MLT 201: INTRODUCTION TO MEDICAL LABORATORY SCIENCE

Medical Laboratory Science entails the Study of human and animal tissues, and fluid,
excretions and production of biological for the purpose of diagnosis, fluid treatment and
research to the extent that they relate to the state of wellbeing of the person(s) or
animals whose tissues or excretion are involved.

A medical laboratory or clinical laboratory is a laboratory where tests are usually

done on clinical Specimens in order to obtain information about the health of a patient as
pertaining to the diagnosis, treatment, and prevention of disease.

The laboratory plays a major role in the diagnosis and treatment of diseases by
performing qualitative, quantitative or screening test procedures on materials derived
from the body.

Medical laboratory science is an umbrella field comprising a number of different

discipline. The major disciplines of medical laboratory science are.

1. Medical microbiology.

2. Haematology.

3. Histopathology.

4. Clinical chemistry.

1. MEDCAL MICROBIOLOGY.

Microbiology is the study of organisms and agents that are generally too small to
be seen clearly by the unaided eye. These organisms include viruses, bacteria,
algae, fungi, and protozoa. Medical microbiology is a branch of medical science
concerned with the prevention, diagnosis and treatment of infectious diseases. In
addition, this field of science studies various clinical applications of microbes for
the improvement of health. Sub specialties in medical microbiology include
medical virology, bacteriology, parasitology, immunology, mycology.

Specimen analysed in medical microbiology urine, blood, sputum, stool, semen,

urogenital specimen, cerebrospinal fluid, throat and mouth Species, Pus, ulcer
material, skin specimen e.t.c.

Investigations carried out in medical microbiology laboratory includes; microscopy,

culture and sensitivity of clinical specimen as Listed above.

Also, serological tests for rapid diagnosis of microbial diseases are carried out in medical
microbiology laboratory. Such serological tests include Widal test, VDRL, antibody
screening for HIV, Hepatitis C Virus, Hepatitis B antigen test (HBsAg, HBeAg, HBcAg)
e.t.c.

Equipment in Medical microbiology laboratory includes: microscope, incubator, hot air

oven, autoclave, anaerobic jars, Bunsen burner, wire loops, refrigerator, centrifuges e.t.c
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Specimen Collection in Microbiology Laboratory

Specimens for microbiological investigation must be collected correctly if pathogens are

to be successfully isolated and identified, reports are not to be misleading and resources
are not to be wasted. The value and reliability of microbiological reports are directly
affected by the quality of the specimen received and the length of time between its
collection and processing.

The collection of specimen must form part of the department’s SOPs (Standard
Operating Procedure) and the laboratory should issue written instructions to all those
responsible for collecting specimen for microbiological examination. Such instructions
should include:

 The amount and type of specimen required, container to use, and need for any
preservative or transport medium. The correct type of specimen depends on the
type of pathogen to be isolated, e.g sputum not saliva is essential for the isolation
of respiratory pathogens.

 Best time to collect a specimen. Specimens such as urine and sputum are best
collected soon after the patient wakes when organisms have the opportunity to
multiply over several hours. Efforts must be made to collect specimen for
microbiological examination on before antimicrobial treatment starts.

 Aseptic method of collection to avoid Contamination and accidental Labeling of

the specimen container. Each specimen must be Clearly labeled with the date and
time of collection and the patient 's name, number, ward or health Centre.

Condition in which specimen must be kept prior to and during transportation.

Specimen Reception in Microbiology Laboratory

Each specimen must be accompanied by a request form which provides details of the
patient (name, age, gender, inpatient or outpatient number, ward, home area).
provisional diagnosis, clinical note, name of requesting doctor and the date and time the
specimen was collected.

The laboratory staff must check that the name/number on the specimen container is the
same, as that on the request form. A specimen must not be accepted if it is unlabeled,
incorrectly labeled, or is obviously not suitable for testing e.g saliva instead of sputum,
any specimen not collected in appropriate container; Most specimens not microbial
culture requires sterile Containers and aseptic collection technique.

Specimens that meet the required criteria are received and registered. Columns in a
reception register should include date, serial number, lab number, name of patient, age,
sex, Hospital/ward, investigation required, nature of specimen, requesting doctor,
amount paid and receipt number.

2. HAEMATOLOGY.

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“Haematology" means study of blood. In clinical set up, a haematology laboratory is
concerned with the abnormalities of the constituents of blood, namely the plasma and
red blood cells (red blood cells, white blood cells and platelets) many diseases in tropical
countries produce blood cell changes which can be detected in laboratory tests.

Haematological tests include the following depending on the local health needs,
resources and availability of staff.

 Measurement of haemoglobin.

 Measurement of pack cell volume (haematocrit) and calculation of red cell indices.

 White blood cell (WBC) count.

 Platelet count.

 Differential WBC count.

 Reporting blood films: red cells, white cells, platelets.

 Erythrocyte sedimentation rate.

 Reticulocyte count.

 Investigation of sickle cell disease.

 Tests to screen for bleeding disorders.

 Haemoglobin electrophoresis e.t.c.

Specimen Collection for Haematological Investigation.

Blood must be collected with care and adequate safety precautions to ensure test results
are reliable contamination of the sample is avoided and infection from blood
transmissible pathogen is prevented. Protective gloves should be worn when collecting
and handling blood samples. Lancets, needles and syringes must be sterile and dry.

Capillary blood collection: capillary blood is mainly use when the patient is an
inventor young child and the volume of blood required is small e.g to measure
haemoglobin, perform a WBC count and to make thick and thin blood films.

Capillary blood can be obtained from the tip off the finger from adults, and from the heel,
or the large toe from infants.

However, the use or capillary blood should be avoided as far as possible because the
high risk of sampling error and of infection. Repeat testing is usually restricted because
quantity of blood collected is usually small.

Technique.

 Select an appropriate site for puncture.

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  Clean the area vigorously with 75% alcohol and allow it to dry.

 Make a firm quick stab with the lancet.

 Wipe away the first drop of blood using a clean dry cotton swab.

 Carefully draw blood into an appropriate pipette.

 Deliver the blood for processing.

Venous blood collection: the most common sites for venipuncture are the veins inside
the bend of the elbow (the antecubital fossa). Other sites include veins in the wrist or
ankle if necessary.

Technique.

 Apply a tourniquet to the upper arm sufficiently tightly to restrict the venous flow
and make the veins stand out. The patient should keep the arms Straight with a
clenched fist.

