MASENO UNIVERSITY

SCHOOL OF PUBLIC HEALTH AND COMMUNTY DEVELOPMENT

DEPARTMENT OF BIOMEDICAL SCIENCE & TECHNOLOGY

PMT337: ATTACHMENT REPORT

AN ATTACHMENT REPORT CONDUCTED AT KENYATTA NATONAL

HOSPITALF OR THE FULLFILLMENT OF BACHELORS DEGREE IN

BACHELOR OF SCIENCE IN MEDICAL LABORATORY

ATTACHMENT PERIOD: 1ST NOV -19TH DEC 2014.

REPORT WRITTEN & SUBMITTED BY:

NAME: NYANDO COMFORT.

REG NO: PH/00109/2011.

DECLARATION.
I Nyando Comfort declare that this attachment report is my original work and has never been
submitted on any other university for academic credit whatsoever.

STUDENT.

MR NYANDO COMFORT

Signature……………………………………… Date…………………………………

SUPERVISOR.

Mr. Elly Munde

Signature…………………………………….. Date…………………………………….

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ACKNOWLEDGEMENT

I would like to appreciate the guidance and knowledge I sourced from Prof Collins Ouma, my

professional colleagues Miss Anna Bwalya and Miss Sylvia Nzula who helped me and gave

me the support throught my attachment period.

My efforts could have been useless without the help of Mrs. Priscilla Okumu who introduced

me to Kenyatta National Hospital; She took us through all the departments under Laboratory

Medicine department.

Also numerous basic scientific empowerments were made success by the staffs of all

departments under Laboratory Medicine in Kenyatta National Hospital. Their need to teach

and help us was endless; they spent most of their free time on students and guided us to carry

out tests and report results on our own.

My special appreciation goes to my parents, for moral, spiritual and financial support that

they granted me.

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Table of Contents

DECLARATION......................................................................................................................iii
ACKNOWLEDGEMENT........................................................................................................iv
1.0 INTRODUCTION................................................................................................................1
1.1 BACKGROUND INFORMATION OF KNH.................................................................4
Legal Framework.......................................................................................................................4
Institutional Framework.............................................................................................................4
1.2 MAIN ACTIVITIES OF KNH.........................................................................................6
Introduction................................................................................................................................6
Vision.........................................................................................................................................6
Mission.......................................................................................................................................6
Motto..........................................................................................................................................6
Core Values................................................................................................................................7
Strategic Goals...........................................................................................................................7
Strategic Objectives...................................................................................................................8
1.3 ATTACHED SECTION (LABORATORY MEDICINE.)..............................................9
Attachment objectives................................................................................................................9
Quality control & quality assurance...........................................................................................9
Purpose.....................................................................................................................................10
Objectives.................................................................................................................................10
Areas of Interest.......................................................................................................................10
1.4 RECEPTION..................................................................................................................11
CHAPTER TWO.....................................................................................................................13
2.0 BLOOD TRANSFUSION UNIT.......................................................................................13
2.1 Blood Grouping..............................................................................................................13
2.2 Cross matching...............................................................................................................14
2.3 Indirect coombs test........................................................................................................15
2.4 Weak D test (Du test).....................................................................................................15
2.5 Preparation of blood components...................................................................................16
2.7 Blood donation...............................................................................................................17
CHAPTER THREE..................................................................................................................19
3.0 PARASITOLOGY.............................................................................................................19
3.1 Stool analysis..................................................................................................................19

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 3.2 Stool analysis report.......................................................................................................20
3.3 Urine analysis.................................................................................................................22
3.4 Blood smear for malaria parasites..................................................................................23
CHAPTER FOUR....................................................................................................................25
4.0 HAEMATOLOGY.............................................................................................................25
4.0 Routine tests...................................................................................................................25
4.1 Full hemogram................................................................................................................25
4.2 Peripheral Blood Film....................................................................................................26
4.3 Erythrocyte sedimentation Test......................................................................................28
4.4 Reticulocyte count..........................................................................................................30
4.5 Haemoglobin Electrophoresis........................................................................................32
4.6 Coagulation Tests...........................................................................................................34
CHAPTER FIVE......................................................................................................................38
5.0 BIOCHEMISTRY..............................................................................................................38
5.0 Renal Function tests.......................................................................................................38
5.2 Liver Function Tests.......................................................................................................41
5.3 Pancreatic Function Tests...............................................................................................44
5.4 Blood Glucose Tests.......................................................................................................45
CHAPTER SIX........................................................................................................................48
6.0 MICROBIOLOGY.............................................................................................................48
6.1 Specimens for culture.....................................................................................................48
6.1.2 Microscopy..................................................................................................................48
6.2 Culture Techniques.........................................................................................................49
6.3 Biochemical Tests..........................................................................................................49
6.4 Staining Techniques.......................................................................................................50
CHAPTER SEVEN..................................................................................................................52
7.0 IMMUNOLOGY LABORATORY...................................................................................52
7.1 Enzyme-linked Immunoassays.......................................................................................52
7.2 Slide agglutination Tests................................................................................................52
CHAPTER EIGHT...................................................................................................................54
HISTOPATHOLOGY & CYTOLOGY LAB.........................................................................54
8.0 Tissues Processing..........................................................................................................54
8.2 Hematoxylin and eosin staining.....................................................................................55
8.3 Pap staining Technique...................................................................................................56
REFERENCES.........................................................................................................................57

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 CHAPTER ONE

1.0 INTRODUCTION.

KENYATTA NATIONAL HOSPITAL

The Hospital was established in 1901, as a Native Civil Hospital, with a two-ward, 40 bed

facility at the junction of Government road (the present Moi Avenue) and Kings Way (the

present University way). It was later relocated to the present Kenya Medical Training College

site in 1922. The facility had a bed capacity of 423 for Africans and 41 for Asians and offered

in-patient services only. The construction of a combined Group Hospital to cater for all races

began in 1937 and was completed in 1947.

The Hospital was renamed King George VI in 1952. The Ismail Rahimtullah Wing was

constructed in 1953 exclusively for the Asian community. The Infectious Diseases Hospital

(IDH) was opened in 1956 as part of King George VI hospital. Following attainment of

Kenya’s independence in 1963, the King George VI Hospital was renamed Kenyatta National

Hospital in honor of the first president of the Republic of Kenya, Mzee Jomo Kenyatta.

Increase in demand for health care services necessitated the introduction of training for

medical personnel and mid-wives in the Hospital in 1965. The University of Nairobi Medical

School located at the hospital was established in 1967. In 1971, the construction of a ten-

storey Tower Block was started and completed in 1981. The Renal unit started operation in

1984, while the Dental and the Orthopedics units were relocated from Kabete in 1985 and

1993 respectively.
Since its inception, the Hospital operated as a department of the Ministry of Health until 1987

when the hospital changed its status to a State Corporation through Legal Notice No. 109 of

6th April 1987.

The Hospital is the major training facility for health care personnel in various disciplines both

at undergraduate and post-graduate levels. The institution works closely with University of

Nairobi’s College of Health Sciences (CHS), Kenya Medical Training College (KMTC),

Kenya Medical Research Institute (KEMRI), Government Chemist Department, National

Radiation Protection Board, National Public Health Laboratories, National AIDS and STDs

Control Programme (NASCOP) and National Blood Transfusion Services (NBTS). It has also

formed linkages with other institutions in providing various clinical services, research and

outreach programs. Collaborations have been established with Operation Smile International,

Operation Heal the Child, Neurosurgical Mission of St. Louis USA, Plastic Surgical Project

of Prof. Platt, Heart to Heart Foundation, Medical and Educational Aid to Kenya (MEAK),

National AIDS Control Council, among others.

