Arsi University

College of Health Sciences Department of Medicine

Course: Clinical Laboratory Method

By:Tafese B Tufa (Medical Microbiologist)

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 Unit- One
Introduction To Clinical Laboratory
Methods

What is Clinical laboratory Methods?

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• To perform qualitative and quantitative
analysis of body specimens such as: urine,
blood, CSF, feces and other specimens.

• Related with all fields such as chemistry,

microbiology, hematology, immunology,
genetics, instrumentation, and electronics.

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 Role of the clinical laboratory
• Role of laboratory in the health care system are:-
 Patient diagnosis, screening and treatment
follow-up.
 Public health aspects such as:
 Epidemic control
 Surveillance
 Delivery of heath information.
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 Outcomes

1) Improvement in the quality of health system

2) Reduce disease transmission

3) Reduce health care costs

4) Greater patient satisfaction

5) Greater motivation of the health worker

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 Selection of laboratory test’s
• Depends on:-

 The value of the information they provide

 Cost of the test

 Availability of the necessary laboratory materials

 The availability of well trained and experience laboratory

staff

Note:- Order correct test from correct sample, at correct

time, and from correct patient.
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 Interpretation of laboratory test Results
• The laboratory tests often performed for:
 The purpose of diagnosing disease
 For monitoring therapy
 Screen for disease

• Result must be both accurate and precise.

• Result must indicate the present condition of the patient.

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 • Quality diagnostic test to indicate disease is present or absent
which described in terms of:-

a) Sensitivity

b) Specificity

c) Predictive value (PPV & NPV test)

d) Test efficiency

1) Sensitivity:- the ability of the diagnostic test to detect very small

amount of the analytic

Sensitivity = TP x 100% = 20/30x100%=67%

TP+ FN
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 2) Specificity:-ability of the method to identify non- infected
individuals correctly
Specify= TN x 100% =D/(D+B)= 37/70 x100=53%
TN + FP
3) Positive Predictive value ( PPV):- is the probably that an
individual with a positive test result has the disease.
PVP = TP X 100% = A/(A + B) =20/53 × 100=37.7%
TP+ FP

4) Negative Predictive value ( NPV):- is the probably that an

individual with a negative test result does not have the disease
PVN = TN X 100%= D/(D + C)= 37/47 ×100 = 79%
TN+FN

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5) Test efficiency:- the ability of test to say positive form all test
results.
Test efficiency= TP x 100%
TP+ FP+TN+FN
Note: - for effective diagnosis and management of patients a method
with high precision and accuracy must be used.
• Accuracy:- is the closeness of measurements to the true value.
• Precision:- the closeness of repeated measurement to each other.

Note:- Precise method may not accurate.

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 Reference Ranges (Normal Range)
• Range that expected to found in normal individual.
• Used to compare with patient test result
• Can be affected by:

– Age
– Sex

– Environment(location)
• Each laboratory should do their own ranges based on their own
instrument and on their specific patient population (might not be
the same as another).
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 Unit Two
Laboratory Equipments 

2.1. Microscope
• Instrument that enables us to visualize minute objects
(animate and inanimate) that cannot be seen by our naked
eye.
Types of microscope
– Compound (simple) microscope (routinely used in medical
laboratories)
– Phase contrast microscope
– Dark field microscope
– Fluorescence microscope
– Electron microscope
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 2.2. Laboratory Glass and Plastic Wares
• Widely used in medical laboratories.
• Glass wares made from boro-silicate glass.
• Boro - silicate glass is:
– Resistant chemicals(except HF acid and H2PO4)
– Withstand mechanical breakage
– Withstand sudden change of temperature.
• Soda lime type of glass does not fit the above
requirements

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 2.2.1 Volumetric Wares
• Used for the measurement of liquid volume.
• Either glass or plastic wares such as:-
– Pipettes
– Volumetric flasks
– Cylinders
– Burettes
2.2.2 Pipettes
• Used for measuring reagent and sample in lab
• Pipettes are designated as class “A” or “B” according
to their accuracy
– Class “A” pipettes are the most accurate
– Class “B” pipettes are less accurate
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Types of pipette can be:
1. Volumetric pipettes
2. Graduated or measuring pipettes
3. Micropipettes
2.2.3 Burettes
• Used for measuring variable quantities of liquid that are used
in volumetric titrations.
2.3.4 Flasks
• Conical flasks
• Flat bottomed round flasks
• Round bottomed flasks
• Volumetric flasks

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 2.2.5 Beakers
• Have capacities from 5 to 5,000 ml
• Available in different shapes.
2.2.6 Cylinders
• Cylinders are supplied in 10 to 5,000 ml
capacities.

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 2.2.7 Test tube
• Harder and withstand actions of chemicals, thermal
shock and centrifugal strains.
• They are used to hold samples and solutions.
• Come with varying size, depending the use in which
they are intended.
2.2.8 Petridishes
• Petridishes are flat glass or plastic containers.
• Used predominantly for the cultivation of
organisms on solid media.
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 2.3 Centrifuges
• Used to separate solid matter from a liquid suspension by
means of centrifugal force.
• They sediment particles (cells, bacteria, casts, parasites, etc.)
suspended in fluid by exerting a force greater than that of
gravity.
• The suspended materials are deposited in the order of their
weight.
• Kinds of centrifuges
– Micro-centrifuges or serofuges
– Medium size centrifuges
– Large centrifuges
2.4 Refrigerators
• Physical means of preserving various laboratory specimens and
reagent.
•Saturday,
It works at a temperatureTB of
September 18, 2021
2 to 80 C.
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 2.5 Ovens
• Used for drying of chemicals and glass wares.
• Used for the sterilization of various materials (works at 160oc
for 1hr).
2.6 Water Bath
• Instrument where water is heated and the set temperature is
maintained at a constant level.
• Used to incubate liquid substances.
• Works at variable temperature.
2.7 Incubator
• Incubation at controlled temperature is required for
bacteriological cultures.

