to work methods and techniques in handling equipment and

machinery that may not be available in their institutions.

To make transition from university to the world of work easier and thus enhance students

contact for later job placement after graduation.

Page 9: Department of Biochemistry

To enlist and strengthen employers’ involvement in the entire educational process of

preparing university graduates for employment in industries.

To prepare students for the work situation they are to meet after graduation (ITF, 2010)

IMPORTANCE OF SIWES

SIWES have a lot of importance among which are:

It exposes students to real life situation, thus supplementing the theoretical lesson.

It helps to improve the quality of skilled manpower of the students.

It gives students practical knowledge of course of study.

It provides a forum for industries to evaluate prospective employers and gives feedback

to institutions.

It establishes a close collaboration between institutions and industries, a factor which is

essential for preparing student for the workforce.

Page 10: Department of Biochemistry

CHAPTER ONE

THE HISTORICAL BACKGROUND OF UMARU SANDA NDAYAKO

HOSPITAL BIDA

Umaru Sanda Ndayako General Hospital Bida was established and commission by

His Excellency former head of the state and commander in chief of arm forces.

General Yakubu Gawon G.C.F.R. on the 28th day of March 2009.

The General Hospital also known as Umaru Sanda Ndayako is located along Minna

Road opposite Zamfara Mosques Bida, at the point of commission the hospital is well equipt

with hospital material and well qualified staff.

Page 11: Department of Biochemistry

CHAPTER TWO

EQUIPMENTS, REAGENTS AND THEIR PREPARATION

2.0 EQUIPMENTS

The Equipment used in the laboratory throughout the SIWES period is as follows:

2.1.1 Ethylene Diamine Tetracetic Acid (EDTA) Bottles

Ethylene Diamine Tetracetic Acid (EDTA) bottles with purple caps, Serum Separation Tube

(SST) with (Red Cap,) lithium heparin Tubes (Green Cap), Erythrocyte Sedimentation Rate

(ESR) Tube (Black cap) and Sterile Universal Container (red and White Caps) as shown in

the figure 1.1 below

Fig 2.1: Ethylene Diamine Tetracetic Acid Bottles

2.1.2 Genotype Electrophoresis Machine

Fig 2.2: Genotype electrophoresis Machine

Page 12: Department of Biochemistry

2.1.3 Reflotron Machine

Fig 2.3: Reflotron Machine

2.1.4 Accu-check

Fig 2.4: Accu – check

2.1.5 Centrifuge Machine

Fig 2.5: Centrifuge Machine

Page 13: Department of Biochemistry

2.1.6 Autoclave

Fig 2.6: Autoclave

2.1.7 Incubator

Fig 2.7: Incubator

Page 14: Department of Biochemistry

2.1.8 Thermostatic Drying Oven

Fig 2.8: Thermostatic Drying Oven

2.1.9 FACS Count Machine (CD4 Machine)

Fig 2.9: FACS Count Machine (CD4 Machine)

Page 15: Department of Biochemistry

Fig 2.10: Microscope

2.2 REAGENTS AND THEIR PREPARATION

Leishman's stain

Leishman's stain is used in microscopy for staining blood smears. It provides excellent stain

quality and is generally used to differentiate and identify leucocytes, malaria parasites, and

trypanosomas. It is based on a mixture of methylene blue and eosin.

Preparation

To make about 400ml:

Leishman stain powder……………………………………………………………………..0.6g

Methanol (methyl alchohol……………………………………………………………....400ml

Note: The methanol MUST be water free

1. Weigh the Leishman powder and transfer it to a completely dry brown bottle. Add a

few dry glass breads (to assist in dissolving the dye)

2. Using a dry cylinder, measure the methanol and add this to the stain. Mix well at

intervals until the dye is completely dissolved. Warming the solution in a 370C water

bath will help the dye to dissolve.

Caution: Methanol is toxic and highly flammable; therefore handle it with care well

away from an open flame.

3. Label the bottle and mark it Flammable and Toxic. Store it at room temperature in the

dark. When kept tightly stoppered, the stain is stable for several weeks. Moisture must

not be allowed to enter the stain.

Page 16: Department of Biochemistry

For use: Filter 50-100ml of the stain into a stain dispensing container which can be closed

when not in use.

Sodioum citrate anticoagulant, 32g/l

To make 100ml

Tri-Sodium citrate, dehydrate………………………………………………………………3.2g

Distilled water…………………………………………………………………………to 100ml

1. Weigh the chemical and transfer it to a container pre-marked to hold 100ml (or to a

100ml volumetric flask).

2. Add about half the water and mix until the chemical is completely dissolved.

3. Add water to the 100ml mark and mix well.

4. Label the container and store it at 2-80C. Discard the reagent if it becomes

contaminated (turbid appearance).

Reagent for Genotype Test

TRIS Boric Acid Buffer Salts: TBE or Tris/Borate/EDTA, is a buffer solution containing a

mixture of Tris base, boric acid and EDTA. In molecular biology, TBE and TAE buffers are

often used in procedures involving nucleic acids, the most common being electrophoresis.

Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA de-

protonated and soluble in water. EDTA is a mixture of divalent cations, particularly of

magnesium (Mg2+). Because these ions are necessary co-factors for many enzymes, including

contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic

degradation (Wikipedia.org)

Preparation

The already prepared salt was dissolved in 500ml of distilled water

Normal saline

Preparation

Nacl salt was dissolved in 500ml of distilled water

Reagent for Blood Group Test

Page 17: Department of Biochemistry

Anti sera

The reagents listed above have been already prepared by the manufacturer. They are stable

and valid when used before the expiry date and stored under their stated temperatures.

NUTRIENT AGAR

Nutrient agar is a microbiological growth medium commonly used for the routine cultivation

of non-fastidious bacteria. It is useful because it remains solid even at relatively high

temperatures. Also, bacteria grown in nutrient agar grows on the surface, and is clearly

visible as small colonies. . It is used for the sensitivity of bacterial colony. The medium is

best prepared from ready to use dehydrated powder, available from most suppliers of culture

mediea. The various component of nutrient agar solution is shown in the table below.

Table 1.1: Nutrient Agar Compositions

INGREDIENTS gm/ltr

Agar 15.00

Peptone 5.00

Sodium chloride 5.00

Beef extract 1.50

Yeast extract 1.50

pH 7.4

Distilled water 1ltr

Preparation

28.0gm was dissolved in 1000ml distilled water. It was heat gentle to dissolve the medium

completely. Sterilized by autoclaving at 15 psi (1210C) for 15 minutes and was cool to 47C,

and mixed well before pouring plates.

Cystine Lactose Electrolyte Deficient Medium (CLED)

CLED Agar is a valuable non-inhibitory growth medium used in the isolation and

differentiation of urinary organisms. Because CLED is electrolyte deficient, it prevents the

swarming of Proteus species. Cystine promotes the formation of cystine-dependent dwarf

Page 18: Department of Biochemistry

colonies. Lactose fermenters produce yellow colonies on CLED agar; non-lactose fermenters

appear blue. It has a pH of approximately 7.3 (Wikipedia.org). CLED agar contains:

Table 1.3: CLED Agar Composition

Content Concentration

Bromo thymol blue 20mg/l

Beef extract 3g/l

L-cystine 128mg/l

Peptone 4g/lAgar 15g/l

Lactose 10g/l

Hydrolysed Cassin 4g/l

Tryptone 4.0g/l

pH 7.3

Preparation

36 grams of powder was weighed and added to 1 litre of cold de-ionized water and

autoclaved at 121C for 15 minutes. Cool to 470C and was mixed thoroughly before pouring

plates. Stored at 2-80C, preferably sealed in plastic bags to prevent loss of moisture.

Salmonella Shigella (S.S Agar)

Table 1.4: Salmonella Shigella Agar Composition

Content Concentration gm/ltr

Beef extract 5.00

Animal tissue 5.00

Sodium citrate 10.00

Sodium thiosulphate 8.50

Agar 15.00

Lactose 10.00

Ferric citrate 1.00

Brilliant green 0.00033

Neutral red 0.025

pH 7.00

Bile salts mixture 8.50

Preparation

Page 19: Department of Biochemistry

63.0 grams of powder was weighed and added to one (1) litre of distilled water and allowed

to soak for 10 minutes, it was swirled to mix and then heat to the boiling with agitation to

dissolve the medium completely. It was allowed to cool to 47C and then mixed thoroughly

and poured into sterile petri plates. No autoclaving was needed for autoclaving may destroy

the selective of the medium.

MacConckey agar

The medium is best prepared from ready to use dehydrated powder, available from most

suppliers of culture media. The medium is usually used at a concentration of 5.2g in every

100ml distilled water. The various component of nutrient agar solution is shown in the table

below:

Table 1.5: MacConckey Agar Composition

Content Concentration (g/l)

Peptone 20

Lactose 10

Bile salts 5

Sodium chloride 5Neutral red 0.075

Agar 12

Ph 7.4

Preparation

52g of powder was suspended in 1 litre of distilled water. It was bring boil to dissolve

completely. Sterilized by autoclaving at 1210C for 15 minutes. Poured into sterile petri plates

and then store in a cool dark place.

Table 1.5: Reagents for Glucose

Content Concentration

Peroxidise (horse raddish) 3.2 U

3,3’ ,5,5’-tetramethylbenzidine (indicator) 72.6µg

Glucose oxidase ≥3.2 U

Preparation

Page 20: Department of Biochemistry

The reagent was already automated. Stored at 2 to 30C, this can be used up to the expiry date

printed on the pack of the container.

Table 1.6: Reagents for Glutamate Oxaloacetate (GOT), (aspartate aminotransferase)

Content Concentration

Pyruvate peroxidise 1.5 U

Peroxidise 18 U

Alanine sulfinate 792µg

Thiamine pyrophosphate 1.49µg

α ketoglutarate 16.2µg

imidazole dihidrochloride 16.4µg

Buffer

Preparation

The reagent was already prepared by the manufacturer. Stored at 2 to 30°C, this is valid and

stable up to the expiry date given by the manufacturer.

Table 1.7: Reagents for Glutamate Pyruvate Transaminase (GPT), (Alanine amino transferase)

Content Concentration

Pyruvate peroxidise 1.5 U

Peroxidise 18 U

Alanine 230µg

Thiamine pyrophosphate 1.49µg

α ketoglutarate 16.2µg

imidazole dihidrochloride 16.4µg

Buffer

Preparation

The content is valid when stored at 2 to 30°C and is used before the expiry date.