 Swab the selected vein and site with 75% alcohol and allow it to dry.

 Prepare the syringe and appropriate containers.

 Using the left thumb, press just below the puncture site to anchor the vein.

 Insert the needle smoothly with bevel facing upwards, at an angle of 20° to 30° to
surface of the arm, and in direct line with the vein.

 When the needle has entered the vein, blood is withdrawn into the syringe and
tourniquet released.

 When sufficient quantity of blood is collected, loosen the tourniquet, place a wad of
cotton wool at the punctured site and withdraw needle gently.

 The punctured site should be kept pressed to stop the flow of blood.

 Ask the patient to release the clenched fist.

 Detach the needle and discard in an appropriate disposal container.

 Dispense the blood in the sample bottles as required.

 Mix the blood to be anticoagulated.

 Apply a strip dressing at the punctured site.

Anticoagulants in Haematology.

Many haematological tests require unclotted blood specimens. Such a sample can be
obtained by collecting it in a tube containing an anticoagulant. There are various types of
anticoagulants used in haemotology, each having its advantages and disadvantages. A

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 suitable anticaogulant should be selected according to the investigation required.
Anticoagulants commonly used in Haemotology include EDTA (Ethylenediamine Tetra
acetic Acid), Trisodium citrate and heparin.

Colour Substance Amount Mode of Uses Advantage Disadvantag

Code action s es

EDTA Pink Di or 1.5mg/ Chelatio Routine & Preserves Not suitable

Tripotassiu ml n of Cellular morpholog for
m EDTA Calcium Haemotolog y of RBC coagulation
y and WBC. studies
No platelet
clumping

Trisodiu Purple 32.0 g/l 1 Binds Studies of Coagulatio Not suitable

m citrate volume calciium Haemostasi n factors for cell
to 9 s stable. counting due
volumes Platelet to dilution
of blood function
retained

Trisodiu Purple 30.8 g/l 1 Binds Wester gren Reference

m citrate and trisodium volume calcium ESR method
white citrate to 4
solution volumes
of blood

Heparin Orang Lithium 1-5 Anti-Xa Osmotic Ca2+ still Unsuitable

e heparin mg/ml and IIa fragility cell present for blood
factors culture films, WBC
and platelet
counting and
coagulation
studies

Specimen Reception in Haematology

Each specimen must be accompanied by a request form which provides details of the
patient (name, age, gender, inpatient or outpatient number, ward, home area).
provisional diagnosis, clinical note, name of requesting doctor and the date and time the
specimen was collected.

The laboratory staff must check that the name/number on the specimen container is the
same, as that on the request form. A specimen must not be accepted if it is unlabeled,
incorrectly labeled, or is obviously not suitable for testing e.g clotted blood in an
anticoagulated container.
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Equipment in Haematology Laboratory:

Equipment required for routine haematology investigations include microscope, water

bath, ESR stand, electrophoretic tank, refrigerator, mechanical differential white cell
counter, bench centrifuge, microhaematocrit centrituge, weighing balance/ scale,
pipettes, mixers and rotators, test tubes, grouping tiles, auto haematology analysers
e.t.c.

3. CHEMICAL PATHOLOGY.

Chemical pathology, also known as clinical chemistry is the division of pathology that
deals with observation and investigation of biochemical changes in the body. It involves
the detection and measurement of biochemical constituents of body fluids and
excretions.

Specimen collected for analysis in clinical chemistry laboratory includes: blood, urine,
cerebrospinal fluid (CSF), aspirates/other body fluid and faecal specimen.

Chemical parameters tested for in clinical chemistry laboratory include; glucose, protein,
urea, creatinine, electrolytes, enzymes, hormones etc.

Equipment in clinical chemistry laboratory includes: colorimeters, spectrophotometer,

flame photometers, water bath, auto analyzers, Pipettes, test tubes, centrifuge,
Glucometer e.t.c.

Specimen Collection in Clinical Chemistry Laboratory

Collection of blood specimens

Venipuncture is the accepted method of blood collection. Factors that affect the
correctness of chemistry test results do originate from blood collection. These factors
include:

i. Incorrect venepuncture technique.

ii. Haemolysis.

iii. Use of wrong specimen container.

iv. Instability of some chemical substances.

I. Venepuncture Technique:

 Do not apply the tourniquet too tightly or for too long a period because this Will
cause venous stasis leading to a concentration of substances in the blood such as
haemoglobin, plasma proteins, potassium and calcium.

 Do not collect blood from an arm pit which an intravenous infusion is being given.

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  If an anticoagulated blood is required, add the correct amount of blood to the tube
or bottle and mix the blood with the anticoagulant by gently inverting the container
several times.

II. Avoiding haemolysis:

The haemolysis (rupture) of red cells can be a serious source of unreliable test results. If
red cells are haemolyzed, substances from cells are released into the serum or plasma
leading to a false increase in the concentration of the analytes, e.g potassium,
Haemolysis therefore interferes with many chemical reactions.

Haemolysis can be avoided by

 Checking that needle and syringe are dry and that the plunger of the syringe fit
well.

 Not using a needle with too fine a bore.

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

 Removing the needle from the syringe before dispensing the blood into the
specimen container. Allow the blood to rundown the inside wall of the container.

 Adding the correct amount of blood to anticoagulant. Do not shake the blood but
gently mix it with the anticoagulant.

 Using clean dry tubes for the collection of blood from which serum is to be required
and by allowing sufficient time for the blood to clot and clot a refrigerator to take place.

 Centrifuging blood sample for a minimum period of time.

 Not storing whole blood samples in, or next to the freezing compartment of
refrigerator.

III. Specimen containers and Anticoagulants. Specimen containers for clinical

Chemistry tests must be leak proof and chemically clean. For non-urgent blood tests, dry
tube or bottle is used for blood collection while for urgent blood tests; bottle containing
lithium heparin anticoagulant can be used.

Use of tube containing fluoride-oxalate is required for analysis of blood glucose. Fluoride
prevents the breakdown of glucose.

IV. Stability of analytes in blood specimens: Some chemical changes occur in

blood specimens within a few hours of being collected. Some of the changes can be
prevented by

 Separating the plasma or serum from the red cells as soon as possible (within 1
hour) after the blood has been collected.

 Not refrigerating the blood before separating serum or plasma.

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 Gvcolysis can be prevented by using fluoride oxalate anticoagulant.