KNH is the public hospital of choice in Kenya and beyond. It offers quality specialized health

care to patients from the great lakes region, southern and central Africa including Namibia.

These services include cardiothoracic surgery, neurosurgery, orthopedic surgery, plastic and

reconstructive surgery and burns management; radiotherapy, critical care services, new born

services, renal services (including kidney transplantation) besides other services. Training of

medical personnel from these countries is also undertaken.

The Hospital is in the process of reorganizing and restructuring in response to the global

health care needs. It is envisaged that the Hospital will continue to grow into a world class

institution, providing quality specialized health care, training, research and medical tourism.
1.1 BACKGROUND INFORMATION OF KNH.

Legal Framework.

The Hospital was established under Legal Notice No.109 of 6 th April 1987 that spells out its

mandate and responsibilities as follows.

 Receive patients on referral from other hospitals or institutions within or outside Kenya

for specialized health care;

 Provide facilities for medical education for the University of Nairobi Medical School,

and for research either directly or through other co-operating health institutions;

 Provide facilities for education and training in nursing and other health and allied

q`qprofessions;

 Participate as a national referral hospital in national health planning.

Institutional Framework.

Board Composition.

The Board consists of eleven members including a non-executive Chairperson and the CEO.

The Board and its committees oversee the corporate governance issues, advice the

management on developing of financial plans, corporate strategies, goals and objectives as

well as evaluating management’s performance in pursuing and achieving those goals. There

are five committees of the Board and include: Audit Committee; Finance and All Purpose

Committee; Tender Committee; Terms and Conditions of Service and Staff Welfare

Committee; and Standards, Quality, Ethics and Research Committee. The committees
reinforce the Board’s independence and legitimacy in areas where there is potential for

conflict of interest.

Organization Structure and Management.

The Hospital is headed by a Chief Executive Officer (CEO) who is responsible to the Board

of Management for planning and day-to-day activities. There are three (3) positions of

Deputy Director, one for Clinical services, for the nursing services and the other for

Administration and Finance. The two Deputies superintend over forty-five (45) departments

and units. This has inherent problems of span of control and effective chain of command.

These limitations have been addressed in the proposed organizational structure.

The typically hierarchical arrangement of lines of authority, communications, rights and

duties of an organization.

Organizational structure determines how the roles, power and responsibilities are assigned,

controlled, and coordinated, and how information flows between the different management
1.2 MAIN ACTIVITIES OF KNH.

Introduction

To realize its objectives, the Hospital strategically focuses on its functions and operations as

stipulated in its Vision, Mission, and Core Values which are the guiding principles. The Vision is

a pre-requisite for effective strategic leadership while the Mission is the overriding raison d’être

that gives our identity and unique purpose. The Core Values reflect our institution culture and

common beliefs to which all members of staff subscribe to. Our strategic focus addresses four

strategic goals and ten strategic objectives with specific strategies on how to realize each

objective.

Vision

To be a world class patient-centered specialized care hospital.

Mission

To promote specialized healthcare through effective strategic planning, performance

contracting, monitoring and evaluation of performance

Motto

We listen, we care.
Core Values

We recognize that Core Values form the glue that holds an organization together. The Hospital

commits itself to the following Corporate Values:

1. Customer focus

2. Professionalism and integrity

3. Team work

4. Equity and equality

5. Employee empowerment

6. Environmental safety

Strategic Goals

 Enhance Provision of Specialized Quality Healthcare Services

 Improve Institutional Capacity

 Enhance Financial Sustainability

 Participate in National Health Planning and Policy

Strategic Objectives

1. To provide quality specialized health care

2. To acquire international accreditation

3. To promote institutional linkages and collaborations

4. To strengthen training and research capacity

5. To participate in national health planning and policy

6. To improve and diversify revenue generation and cost efficiency

7. To improve human resource management and development

8. To implement reliable information and communication technology

 1.3 ATTACHED SECTION (LABORATORY MEDICINE.)

Laboratory Medicine department has 9 main sub-departments. They include:

a) Phlebotomy

b) Parasitology

c) Biochemistry

d) Immunology

e) Hematology

f) Blood Transfusion Unit

g) Histopathology and Cytology

h) Microbiology

Attachment objectives.

 To gain hands on experience on processing of clinical specimen in hospital laboratories.

 To appreciate and gain deeper understanding of theory knowledge attained in class.

 To fulfill the requirement for the award of the Bachelors of Science Degree in medical

laboratory science.

Quality control & quality assurance.

The KNH Laboratory Department in conjunction with ISO standards has established a quality

policy to ensure that their laboratories meet international standards.

Purpose.

The laboratory quality assurance policy was designed to monitor, evaluate and improve the

quality of laboratory performance and ensure reliability of test data and to evaluate the

competency of the laboratory staff.

Objectives.

 To ensure that the quality assurance activities are comprehensive and coordinated to

ensure accurate and reproducible results are reported.

 To establish, maintain, support and document a quality assurance program and identify

opportunities for improving patient healthcare.

 Improving healthcare and identifying problems through monitoring, assessment and

follow up of problems hindering laboratory performance.

 To follow up identified problems to assure improvement and resolution in a timely

manner with documentation of corrective action.

Areas of Interest.

Quality Assurance Monitors: Include proficiency testing, specimen management, reporting of

results, technical delays, SOPs, Complaints and turnaround time. Quality assurance monitors are

improved and maintained regularly to suit the current ISO standards.

Training: Laboratory staffs are retrained on new procedures or new equipments to ensure

successful operations are done. Through seminars, workshops and SMEs staffs are refreshed on

new applications in the laboratory.

Quality Control: Each test procedure outlines the required quality control materials for the test.

The laboratory staff should ensure that quality control should be run parallel to the tests for

validation of results.

Calibration and Maintenance: Each instrument in use must have a file with procedures for

maintenance and calibration of test parameters. Maintenance log sheets are kept on daily,

weekly, monthly, quarterly, semi-annually and annually basis as described for each instrument.

These records are reviewed and signed by the laboratory manager and retained for two years.

Any preventive maintenance, repairs or part of replacement records are kept for the life span of

the equipment.

1.4 RECEPTION.

Laboratory reception serves vast of activities that are not limited to; receiving specimens and

dispatching results.
Receiving Specimens.

The samples are received in the laboratory and confirmed that they match with the information

on the request form, they should also be in the right container, the details are entered into the

specimen reception book. The samples are given a laboratory number which acts as a reference

for a follow up.Samples that satisfy the acceptance criteria are then ready to be processed.

Samples that do not meet the required criteria are rejected.

Dispatching results.

Results are supposed to be dispatched under the turnaround time for patients care and

management.
 CHAPTER TWO

2.0 BLOOD TRANSFUSION UNIT.

Tests carried out in BTU include: Blood grouping, Cross matching, Indirect coombs test, Direct

coombs test, Weak D test (Du test) and Preparation of Blood Components. Other activities

include; Blood donation.

2.1 Blood Grouping.

Blood was grouped according to the ABO and Rhesus grouping systems. ABO grouping is

divided into two; forward grouping or cell grouping and reverse grouping or serum grouping.

Forward grouping red cells are tested or antigens A and B using Antisera A and B while Reverse

grouping serum is tested for anti-a and anti-b antibodies using known A and B cells. KNH used

the forward blood grouping method.

Method.

A drop of Antisera A, B and D was placed in three different wells respectively. One drop of

Cells of Sample was added to each well. Contents were mixed and left to stand for 2minutes at

room temperature. Report for agglutination or no agglutination in each well. If agglutination fails

in well D, Du test was carried to determine the variant D type. The Blood group table was used

to deduce the blood group type.