Table 1.11: Reagents for UREA

Content Concentration

Page 21: Department of Biochemistry

Urease (jack beans) ≥4.7U

Tetrachlorophenoltetrabromosulfophthalein (indicator)

20.4µg

Buffer

Preparation

The reagents were already prepared by the manufacturer and normally stored at 2 to 30.

Under this condition, the tests can be used up to the expiry date printed on the pack or

container.

Table 1.12: Reagents for Creatinine

Content CompositionCreatinine iminohydrolase ≥ 1.38U

N-methylhydantoinase ≥ 0.108U

Carbamoyl Sarcosine hydrolyse ≥ 0.439U

Sarcosine oxidase ≥ 0.475U

Peroxidase ≥ 2.29U

Ascorbate oxidase ≥ 0.285U

ATP 82.8µg

Hydrochloride (indicator) 19.5µg

Buffer

Preparation

The reagents were already prepared by manufacturer. The test can be used up to expiry date

printed on the pack or container when stored at 2 to 30°C.

Page 22: Department of Biochemistry

Table 1.13: Reagents for Billurubin

Content Concentration

Dyphylline 0.84mg

2-methoxy-4-niyrophenyldiazoniumtetrafluoroborate

10.4µg

Preparation

The reagents were already prepared by manufacturer. The test can be used up to expiry date

printed on the pack or container when stored at 2 to 30°C.

Table 1.14: Reagents for Alkaline Phosphatase

Content ConcentrationO-cresolphthalein phosphate ≥ 394µgN-methyl-D-glucamine ≥ 1005µg

Magnesium ≥ 0.56µg

Preparation

The reagents were already prepared by manufacturer. The test can be used up to expiry date

printed on the pack or container when stored at 2 to 30°C.

Table 1.15: Reagents for Sodium ions

Content ConcentrationUranyl acetate 2.1MmMagnesium acetate 20Mm

Sodium standard 150mEq/L

Preparation

The reagents were already prepared by manufacturer. Reagent set were all stored at room

temperature (15-300C). The reagents are stable until the expiration date indicated on the label.

Page 23: Department of Biochemistry

Table 1.15: Reagents for Bicarbonate ions

When reconstituted according to the direction, CO2 reagent contains the concentrations on the

table below:

Content ConcentrationPEP 1.8MmMagnesium Sulfate 10mM

NADH 0.40mM

MDH (porcine) 1,250U/L

PEPC (microbial) 200U/L

Sodium Oxamate 2.5mM

Buffer (pH) 7.0

Non-reactive fillers and Stabilizers 0.1%

Preparation

Reconstitute CO2 reagent with the 6ml of CO2-free water. Mix by inversion 5-6 times. Do not

shake. Reconstituted reagents stored in the closed tightly stoppered bottle are stable for 1 day

at room temperature (18-260C) and 7 days refrigerated (2-80C).

Page 24: Department of Biochemistry

CHAPTER THREE

METHODS/ANALYSIS OF ROUTINE WORK DONE IN THE

VARIOUS SECTION OF THE LABORATORY

USGH laboratory is divided into five (5) main units, these are:

Haematology unit

Medical microbiology/parasitology unit

Chemical pathology unit

Immunology unit

Transfusion unit

3.0 CHEMICAL PATHOLOGY UNIT

This is a unit of the laboratory that deals with the metabolism of the constituents of the blood.

Activities carried out in this unit include:

Cleaning work surfaces

Performing daily cleaning of the Reflotron machine

Carrying out urinalysis using the combi 9

Spinning and separating serum for use in the centrifuge machine

Carrying out blood sugar test using the accu-chek

Carrying out liver profile test using the Reflotron machine or working reagents

Carrying out electrolyte test using the Reflotron for potassium and creatinine or working

reagents for all electrolytes

Registering all tests carried out in their respective ledgers

3.1 COLLECTION OF BLOOD SAMPLES AT PHLEBOTOMY

Factors regarding the collection of blood specimens that can affect the correctness of clinical

chemistry test results include:

- Incorrect venepuncture technique

- Haemolysis of red cells,

- Collection into the wrong container

- Instability of some chemical substances in whole blood, serum, and plasma

Page 25: Department of Biochemistry

VENEPUNCTURE TECHNIQUE

The following precautions need to be followed when collecting venous blood in chemical

pathology:

* Do not apply the tourniquet too tightly or for too long a period because this will cause

venous stasis leading to a concentration of substance in the blood such as

haemoglobin, plasma proteins, potassium and calcium.

* Do not collect the blood from an arm into which an intravenous (IV) infusion is being

given.

* If an anticoagulated specimen is required, add the correct amount of blood with the

anticoagulant by gently inverting the container several times.

* Follow a safe technique and wear protective gloves.

AVODING HAEMOLYSIS

The haemolysis (rupture) of red cells can be a serious source of unreliable test results. If red

cells are haemolysed, substances from the cells are release into the serum or plasma leading

to a false increase in the concentration of analytes, e.g. potassium. Haemolysis also interferes

with many chemical reactions.

Haemolysis can be avoided by:

* Checking that the syringe and needle are dry and that the barrel and plunger of the

syringe fit well.

* Not using needle with too fine a bore.

* Not withdrawing the blood too rapidly or moving the needle once it is in the vein.