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 2.8 Colorimeter (Photometer)
• Used to measure the concentration of a substance in the
colored sample.
• Biological samples contain many substances that can be
determined quantitatively or qualitatively.
2.9 PH Meter
• Used to measure the pH or hydrogen ion concentration of a
given solution.
2.10 Distiller
• The quality of water used in the lab is very crucial.
• Distill water uses in reagent and solution preparation.
• All water used in lab should be free from extra chemicals.

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Congratulations
!

• Now that you’ve learned the names and uses of these pieces
of lab equipment, you are almost ready to work in the
laboratory!

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 Unit-Three
Basic Hematological Tests
3.1. Blood collection
 Blood is a liquid form in vivo and clots in vitro.
 If it is left undisturbed in a tube, serum will be produced.
 The fluid part of anticoagulated blood is plasma.
 Blood for analysis may be obtained form veins, arteries or
capillaries.
Quiz: what is the difference b/n serum and Plasma? Which one is
better for chemistry test? Why?

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 3.1.1. Venipuncture
• It is a procedure of collecting blood sample from veins.
• Performed for tests that require anticoagulant and
large quantities of blood.
• Commonly used site for vein puncture are the vein of
antecubital fossa. Such as:
– Cephalic
– Median cephalic
– Median basilica veins
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Procedures of blood vein collecting
1. Applied the tourniquet to the selected arm.
2. Clean the site with alcohol and cotton.

3. Insert the needle by keeping 30-45 degree.

4. Drawn the needed amount of blood.
5. Release the tourniquet before draw out the needle.

6. Apply the cotton on the site of punctured.

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 Blood collection errors

Error Solutions

Labeling error Don`t pre- label test tubes

Use of wrong anticoagulant Have to check which anticoagulant for which

/tubes test
Wrong ratio anticoagulant e.g. 1/5 Na-citrate

Partially clotted specimens Mix specimen 8-10 times

Hemolysis Use proper specimen techniques

Hemodilutions Always draw specimen from an arm opposite

of IVs
Hemoconcentrations Don`t leave on the tourniquet > 1min

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• Anticoagulants may or may not be used depending upon the
type of tests.
• The common anticoagulants used for hematological tests are:-

– Ethylene diamine tetra acetic acid ( EDTA)

– Sodium citrate=> used for ESR. (Mixing one part of
citrate with four part of blood)
– Heparine
– Oxalates

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 3.1.2. Skin (capillary) puncture
• Used when only small quantities of blood are
required e.g. Hgb, WBC, diff. count, etc.
• Capillary blood is obtained from:-
– The tip of a finger or earlobes in adults.
– The great toe or heel in infants.

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 3.2. Hematological Tests

• Most of hematological tests can be performed in small lab.

• The frequently encountered hematological tests include: CBC,
ESR, and B/F
a. Complete blood count
• It includes RBC, WBC, and platelets
• A complete blood count can be done to:-
– Investigate the cause of certain symptoms such as fatigue, weakness,
fever, or weight loss
– Detect anemia or determine the severity of blood loss.
– Diagnose polycythemia / increase in RBC/
– Help diagnose an infection.
– Help diagnose disease of the blood, such as leukemia.
– Monitor the response to some types of drug or radiation treatment.
– Investigate a history of abnormal bleeding.
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• CBC consists of:-

a) Total WBC count(TWBC)

b) Differential white blood cell count
c) Red blood cell ( RBC) count
d) Hematocrit ( packed red cell volume)
e) Estimation of hemoglobin(Hgb)
f) Reticulocyte count
g) Red blood cell indices ( MCV,MCH,MCHC)
h) Platelet count

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 3.2.1. Packed cell volume (Hematocrit)
• Is the volume of RBC expressed as a % of the volume of whole
blood in a sample.
• Expressed as percent or decimal.
• Dried heparin, balanced oxalate, or EDTA is used as an
anticoagulant.
• To determine the red cell mass by measuring space occupied
by packed red cells in a volume of whole blood.

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• PCV can be used as:-
– A single screening test of anemia.

– A reference method for calibrating automated blood count

systems.
– Rough estimation of haemoglobin value(PCV= 3xHgb)
– In conjunction with Hgb value and RBC count.

Quiz what is the relationship between Hgb, Hct, RBC? Which

can be done easily when electric power is not present?

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 Normal Values
Men: 35% - 47% or 0.35 - 0.47
 Women: 32% - 44% or 0.32 - 0.44
Infants: 34% -58% or 0.34 - 0.58
Test significance
• Causes of reduced HCT- anaemia.
• Causes of raised HCT- poliycythemia.