 Protecting specimen from direct sun light will prevent decomposition bilirubin.

Collection of urine specimen

Specimen container for collecting urine specimen should be clean, dry, leak-proof, and
sufficiently wide-necked for the patient to use. lt must be free from all traces of
disinfectants. Whenever possible samples should be the first urine passed by the Patient
at the beginning of the day because this will generally contain the highest Concentration
of substances to be tested.

Chemical changes that occur after urine sample has been voided can be slowed down by
refrigerating the sample and also by adding preservatives such as hydrochloric acid and
thymol.

Specimen Reception and Registration in Clinical Chemistry:

Specimen Reception in Clinical Chemistry Laboratory

Each specimen must be accompanied by a request form which provides details of the
patient (name, age, gender, inpatient or outpatient number, ward, home area),
provisional diagnosis, clinical note, name of requesting doctor and the date and time the
specimen was collected.

The laboratory staff must check that the name number on the specimen Container is the
same as that on the request form, A specimen must not be accepted if it is unlabeled,
incorrectly labeled, or is obviously not suitable for testing e.g containing the incorrect
anticoagulant.

4. HISTOPATHOLOGY

Histopathology is the microscopic study of diseased or abnormal tissues. It is the study

of microscopic changes in tissues as a result of disease. It is derived from the
Combination of three Greek words: histos neaning tissue; pathos meaning disease or
suffering and logos meaning study. Histopathological examinations are carried out for
diagnostic purpose (biopsy) or as post mortem examination to ascertain cause of death.

Reception of Specimen in Histopathology Laboratory

On arrival in the departmental reception the specimen is checked at the earliest

opportunity for the following:

1. That the specimen is for histopathologica| examination.

2. That the container is clearly labeled and accompanied by a request form.

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 3. That sufficient fixative is in the Container or if the specimen is not in a fixative or is
in a wrong fluid.

After the above checks, the form is dated and stamped; the specimen is registered
given laboratory number which remains with the specimen until all the investigations
have been carried out.

All specimens sent to histopathology laboratory should be regarded as pathogenic

and handled with necessary safety precautions.

QUALITY ASSURANCE (QA).

QA includes all activities both in and outside the laboratory, performance standards,
good laboratory practice, and management skills that are required to achieve and
maintain a quality service and provide for continuing improvement. The purpose of
QA in laboratory practice is to provide test results that are relevant; reliable and
reproducible; timely and interpreted correctly.

Quality Assurance has been defined by WHO as the total process whereby the quality
of laboratory reports can be guaranteed. It has been summarized as the:

Right result at the

right time, on the

right specimen, from the

right patient, with result interpretation based on

Correct reference data, and at the

right price.

QA encompasses factors in three different areas namely:

1. Preanalytical Factors.

2. Analytical Factors.

3. Postanalytical Factors

Preanalytical Factors: These factors occur outside the laboratory and include patient
preparation and collection, handling and storage of specimen before processing.

i. Patient identification and Preparation: Before a sample is taken the patient

should be positively identified by verifying his name and identification number. The
laboratory should provide a procedure manual containing information on regarding
each test which should be made available to all medical and nursing staff involved

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 in specimen collection. Patient preparation may include instructions about
nutritional status, alcohol, drugs, smoking, exercise and sleep.

ii. Specimen Collection, Transport and Storage: The method and time of
specimen collection depend on the nature of the test requested. Guidelines on the
collection, transportation and storage should be included in the procedure manual.

iii. Specimen Reception and Recording: Each specimen must be accompanied by

request form which details patient information type of Specimen, clinical note and
clinical diagnosis Procedure manual Should include procedure to be followed for
each specimen prior to acceptance and recording in the laboratory. Criteria for
specimen rejection should be spelt out.

Analytical Factors: Analytical system checks are implemented to ensure that

results are accurate. It is made of internal quality control and external quality.

1) Internal Quality Control: Monitors daily quality control in a laboratory. It

includes staff development; maintenance of equipment and glass ware, quality control of
reagents, standards, control materials and reference ranges; and method selection and
evaluation.

2) External Quality Control: It is also referred to as proficiency surveys It is done

by comparing the performance of a laboratory with reference to others. Proficiency
testing programs send out samples of unknown result periodically to participating
laboratories. Samples are analyzed and results returned within a specified period.
Results are then compared with those of other laboratories and the performance of the
laboratory quality controlled or assessed.

3) Post Analytical Factors: Post analytical factors include recording and

transmitting patients results to the clinical within reasonable time interval. Results
should include reference ranges, critical and technical limits. Clerical mistakes must be
provided.

LABORATORY HAZARDS

The different hazards and accidents in the laboratory can be discussed under the
following areas:

1. Infection

2. Burns

3. Cuts and pricks

4. Hazards of toxic chemicals

5. Electric shocks.

Infection.

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Infections in the laboratory can occur in different ways, but the commonest causes are:

 Inhalation of pathogens in air-borne droplets which are released during breakage

or spilling of infectious fluid, centrifuging dispensing or pipetting of infectious materials;
and snap opening and closing of specimen containers.

 Ingestion of pathogens from contaminated food or finger or ingestion of pathogens

by mouth-pipetting.

 Pathogens finding their way into the body through pricks, cuts, scratches. insect-
bites, sores or skin lesions.

Burns

Burns sustained in the laboratory may be caused by:

 Inflammable substances catching fire.

 Fires from Bunsen burners, spirit lamps or from faulty or overloaded electric
circuits.

 Swallowing of corrosive Substances during pipetting or spilling Such substances on

the skin.

 Cuts and Pricks.

Cuts and pricks may result from:

 Edges of broken glassware.

 Edges of a knife.

 Accidental pricking with needle or any other sharp instrument.

 Walking on glass chippings.

INTRODUCTION

Medical laboratory science is concerned with the practice involving the analysis of
human or animal tissues, body fluids, excretions, production of biological, design and
fabrication of equipment for the purpose of laboratory diagnosis, treatment and
research.

CADRES IN MEDICAL LARORATORY SCIENCE

A Medical Laboratory Scientist (MLS): this is a healthcare professional that performs

chemical, hematological, histopathological, cytological, immunological, parasitological,
and bacteriological diagnostic analyses on body fluids such as blood, urine, sputum,
stool, cerebrospinal fluid (CSF), peritoneal fluid, pericardial fluid, tissue samples and
synovial fluid as well as other specimens.
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Medical Laboratory Technician (MLT): applies to persons trained to operate
equipment and performs tests under the supervision of a certified MLS in their
department.