Frothing of the blood must be avoided.

* Removing the needle from the syringe before dispensing the blood into the specimen

container. Allow the blood to run gently down the inside wall of the container.

* Adding the correct amount of blood to anticoagulant. Do not shake the blood but

gentle mix it with the anticoagulant.

* Using clean dry tubes bottles for the collection of blood from which serum is required

and by allowing sufficient time for the blood to clot and clot retraction to take place.

Red cells are very easily haemolysed by rough use of an applicator stick to dislodge a

clot.

* Centrifuging blood samples for a minimum period of time. Centrifuging for 5 minutes

at about 1000g is adequate to obtain serum or plasma.

* Not storing whole blood samples in, or next to, the freezing compartment of a

refrigerator.

Page 26: Department of Biochemistry

SPECIMEN CONTAINERS AND ANTICOAGULANT

Specimen containers for clinical chemistry tests must be leak proof and chemically clean.

They should be well washed with detergent, rinsed in several changes of clean water before

being allowed to dry.

To avoid the confusion which often arises from the use of several different types of container,

the following system is recommended:

Non-urgent blood tests: Dispense about 5ml of blood into a dry tube or bottle and allow

clotting. When the clot has retracted, centrifuge the specimen and transfer the serum to a

labelled container. Tightly cap the container to avoid water evaporating from the specimen in

dry warm conditions or moisture entering in humid environments.

Urgent blood tests and paediatric blood tests: Dispense the correct amount of blood into a

tube containing lithium heparin anticoagulant. Gently mix the blood with the anticoagulant.

Centrifuge the specimen and transfer the plasma to a labelled container.

# Lithium heparin does not interfere with most chemical reactions and helps to minimize

haemolysis. It does not contain sodium or potassium. Anticoagulants such as EDTA and

fluride-oxalat contain sodium or potassium salts and therefore these cannot be used when

electrolytes are to be measured.

Blood glucose (non-urgent or urgent): Dispense the correct amount of blood into a tube

containing fluoride-oxalate. Gentle mix the blood with the anticoagulant. Centrifuge the

specimen to obtain plasma.

# Fluoride is an enzyme inhibitor. It prevents the breakdown of glucose to lactic by acid by

enzyme action (glycolysis). Blood collected into fluoride-oxalate can also be used for

measuring protein, urea and bilirubin but not for electrolytes or enzymes. Fluoride-oxalate

must not be used if measuring glucose using a reagent strip test.

STABILITY OF ANALYTES IN BLOOD SPECIMENS

Biochemical substances in blood are found in both plasma and cells but not necessarily in

equal concentrations:

More concentrated in plasma Potassium

Phosphate

Aspartate amino

Transferase (AST)

Lactate dehydrogenase

More concentration in plasma Sodium

Page 27: Department of Biochemistry

Chloride

Carbon dioxide

More concentration in plasma Creatinine

Glucose

Urea

Urate

Some of the chemical changes which may occur in blood specimens within a few hours of

being collected include:

* Diffusion of potassium and some enzymes through the red cell membrane into the

serum or plasma.

* Diffusion of carbon dioxide off the surface of the blood, leading to a lowering in the

concentration of bicarbonate with a compensatory increase in plasma chloride.

* Decrease in the concentration of glucose by glycolysis (when fluoride-oxalate is not

used).

* Reduction or losses in the activity of certain enzymes, for example acid phosphatise.

* Decomposition of bilirubin in daylight or fluorescent light.

Some of these changes can be prevented by separating the plasma or serum forms the red

cells as soon as possible (within 1 hour) after the blood has been collected. The blood should

not be refrigerated before the serum or plasma is separated because this can lead to falsely

raised potassium values. Glycolysis can be prevented by using fluoride-oxalate anticoagulant.

The decomposition of bilirubin can be avoided by protecting specimens from light.

To minimize any alteration in the concentration of substances due to the ingestion of food or

day time variation, most blood specimens are best collected at the beginning of the day before

a patient takes food.

3.1.1 ELECTROLYTES TEST

Electrolytes are substances that become ions in solution and acquire the capacity to conduct

electricity. The electrolytes that are measured include sodium, potassium, bicarbonate and

chloride using their various working reagents.

Sodium Test

Sodium is the major cation of extracellular fluid. It plays a central role in the maintenance of

the normal distribution of water and the osmotic pressure in the various fluid compartments.

Page 28: Department of Biochemistry

The main source of body sodium is sodium chloride contained in ingested foods. Only about

one-third of the total body’s sodium is contained in the skeleton since most of it is contained

in the extracellular body fluids.

Principle

Sodium is precipitated as the triple salt sodium magnesium uranyl acetate; the excess

uranium then reacts with ferrocyanide to form a coloured complex whose absorbance varies

inversely as the concentration in the test specimen.

Procedure

Filtrate preparation

Test tubes were labelled blank, standard and sample.

1.0ml of filtrate reagent was pipette to all tubes.

0.05ml of sample was added to all tubes and distilled water to the blank.

Tubes were vigorously shake and mixed continuously for 3 minutes.

All the tubes were centrifuged at high speed (1,500G) for 10 minutes after which their

supernatant fluids were tested as describe below:

Colour development

The test tubes were labelled corresponding to the above filtrate tubes.

1.0ml of acid reagent was added to all tubes.