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 Red blood cell ( RBC) count
• Carry oxygen from the lungs to the rest of the body.
• Carry carbon dioxide back to lungs so it can be exhaled.
• If the RBC count is low, the body may not be getting the oxygen
it needs.
• Raising a condition called polycythemia, risk that the RBC will
clump together and block tiny blood vessels ( capillaries).
• Counted manually using counting chamber or electronic
devices.
• Counting RBCs by manual method is obsolete.
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 3.2.2. Hemoglobin ( Hgb) determination

 Serves as a carrier of O2 from the lung to the tissue and C02

from the tissue to the lung.
 It is composed of globin and heme.

 Each gram of Hgb can carry 1.34ml of O2.

 The O2 combining capacity of the blood is directly proportional

to the Hgb conc. rather than to the RBC.
 Measures the amount of Hgb in blood is a good indication of
the blood’s ability to carry throughout the body.

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 Estimation of Hgb
 The estimation of Hgb is used to:

1) Screen diseases associated with anemia.

2) Determine the severity of anemia.

3) Follow the response to treatment of anemia.

4) Evaluate polycythemia.
5) Helps in determining changes in the Hgb conc. before and
after operation and blood transfusion.

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  Methods of Determinations are:-
A] Sahali hellige method
B] Cyanomethemoglobin method
C] Oxy hemoglobin method
D] Alkaline hematin method
E] Copper sulphate densitometery
 Expressed as gram per deciliter (gm/dl) of blood.
Normal values
 Men: 13.5-17.5gm/dl
 Women: 12-16gm/dl
 Newborn: 14 – 20 gm/dl

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 High Value
 Caused by a lack of oxygen (which can occur from living at high
altitude).
 Smoking exposure to carbon monoxide.
 Long term lung disease.
 Certain forms of heart disease.
 Kidney disease or polycythemia.
 A rare disorder of the bone marrow
 Dehydration a result of drinking too little water.
 Other causes include:
– Frequent diarrhea
– Vomiting
– Excessive sweating
– Severe burns
– Use of diuretics
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 Sahli Hellige method
 This is a visual method of Hgb estimation, and is not very
accurate.
 Used only in world where electricity is not available.
Specimen:- Capillary blood or anticoagulant venous blood.
 Normal hemoglobin levels vary according to:
– Age
– Gender
– Altitude at which a person lives
Cyanometohemoglobin method
• This is a colorimetric method and more accurate than sahli’s
method.

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 3.2.4 RBC Indices
• Used to define the size and Hgb content of red blood cell.
• These are derived parameters.
• It includes:- MCV, MCH, MCHC

A) Mean Cell volume ( MCV)

• Volume occupied by a single red cell and the best index for
classifying anemia.
• Indicates whether the red cell appears normocytic, microcytic or
macrocytic.
• Calculated by dividing the PCV by RBC or in automated systems
• MCV is measured directly
E.g. MCV = PCV
RBC
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 Normal values:- 87-103 femto-liter (fl= 10-15)
• higher values in infants and newborn.
• A high mean cell volume may indicates:-
 Alcoholism
 Liver disease
 Lack of folic acid or vitamin B12
 Certain bone marrow disorders
• A low mean cell volume may indicates:-
 Iron deficiency aneomia
 Thalassemia
 Lead poisoning
 Long term infection
 Certain chronic cases ( e.g. diabetes or arthritis)

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 B) Mean Cell Hemoglobin ( MCH)
• Measure of the average weight of Hgb in the red blood cells.
• Calculated from the Hgb and RBC
• Its unit is pico-gram(pg=10-12)
MCH= Hgb
RBC
Normal value:- 26 – 34 pg
• Decreased values:
 Iron deficiency anemia
 Microcytic anemia and chronic blood loss anemia
 Thalassemia
• Increase values: usually indicate spherocytosis.

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C) Mean Cell Hgb- Concentration ( MCHC)
• Measure of the average concentration of Hgb in the RBCs
• The smaller the size of red blood cell, the higher the MCHC

• Calculated from Hgb and Hct and unit is gm/dl

MCHC= Hgb ( gm/dl)
Hct (%)
Normal value :- 31- 37gm/dl.

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 3. 2.5. Stained Blood Investigation
• Used in the investigation and management of anemia, infections and other
disorders.
• The test includes:

– Making thin blood film

– Fixing and staining of BF

– Examination under 100x objective.

• Reporting blood film includes:

– Differential and white cell morphological change if present .

– Red cell morphology.

– Comment on platelets- platelet structure, shape and number .

– Presence of haemoparasites (blood parasite) .

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 Stained Red cell Examination

• To determine variation and abnormalities in erythrocytes

size, shape, structure, Hgb content and staining properties.
• It is useful in diagnosing blood disorders such as anemia,
leukemia and blood parasites .

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• Assignment (15%)
1. Antibiotic susceptibility tests (AST)
2. AOB Blood typing
3. C-reactive protein tests (as diagnosis and monitoring tests)
4. Random blood sugar (RBS) tests
5. Electrolyte test(Ca, Na, Cl, K, Mg).
6. Urine microscopic tests
7. Screening tests for ANC attending pregnant mothers.
8. HIV test algorithm
9. HCG Test
10. Screening tests for blood donation
11. ART monitoring laboratory tests for HIV positive patients.
12.Cross-match /compatibility test/
13. Liver function tests (LFT)
14. Renal Function Tests (RFT)
N.B. protocol (introduction, procedures, principles, result interpretation, clinical
significance, summary & references)
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 3.2.6. White Blood Cells (leukocytes) Count
• WBC’s are a heterogeneous group of nucleated cells.
• Leukocytes are divided in to two main groups:
– Granulocytes
– Agranulocytes
• Granulocytes Agranulocytes
– Band neutrophils( 0 - 5%) => lymphocytes ( 20-40%)
– Neutrophils (60 - 70%) => Monocytes ( 2 - 6%)
– Eosinophils (1 - 4%)
– Basophiles (0.5 - 1%)
• The granulocytes contain granules in cytoplasm of cell.
• Used in the diagnosis and prognosis of the disease process.
• Certain disease decrease or increase in specific type of WBC.