Medical Laboratory Assistant: this is the lowest cadre of the medical laboratory
science profession trained to assist Scientists and Technicians and also perform some
minor laboratory test under supervision.

Medical laboratory science is a broad field of study that encompasses a number of

different fields/disciplines all aimed at assisting in the diagnosis and treatment of disease
by performing accurate test procedures or examinations of human samples.

The role of a medical laboratory is a key to health care system by:

 Carrying out laboratory tests ranging from simple to more complex tests to aid
medical diagnosis.

 Confirming the accuracy of test results and reporting findings to physicians.

 Regulating therapy.

 Monitoring of patient’s response to treatment.

 Performing equipment validation, Calibrations, quality controls e.t.c.

 Performs other administrative duties as maybe assigned.

 Training and retraining of laboratory personnel.

 Play important roles in disease prevention surveillance and control through public
health programs.

 Involve in medical and scientific research.

The following disciplines shall be considered in the study of this course:

 Medical Microbiology (Bacteriology, Parasitology and Virology).

 Chemical pathology

 Histopathology

 Forensic science.

 Haematology

 Immunohaematology/Blood Transfusion science.

MEDICAL MICROBIOLOGY

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Microbiology is the study of living organisms that are individually too small to be seen
with the naked eye. It considers the microscopic forms of life and deals with their
reproduction, physiology and participation in the process of nature, helpful and harmful
relationship with other living things, and significance to health.

Technology has contributed greatly to this effect such that these organisms can be
cultured on a media that can allow them to grow; these media have the nutritional
requirements needed by these microorganisms making them to grow as if they are in the
body. Through this means, scientists have been able to isolate, study and classify
microorganisms. Microbiology includes other disciplines like Parasitology, bacteriology
and virology.

Bacteriology

This is the study of bacteria. Bacteria are single cell microscopic prokaryotes that thrive
in diverse environments. They measure between 0.5-2.0m in diameter.

Classification of bacteria.

Morphology and staining of bacteria are the commonly used characteristics to classify
bacteria.

Morphology of bacteria.

When bacteria are visualized under light microscope, the following morphologies are
seen.

1. Cocci (singular coccus): Round or oval bacteria measuring about 0.5- 1.0mb in
diameter. They are found in single, pairs (diplococci. e.g Neisseria gonorrhea). chains
(streptococci) or clusters (staphylococci).

2. Bacilli (singular bacillus): Stick-like bacteria with rounded, tapered, square or

swollen ends: with a size measuring 1-1Oum in length by 0.3-1.Om in width.

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3. Spiral: Spiral shaped bacteria with regular or irregular distance between twisting E.g.
Spirilla and spirochaetes.

Spiral

-3 forms: vibrio, Spirillum, spirochete.

Classification based on staining characteristics

Gram and Ziehl Neelsen (Acid fast) staining methods are the most commonly used
techniques.

Gram's Staining

This method was developed by Christian Gram in 1884. It is used to differentiates

bacteria by the chemical and physical properties of their cell wall.

Principle; Gram positive organisms have a thick layer of peptidoglycan in the cell wall
made of teichoic acid that retains the primary stain- iodine complex while gram negative

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organisms have a thinner peptidoglycan layer in the cell wall that allows the primary
stain-iodine complex to wash out and stain with counter stains.

Required reagents:

 Primary stain (crystal violet, gentian violet, methyl violet)

 Gram's Iodine

 Acetone

 Counter stain (safranin, carbol fuchsine, neutral red)

Procedure:

1. Prepare the smear from the culture or from the specimen.

2. Allow the smear to air-dry completely.

3. Rapidly pass the slide (smear upper most) three times through the flame.

4. Cover the fixed smear with crystal violet for 1 minute and wash with distilled
water.

5. Tip off the water and cover the smear with gram's iodine for 1 minute.

6. Wash off the iodine with clean water.

7. Decolorize rapidly with acetone.

8. Wash off the acetone with clean water.

9. Counter stain with neutral red for 1 minute.

10. Wash off the stain; wipe the back of the slide. Let the smear to air-dry.

11. Examine the smear with oil immersion objective (100x) to look for bacteria.

Result:

Gram-positive bacterium............................... Purple

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Gram-negative bacterium.............................. Pink

Reaction of organisms to Gram stain

Gram Positive Gram Negative

Staphylococci vibrio

Pneumococci Shigellae

Streptococci Salmonellae

Clostridia Spirochates

Bacillus Haemophilus

Corynebacteria Neisseriae

Coliforms

In order to study bacterial, a smear must be made from the medium onto which the
organism has grown. The following care must be taken.

 Use grease free clean slides.

 Label slide with patient's identification infomation

 Use distilled water to emulsify the smear when using a solid medium.

 Use only one smear per slide.

 Smear should not be too thick.

 Never use staining jars when handling acids fast material to prevent the possibility
of transferring of acid fast organisms onto other slides.

 Take aseptic precautionary measures when handling culture plates

Making smear from liquid media.

 Sterilize wire loop in a bursen flame.

 Withdraw a loopful of culture and transfer to a clean grease free slide and spread
the sample with the loop to form a thick film of fluid.

 Allow the smear to air dry without heating and then rapidly pass the slide 3 times
through the Bursen flame to fix the bacteria to the slide.

 Allow the film to cool and stain appropriately

Making Smear from solid media.

 Sterilize wire loop in Bursen flame.

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 Place a drop of distilled water on a clear slide and re-sterilize your wireloop.

 Transfer a small portion of the growth to be examined and emulsify in the drop of
water on the slide until a thin homogenous smear is made

 Allow to dry, heat fix and stain.

Culture media used in bacteriology for the growth of bacteria

Media are composed of artificial substances that contain nutrients required for bacteria
organisms to grow. Media may be solid or liquid.

Media used in microbiology to grow microorganisms includes:

 Differential media: these refers to media that contain substances or indicators

that differentiates one organism from another e.g. MacConkey agar, Blood agar,
Cysteine Lactose Electrolyte Deficient (CLED).

 Selective media: these are solid media which contain substances that inhibit or
slow down the growth of microorganisms other than those for which the media is
devised e.g Shigella Salmonella Agar (SSA), Tellurite media & Deoxycholate citrate
agar (DCA).