 2.2 Cross matching.

Cross match was requested when recipients needs blood in cases of anemia, accident,

hemorrhagic fever, theater and dialysis patients. Cross match was carried to ensure compatibility

between recipient and donors blood and minimize chances of post-transfusion reaction.

Two types of cross match; major and minor cross match.

Major cross match involves mixing of patient’s serum with donor’s red cells while minor cross

match involves mixing of donor’s serum with patient’s red cells.

There are four phases of cross match; saline at room temperature, saline at 37 0C, Bovine albumin

and Antihuman Globulin.

Method

Blood samples of the recipient were grouped. 4% cell suspension of the donor’s cells were

prepared. 4 test tubes were labeled Saline at RT, Saline at 370c, Coombs at 370c and Albumin at

370c. 2drops of patient serum was added into the four test tubes. 1 drop of 4% cell suspension

was added into the four test tubes. Mix well and incubate at respective temperature for 30

minutes. For saline at room temperature and saline at 370C the contents were mixed and

agglutination checked, If no agglutination discard the tubes. For coombs at 37 and albumin at 37

wash the contents of the tubes and add coombs reagent and bovine albumin reagent in their

respective tubes. Agglutination both macroscopically and microscopically was checked.

2.3 Indirect coombs test

Used to detect invitro antibody-antigen reaction, in transfusion science its used to detect very

low concentration of antibodies in patients plasma or serum prior to transfusion. In antenatal

care, ICT is used to screen whether a rhesus negative pregnant woman has been sensitized with

ant-rh antibodies from a rhesus positive fetus, clinically its important to curb chances of

hemolytic disease of the new born.

Method

4% cell suspension of O positive pooled cells was prepared. One test tube was labeled Coombs

at 370C. 1 drop of patient serum into the test tube. Add 2 drops of 4% cell suspension of O

pooled cells. Incubate for 45 minutes. Cells are washed three times. Add 1 drop of Antihuman

globulin.Mix and check for agglutination. Both macroscopically and Microscopically

2.4 Weak D test (Du test)

To test for the presence of weak D antigen in red blood cells who turn out negative. The

procedure has two steps; sensitization of red cells and addition of antihuman globulin.

Method

Cells are washed 3times. 4% cell suspension is prepared. Put 1 drop of Anti-D in attest tube. Add

1 drop of 4% cell suspension. Incubate at 370C for 30 minutes. Check for agglutination both

macroscopically and microscopically. If no agglutination add 1 drop of AHG and spin 1400rpm

for 2minutes. Check for agglutination both macroscopically and microscopically.

2.5 Preparation of blood components.

Components derived from blood in the laboratory include: fresh frozen plasma, packed red cells,

cryoprecipitate/ factor VIII, platelet rich plasma and platelet concentrate. Factors considered

when preparing these components include the speed, temperature and time as cryo-centrifuge is

programmed to carry out this activities.

Blood components are given to patients depending on the type of disease or condition they are

suffering from; patients with burns are given fresh frozen plasma, those with clotting conditions

are given platelet concentrate or platelet rich plasma, those that are anemic, with lower Hb and

bleeding are given packed red cells and hemophilic patients are given are given factor VII/ant

hemophilic factor.

Platelet rich plasma and platelet concentrate.

Platelet rich plasma: Separated 4hrs after collection or donation, preparation conditions include

spin at 1500rpm at 210C for 15minutes, stored at room temperature of 20-24 for 5 days with

constant agitation. Each unit should contain 5.5-9.0 ×10 6 platelets. Preparation involves two

spins; soft and heavy. Soft spins occurs at 1500rpm for 15 minutes and separates RBCs, WBCs

from plasma and platelets. While heavy spin occurs at 2500rpm for 6minutes formation of

platelet rich plasma about 40-60 mls are collected into the separation bag.
Packed Red Blood Cells.

These are units of whole blood with plasma removed; they are prepared at anytime during the

shelf life of the blood. Shelf life of packed red cells is 21-35 days from date of collection

depending on the bag used for donation.

Fresh frozen Plasma (FFP).

Plasma that is frozen within 8hrs of donating blood, preparation conditions include spinning at

2500rpm at 40C for 15minutes and it’s stored under temperatures of 1-6 0C. Before transfusion

FFP should be ABO compatible and thawed between 30-37 0C for 45minutes. Shelf life of FFP is

12 months from date of collection. FFP frozen at -650C is stored for 7 years.

2.7 Blood donation.

Process of giving out blood willingly for the purpose of saving life.

Donor selection criteria.

Donor selection is based on medical history and limited to physical examination and is done

prior to donation to ensure the safety of the donor is guaranteed. Examination is based on

medical history such as treatment of any tropical diseases for the past three months, any chronic

illnesses and physical appearance; the general appearance, must be of 50kgs-100kgs of weight,

should have take food before donation.

Blood collection bags.

Types of bags used at Kenyatta National Hospital include;

Citrate-phosphate dextrose (CPD) preserves for 21 days.

Citrate Phosphate dextrose dextrose (CPD2) preserves for 28 days.

Citrate Phosphate dextrose adenine (CPDA-1) preserves for 35-42 days.

All the bags have a total volume of450ml and contains anticoagulant-preservative solution to

prolong the life span of blood.

 CHAPTER THREE

3.0 PARASITOLOGY.

Specimens proceed in this Laboratory include;

 Stool for ova and cyst examination.

 Blood for blood parasites.

 Urine for urinalysis and microscopy

Other activities carried in this laboratory include;

 Preparation of 5% giemsa stain solution.

Adding 0.5mls of Giemsa stock solution in 9.5 buffered water solution of pH 7.2

 Preparation of 10% formal saline.

Through the formulae of preparation that is applied;

= (Required volume × Required concentration) ÷ Original Concentration.

3.1 Stool analysis.

Depending on the consistency of stool it can be processed either through concentration method

or direct method.

Concentration Method

Principle.
Formalin fixes the eggs, larvae and cysts so that their morphology is preserved. Diethyl ether

emulsifies the fats and float debris. During centrifugation fecal matter is extracted into the other

phase of the solution, freeing the parasitic elements from at least some of the artifactual material

in stool. Iodine stains te nuclear and glycogen vacuole of the cysts.

This method is applied on formed or semi-formed stool.

Procedure.

1 gram of stool sample was emulsified in 10mls of normal saline in a centrifuge tube and spun at

3.000 r.p.m for 10 minutes. The supernatant was discarded. This process was done two times to

wash the stool sample. Then the sediment was resuspended in 7mls of formol saline and 3mls of

petrol (super) was added and the mixture stoppered with a rubber bung and shaken vigorously. It

was spun at 3.000 r.p.m for 10 minutes. It separated into three portions. The first from the bottom

was the sediment, followed by a layer of formol saline in the middle and at the top was coarse

stool particles, petrol and fats. The layers above the sediment were carefully aspirated and

discarded using pasture pipette. The sediment was examined under the microscope for the

presence of parasites.

Direct Method.

This method is applied on loose watery stool.

Procedure.

Pea size stool was placed on a slide. Add few drops of normal saline on the stool and stir.

Observe under microscope(×10 for focusing and ×40 for identification.

 3.2 Stool analysis report.

Macroscopically: Stool is reported on it’s

 Consistency i.e. Formed, Semi formed or loose.

 Color i.e. Normal, Blood stained or Mucoid

 Smell

Microscopically: Stages of different intestinal parasites were reported and Stool non-parasitic

structures were also reported.

Observations.

Examples of Intestinal Parasites Identified.

 Ova of Hookworms were reported, they appear colorless with a thin outer shell that

appears as black microscopically, It’s also oval in shape and contains segmented ovum.