0.05ml of each supernatant was added to their respective tubes and mixed.

0.05ml of colour reagent was added to all tubes and mixed.

Distilled was used to zero the spectrophotometer at 550nm.

Absorbencies of all tubes were red and recorded.

Calculations

Abs of Blank – Abs of Sample X Conc. of Std (mEq/L) = Conc. of SodiumAbs of Blank – Abs Standard

Reference Range

135 – 155mEq/L

Potassium Test

Page 29: Department of Biochemistry

Potassium is the principle cation of the intracellular fluid. It is also an important constituent

of the extracellular fluid due to its influence on muscle activity. Its intracellular function

parallels that of its extracellular function, namely influencing acid-base balance and osmotic

pressure, including water retention.

Elevated potassium levels (hyperkalemia) are often associated with renal failure, dehydration

shock or adrenal insufficiency. Decrease potassium levels (hypokalemia) are associated with

malnutrition, negative nitrogen balance, gastrointestinal fluid losses and hyperactivity of the

adrenal cortex.

Principle

The amount of potassium is determined by using sodium tetraphenylboron in a specifically

prepared mixture to produce a colloidal suspension. The turbidity of which is proportional to

potassium concentration.

Procedure

Test tubes were labelled blank, standard and sample.

1.0ml of potassium reagent was pipette to all tubes.

0.01ml of samples was added to sample, standard reagent to the standard and mixed then

was allowed to stand at room temperature for 3 minutes.

Reagent Blank was used to zero the spectrophotometer at 500nm.

Absorbencies of all tubes were red and recorded.

Calculations

Abs of Unknown X Conc. of Std (mEq/L) = Potassium Conc. (mEq/L)Abs of Standard

Reference Range

3.4 – 5.3mEq/L

Chloride Test

Chloride, a major anion, is important in the maintenance of the cation/anion balance between

intra and extra-cellular fluids. This electrolyte is therefore essential to the control of proper

hydration, osmotic pressure, and acid/base equilibrium. Low serum chloride values are fond

with extensive burns, excessive vomiting, intestinal obstruction, nephritis, metabolic acidosis,

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and in addisonian crisis. Elevated serum chloride value may be seen in dehydration,

hyperventilation, congestive heart valve, and prostatic or other types of urinary obstruction.

Principle

Hg(SCN)2 + 2Cl- HgCl2 + 2SCN-

3SCN- + Fe3+ Fe(SCN)3 (red complex)

Chloride ions form a soluble, non-ionized compound, with mercuric ions and will displace

thiocyanate ions from non-ionized mercuric thiocyanate. The released thiocyanate ions react

with ferric ions to form a colour complex that absorbs light at 480nm. The intensity of the

colour produced is directly proportional to the chloride concentration.

Procedure

Test tubes were labelled blank, standard and sample.

1.5ml of chloride reagent was pipette to each tube.

0.01ml of sample was added to sample tube, standard reagent to standard and distilled

water to blank and mixed.

Tubes were incubated at room temperature for at least 5 minutes

Spectrophotometer was set at 480nm and was zero with reagent blank.

Absorbencies of all tubes were red and recorded.

Calculations

Abs of Unknown X Conc. of Std (mEq/L) = Chloride Conc. (mEq/L)Abs of Standard

Reference Range

98 – 106mEq/L

3.1.2 UREA TEST

Urea is the major end product of protein nitrogen metabolism. It is synthesized in the liver

from ammonia, which is produced by amino acid determination. The determination of serum

urea renal function or increased tissue protein breakdown is associated with increased Urea

Nitrogen levels. Whereas liver damage or pregnancy are associated with decreased levels.

Principle

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urease

The Berthelot reaction has long been used for the measurement of urea and ammonia. The

present method is a modified Berthelot.

Urea + H2O 2NH3 + CO2

NH3 + Salicylate + Hypochlorite 2,2-Dicarboyindophenol

The BUN colorimetric procedure is a modification of the Berthelot reaction. Urea is

converted to ammonium by the use of urease. Ammonium ion then reacts with a mixture of

salicylate, sodium nitroprusside, and hypochlorite to yield a blue-green chromophore. The

intensity of the colour formed is proportional to the urea concentration in the sample.

Procedure

BUN Enzyme Reagent was reconstituted according to the instruction

1.5ml of BUN Enzyme Reagent was pipette into labelled test tubes. It was allowed to

equilibrate to room temperature.

0.01ml (10µ) of each sample was added to its respective tubes and then mixed gentle.

Deionized water was used as the sample for the Reagent Blank.

The tubes were incubated for five minute at 37oC or ten minutes at room temperature

(18-30oC).

1.5ml of BUN Colour Developer was added and then mixed gently.

Tubes were incubated for five minute at 37oC or ten minutes at room temperature.

Reagent Blank was used to zero the spectrophotometer at 630nm.

Absorbencies of all tubes were red and recorded.

Calculations

Abs of Unknown X Conc. of Std = Urea Nitrogen (mg/dL)Abs of Standard

Reference Range

7-23mg/dL

3.1.3 CREATININE TEST

Creatinine is a Nitrogenous waste product formed from the metabolism of creatinine in

skeletal muscle. Creatinine diffuses freely throughout the body water. It is filtered from the

extracellular fluid by the kidney and excreted in the urine. The excretion of creatinine is

mainly renal and its elevation is highly specific for kidney diseases.