 Enriched media: are culture media that are enriched with whole or Iysed blood,
serum, nutrients or extracts to support the growth of organisms that cannot grow
on basal media e.g Blood agar & chocolate agar.

 Enrichment media: they are liquid that are similar in function to the selective
media e.g selenite F broth used for isolation of salmonella organisms.

 Basal media: are simple media that support the growth of most microorganisms
that do not need special nutritional requirements e.g. Nutrient broth.

 Transport media: these are meant for transportation of clinical specimens

containing delicate microorganisms from where they are collected to where they
are processed and analyzed, such media contain substances that prevent the
overgrowth of commensals and prevent bacteria from 000dying e.g. Stuart and
Amies transport media.

Samples used in Bacteriology for Isolation of bacterial are:

 Stool

 Urine

 Blood

 Sputum

 Cerebrospinal fluid (CSF)

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 Body fluids (Pleural, synovial, peritoneal e.t.c.)

 Swabs (throat, Nose, Eye, Genitourinary) e.t.c.

 Semen

After isolating the microorganisms, they are further subjected to sensitivity which
involves placing of various anti-bacteria discs (drugs) to find the appropriate therapy
based on the microorganism’s ability to resist or become sensitive to the antimicrobials.

Parasitology

Parasitology is the study of organisms that are dependent on another living organism for
survival such as dependent organism is referred to as a parasite.

Definition of terms

Parasite: A living organism that resides on or within another living organism (Host) tor
its shelter and derive nutrients for its growth and reproduction.

Host: this is a living organism which a parasite depend on for shelter and derive
nutrients for survival.

Definitive or primary host: this is a host in which sexual, reproduction takes place or
in which most highly developed forms of the parasite occurs. E.g. man is a definitive host
of schistosomes.

Intermediate or secondary host: this is at host in which the development of larvae

stages or asexual reproduction of takes place. E.g. mosquito is an intermediate host
filarial worm.

Reservoir host: this is an animal host infected with parasites and becomes a source of
infection for other animals including humans.

Zoonosis: this in an infection acquired by man from domestic or zoological animals as a

result of man's interaction with animals E.g. balantidiasis

Classifications of parasites

Human parasites of medical are broadly divided into two groups, protozoa (single-celled,
simple organisms consisting of nucleus and cytoplasm) and helminthes (multi-cellular,
more developed Organisms.

Protozoa are grouped into four (4) classes based on their means of locomotion

Amoebae (Sarcodina): move by means of cytoplasmic protrutions (pseudopodia/false

feet)

Flagillates (Mastigophora): possesses the flagella as a locomotive organ which may

vary in number and composition depending on species

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Ciliate (cillophora/ciliata): moves with the help of cilia which are short hair-like
extensions cytoplasm covering the surface of the organism.

Sporozoa: have no specific organ of movement with a complex life Cycle consisting of
asexual and sexual life cycles.

Helminthes are divided into two (2)

 Nematodes which are round worms with unsegmented bodies, elongated and
tapered at both ends.

 Platyhelminthes which are flatworms, platyhelminthes are further divided into

cestodes (tape worms) and trematodes (flukes)

Medical Parasitology

Protozoa (Protozoology) Helminthes (Helminthology)

Kingdom – Protista (Unicellular) Kingdom – Animalia

(Multicellular)

Amoebae Flagellates Sporozoa Ciliates

(Have one or
(Typically, more whip-like (Undergo a (Complex
amoeboid and flagella) complex life Protozoa
use Intestinal and cycle with bearing cilia)
pseudopodia or genitourinary alternating
protoplasmic normal flagellates Balantidum coli
sexual and
 Giardia
flow to move) asexual
 Trichomona
Entamoeba s reproductive
histolytica  Blood and phases)
tissue flagellates
 Leishmonia Plasmodium
 Trypanosom
a Toxoplama

Nematodes (round Platyhelminthes (flatworms)

worms)

They are elongated

Cestodes (tape Trematodes
and tapered at both
worms) (Flukes)
ends, round in cross
section and Have a ribbon like Typically flattened
unsegmented. chain of segments and leaf shaped
Page 19 of 37 with 2 muscular
(proglottids)
 Wuchereria
Taenia solium sucker.
bancrofti
T. saginna They lack cuticle.
 Ascaris sp
Transmission of parasitic infections

Parasites are transmitted through;

 Faecal-oral route; usually by ingestion of infective organisms in contaminated food

and drinks.

 Direct skin penetration (when parasite penetrate the skin or injected through
insect bite.

 By sexual contact.

Samples used in parasitology laboratory

 Blood

 Stool

 Urine

 Skin Snip

 Urogenital samples (endocervical swab (ECS), high vaginal swab (HVS) urethra
swab Cerebrospinal fluid (CSF)

 Ear, nose and throat swabs

 Wound swab

 Exudates

Examination of stool for intestinal parasite involves both the macroscopic (physical) end
microscopic examinations

Macroscopy. -The visual examination includes

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 Color (Normal Color is brown)

 Consistency- Normal stool is formed or semi formed, however stool may be loose
or watery in diarrhoeic cases

 Presence of blood or parasite

Microscopy

The easiest and routinely used technique is wet preparation which is prepared directly
from the faeces in saline and iodine solution.

The saline wet preparation is used for microscopic examination of faeces to detect
protozoan trophozoites and cysts, larvae and egg helminthes based on their
characteristic morphology and motility, Pus cells, RBCs, and other significant materials
can be found in addition. The iodine help in staining the nuclei and glycogen of the cysts
of most parasites though they are not motile here. This help in identifying the parasites.

Procedure for wet preparation

 Place a drop of saline in the centre of the left half of the slide and a drop of iodine
on the right half of the slide.

 pick a small portion of the sample with the aid of an applicator stick and mix with
the saline, do same for the iodine.

 Lower a cover slip gently on the saline and another on the iodine reducing the
formulation of air bubbles.

 Focus on a microscope using 10X objective with the condenser iris sufficiently
closed.

 Examine for parasite by moving slide slowly on the stage.

Other methods have been developed to detect and identify intestinal parasites are: -

I. Concentration techniques using (a) floatation with zinc sulphate solution and (b)
saturated sodium chloride (Brine solution)

II. sedimentation technique using formol ether solution

When the parasite load is not high, direct wet preparation may not be helpful,
concentration techniques may be carried out. They are sedimentation and floatation
methods. Floatation may be done using Brime solution, zinc or sulphate solution.