 Cysts of Giardia lamblia, Entamoeba histolytica, Entamoeba coli, Iodamoeba butscili,

Endolimax nana, Blastocystis hominis,

 Oocyst of cryptosporidium species.

 Trophozoites of Trichomonas vaginalis.

Examples of Non-parasitic structures found in stool.

 Muscle fibers

 Vegetable cells
  Starch ells

 Pollen grains

 Yeast and spores

 Charcot Leyden crystal.

3.3 Urine analysis.

Involve use of reagent strips for quantitative and semi-quantitative detection of the following

analytes in urine; Glucose, Bilirubin, Ketone, Specific gravity, Blood, pH, Protein, Urobilinogen,

Nitrite and Leukocyte.

This assists in diagnosis of different risk diseases e.g. Kidney function, Urinary tract infections,

Carbohydrate metabolism defects, Aid-base balance and Urine concentration. It also determines

if microscopic examination is required in cases where Blood and leukocytes are present.

Steps in Urine analysis;

Microscopic examination.

Describe the appearance of urine sample based on; color and turbidity.

Urine analysis using Dipstick method.

Principle of Dipstick method.

Dipsticks are plastic strips impregnated with chemical reagent which react with specific in urine

to produce color-coded results. The depth of the color produced correspond to the concentration

of the substance in urine.

Procedure.

Urinalysis strip was dipped in fresh urine sample. Parameters were read with Dipstick meter or

manually against the provided analyzed chart. Parameters with abnormal values were noted
Microscopic analysis.

This is done on urine samples that are presented with deposits of leukocytes, blood and Glucose

Procedure.

Mix the urine sample gently. 2mls of urine are placed in a centrifuge tube.Centrifuge at

1500Rpm for 2 minutes. Pour out the supernatant. Place the urine deposits on a glass slide and

cover slip. Observe under microscope(×10 for focusing and ×40 for identification)

Observations.

Appearance of urine may be altered by conditions such as Urinary tract infections, Jaundice,

Urinary schistosomiasis and malaria haemoglobinuria.

Urine deposits that may indicate abnormality of the kidney role include; pus cells, red cells,

casts, crystals, ova and epithelial cells.

3.4 Blood smear for malaria parasites.

Routine malaria diagnosis was done on both thin and thick films and staining was done by

giemsa stain.

Thin film preparation.

A drop of capillary blood was placed on a glass slide. Using a glass slide spreader at an angle of

450 touch the drop on the glass slide and allow the blood to run along its edge. Firmly push the
slide along the slide keeping an angle of 450.Label the patient’s number on the slide using a

pencil.Allow the slide to air dry.

Thick film preparation.

A drop of capillary blood was placed on a glass slide. Using an applicator stick gently spread the

drop. Label the patient’s number on the slide using a pencil. Allow the slide to air dry.

Principle of Giemsa stain.

The cytoplasm of the parasites and takes the basic part of the stain i.e. stains blue while the

nucleus takes the acidic portion and stains red.

Giemsa staining procedure.

The slide must be air dried. Then the slide is flooded with 5% Giemsa solution. Allow the slide

to stain for 20 minutes. Wash the slide wit running tap water. Wipe off the slide, air dry and

observe under the microscope under ×100 objective lens

Observations.

Different stages of Malaria parasites were noted they included; trophozoites, schizonts and

gametocytes. Commonly trophozoites of plasmodium falciparum parasites were seen

microscopically.
 CHAPTER FOUR

4.0 HAEMATOLOGY.

Haematology was divided into 3 major sub-departments namely: Routine test, Hemostasis and

Special Haematology.

4.0 Routine tests.

They included; full hemogram, Reticulocyte count, Erythrocyte sedimentation test , Peripheral

blood film.

4.1 Full hemogram.

This is an automated test that runs all blood parameters, blood volumes and cells sizes.

Full hemogram entails a report on percentages of; red cells, white blood cells and platelets.

Abnormal ranges in their indices are an indication of a blood disorder depending on the type of

blood.
Normal values for full hemogram.

Parameter Males Females

Red blood cells(*1011/L) 4.5-6.5 3.9-5.6
Hemoglobin gm/dl 13.5-17.5 11.5-15.5
MCV fl 80-95 80-95
MCH 27-34 27-34
MCHC 30-35 30-35
Hematocrit % 40-52 36-48
Platelet *109/L 105-450 105-450

Full hemogram results are confirmed by a peripheral blood smear that distinctively tells one on

how these cells appear under a microscope.

4.2 Peripheral Blood Film

Value of blood films.

Examination of thin blood films was important in the investigation and management of anaemia,

infections, and other conditions which produce changes in the appearance of blood cells and

differential white cell count. A blood film report can provide rapidly and at low cost, useful

information about a patient’s condition.

Staining thin blood films.

In KNH laboratories, thin blood films are usually stained manually using Leishman or Wright’s

stain. These stains are examples of alcohol containing Romanowsky stains which stain blood

cells differentially.

Romanowsky stains.

These stains contain eosin Y which is an acidic anionic dye and azure B and other thiazine dyes

(derived from the oxidation, or polychroming, of methylene blue) which are basic cationic dyes.

When diluted in buffered water, ionization occurs. Eosin stains the basic components of blood

cells, e.g. haemoglobin stains pink-red, and the granules of eosinophils stain orange-red. Azure B

and other methylene blue derived dyes, stain the acidic components of cells. Nucleic acids and

nucleoprotein, stain various shades of mauve-purple and violet, the granules of basophils stain

dark blue-violet, and the cytoplasm of monocytes and lymphocytes stains blue or bluegrey. The

staining reactions of Romanowsky stains are pH dependent which is why the stains are diluted in

buffered water of specific pH.

Reporting blood films.

Reporting Romanowsky stained thin blood films includes:

– Differential white cell count and white cell morphology

– Red cell morphology

– Comments on platelets (venous EDTA anticoagulated blood sample)

– Other abnormalities, e.g. presence of malaria parasites, trypanosomes, microfilariae,

Bartonella, and Borrelia

4.3 Erythrocyte sedimentation Test.

Value of test: 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 (see later

text). Moderately raised sedimentation rates can sometimes be found in healthy people,

particularly those living in tropical countries and a ‘normal’ ESR cannot exclude disease. In

many tropical countries, ESR measurements have been discontinued because they add little to

diagnosing disease, assessing its progress and monitoring response to treatment. When

performed, test results must be interpreted in conjunction with clinical findings and the results of

other laboratory tests.

Principle of test

When citrated blood was placed in a vertically positioned Westergren pipette and left

undisturbed, red cells aggregate, stack together to form rouleaux, and sediment through the

plasma. The ESR is the rate at which this sedimentation occurs in 1 hour as indicated by the

length of the column of clear plasma above the red cells, measured in mm. Fibrinogen,

immunoglobulins, and C reactive protein increase red cell aggregation. Sedimentation is

increased when the ratio of red cells to plasma is altered, e.g. in anaemia. Sedimentation is

reduced when the red cells are abnormally shaped, e.g. sickle cells. High temperatures (over 25

_C) increase sedimentation.

Specimen: Either venous blood collected directly into sodium citrate and tested within 2 hours,

or EDTA anticoagulated blood diluted in sodium citrate can be used. If EDTA blood is used and

kept refrigerated at 4–8 _C, citrate dilution of the blood and testing can be delayed for up to 6

hours.

Test method
Pipette 0.4 ml of sodium citrate anticoagulant into a small container. Add 1.6 ml of venous

blood or EDTA anticoagulated blood and mix well. Remove the cap of the container and place

the sample in the ESR stand. Insert a Westergren pipette and ensure it is positioned vertically.