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alkali

Principle

Creatinine + Sodium Picrate Creatinine + Picrate complex

Creatinine reacts with picric acid in alkaline conditions to form a colour complex, which

absorbs at 510nm. The rate of formation of colour is proportional to the creatinine

concentration in the sample. In the endpoint method the difference in absorbance

measurements after colour formation yields a creatinine value corrected for interfering

substances.

Procedure

Equal volumes of creatinine picric acid reagent and creatinine buffer reagent were

combined and mixed well.

Test tubes were labelled blank, standard and sample

3.0ml of working reagent was pipette into the tubes

0.1ml of sample was transferred to its respective tube, standard reagent to the standard

and distilled water to reagent blank and mixed.

Tubes were incubated at 37oC for 15 minutes.

Reagent Blank was used to zero the spectrophotometer at 510nm.

Absorbencies of all tubes were red and recorded.

Absorbencies of all tubes were red and recorded.

Calculations

The creatinine value of the unknown is determined by comparing its absorbance change with

that of a known standard.

Abs of Unknown X Conc. of Std = Conc. of CreatinineAbs of Standard

Reference Range

Male: 0.9-1.50mg/dl

Female: 0.7-1.37mg/kl

3.1.4 UREA AND CREATININE USING REFLOTRON MACHINE

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Urea samples were collected for the evaluation of renal function. The test is often used

together with the determination of Creatinine for differentiation between pre-renal and post-

renal azotaemia on the basis of the urea/Creatinine ratio. These are products of

osmoregulation and are analyzed using the Reflotron. The reference ranges for urea and

Creatinine is 10 – 50mg/dl and 0.5 – 1.1mg/dl respectively.

Test Principle

After application to test strip, the sample flows into the reaction zone, in the case of blood

after separation of the erythrocytes from the plasma. The urea contained in the sample is

hydrolyzed to ammonium carbonate from which ammonia is expelled due to the alkaline

buffering. This leads to the partial colour change of a buffered indicator to green /blue, the

intensity of which is proportional to the urea concentration of the sample:

(NH2)2 CO + H2O Urease 2NH3 + CO2

NH3 + indicator (yellow) NH4+ + indicator (blue)

At a temperature of 37OC, the depth of colour formed is measured at 642nm and the urea

concentration displayed after about 190 seconds in mg/dl depending on how the instrument

has been set.

Sample Collection

Blood sample was collected in the Lithium Heparin tubes or Ethylene Diamine tetra acetic

tube.

Testing Procedure

Additional materials required are Reflotron instrument; Reflotron pipette and pipette tips or

capillary pipette and controls. The procedure used is as follows:

The instrument (Reflotron) was switched on.

When the display shows “ready”, the test strip was removed from the container

The strip was unwrapped, taking care not to bend it.

Using the Reflotron pipette, sample material was drawn into the pipette (avoiding

bubbles) and applied a drop to the centre of the red application zone carefully in order

not touch the application zone with the pipette tip. (The required volume of specimen for

application was 30nl).

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With the sliding cover or flap opened, the test strip was placed on the guide within 15 sec

and slide forward horizontally until it locks into place, and the sliding cover/flap was

then closed.

The instrument displays urea/Creatinine to confirm that it has correctly read in the test-

specific magnetic code. The display shows the number of seconds left before the result

was displayed. The concentration was calculated automatically from the readings taken

using a function and conversion factors that were entered in the instrument via the

magnetic strip on the underside of each test strip. The concentration was displayed in

mg/dl or mmol/l depending on whether the instrument has been set to show conventional

or S.I units.

The test strip used was then removed from the Reflotron and disposed of according to

laboratory’s procedure.

3.1.5 LIVER FUNCTION TEST (LFT)

The liver function test was carried out using the Reflotron. The machine determines the

concentration of particular enzyme and proteins in the liver by the use of strips. Liver

function test comprises of the Total Billurubin, glutamate oxaloacetate transaminase (GOT),

Glutamate Pyruvate transaminase (GPT), and Alkaline phosphate (ALP).

Glutamate Oxaloacetate Transaminase (GOT)

Glutamate oxaloacetate transaminase (aspartate aminotransferase) belongs to the group of

transaminase which catalyze the conversion of amino acids to the corresponding α-keto acids

and vice versa by transfer of amino groups. Aspartate aminotransferase is found in many

tissues of the body.

Test Principle

After application to the test strip, the sample flows into the reaction zone, in the case of blood

after separation of the erythrocytes from the plasma. In the presence of GOT, α-ketoglutarate

and alanine sulfinate are converted to Pyruvate oxidase, the resulting Pyruvate is cleaved into

acetyl phosphate, carbon dioxide and hydrogen peroxide. In the presence of POD the

hydrogen peroxide converts an indicator into its oxidised blue form:

α-ketoglutarate + alanine sulfinate GOT glutamate + Pyruvate + SO32-

Pyruvate + PO43- + O2 + H20 PyOD acetyl phosphate + H202 + CO2

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H2O2 + indicator (red) POD indicator (ox.) + H2O

Endogenous Pyruvate was eliminated in a preliminary reaction.

At a temperature of 370C the formation of the dye was measured kinetically at 567nm as a

measure of the enzyme activity of GOT and the result was displayed after 124 seconds. The

result displayed for a temperature of 370C, 300C or 250C and in u/l.