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Value of test
• Used to investigate infections and unexplained fever and to
monitor treatment which can cause leucopenia.

Principle: - Whole blood is diluted 1 in 20 in an acid reagent

which hemolyzes non-nucleated red cells, leaving the white
cell to be counted.
• White cells are microscopically using an improved counting
chamber, and the number of WBC’s per liter of blood
calculated.

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 Calculation
WBC count (per mm3)= Cell counted x dilution factor(DF)

Area counted
Normal Range of WBC count
– Children ≤ 1yr----------------- 6.0 – 18.0x103/mm3
– Children 4- 7 years------------5.0 – 15.0x103/mm3

– Adults----------------------------4.0 - 10.0x103/mm3
– Pregnant women---------------up to 15x103/mm3

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 Clinical significance
• Increased value indicates:-
– Infection, inflammation, damage to body tissues, severe physical or
emotional stress, kidney failure, or disease such as cancer.
– Very high levels of WBCs may indicate leukemia
• Decreased value indicates:-
– The body is not producing enough white blood cells.
– Pernicious anemia or after cancer chemo therapy or radiation
therapy, allergic reaction to a medication, bone marrow failure ( a
plastic anemia), viral infection, malaria, alcoholism, AIDs, and
other condition.
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 3.2.7. The differential WBC count
• There are five major kinds of white blood cells:-
– Neutrophils
– Lymphocytes
– Monocytes
– Eosinophils
– Basophils
• Each type of cell plays a different role in protecting the body.
• Test performed by staining thin blood film with Wright stain or
Giemsa stain.
Steps
• With Wright stain
– Labeling=> smearing=> air drying=> staining
• With Giemsa stain
– Labeling=> smearing => Air drying=> fixing=> staining
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 Normal range ( values)
Cells Relative values
– Neutrophils----------60 - 70%
– Eosinophils-----------1 - 4%
– Basophiles------------0.5 - 1%
– Lymphocyte----------20 - 40%
– Monocytes------------2 - 6%
• Absolute value (WBC/mm3) = Relative(%) x Total WBC
count/mm3
• Staining for differential WBC count can also be used for blood
parasite investigation, cell morphology, and platelet estimation. 

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 3.2.8. ESR
• Not a part of complete blood count, but it is usually requested with
it.
• When whole blood is mixed with an anticoagulant and placed in a
perpendicular tube, the red blood cells sink to the bottom.
• B/c they are heavier than the plasma in which they are suspended.
• The sedimentation rate measures how quickly RBCs (erythrocytes)
settle in a test tube.
• The more red cells that fall to the bottom of a special test tube in
one hour, the higher the sedimentation rate.

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• Sequentially there are three stages in erythrocyte
sedimentation:-
1) Rouleaux formation stage: 1st 10min
2) Period of sedimentation: the next 40min

3) Period of packing: the last 10min

• These proteins are produced by the liver and immune system
under many abnormal conditions, such as an infection,
autoimmune disease, and cancer.
• There are many possible causes of an elevated sedimentation
rate.
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 Greater than normal value indicates:
• It may include:-
• Pneumonia
• Pelvic inflammatory disease
• Appendicitis
• Kidney, bone, joints, skin, or heart valve infections
• Cancer
• Autoimmune disease

• A extremely high ESR (>100mm/hr) is often found in:

• Some severe infections ( such as osteomyelitis or endocarditis).
• Certain inflammatory disease ( such as temporal arthritis)

• Cancer ( such as multiple myeloma or lymphoma)

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 Normal Range
Men: 0-15mm/hr
Women:0-25mm/hr

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 Platelet count
• It is helpful in evaluating bleeding disorders.
• Platelets are difficult to count because:
– they agglutinate, fragment and break down readily and
quickly.
– their small size.

– they adhere to glass.

– Platelets are not evenly distributed through out the blood and
have a tendency to clump together .
• Plt count can be performed by: indirect method & direct method.
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 3.4. Anemia
• Definition: - anemia is defined as the lowering of the circulating red cell
mass below the normal level.
• It is a clinical sign and not a diagnostic only.
• The oxygen carrying capacity is reduced below the normal.

Classification of Anemia
• Depending on the morphological changes of RBC, it can be classified in to:

1) Normocytic normochromic anemia:- the red blood cells have normal

size and hemoglobin content.
 The problem may be due to decrease in RBCs production or increased in
destruction of RBCs i.e. hemolytic anemia, blood loss anemia.

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2) Microcytic hypochromic anemia:- the size of RBCs will be
less than normal and the haemoglobin content is low.
 It is commonly seen in iron deficiency anemia, thalassemia and
lead poisoning.
3) Macrocytic hyperchromic anemia:- the RBCs size is bigger
than normal cells.
 Such blood cells could be produced due to difference in the rate
of maturation of nucleus and cytoplasm.