Using a safe suction method, draw the blood to the 0 mark of the Westergren pipette, avoiding

air bubbles. Check that the ESR stand is level by making sure that the bubble in the spirit level is

central. If required, adjust the screws on the bottom of the stand. Re-check that the pipette is

vertical. Set the timer for 1 hour. Ensure the ESR stand and pipette will not be exposed to direct

sunlight. After exactly 1 hour, read the level at which the plasma meets the red cells in mm.

After reading the ESR, return the blood to its container, remove carefully the pipette and soak it

in sodium hypochlorite 2 500 ppm av Cl (0.25%) disinfectant.

ESR test results

Reference range

Men . . . . . . . . . . . . . . . . . . . . . Up to 10 mm/hour

Women . . . . . . . . . . . . . . . . . . . Up to 15 mm/hour

Elderly . . . . . . . . . . . . . . . . . . . . Up to 20 mm/hour
4.4 Reticulocyte count.

Value of test:

Reticulocytes are immature red cells normally present in small numbers in the blood (up to 2%).

Reticulocyte numbers increase when there is an increase in erythropoietic activity. A reticulocyte

count assesses bone marrow activity, e.g. whether there is an effective erythropoietic response

when there is a reduction in the number of red cells due to haemolysis or haemorrhage. A

reticulocyte count is also of value in monitoring the erythropoietic response of an anaemic

patient following treatment.

Principle of test:

An isotonic solution of a supravital stain (i.e. one that stains living material) such as New

methylene blue or brilliant cresyl blue is incubated with a few drops of blood. To detect

ribosomal RNA in reticulocytes, the red cells must be stained while they are still living (not

fixed). A thin preparation is made and the reticulocytes counted microscopically. Reticulocytes

are recognized by the violet-blue stained granules of ribosomal RNA (reticulin) they contain.

The reticulocyte count is expressed as a percentage, or preferably in absolute numbers when an

electronic analyzer RBC count is available.

Specimen:

Use well-mixed EDTA anticoagulated blood or if the patient is a young child, use free flowing

capillary blood.

Test method
Filter 3 drops of the stain into a small tube or vial. Add about 4 drops of EDTA anticoagulated

blood or capillary blood and mix well. Incubate at room temperature for 20 minutes or 15

minutes at 35–37 C. Mix gently to resuspend the red cells and using a capillary or plastic bulb

pipette, transfer a drop of the stained blood to each of two slides. Spread to make two evenly

spread thin films. Wave the slides back and forth to air-dry the films. Protect the films from dust

and insects until the count can be performed. Count the reticulocytes microscopically. Use the 10

objective to check the distribution of the red cells. Select an area where the red cells can be seen

individually, add a drop of immersion oil, and examine using the oil immersion objective. Count

systematically (i.e. consecutive fields), 500 red cells (1 000 if there are very few reticulocytes),

noting the number that are reticulocytes. Calculate the percentage of reticulocytes

Appearance of reticulocytes

Reticulocytes appear as pale green-blue stained cells containing dark blue-violet inclusions in the

form of small granules, distributed irregularly as shown in colour Plate 110. Mature red cells

stain pale green-blue.

Counting reticulocytes:

A convenient method of counting reticulocytes is to reduce the size of the microscope field by

inserting in each eyepiece a circular piece of black (opaque) paper which has a punched out hole

of about 5 mm.
To calculate % of reticulocytes: Using a hand tally counter, count a total of 500 red cells, noting

on paper the number of cells that are reticulocytes (alternatively use two hand tally counters or a

white cell differential counter). Multiply the number of reticulocytes counted by 2. Divide the

figure by 10 to obtain the percentage figure.

Reticulocyte counts
Reference range

Infants at birth . . . . . . . . . . . . . . . . . . . . . . . . . 2–5%

Children and adults . . . . . . . . . . . . . . . . . . 0.5–2.5%

Raised reticulocyte counts:

Found when there is an increase in red blood cell production as occurs in:

– Haemolytic anaemias (with effective erythropoiesis).

– Following acute blood loss

– After iron therapy for iron deficiency anaemia or specific therapy for megaloblastic anaemia

Reticulocyte responses are higher with haemolysis than haemorrhage.

Decreased reticulocyte count:

Associated with ineffective erythropoiesis or decreased production of red cells.

4.5 Haemoglobin Electrophoresis.

Haemoglobin electrophoresis is used to separate and identify the different haemoglobins by their

migration within an electric field. Haemoglobin variants separate at different rates due to

differences in their surface electrical charge as determined by their amino acid structure.
Alkaline cellulose acetate electrophoresis.

Several techniques are available to separate haemoglobin variants by electrophoresis. For routine

work, electrophoresis in an alkaline buffer at pH 8.4–8.6 using a cellulose acetate membrane is

adequate. This gives good separation of HbA, HbF, HbS, and HbC. On alkaline electrophoresis

HbD and HbS have the same mobility and HbC, HbE and HbO also co-migrate. In specialist

laboratories agarose gel electrophoresis at an acid pH (6.0) can be used to separate these

haemoglobins and also to provide a clear separation of HbF from HbS and HbC.

Method

The procedure for performing alkaline cellulose acetate haemoglobin electrophoresis using

Helena BioSciences equipment and Mylar-backed supported cellulose acetate membranes (Titan

111 cellulose acetate plates) is supplied with the membranes. The following is a summary of the

method used to separate the different haemoglobins (e.g. Hb A, F, S, and C), excluding staining

and densitometry to quantitate the relative percentage of each haemoglobin band.

Prepare the cellulose acetate membrane (Titan 111 cellulose acetate plate) exactly as described

in the Helena BioSciences procedure. Pour 100 ml of the Tris-EDTA-borate buffer into each of

the outer sections of the Zip-Zone electrophoresis chamber. Wet two wicks (as supplied) in the

buffer and drape one over each support bridge, ensuring each makes contact with the buffer and

that there are no air bubbles under the wicks. Cover the chamber to prevent evaporation. Transfer

1 of each haemolysate sample (tests and controls) into the Zip-Zone well plate. Place a cellulose

acetate membrane (plate) in the Zip-Zone aligning plate and apply the samples using the 8 unit

applicator exactly as described in the Helena BioSciences procedure. Immediately place the
cellulose acetate membrane (plate) in the electrophoresis chamber, cellulose acetate side down.

Connect the chamber to the Power Supply and electrophorese the plate for 25 minutes (or

shorter) at 350 volts and 50 mA.

Results

The relative mobilities of haemoglobins A, F, S, D, E, C and H after alkaline electrophoresis.

In alkaline buffer, HbS and HbD have similar mobility and HbC, HbA2, HbE, and HbO

have similar mobility.

4.6 Coagulation Tests

These are tests done to investigate coagulation disorders. They include; activated partial

thrombine (aPPT), prothrombine time test (PT) and thromnin time (TT) test.

Prothrombin time (PT) test.

The PT is a screening test for the extrinsic clotting system, i.e. factor VII. It will also detect

deficiencies of factors, prothrombin, V, X, and fibrinogen. It is mainly used to monitor patients

receiving warfarin anticoagulation.

Principle of the test.

Plasma or capillary blood is added to a thromboplastin and calcium chloride reagent at 37 _C and

the time taken for a clot to form is measured. The clotting time in seconds is converted

to the International Normalized Ratio (INR), usually by reference to a table provided by the

manufacturer of the reagent or from the formula.

Reference PT range.

Normal plasma samples (patients not on anticoagulant) clot in 11–16 seconds. Each laboratory

should establish its own normal reference range. Main causes of a prolonged PT test are:

Treatment with oral anticoagulant drugs (vitamin K antagonists) such as warfarin.