Urine

Parasites found in urine include Micro filariae like wuchereria bancrofti, Schistosoma
haematobium and trophozoites of Trichomonas vaginalis. In addition to pus cell,

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 epithelial cells. Hyaline cast. Granular casts, yeast cells, bacteria., spermatozoa, RBCs
and crystals such as cystine, tyrosine, uric acid, calcium phosphate, triple phosphate
and calcium oxalate crystal.

Examination of Urine

A. Physical examination (macroscopy). This deals with the visual examination of the
urine appearance for

 Color

 Transparency and in some cases odor and formy nature of the urine sample.

Microscopy

This is the examination of the urine sample on the microscope.

Procedure:

 Collect the urine sample in a centrifuge tube and centrifuge

 Decant the supernatant

 Transfer a small portion of the sediment to slide and mix.

 View under the microscope using X10 and X40 objective lenses with the condenser
iris sufficiently closed.

Blood: this is examined in the parasitology laboratory for micro filaria, leishmaria
Species. Trypanosomes and plasmodium species. The plasmodium organisms are
causative agents of malaria to man. 4 species are known:

P. falciparum

P. ovale

P.vivax

P. malaria

Diagnosis of malaria parasites is done using Rapid Diagnostic Test (RTD) kits or
microscopically after staining with one of the Romanowsky stains such as Giemsa stain
and Field stain.

Chemical Pathology

this is a branch of MLS that deals with observation and investigation of biochemical
changes in the constituents of body fluids and excretions. Samples used in chemistry
includes:

Urine (for urinalysis e.t.c.) samples are collected in a universal container or Jerican (4L)
if it is 24-hour urine collection chemical preservatives such as HCl thymol, toluene e.t.c.
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 Can be added depending on the investigation required or the urine sample can be stored
in the refrigerator if investigation is to be delayed. This is aimed at stopping,

 Precipitation of urates in acidic urine

 Breakdown of urea to ammonia by tie action of bacteria resulting in a rise in urine

PH

 Oxidation of urobilinogen to urobin.

Constituents of Urine:

Normal Abnormal

Urea, uric acid, water, electrolytes, creatinine. Blood, proteins glucose, ketones, nitrite,
Leukocytes.

Cerebrospinal fluid (CSF)

This is collected in an Ordinary clean container and kept. in the refrigerator if there is
delay in estimation or can be collected in fluoride Oxalate container, CSF is sent to the
chemical pathology laboratory for the estimation of glucose and total proteins.

III. Serum. This is gotten from a clotted whole blood that has been collected in a plain
container and is used for a vast array of test including

 Euer (electrolytes, Urea & creatinine)

 Liver function (Bilirubin. Alkaline Phosphatase (ALP), Alanine aminotransferase

(ALT), Aspartate amino transferase (AST).

 Protein

 Lipid profile (Total Cholesterol, low density lipoprotein (LDL). Very low-density
Lipoprotein (VLDL)) High density lipoprotein (HDL) & Triglyceride (TG).

IV. Plasma

This is used for blood glucose test which can be

 Fasting blood sugar-glucose (FBS/FBG)

 2 hours post prandial (2HPP)

 Oral glucose tolerance test (OGTT)

 Random blood sugar (RBS).

Sample containers used in clinical chemistry

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 Fluoride oxalate

 Universal containers

 Plain containers

 Lithium heparin

 EDTA

Histopathology

This division of MLS deals with the study of abnormal or diseased tissues. Types of
tissues used in Histopathology

Biopsy- This is a type of tissue that is removed from a life animal to detect a disease.

Autopsy- This type of tissue is gotten from a dead animal. In most cases, autopsies are
used to examine the cause of death.

The arrival of these tissues at the reception is met with a serious check for the following
before specimen can be accepted

 That the specimen is right for the histological examination requested

 That the container is clearly labeled and accompanied by a completed request

from

 That the right fixative is in the container and is sufficient

Soon after death or removal from the body, tissues and cells begin to undergo
destruction which goes a long way to interfering with examination. Such changes are:

a) Atolysis: This occurs due to the action of lysosomal enzymes that are normally
present in the tissue, as a result of the loss of the body's ability to control their actions.

b) Putrefaction: This change in tissue architecture occurs as a result of external

influence such as the action of bacteria which result in decomposition of the tissue.

These changes are arrested by using a fixative.

A fixative is a chemical used to preserve the architecture and chemical constituents of

tissues and cells soon after death or removal from the body

The process of using fixatives for this purpose is termed fixation.

Qualities of a good fixative:

 Should be cheap and easy to prepare

 Prevent autolysis and putrefaction

 Should not cause tissue shrinkage or Swelling

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 Should be non-toxic non-corrosive and non-flammable

 Prevent distortion by any subsequently used reagent

 Should not add to or remove from the tissue constituents.

 Should penetrate the tissue and cells rapidly, evenly and deeply.

 Should preserve specimens for a long time

 Allow restoration of some natural colors for museum work and photography

There is no single fixative that fulfils all the above-mentioned qualities. Some fixatives on
their own are used as fairly good fixatives because of their individual properties; such
fixatives are called simple fixatives. Others work better when combined and are
referred to as compound fixatives

Simple fixatives compound fixatives

 Mercury chloride Zenker’s fluid

 Picric acid Helly’s fluid

 Formaldehyde Boun’s fluid

 Acetic acid 10% formal saline

 Osmium tetroxide Buffered formalin

 Chromic acid Carnoy’s fluid

 Ethyl alcohol Flemming’s fluid

 Potassium dichromate Gendre’s fluid

Decalcification: This is the removal of calcium from hard tissues so that they can be
easily sectioned. This involves the use of decalcifying fluids such as:

 Formic acid

 Nitric acid

 Ebner’s fluid

 Perenyi’s fluid

 Chelating agents such as EDTA

Tissue processing

Processing is term used to describe the various Stages between fixation and sectioning.
The stages are:

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a) Dehydration: This s the removal of water from issues using ascending grades of
alcohol.

b) Clearing/De-alcoholisation: This involves the removal of alcohol from tissues by

immersing them in a fluid that will dissolve the wax which the tissue must be
impregnated

c) Infiltration: This is the impregnation of the tissues with a medium that fills all-
natural cavities, spaces, and interstices of the tissue. e.g paraffin wax.

Tissue processing can be manually or automatic using an automated tissue processor.