Glutamate Pyruvate Transaminase

Glutamate pyruvate transaminase (alanine aminotransferase) belongs to the group of

transaminase which catalyzes the conversion of amino acids to the corresponding α-keto

acids and vice versa by transfer of amino groups.

Test Principle

After application to the test strip, the sample flows into the reaction zone, in the case of blood

after separation of erythrocytes from the plasma. In the presence of GPT, α-ketoglutarate and

alanine are converted to glutamate and pyruvate. In the second reaction step, catalyzed by

pyruvate oxidase, the resulting pyruvate was cleaved into acetyl phosphate, carbon dioxide

and hydrogen peroxide. In the presence of POD the hydrogen peroxide converts an indicator

into its oxidised blue form:

α-ketoglutarate + alanine GPT glutamate + pyruvate

Pyruvate + PO43- + O2 + H2O PyOD indicator (ox.) + H2O

H2O + indicator (red.) POD indicator (ox.) + H2O

Endogenous pyruvate was eliminated in preliminary reaction.

At a temperature of 370C the formation of the dye is measured kinetically at 567 nm as a

measure of the enzyme activity of GPT and the result displayed after 140 seconds. The result

displayed for temperature of 370 C , 300C or 25oC and in u/l

Alkaline Phosphatase (ALP)

Alkaline Phosphate (ALP) is present in various human tissues e.g. liver, bone, intestine and

placenta.

Test Principle

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After application to the test strip, the sample flows into the reaction zone, in the case of blood

after separation of the erythrocytes from the plasma. ALP hydrolyzes o-cresol-phthalein and

transfers the phosphate group to the acceptor molecule methylglucamine. The coloured

hydrolysis product o-cresolphthalein that is produced per unit of time under alkaline

conditions is directly proportional to alkaline phosphate activity:

O-cresolphthalein phosphate + methylglucamine o-cresolphathlein + methylglucamine

phosphate.

Dye formation is determined kinetically at 370C as a measure of the enzyme activity of ALP.

The result is displayed after approximately 135 seconds in U/l.

Sample collection for LFT

Blood sample was collected into the lithium heparin tube or Ethylene diamine tetra acetic

tube.

Testing procedure for LFT (GOT, GPT, ALP)

Additional material required (not supplied): Reflotron instrument; Reflotron pipette and

pipette tips or capillary pipette, controls and usual laboratory equipment were used for

collecting blood. The procedure used is as follows:

The instrument was switched on

When the display showed “READY”, the test strip was removed from the container.

The strip was unwrapped carefully so as not to bend it.

Using the Reflotron pipette, the sample material drawn into the pipette (avoiding

bubbles) and applied as a drop to the centre of the red application zone, taking care

not to touch the application zone with the pipette tip. Required volume of specimen

for application was 30nl

With the sliding cover or flap open, the test strip was placed on the guide within 15

seconds and slide forward horizontally until it locks intro place

The sliding cover/flap was then closed.

The instrument displays “ALP” to confirm that it has correctly read in the test-n

specific magnetic code. The display shows the number of seconds left before the

result was displayed.

ALP activity was calculated automatically from the readings taken using function and

conversion factors that are entered in the instrument via the magnetic strip on the

underside of each test strip. The enzyme activity is shown in U/l

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The test strip used was then removed from the Reflotron and disposed of according to

your laboratory’s procedure. The table below shows the reference range for male and

female:

Table 3.3: Reference Range for Male and Female

Component Male (mg/dl) Female (mg/dl)

Glutamate pyruvate transaminase(GPT) Up to 41 Up to 32

Alkaline Phosphate(ALP) Up to 270 Up to 240

Total Billurubin 0.5-1.0 0.5-1.0

Glutamine Oxaloacetate Transaminase (GOT) Up to 40 Up to 33

3.2 HAEMATOLOGY UNIT

Haematology is unit in the main laboratory which is concerned with the study of blood, the

blood forming organs and blood diseases. The activities carried out in this unit include:

Cleaning the work bench with normal saline and methylated spirit

Making forms for the patients’ tests

Running full blood count using stained film on microscope and counting chamber

Making thin slides of blood films

Staining the slides with liechman’s stain

Carrying out malaria parasite tests using MP strip

Viewing the stained films under microscope for detection of malarial parasites

Carrying out erythrocyte sedimentation rate ESR test

Carrying out genotype test using the cellulose acetate electrophoresis technique

Carrying out clotting profile test

Registering the results onto the ledger and the forms

2.4 MEDICAL MICROBIOLOGY UNIT

The medical microbiology section deals with the study of organisms of microscopic size,

including bacteria, protozoan, viruses, and certain algae and fungi. The unit is divided into

two; microbiology section and parasitology

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The medical microbiology section has media room as well as general microbiology section

where basic tests are being carried out.