● Liver disease

● DIC

● Haemolytic disease of the newborn

● Rarely a deficiency of factor VII, X, V, or prothrombin.

Thrombin time (TT) test.

The TT test is sensitive to a deficiency of fibrinogen or inhibition of thrombin. It measures the

formation of a fibrin clot by the action of thrombin on fibrinogen.

Principle of test.

Thrombin is added to citrated plasma at 37 _C. The time taken for the mixture to clot is

measured and the appearance of the clot noted.

Reference TT range.

Normal plasma samples clot within 12–15 seconds.The thrombin time is prolonged in the

following Conditions;

DIC and other conditions which produce a low fibrinogen level.

 Abnormal fibrinogen.

Treatment with heparin

Liver failure.

Presence of inhibitors of thrombin such as FDPs.

Activated partial thromboplastin time (APTT) test

The APTT is a screening test of the intrinsic clotting system. It will detect the inhibition or

deficiency of one or more of the following factors: prothrombin, V, VIII (antihaemophilic

factor), IX, X, XI, XII and fibrinogen. The APTT is also used to monitor patients being treated

with heparin.

Principle of test.

Kaolin (surface activator) and platelet substitute (phospholipid) are incubated with citrated

plasma at 37 _C for the time specified in the test method. Calcium chloride (CaCl2) is added and

the time taken for the mixture to clot is measure

Reference APTT range.

Normal plasma clots in 36–50 seconds

Causes of a prolonged APTT include:

● DIC (involving several clotting factors)

● Deficiency of clotting factors: prothrombin, V, VIII, IX, X, XI or XII due to vitamin K

deficiency, liver disease, heparin or warfarin anticoagulation, or less commonly an inherited

coagulation disorder.
 CHAPTER FIVE

5.0 BIOCHEMISTRY

Specimens handled include: Blood and serum, Tests: Liver function tests, Glucose tests, Renal

function Test, Lipid profile and Bone profile

5.0 Renal Function tests.

Blood urea.

Value of the test.

Urea is synthesized in the liver as a by product of the deamination of aminoacids. Its eliminatin

in the urine represents the major route of nitrogen excretion. Elevated urea in plasma is found as

a result of high protein diet, increased protein catabolism, gastrointestinal hemorrhagae, mild

dehydration, shock and heart failure.

Principle of the diacetyl monoxime urea

method

Urea reacts with diacetyl monoxime at high temperature in an acid medium in the presence

of cadmium ions and thiosemicarbazide. The absorbance of the red colour produced is measured

in a colorimeter using a green filter 520 nm.

Test results

Approximate urea reference (normal) range

Adults: 3.3–7.7 mmol/l (20–46 mg%)

Infants: 1.3–5.8 mmol/l (8–35 mg%)

Serum electrolytes.

Value of tests

The measurement of sodium, potassium, or both electrolytes is usually requested in the

assessment of renal function, to assist in the management of a

patient that is unconscious or confused or a diabetic patient with ketoacidosis, to assess and

monitor states of dehydration (particularly an infant losing fluid), to monitor diuretic therapy and

to assist in fluid replacement therapy.

Test Results

Approximate reference (normal) ranges

Sodium:

134–146 mmol/l (134–146 mEq/l)

Potassium:

Adults: 3.6–5.0 mmol/l (3.6–5.0 mEq/l)

Newborns: 4.0–5.9 mmol/l (4.0–5.9 mEq/l)

Values are highest immediately after birth.

Serum Creatinine.

Value of test

Measurement of serum or plasma creatinine is an important test of kidney function. It is

recommended in preference to the measurement of serum or plasma urea because it is a better

indicator of overall renal function and progress in renal failure. Serum creatinine levels are less
affected than urea levels by age, dehydration, and catabolic states, e.g. fever, sepsis, and internal

bleeding. Creatinine levels are also less influenced by changes in diet such as low intake of

protein (providing this is not prolonged). Increasingly the measurement of serum creatinine

is being used to investigate HIV associated renal disease and to monitor patients being treated

with nephrotoxic antiretroviral drugs, e.g. tenofovir.

Principle of the Jaffe-Slot alkaline picrate creatinine method

Creatinine reacts with picric acid in an alkaline medium. The absorbance of the yellow-red

colour produced is measured in a colorimeter using a bluegreen filter 490nm.A number of other

compounds similarly react with picric acid giving artificially high results for creatinine if one

simply measures the total yellow-red colour produced. A second reading is thereforemade after

making the solution acid. The colour produced by creatinine is quickly destroyed by acid

whereas that given by non-creatinine chromogens is destroyed more slowly. By subtracting the

second reading which is due to non-creatinine substances from the first reading (due to creatinine

and noncreatinine substances), the colour produced by the true creatinine can be obtained.

Test results

Approximate creatinine reference (normal) range

Males: 60–130 _mol/l (0.7–1.4 mg%)

Females: 40–110 _mol/l (0.4–1.2 mg%)

5.2 Liver Function Tests

Serum Bilirubin.

Value of test.

The measurement of serum or plasma bilirubin is usually performed to investigate the causes of

liver disease and jaundice, and to monitor a patient’sprogress, e.g. an infant with serious neonatal

jaundice (high levels of unconjugated bilirubin).

Principle of the Jendrassik and Grof bilirubin method.

Sulphanilic acid is diazotized by the nitrous acid produced from the reaction between sodium

nitrite and hydrochloric acid. Bilirubin reacts with the diazotized sulphanilic acid (diazo reagent)

to form azobilirubin. Caffeine is an accelerator and gives a rapid and complete conversion to

azobilirubin. The pink acid azobilirubin is converted to blue azobilirubin by an alkaline tartrate

reagent and the absorbance of the blue-green solution is read in a colorimeter using an orange

filter 590 nm. Measurement of the azobilirubin in an alkaline medium removes turbidity and

increases specificity. There is very little interference by other pigments at 600 nm wavelength.

Conjugated (direct) bilirubin: This is measured in the absence of the caffeine-benzoate catalyst

and at an acid pH. Under these conditions only the conjugated bilirubin will react. The reaction is

terminated by ascorbic acid and alkaline tartrate is added. The concentration of unconjugated

bilirubin can be calculated by subtracting the conjugated bilirubin value from the total bilirubin

value.

Test Results.
Approximate total bilirubin reference (normal) range

Adults: 3–21 _mol/l (0.2–1.3 mg%)

Newborns: 8–67 _mol/l (0.5–4.0 mg%)

Serum Albumin.

Value of test

Serum albumin is mainly measured to investigate liver diseases, protein energy malnutrition,

disorders of water balance, nephrotic syndrome, and proteinlosing gastrointestinal diseases.

Principle of the BCG albumin method.

Bromocresol green is an indicator which is yellow between pH 3.5–4.2. When it binds to

albumin the colour of the indicator changes from yellow to bluegreen. The absorbance of the

colour produced is measured in a colorimeter using an orange filter

590 nm.

Test Results.

Approximate albumin reference (normal) range

30–45 g/l

Serum Alanine amino transferase.

Value of test
Measurement of ALT activity is mainly performed to investigate liver disease. Increasingly ALT

is being measured to monitor patients receving antiretroviral drugs associated with

hepatotoxicity such as nevirapine (NVP) and stavudine (d47). While both ALT and AST are

raised with hepatocellular injury, ALT is more specific for detecting liver cell damage.