After processing, tissues can then be cut into thin sections using a microtome, a process
generally referred to a tissue sectioning. The sections can then be mounted on a slide
and stained appropriately using either the Haematoxylin &eosin (H&E) staining method
or other histological staining techniques

Forensic science

Forensic Science deals with the problem of identity. It plays a vital role in paternity
dispute and crime detection such as rape cases and finger printing.

 Paternity disputes- Forensic science has contributed greatly to solving problems

involving more than one person claiming a child, this is made possible by means of
family studies involving genes and blood group inheritance patterns which obeys
Mendelian law which state that for a gene to be present in an offspring, its product must
be present in one or both of the parents.

 Rape: This is examined by checking for the presence of sperm cells from a vaginal
smear from the victim

 Crime detection- forensic science can identify culprits by means of using a

special photograph to capture the finger prints on items believed to have been touched
by the culprit during the time of committing a crime, the finger prints can then be
matched with those contained on the database already captured by the Government for
the entire population of a particular region.

Haematology

Haematology is the Science or study of blood. It encompasses the study of blood cells
and coagulation, it also includes the analysis of the concentration, structure and function
of cells in blood, their precursors in the bone marrow, chemical constituents of plasma or
serum ultimately linked with cell structure and function.

Blood is formed through a process generally termed Hematopoiesis.

Organs of hematopoiesis include

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 York sac at foetal stage

Liver, spleen and bone marrow at infant stage

All bones in children

Long bones in adult

The functions of blood

The functions of blood are broadly grouped into three (3) which can further be splited,
they are:

Transport function

Protective function

regulatory function

Cells studied in haematology

i. Red Blood cells (Erythrocytes) RBCs

ii. Platelets (Thrombocytes)

iii. White Blood cells (Leucocytes) WBC which are divided into granulocytes and
agranulocytes.

Granulocytes: Neutrotrophils, Eosinophils and Basophils, these cells have granules

within their cytoplasm

Agranulocytes: Monocytes and Lymphocytes

These cells have their unique morphologic characteristics that Make them easy to
identify on microscope.

Other parameters that are carried out in the Haematology laboratory include.

 Packed cell volume (PCV)

 Full blood count (FBC)

 Erythrocyte sedimentation Rate (ESR)

 Hemoglobin electrophoresis (Genotype)

 Clottology which encompasses

a) Prothrombin time (PT)

b) Activated partial thromboplastin time (APTT)

c) Bleeding time

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d) Whole blood clotting time.

All these parameters have their reference ranges which tend to increase or decrease in
disease conditions. Those reference ranges have gender, age and racial variations.

Disease may also alter the concentration, composition plasma or serum in the body as
well as affect the morphology of blood cells, such disease include.

 Anemia

 Leukenia

 Infections with bacteria, Viruses and parasites

 Autoimmune disease

 Bleeding disorders

 Organ related disease e.t.c.

Packed cell volume (PCV)

the packed cell volume (PCV), also called hematocrit (Hct) is the proportion of whole
blood occupied by red cells, expressed as a ratio (liter/liter) or as a percentage. It is one
of the simplest. most accurate and most valuable of all hematological investigations. It is
of greater reliability and usefulness than the red cell count that is performed manually.
In conjunction with estimation of hemoglobin and RBC count, knowledge of PCV enables
the calculation of the red cell indices (absolute values that indicate red cell volume,
hemnoglobin content and concentration) that are widely used in the classification of
anemia.

The PCV is also used to screen for anemia when it is not possible to measure
hemoglobin, and to diagnose polycythemia vera and to monitor its treatment. It is
suitable for screening large clinic populations, e.g. antenatal clinics. To measure the PCV,
a well-mixed well EDTA anticoagulated blood can be used or capillary blood collected
inte a heparinized capillary tube. There are two methods of determination:
microhematocrit method and microhematocrit (Wintrobe) method.

Principle: when a known quantity of blood is centrifuged at 12000g for 3-5 minutes, the
cells settles according to their weight.

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Microhematocrit method Materials required

 Capillary tubes, these need to be plain or heparinized capillaries, measuring 75mm

in length with an internal diameter of 1mm and wall thickness of 0.2-0.5mm. Plain
capillaries are often blue-tipped and heparinized capillaries, red-tipped. The plain ones
are used for anticoagulated venous blood while the heparinized ones (inside coated with
2 10 heparin) are used for direct collection of capillary blood from skin puncture.

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 Microhematocrit centrifuge

 Reading device. There are two types of microhematocrit PCV reader, i.e. an
integral Spiral reader which fits inside the centrifuge allowing PCV measurements to be
made after centrifuging with the capillaries in place in the rotor, and a hand-held scale or
graph A hand-held PCV reader can be used to read samples centrifuged in any
microhematocrit centrifuge, whereas an integral PCV reader can usually be used only
with the centrifuge for which it has been designed.

 Sealant. Although the end of capillary can be heat-sealed this often distorts the
end of the tube resulting in breakage, or the heat damages the red cells resulting in an
incorrect PCV. Capillaries are best sealed using a plastic sealant, modeling clay, or
plasticine.

 Blood sample.

Test Method.

1. Allow the blood to enter the tube by capillarity (if anticoagulated venous blood,
adequate mixing is mandatory) leaving at least 15mm unfilled (or fill 3/4th of the
capillary tube)

2. Seal the capillary tubes by vertically placing the dry end into a tray of sealing
compound (wax or plasticin). Rotate the capillary tube slightly and remove it from the
tray. The sealant plug should be 4-6mm long. Inspect the seal for a flat bottom.

3. Place the filled, sealed capillary tube in the grooves (slots) of the centrifuge with
the sealed end toward the periphery.

4. Set the timer of the centrifuge at 5 minute and spin at 10,000-15,000g.

5. Read the PCV using a reading device that is either part of the centrifuge or
separate from it. Alternatively, the ratio of the red cell column to whole column (i.e.,
plasma and red cells) can be calculated from measurements obtained by placing the
tube against arithmetic graph paper or against a ruler.