E.g. folic acid and vitamin B12 deficiency anemia

 
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 Unit Four
Basic Clinical Chemistry Tests
4.1. Liver Function Tests
• Liver is the largest organ in humans.
• Located in the right upper quadrant of the abdomen.
• Have the following function:
– Directly receives, processes and stores materials absorbed from the
digestive tract.
– Produce plasma proteins, clotting factors, transport proteins, etc.
– Main site of metabolic conversion of endogenous and exogenous
compounds.
– Synthesizes bile acid from cholesterol.
– Regulate blood glucose concentration.
– Participates in immune function and clear immune- complexes from the
circulation

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 Laboratory assessment of liver function ( LFT)
• LFTs are used for the recognition of hepatic dysfunction
• Used for:
• Screen of abnormalities in liver function
• Document on abnormality
• Determine the type of injury
• Facilitate prognosis and follow-up of patient with hepatic disease.

• Any single laboratory test will not satisfy all of the objectives.
• So tests are used in combination.

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• Tests of hepatic function can be classified in to:
1) Tests based on substances produced by the liver:
– Albuminuria, coagulation factors, cholinesterase
2) Test based on substances metabolized by the liver:
– Drugs, antibiotics, bilirubin, cholesterol
3) Test based on substances released from damaged tissue
 Endogenous substance released from damaged hepatocytes – enzyme
( AST, ALT)
 Endogenous substance synthesized at increased rate or reduced, ( such
as ALP, Gamma – GT, etc)
4) Tests based on substances cleared from plasma by the liver
 Endogenous substance ( bile – acids, bilirubin, ammonia)
 Exogenous substance, (lidocaine, indocyanice green, caffeine,
galactose)

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1) Hepatic Excretory Function
Bilirubin
• Assessment of this excretory function of liver can provide
important clinical information.
Metabolism of Bilirubin
• It is orange-yellow pigment produced from heme catabolism.
• Each mole of heme produces one mole of co-bilirubin and ferric
iron.

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 Note:- 250 – 300mg/day bilirubin is produced.
– The normal concentration of bilirubin in the serum is 0.2 –
1.0mg/dl.
– Out of this 95% is un-conjugated and 5% is conjugated

Note:-Total bilirubin= conjugated + un-conjugated

– Un-conjugated(indirect) bilirubin which is not water soluble so
not excreted in urine and bound to albumin for transport.
– Conjugated(direct) bilirubin is water soluble and pass through
urine.

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 Disturbance of Bilirubin Metabolism
• A number of inherited and acquired diseases affect in the production,
uptake, storage, metabolism and excretion of bilirubin.
• Bilirubinemia is direct result of these disturbances.
• Depending on the disorder, unconjugated bilirubin, conjugated
bilirubin or both are major contributors in hyperbilirubinemia
i) Unconjugated hyperbilirubinemia: can be
a) Physiological Jaundice
– It is seen in new born due to hemolysis of RBC.
– Unconjugated bilirubin can reach 40-50mg/dl by 48hrs and
decrease to normal value by 7-10 days.
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b) Hereditary Defect’s
• Crigler- Najar syndromes:

• Gilberts syndrome(pre-conjugation transport failure)

ii) Conjugated hyperbilirubinemia

• In hepatobiliary diseases(post-hepatic jaundice).

• Dubin- Johnson syndrome(post-conjugation transport failure).

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 Calculations
• Values for unconjugated bilirubin are obtained by subtracting
the conjugated from the total bilirubin concentration
Reference range:
• for infants >1month and adults
• Conjugated= 0 - 0.2mg/dl

• Unconjugated= 0.2 - 0.8mg/dl

• Total bilirubin= 0.2 - 1mg/dl

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2). Tests measuring hepatic synthetic ability
• The measurement of the end synthetic products of liver activity is used
to assess liver disease.
1) Plasma proteins:- it indicates sever and long standing hepatic disease.
At this time decrease the synthesis capacity of liver and decrease
plasma protein concentration.
2) Coagulation proteins:- most of plasma coagulation factors are
synthesized in the liver. coagulation proteins decrease in case of long
standing hepatic disease.
3) Lipid and lipoproteins:- liver is the major source of plasma
lipoprotein production and metabolism.
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3) The test based on substance released form damaged liver
tissue
a) Aspirate amino Transferase (AST):- This enzyme is also
known as SGOT
b)Alanine amino Transferase ( ALT):- This enzyme is also
known as SGPT
c) ALP- membrane bound glycoprotein enzyme
d)Gamma Glutamate ( -GT)
a) Aspartate amino transferase ( SGOT)
• The tissue source of increased concentration is found in liver,
cardiac tissues and skeletal muscles.

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Principle:- L- Aspartate + -oxoglutarate AST Oxaloacetate + L- L-
glutamate
 
• There is decrease in absorbance during reaction progress which is
proportional with AST activity.
• Measured at 340nm
Normal range:-
– Men= up to 37IU/L
– Women =up to 31IU/L
– On some literatures the average reference for adults =0 - 60IU/L
Interpretation of the result: Increased activity of AST may be due
to:
– Myocardial infraction
– Hepatic disorder
– Skeletal muscle problem
– Liver hepatitis
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 b) Alanine aminotrasferase (SGPT)
• Involved in the transfer of amino group from alanine to 2-
ketoglutrate.
• High concentration is comparatively found in liver
• Principle:
L- alanine + -ketoglutarate ALT pyruvate + L glutarate

Pyruvate + NADH LD lactacte + NAD+ + H+

• As NADH is converted in to NAD+ there is a decrease in

absorbance which is proportional with the activity of ALT
measured at 340nm.