Principle of Reitman-Frankel ALT method

ALT is incubated at 37 °C for exactly 30 minutes in a pH 7.4 buffered substrate containing

alanine and -ketoglutarate. ALT catalyzes the transfer of the amino group from alanine to

ketoglutarate, forming pyruvate and glutamate. The pyruvate reacts with 2,4-

dinitrophenylhydrazine (DNPH) to form 2,4-dinitrophenylhydrazone which in an alkaline

medium gives a red-brown colour. The absorbance of the colour produced is measured in a

colorimeter using a green filter 520 nm (Ilford No. 604) or in a spectrophotometer

at 505 nm wavelength.

Test Results

Approximate ALT reference (normal) range

5–35 IU/l
5.3 Pancreatic Function Tests.

Serum Alpha-amylase

Value of test

Measurement of serum or plasma amylase activity is usually requested to assist in the

differentiation of acute pancreatitis from other acute abdominal disorders. It is an early indicator

of acute pancreatitis.

Principle of the Caraway Somogyi amylase method

The test is based on the hydrolysis of starch by amylase and the blue-black complex that forms

when iodine reacts with starch. Amylase is incubated at 37 °C for exactly 7 minutes in a pH 7.0

phosphate buffered starch substrate. The enzyme hydrolyzes the starch to maltose and other

fragments. The amount of starch which remains at the end of the incubation period is shown by

the addition of an iodine solution, which produces a blue-black colour. A reagent blank is used

which contains starch and iodine but no serum. The absorbances of the test and blank solutions

are read in a colorimeter using a red filter 680 nm or in a spectrophotometer

set at 660 nm.

The amylase activity is measured by the difference in absorbance of the starch-iodine complex of

the test against that of the reagent blank in which there is no hydrolysis. The result is expressed

in international units per litre (U/l).

Test Results
Approximate amylase reference (normal) range

70–340 U/l

5.4 Blood Glucose Tests

Value of test

Plasma or blood glucose is measured mainly in the diagnosis and management of diabetes

mellitus. Good control of blood glucose levels in diabetic patients helps to prevent or delay the

development of complications which may lead to premature disability or death from blindness,

kidney failure, coronary thrombosis, stroke, bacterial infections (particularly mycobacterial and

anaerobic infections), and fungal infections.

Terms used to describe the collection of blood glucose specimens.

Fasting specimen: This refers to blood collected after a period of no food intake. For adults the

fasting time is usually 10 to 16 hours. For children the fasting time is 6 hours unless a longer

time is indicated, e.g. when investigating hypoglycaemia. The drinking of plain water is

permitted.

Post-prandial specimen: This describes blood collected after a meal has been taken. The sample

is usually taken as a 2 h postprandial specimen.

Random specimen: This refers to a blood sample collected at any time, regardless of food

intake.
Test Results

Approximate glucose reference (normal) range

Adults

– Fasting (plasma) 3.6–6.4 mmol/l.

– Random (plasma) 3.3–7.4 mmol/l.

Children

– Fasting (plasma) 2.4–5.3 mmol/l.

Newborn values are slightly lower, i.e. 1.1–4.4 mmol/l

Glucose tolerance test (gtt)

Glucose tolerance: A GTT measures the ability of the body to tolerate, or cope with, a standard

dose of glucose. The degree of tolerance to the glucose, as shown by a change in the blood level,

is mainly dependent on the rate of glucose absorption and on the insulin response. As the glucose

is absorbed, the level of glucose in the blood rises and the normal response is for insulin to be

released from the pancreas to lower the glucose level. Tolerance is reduced when insulin is

insufficient or absent. A glucose tolerance test (GTT) is usually requested to investigate

glycosuria or when a random or fasting blood glucose is suggestive but not diagnostic of

diabetes. It happens only rarely however that the result of a fasting or random blood glucose is

difficult to interpret and a GTT is necessary. If glycosuria is found, measurement of fasting

glucose should be performed before the patient is subjected to a GTT. A GTT should not be

necessary in children.
Glycated haemoglobin in monitoring blood glucose control in diabetes.

Red cells normally contain some haemoglobin A in glycated form, i.e. attached to a glucose

residue. Glycated (glycosylated) haemoglobin A is referred to as HbA1. The main glycated

fraction is HbA1c (forms about 5% of circulating Hb). HbA1c is specifically glycated by

glucose. The glucose remains complexed to the haemoglobin molecule for the lifetime of the red

cell and therefore the concentration of glycated haemoglobin circulating in red cells is a guide to

the average blood glucose level over a period of the previous 8–12 weeks (lifespan of red cells).

Measurement of the glycated haemoglobin can help in assessing the control of diabetic patients,

particularly patients with type 2 diabetes whose blood glucose levels do not change markedly.

The test does not detect swings from high to low blood glucose levels, as it reflects an average of

preceding glycaemia. Nevertheless, HbAlc levels are closely related to the risk of complication

development in diabetes. Levels of 7.0% or below are ideal, though these are often

hard to achieve. Microchromatographic and ion-exchange resin colorimetric methods for

measuring glycated haemoglobin are available but expensive. HbA1c results are expressed as a

percentage of total Hb. In poorly controlled diabetes the level of HbAlc may be greatly

increased. Reference ranges are assay specific and to some extent are not relevant to the clinical

situation, as “target levels” (based on previous clinical research) are of more use. With some

assays, the presence of abnormal haemoglobins such as Hb S, C, D and increased levels of HbF

can affect test results.

 CHAPTER SIX

6.0 MICROBIOLOGY.

6.1 Specimens for culture.

Specimens for culture include; Stool, urine, blood, CSF, high vaginal swab, wound swabs.

The laboratory investigation of microbial diseases involves: Examining specimens to detect,

isolate, and identify pathogens or their products using:

– Microscopy

– Culture techniques

– Biochemical methods/tests

6.1.2 Microscopy.

To assist in the diagnosis of microbial infections, microorganisms can be examined

microscopically for their motility, morphology, and staining reactions.

Examples

Motile Vibrio cholerae in a rice water faecal specimen from a person with cholera.

Treponema pallidum in chancre fluid (using dark-field microscopy), establishing a diagnosis of

primary syphilis.

Fungal hyphae and arthrospores in a sodium hydroxide preparation of skin from a person with

ringworm.
Gram negative reaction and characteristic morphology of Neisseriae gonorrhoeae (intracellular

diplococci) in a urethral discharge from a man with gonorrhoea.

Gram positive reaction and morphology of pneumococci in cerebrospinal fluid from a patient

with pneumococcal meningitis.

Gram positive reaction and morphology of yeast cells in a vaginal discharge from a woman with

vaginal candidiasis.

Acid fast reaction of Mycobacterium tuberculosis in Ziehl-Neelsen stained sputum from a person

with pulmonary tuberculosis.

6.2 Culture Techniques.

The culture of pathogens enables colonies of pure growth to be isolated for identification and,

when required, antimicrobial susceptibility testing.

6.3 Biochemical Tests.

Following culture, biochemical tests are often required to identify pathogens including the use of

substrates and sugars to identify pathogens by their enzymatic and fermentation reactions.

Examples

Catalase test to differentiate staphylococci which produce the enzyme catalase from streptococci

which are noncatalase producing.

Oxidase test to help identify Vibrio, Neisseria, Pasteurella and Pseudomonas species, all of

which produce oxidase enzymes.

Coagulase test to help identify Staphylococcus aureus which produces the enzyme coagulase

(coagulates plasma).

Fermentation tests to differentiate enterobacteria, e.g. use of glucose and lactose in Kligler iron

agar medium to assist in the identification of Shigella and Salmonella organisms.

Indole test to detect those organisms that are able to break down tryptophan with the release of

indole. It is mainly used to differentiate Escherichia coli from other enterobacteria.