Example

Height of red cell column 19mm

Height of total blood column 49mm

=PCV =19mm49mm = 0.388 (VI) or 38.8%

Anticoagulants in Haematology

Anticoagulants are chemical substances added to blood to prevent it from clotting. They
include:

 Ethylene diamine tetra acetic acid (EDTA)

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 Heparin

 Trisodium citrate

 Balanced/double oxalate

Sample collection for Haematological examination

Basically 2 types of samples are collected

1) Capillary blood: This is frequently used when only a small quantity of blood is
required when venipuncture is impossible, sites for collection of capillary blood
include:

 Palmar surface of the tip of the ring finger or middle finger (for adults and children)

 Plantar surface of the big toe or heel (for infants)

Procedure for capillary blood sample collection

 Rub the site vigorously with a gauze pad of cotton wool moistened with 70%
alcohol

 After the site has dried, make a puncture 2-3 mm deep with a sterile lancet

 Clean the first drop of blood and use a capillary tube to collect the sample, avoid
undue squeezing of the site so that the sample is not diluted with tissue juices.

Advantages of capillary blood.

 It is obtained with ease.

 It is the preferred specimen for making peripheral blood film since no

anticoagulant is added that Can affect cell morphology.

Disadvantages.

 Repeated examination requires a new specimen.

 Precision is poor because of the variation in blood flow and dilution with interstitial
fluid

 Samples are easy to haemolyse.

 Platelet count cannot be performed on capillary sample as some of platelets are

unavoidably lost by adhering onto the wound.

 Venous Blood collection: This is used or collected when test require a larger
quantity of blood. Site of collection include the 3 veins in the cubital fossa (Cephalic,
Basilic and median cephalic veins) and veins on the ankle and dorsum of the hand.

Procedure

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 Assemble the required materials

 Palpate the veins and use the most prominent

 Swab the vein with a gauze pad or cotton wool moistened in 70% alcohol

 Apply a tourniquet at a point 6-8 cm above the vein

 Using the vein with the bevel of the needle facing up. If it's a vacutainer, blood will
be begging to enter the syringe spontaneously if not the piston is gently withdrawn

 Release tourniquet, apply a dry cotton wool to the puncture -site and gently
withdraw the needle.

 Cover the needle with its cap, remove it from the nozzle of the syringe and gently
expel sample into a tube.

Advantages of venous Blood

 In case of accident or breakage or doubtful result, test can always be repeated

 Aliquots of the specimen may be preserve for future reference

 It reduces the possibility of error resulting from dilution with interstitial fluid

Disadvantages

 It is a bit lengthy procedure

 It is technically difficult in children, obese individuals and in patients with shock

Blood Transfusion Science

This deals with the provision of safe blood for transfusion. It covers the screening of
donors for transmissible disease and crosshatching as well as grouping of donors and
patient’s samples

Donor Screening/ Assessment

 Donors must be free from transmissible infections (HIV, HBSAg, HCV & syphilis
(VDRL)

 Must not have donated in the last 3 months

 Must be between the ages of 18-65

 Must weigh at least 55 kg

 Must not be a dwarf or an albino

 Female donors must not be breast feeding mothers, menstruating or pregnant

 Must have a PCV of 40% and above

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  Must not be a drunkard.

Types of Blood Donors

 Voluntary donor

 Family replacement

 Autologous donor

 Commercial donor

Types of Blood Bag

 Adult: Which contains 450 ml of blood

 Pediatric blood bag:

Types of anticoagulants used in blood bags

 Acid citrate dextrose (ACD) 21 days

 Citrate phosphate dextrose (CPD) 28 days

 Citrate phosphate dextrose adenine (ČPDA) 35

Blood grouping

Basically, two types of grouping exist

i. ABO grouping

ii. Rhesus grouping

They can either be performed in a tube or no a time,

ABO grouping can be done using cells

(Cell grouping/Direct grouping) or serum

(Serum/indirect grouping)

ABO cell grouping

Sample Anti A Anti B Anti AB Anti D Blood group

E + - + + A Rh D positive

F - + + - B Rh D negative

G + + + + AB Rh D positive

H - - - - O Rh D Negative

Serum Grouping
Page 33 of 37
Samples A cells B cells O cells Blood group

E + - - B

F - + - A

G + + - O

H - - - AB

Phenotype Antigen Antibody

A A Anti B

B B Anti A

AB AB ------

O ----- Anti A & Anti B

Washing of cells

This involves adding a solution of normal saline to blood in a tube and centrifugating
after which the supernatant is decanted

Reasons for washing cells

 To expose antigenic site on the Red cells

 To remove obstructive proteins

Tests done in blood transfusion service laboratory include;

 Blood grouping and PCV

 Combs’ test (Direct & Indirect)

 Cross match

 Donor screening

Virology

Virology is the study of viruses which are biological agents that reproduces inside the
cells of living host. When infected by a virus, a host cell is forced to produce thousands
of identical copies of the original virus at a faster rate. Viruses are the Smallest infections
disease causing agents measuring between 20nm-300nm in diameter. They lack the
enzymes that are necessary for metabolism and are complete intracellular parasites.
They are entirely dependent on host cells for survival They have the Capacity to cause a
wide variety of human diseases ranging from mild to highly fatal ailment.

Classification of Viruses of Medical Importance

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Scheme of classification of viruses is dependent upon viral Structure (envelope and
shape) antigenic composition and other important properties.

RNA Virus DNA Virus

Polio virus Parvoinus

Coaxackie virus Papilloma Virus

Rhino viruses Polymoma virus

Echo Virus Adeno Virus

Reo Virus Hespes simplex virus

Rota virus Varicella/zoster (chicken pox)

Influenza virus Espstern-Barr-virus

Mumps virus Cytomegalovirus

Respiratory syncytial virus (RSV) Variola (small pox)

Rubeola virus Hepatitis B virus

Rabies virus

Human corona virus

Arbovirus

Lassa fever viruses

HIV and Hepatitis C virus

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Transmission of viruses

Viruses are spread through;

 Sexual contact

 Contaminated food,

 water and air droplets

 Contact with body fluids of infected persons

 Sharing of contaminated body objects such as needles and blades

 Insect and animal bites

 Waste products such as droppings and urine of rodents and mammals

Diseases caused by viruses

 Hepatitis

 Flu

 Pox

 Polio

 Common cold

 Rabies

 Hemorrhagic fever

 AIDS

 Mumps

 SARS

 Meningitis

 Yellow fever

 Herpes

 Lassa fever etc

Diagnosis of viruses

 Immunological techniques (BLISA. Radio Immunoassay RIA)

 Demonstration of viruses’ specific DNA or RNA by acid hybridization technique

(PCR)

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 Demonstrations of viral particles in specimen by electron microscopy

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