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 Reference values:
– Men= up to 45IU/L
– Women= up to 34IU/L
– Note:- on some literature normal range for adults ranges 0 -
50IU/L
Interpretation of Results: Increased activity of ALT or GPT
may be due to:
– Liver disorder
– Renal problem
– Heart problem

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 c) Alkaline phosphatase
• Catalyze the hydrolysis of phosphomonoesters.
• Found mainly in bone, liver and placenta in small activity in the
intestine and kidney.
• is used as an index of liver and bone disease when correlated with
other clinical findings.

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Normal value
– Men= 53 – 128IU/L
– Women= 42 – 98IU/L
– Average reference value= 40 -150IU/L

Interpretation:- Increased value is mainly due to:

– Hepatobiliary disease
– Bone disease

N.B. Reading assignment read about cholesterol, lipid and

electrolyte test.
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4.2. Renal Function Tests
• Two highly vascular organs located on side of the
abdomen in the region of lower back.
• Selectively filter blood and remove metabolic wastes and
understood materials.
 The following process takes place inside the kidney
– Filtration process
– Reabsorption
– secretion
– Excretion
• Generally the urinary system is made up of
– A pair of kidneys
– A pair of ureters
– The bladder, and
– The urethra
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• Renal function
– Maintenance of blood volume and concentration
– Maintenance of blood pH buffering system

– Removal of waste products.

Note:- the average pH of human blood is 7.4 any change larger than
0.1 pH units in any direction is pathological and if the PH goes <
6.8 or > 7.8 deaths can result due to:
– Acidosis - pH below normal

– Alkalosis - pH above normal.

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 Factor that affect evaluation of renal test
• Renal function test can be affected by three factors
1) Pre-renal factor
• Are not directly related with renal function
• This factor affects the test before the substances reach the kidney.
Example:
– Dehydration
– Excessive loss of blood
– Cardiac failure
– High protein intake
Note:- generally is not problem of kidney.
2) Renal factors
• These are directly associated with kidney problems.

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 3) Post – renal factors
• Can be caused by obstruction to urine out follow (the problem the
prevent urine out follow)
– Prostate enlargement

– Stones tumor or carcinogens.

Aim of renal function Tests

– To detect the presence of a kidney lesion ( preferably at an early stage)
– To localize the site of a disease whether pre-renal, tubular, glomerular or
post renal.
– To quantitative the extent of damage (severity of the disease).

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 Renal function Test
• Test’s that used to evaluate kidney includes:-
– Urea tests
– Creatinine tests
– Uric acid tests
A) Blood Urea Nitrogen ( BUN)
• Urea is synthesized in the liver from ammonia produced as a
result of diamination of amino acids.
• The structure of urea is NH2-CO-NH2 and its molecular mass is
60gm.
• It contains two nitrogen atoms with combined weight of 28gm.
• Therefore, a urea nitrogen value can be converted to urea by
multiplying the value by 60/28= 2.14.

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 Determination of urea nitrogen
• Azotemia- is increased blood urea nitrogen.

• Uremia- is increased urea in blood.

Enzymatic colorimetric method (endpoint)

Sample:- serum, plasma, urine

Principle:-Urea is hydrolyzed by urease in to ammonia and CO2.

• The ammonia generated reacts with alkaline hypochlorite and

sodium salicylate yield a blue color.
• The intensity of colored formed is proportional to the
concentration of the urea present in the sample
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Normal Value
– New born ( <10 days) ------- 6.43 - 53.5 mg/dl.
– Adult------- 26-40mg/dl
– Urine urea: Adult(normal diet)----------- 26 – 43mg/dl
Significance for Determination of BUN ( urea)
– For evaluation of kidney
– requested with serum Creatinine test to help for differential
diagnosis of pre-renal and post-renal hyperuremia.
• Increasing in serum urea nitrogen may be due to
1) Pre-renal causes
– Increased blood supply
– Cardiac decompensation
– Water depletion due to decrease intake or excessive loss
– Increased protein catabolism - Urea increase but creatinine don’t
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 2) Renal causes
• Any disease that affects kidney example
– Glomerulonephrities ( acute or chronic)
– Kidney tumors
– Renal tuberculosis
3) Post renal cause
• Caused by condition that prevent the smooth flow of urine
/obstruction to urine out flow/
• Both creatinine and urea increases.

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 B) Creatinine Determination
• Creatine phosphate acts as storage of high energy readily
convertible to ATP in the muscle tissues.
• It is synthesized in the liver and pancreases form three amino
acids ( arginine, glycine, and methionine)
• Creatine is converted to creatinine at the rate of 2% per day.

• Creatinine is waste product derived from creatine and excreted

by kidneys.

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 Method of determination
Jaffe reaction ( kinetic fixed time ) method
Sample:-serum, plasma, whole blood, urine and other body fluid
Principle:- based on a modification of the original picrate rxn
( jaffe).
• Creatinine under alkaline conditions reacts with picrate ions
forming a golden brown ( redish) complex color.
• The absorbance is proportional to the concentration of the
creatinine present in the sample.
Normal value
• In serum/plasma-----Men----- 0.7 - 1.2mg/dl
Women----- 0.5 - 0.9mg/dl

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 Clinical utility / significance
• In severe renal disease, serum creatinine is elevated to 2-4 mg/dl.
• The plasma creatinine level is constant and not affected by diet.
• This makes creatinine an excellent analyte for assessment of
renal function.
• The creatinine clearance test is one of the most sensitive test to
diagnose renal function test specially the glomerular filtration
rate.
• Elevated level of creatinine in the serum is usually associated
with disease of glomerular nephritis.
• It is determined in conjugation with plasma urea.
• Creatinine and urea will increase in post renal diseases.
• Creatinine in urine may be assayed on fresh random urine
sample.