Media Room

The media room is where all the media used for culturing are prepared and stored. The most

common media used in the laboratory are; nutrient agar, chocolate agar, Cystine Lactose

Electrolyte Deficiency (CLED) agar, Salmonella Shigella Agar (SSA) and Mac Conkey agar

Activities carried out in this unit include:

Cleaning my work surfaces every beginning and ending of work

Drying culture and sensitivity plates in the incubator

Reading sensitivity plates

Culturing samples like stool, urine, sputum, and swab

Reading the cultured plates

Carrying out seminal fluid analysis SFA using the sperm quality analyser

Carrying out microscopy of various samples

Carrying out tests like the hepatitis b surface antigen and hepatitis c virus tests

respectively

Running tests like the widal, pregnancy

Performing tests like the VDRL

Preparation of media using the autoclave

Registering all results of tests carried out for future reference

2.5 IMMUNOLOGY UNIT

This is a unit of the laboratory where the immune system of HIV patients is assay and

counted using the BD FACSCount machine (CD4 Machine). The activities carried out in this

unit include:

Cleaning the work bench with jik and methylated spirit to disinfect it

Giving the samples numbers

Cleaning the FACSCount machine

Preparing the samples using the appropriate reagents

Running the samples on FACSCount machine

Cleaning the FACSCount machine after running the samples

Shutdown the machine

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Registering all results of tests carried out for future reference

2.6 TRANSFUSION UNIT

This is a unit of the laboratory where blood samples are screen for infectious diseases before

been transfuse into the patient. The activities carried out in this unit include:

Cleaning work surfaces

Carrying blood grouping test and cross matching

HIV screening test

HIV Counselling

Registering all results of tests carried out for future reference

ANALYSIS

3.0 CHEMICAL PATHOLOGY UNIT

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IMMUNOLOGY UNIT

This is a unit of the laboratory where the immune system of HIV patients is assay and

counted using the BD FACSCount machine (CD4 Machine).

SPECIMEN COLLECTION AND STORAGE

FACSCount technique is designed to be used with human whole blood samples. Whole blood

is collected in a K3 EDTA vacutainer tube and stored no longer than 6 hours at room

temperature (20-250C).

Test procedure

All reagents were brought to ambient temperature

Procedure for preparing and running controls

Normal blood is needed to run controls. Blood sample must be collected in a K3 EDTA

vacutainer tube from a normal donor and run immediately or could be stored no longer than 6

hours at room temperature (20-250C).

Controls are prepared by adding normal blood, then fixative solution to the CD4 and CD8

reagents tubes followed by addition of control beads before reagent tubes are run. Controls

beads are packed in tube pairs similar to reagent tube pairs.

The normal blood donor was placed in the workstation. Two reagent tube pairs from foil bag

were removed. The foil bag was sealed and the unused reagent pairs were returned to the

refrigerator.

The tabs of the two reagent tube pairs were labelled:

Pair one: Label the CD4 (green top) – Zero

Label the CD8 (clear top) – Low

Pair two: Label the CD4 (green top) – Medium

Label the CD8 (clear top) – High

* The pairs were vortex upside down for 5 seconds. The pairs were vortex upright for 5

seconds.

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* The reagent tubes was opened with coring station and closed to protect the reagents

from light.

* The whole blood was mixed by inverting the tube five times.

* 50µl of normal whole blood was pipette into each of the four reagent tubes. Each was

changed between tubes.

* Tubes were caped and vortex for seconds.

* Tubes were incubated for 60 to 120 minutes at room temperature (20-250) in the dark.

* After which tubes were uncapped and 50µl of fixative solution was pipette into each

reagent tube. Tips were changed between tubes.

* Reagent tubes were recapped with new caps and vortex upright for 5 seconds.

* Incubated for at least 30 minutes at room temperature in the dark.

* One pair of Zero/Low control beads and one pair of Medium/High control beads were

removed from the control kit and placed in the control area of the workstation.

* Tubes labelled zero, low, medium and high were uncapped.

* The zero/low control beads pair was vortex for 6 seconds and reverse pipette of 50 µl

of low control beads into the tube labelled low.

* The medium/high control bead pair was vortex for 6 seconds and reverse pipette of

50lµ of medium control beads into the tube labelled medium.

* Reverse pipette of 50µl of the high control beads into the tube labelled high.

* The controls were then run on the BD FACSCount Machine within 24 hours of

adding the control beads.

Procedure for preparing and running controls

Patient blood sample must be collected in a K3 EDTA vacutainer tube from a normal donor

and run immediately or could be stored no longer than 6 hours at room temperature (20-

250C).

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Patient samples were prepared by adding blood, then fixative solution to the CD4 reagent

tube.

The normal blood donor was placed in the workstation. One reagent tube pair was removed

for each patient. The foil bag was sealed and the unused reagent pairs were returned to the

refrigerator.

The tabs of one reagent tube pair was labelled with the patient laboratory

number

The pairs were vortex upside down for 5 seconds. The pairs were vortex upright for 5

seconds.

The reagent tubes were opened with coring station. The workstation was closed to

protect the reagents from light.

The patient whole blood was mixed by inverting the tube five times

50µl of whole blood was pipette into each two reagent tubes.

The tubes were capped and vortex for 5 seconds.

Then incubated for 60-120 minutes at room temperature (20-250) in the dark. The

reagent pairs were placed in the workstation and the cover was closed to protect the

reagents from the light.

The tubes were uncapped and 50µl of fixative solution was pipette into each reagent

tube.

The reagent tubes were recapped with new caps and were vortex upright for 5

seconds.

The tubes were run on the FACSCount Machine within 48 hours of preparation.

Samples were stored at room temperature in the workstation until they were run on

the Machine. They were vortex upright for 5 seconds immediately before running.