Urease test to assist in the identification of organisms such as Proteus species which produce the

enzyme urease.

6.4 Staining Techniques.

Gram technique

The Gram staining reaction is used to help identify pathogens in specimens and cultures by their

Gramreaction (Gram positive or Gram negative) and morphology. Pus cells can also be identified

in Gram smears.

Gram positive bacteria: Stain dark purple with crystal violet (or methyl violet) and are not

decolorized by acetone or ethanol. Examples include species of:

Staphylococcus Actinomyces

Streptococcus

Clostridium

Corynebacterium
Gram negative bacteria: Stain red because after being stained with crystal violet (or methyl

violet) they are decolorized by acetone or ethanol and take up the red counterstain (e.g. neutral

red, safranin, or dilute carbol fuchsin). Examples include species of:

Neisseria Klebsiella

Haemophilus Brucella

Salmonella Yersinia

Shigella Coliforms

Vibrio

Method

Fix the dried smear it should be fixed with methanol for 2 minutes (avoids damaging pus cells).

Cover the fixed smear with crystal violet stain for 30–60 seconds. Rapidly wash off the stain

with clean water. Tip off all the water, and cover the smear with Lugol’s iodine for 30–60

seconds. Wash off the iodine with clean water. Decolorize rapidly (few seconds) with acetone–

alcohol. Wash immediately with clean water. Cover the smear with neutral red stain for 2

Minutes. Wash off the stain with clean water. Wipe the back of the slide clean, and place it in a

draining rack for the smear to air-dry. Examine the smear microscopically, first with the 40_

objective to check the staining and to see the distribution of material, and then with the oil

immersion objective to report the bacteria and cells.

 CHAPTER SEVEN

7.0 IMMUNOLOGY LABORATORY.

Immunology Lab carried out enzyme linked immunorsobent tests and slide agglutination tests.

7.1 Enzyme-linked Immunoassays.

Routine Elisa tests were for HIV, HBV, HCV, Toxoplasma, herpes virus, rubella virus and

cytomegalovirus.

General principle of ELISA.

These tests employs the principle of antigen –antibody reactions whereby recombinant

antigens and antibodies are coated onto micro titer wells then test serum is added into the wells

and incubation takes place to ensure that if the antigen or antibody is looked for in the patients

serum is present it will bind to the solid phase of the well plate. The wells are then washed to

remove any unbound IgGs or other proteins which might lead to false positive results. The

enzyme conjugate is then added so that if there was an Ag-Ab reaction it will bind to the second

antibody. The wells are washed again to remove any unbound enzyme conjugate. Substrate is

added followed by a an incubaton step and later a stop solution is added. Color development is

stopped by the addition of stop solution and its intensity is measured spectrophotometrically at

450nm.

7.2 Slide agglutination Tests.

Rapid plasma regain test

Syphilis is transmitted by the spirochete of T. pallidum. After infection there is a formation of

treponemal antibodies to T.pallidum and the body also forms non-treponemal anti-lipoidal

antibodies in response to the lipoidal material released from the damaged host cell. The RPR test

is a macroscopic non-treponemal flocculation test for the detection of antilipoidal antibodies.

Principle.

The specimen is mixed with RPR reagent and allowed to react for 8 minutes. The presence of

antilipoidal antibodies is detected is detected by the formation of visible floccules. Negative

samples show no flocculation.

Procedure.

Place one drop of test sample, positive and negative controls onto separate reaction circles of the

disposable slide using a simple dispensing pipette. Add one drop of well mixed RPR reagent

thoroughly spreading uniformly over the entire circle. Immediately start a stopwatch. Rotate the

slide gently and continuously manually or using a mechanical rotor at 100rpm. Observe for

flocculation macroscopically after 8 minutes.

CHAPTER EIGHT

HISTOPATHOLOGY & CYTOLOGY LAB.

Histopathology is the microscopic study of the tissues of the body affected by disease.

Specimens handled include; tissues.

8.0 Tissues Processing.

In tissue processing the tissues specimen are treated with various agents to enable the production

of thin of thin sections that can be observed under a microscope.

Fixation; the tissues are fixed in 10% formal saline for 1 hr. Fixation is to maintain the

morphology of cells .

Dehydration; is the removal of water from the tissues. It’s necessary because many embedding

media are immiscible with water. The tissues are dipped in 70% alcohol for 1hr, then in 90%

alcohol for 1 hr and 100% for 1 hr.

Clearing; the tissue id dipped in a clearing solution for 1 hr. The clearing agent used was xylene.

Clearing is the replacement fluid with substances that are miscible with the embedding medium.

Impregnation; the tissues are dipped a paraffin wax well for three hours, they are immersed in

another paraffin wax for another 3 hrs. Impregnation is completely is the process of completely

saturating the tissues with the medium to be used for embedding.

Embedding; Tissues are embedded in wax using a wax dispenser. The wax is then cooled and

solidifies thus providing external suppor for the tissue and this will facilitate easy sectioning of

tissues.

Microtomy; after the tissue has been embedded its cut into sections that can be examined under

the microscope. Routinely sections between 5-10 microns are cut. The sectioning is done using a

specialized machine called a microtome. Staining; Histological stains include Hematoxyllin and

eosin.

8.2 Hematoxylin and eosin staining.

Principle

The oxidation product of haematoxylin is haematin, and is the active ingredient in the staining

solution.  Haematoxylin is not classified as a dye since the molecule possesses no chromophore. 

Thein situ oxidation of haematoxylin is effected by the addition of a strong oxidant to the stain, in

this case sodium iodate. 

Staining Procedure.

Deparaffinize sections, 2 changes of xylene, 10 minutes each. Re-hydrate in 2 changes of

absolute alcohol, 5 minutes each. Dip in 95% alcohol for 2 minutes and 70% alcohol for 2

miuntes. Wash briefly in distilled water. Stain in Harris hematoxylin solution for 8 minutes.

Wash in running tap water for 5 minutes. Differentiate in 1% acid alcohol for 30 seconds. Wash

running tap water for 1 minute. Bluing in 0.2% ammonia water or saturated lithium carbonate
solution for 30 seconds to 1 minute. Wash in running tap water for 5 minutes. Rinse in 95%

alcohol, 10 dips. Counterstain in eosin-phloxine solution for 30 seconds to 1 minute. Dehydrate

through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each. Clear in 2 changes of

xylene, 5 minutes each. Mount with xylene based mounting medium.

8.3 Pap staining Technique.

Procedure 1 (Standard Method):

Dip in 95% Ethanol 15 minutes (fixation). Rinse in tap water. Place in Harris or Gill

Hematoxylin 1-3 minutes (Time vary with selection of hematoxylin solution). Rinse in tap water

or Scott's tap water. In 95% Ethanol make 10 dips. Dip in OG-6 stain for 1.5 minutes. Dip in

95% Ethanol 10 dips. EA-50, or Modified EA-50, or EA-65 stain for 2.5 minutes. 95% Ethanol

10 dips, 2 changes. 100% Ethanol 1 minute. Clear in 2 changes of xylene, 2 minutes each. Mount

with permanent mounting medium.

REFERENCES.

1. Howard University Department of Pathology Standard Operating Procedures/

Hematology.

2. Mc Kenzie, S. Clinical Laboratory Hematology, Pearson Prentice hall 2004, p.142

Hematology procedures and Job Aids.

3. Henry, J.B Clinical Laboratory & Management by Laboratory Methods, 20th Edition. WB

saunders, 2001.

4. Monica Cheesbrough District-Laboratory-Practice-in-Tropical-Countries Part-1 & 2, 2 nd

Edition,2006.

5. General Clinical Laboratory Manual for reference and diagnosis.g