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 C) Uric acid determination
• Uric acid is a waste product which is either
– Derived from diet, or
– Synthesized in the body.
• Uric acid is the end product of purines metabolism.
• Normally about one half of the total uric acid is eliminated and
replaced each day.
• Elimination of uric acid can be
– by way of urinary excretion
– Through destruction in the intestinal tract by microorganisms.
Method of determination
Uricase peroxidase method
Sample:- serum, plasma, and diluted urine

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 Clinical utility (significance)
• Increased formation of uric acid
• Increased purine synthesis
• Inherited metabolic disorder of uric acid
• Excess dietary purine intake
• Increased metabolic turn over
• Malignancy
• Joint pain (Gout)
• Decreased excretion
• Idiopathic
• Chronic renal failure
• Increased renal absorption of uric acid
• Reduced secretion of uric acid

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4.3. Carbohydrates
• Carbohydrates are the major food supply and energy source for
the people of the world.
• Carbohydrates are aldehyde and ketone derivatives of polyhydric
alcohol and they are hydrates of carbon.
Classification of carbohydrates
a) Monosaccharide
• Example of some monosaccharide
– Trioses- contain three carbon atoms e.g. Glycerol, dihyroxyacetone
– Tetroses- contain four carbon atoms e.g. Erythrose
– Pentoses- contain five carbon atoms e.g. ribose xylose
– Hexoses- contain six carbon atoms e.g. glucose, fructose, Galactose

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b) Disaccharides
• Interaction between two monosaccharides with a lose of a
molecule of water
– maltose= Glucose + glucose
– Lactose= Glucose + galactose

– Sucrose+ Glucose + fructiose

C) Polysaccharides
• Linkage of many monosacharides e.g. starch and glycogen

• Contain from about 25 to 2500 monosaccharides

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 Metabolism of carbohydrates
• How carbohydrate metabolized

Terms applied in carbohydrate metabolism

• Glycogenesis => Conversion of glucose to glycogen
• Glycogenolysis => break down of glycogen in to glucose

• Gluconeogenesis => synthesis of glucose from non carbohydrate

substances ( amino acids, lipids, and etc) by liver
• Glycolysis => conversion of glucose in to lactate or pyruvate

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A. Factors that add glucose to the blood
– Glycogenolysis
– Gluconeogenesis
– Intestinal absorption of glucose

B. Factors that remove glucose from the blood

1. Glycogenesis

2. Glycolysis
3. lipogenesis

4. Amino acid synthesis

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Blood glucose tests may be done at different times
Fasting blood sugar ( FBS):
 Measurement of blood glucose taken after you have not eaten for
12 to 14 hours.
 It is often the first test done to detect diabetes.

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2) 2- hour post prandial blood sugar (2 hour ):
• measurement of blood glucose taken exactly after 2 hrs you
eat a meal.
3) Random blood sugar (RBS):
• measurement of blood glucose that is taken regardless of when
you last ate.
• It is also called a casual blood glucose test.
• It is useful b/c glucose levels in healthy people do not vary
widely throughout the day.
• Blood glucose levels that vary widely may indicate a problem.
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4) Oral glucose tolerance test:
• done to confirm a diagnosis of diabetes by drinking a
solution containing a specific amount of glucose.
• is most commonly used to diagnose gestational diabetes.
• It is not recommended to diagnose diabetes in a person who
is not pregnant.

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Normal value
– Fasting blood glucose: less than 110mg/dl (6.1mmol/L)
– 2-hour post prandial: less than 140mg/dl (7.8mmol/L)

– Random( casual): less than 126 mg/dl (7.0mmol)/L)

Clinical significance
 Greater- than- normal value
• Due to insulin deficiency (hyperglycemia)
• Other conditions that can cause high blood glucose levels include:

– severe stress, heart affect, stroke, medications such as corticosteroids,

rare cancers, or excess production of growth hormone.

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Lower- than-normal values
• Due to excess production of insulin(hypoglycemia)
• Can be caused by:-
– A tumor in the pituitary gland
– Liver disease, such as cirrhosis

– Kidney failure
– Malnutrition, etc
• Reading assignment:- glucose tolerance diagnostic test.

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 Unit five
Body Fluid Analysis
5.1- Cerebrospinal fluid analysis
• Cerebrospinal fluid ( CSF) is a clear color less liquid formed
within the cavities or ventricles of the brain.
• About 500ml are formed per day.
Function of CSF
• Physical support and protection.
• Regulate intracranial pressure.
• Transport nutrients and waste products.
• Provision of a controlled chemical states.

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Clinical interpretation
1) Abnormal color
a) Blood CSF: -
 if the blood evenly mixed in all three tubes can be subarachnoid
or cerebral bleeding.

 If the blood is present only in the first test tube and decrease in
the 2nd and 3rd test tube may be artificial during LP trauma.

b) Turbid CSF:
 usually indicate
– Increased WBCs, RBCS, microorganism such as bacteria or yeast, Ab-Ag
complex increase turbidity of CSF.
– If it is purulent- make immediately smear for gram’s staining.

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C) Xanthochromia:- pale to dark yellow color
– Mostly occur in chronic bleeding.
– In jaundice ( bilirubin)
• Conjugated in adults and un conjugated in infants.

d) protein level (> 150mg/dl):

 Clot formed in CSF with high level of proteins (fibrinogen).

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 Collection of CSF

• CSF is usually obtained by lumbar puncture ( LP) for analysis , at the site
of L4 - L5 or L3 – L4

• Indications of LP are:-

– To examine the CSF

– To determine the intracranial pressure

– To introduce – Anesthetics
- Drugs
- Radiographic contrast media

• Adult total volume 140-170 ml.

• Neonate total volume 10-60 ml.

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 Laboratory investigation of CSF

a) Macroscopic examination of CSF

b) Microscopic examination of CSF such as cell count and

differential

c) Biochemical analysis

d) Serological and Bacteriological tests

e) Tumor markers studies

• Specimens of CSF collected by LP is usually a liquated in to 3 test
tubes
– 1st- for biochemical and serological examination
– 2nd- for bacteriology and fungal study
– 3rd- for microscopic examination (hematology test).

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• The color change in macroscopic examination in each test tube
can give either artificial bleeding during LP or pathological
bleeding or chronic bleeding.
i) Macroscopic Examination of CSF
• Visual examination done to observe
– General appearance
– Consistency
– Tendency to clot

• Normal CSF: is clear, free of clot, color less, free of blood cells.

• There is no turbidity, a newspaper can be read through the tube.

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ii) Microscopic examination of cells

1) Total cell count and differential cell count of CSF

• Normal CSF is free of cells.
• When the cells are counted they are identified by cell type.
• Increased cell count in CSF may indicate inflammatory
disease, hemorrhage, neoplasm’s and trauma.

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Clinical Interpretation
• Pleocytosis is an increases in the number of WBC in CSF.
– > 50WBCs mostly occur in purulent infection & dominant cells
are granulocytes.
– Between 300-500 WBC with increased mononuclear cells
( lymphocytes) are indicative of viral infection, syphilis of CNS
and TB.
– WBC’s count with 40% or more of monocytes indicates
subarachnoid hemorrhoid.
– Other cells which can be formed in CSF
– Malignant cells, Leukemic cells, Plasma cells

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 iii) Biochemical analysis of CSF
• The tests of interests are:
– Chloride - lactate
– Glucose - LDH
– Protein - Glutamine
• Most frequently performed biochemical tests are glucose and
protein analysis.
• Both have great diagnostic values.
a) Measurement of glucose in CSF
• Normally CSF glucose level is about 60% to 70% of blood
glucoses.
Reference value
– Adult= 40- 70mg/dl
– Child= 60-80mg/dl

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 Decreased CSF- glucose- can a result of
1. Disorder in carrier mediated glucose transport into CSF.
2. Active metabolism of glucose by cells or microorganisms.
• Normal in viral and aseptic meningitis.
• Increased level are associated with DM
b) Examination of CSF total and specific protein
• The total protein concentration in CSF is about 5% or 1/20 that
of plasma.
• Clinical significance of CSF protein analysis is used mainly
– To detect increased permeability of the blood brain barrier to plasma
protein.
– To detect increased production of immunoglobilins or tissue
degeneration in CNS.

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iv) Bacteriological and serological tests
1) Bacteriological ( fungal ) examination of CSF;
– Gram stain

– AFB stain
– Culture and drug sensitivity
– India ink preparation.

2) Serological test of CSF

– VDRL ( RPR) test for syphilis
– Latex cryptococcal Ag test or lateral flow assay.

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 Procedures
•Drop the CSF sediment or colony on the slide
•Add one drop of India ink
•Mix it with normal saline
•Put on it cover slide and observe under 40x objective.

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5.2. Laboratory Examination of Effusion
• Effusions are accumulation of fluids.
• They may be exudates, which generally accumulate as the result
of inflammation or triangulates ( transudates) which are fluids
not associated with inflammation.
• Generally transudate:
– Has low WBC content
– Will not clot

– Malignant cell may be present.

– Specific gravity is less than 1.015.

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• Effusions are found in the pericardial sac, pleural cavities and
abdominal cavities
• Routine analysis of effusions of known etiology are:-
– Gross examination

– Microscopic
– Chemical

– Cylological studies

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 Unit Six
Parasitological Techniques
6.1. Stool Specimen
• Specimen submitted to the laboratory for examination of parasites
must be collected & transported to the laboratory without delay.
Collection and Handling of fecal specimen
• Collected in clean dry wide mouthed containers.
• Collected prior to any medications & avoid contamination with
urine.
• Approximately 5-7 gms of feces collected.
• Safety should be considered (gown, gloves, etc)
Number of specimens
• At least three specimens should be submitted for routine
examination of intestinal parasite before reporting <No ova of
parasite seen>
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Fresh versus preserved specimens
• Liquid stool should be examined within 15 minutes of
passage or be preserved.
• Semi-formed specimens should be examined within 1
hour passage or preserved.
• The three most common preservatives are:
– formalin,
– PVA (polyvinyl alcohol), and
– sodium acetate formalin

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• Formalin: is an effective for long term preservation
of protozoan cysts, helminthes eggs and larvae.
– The proportion is 3 parts of formalin in 1 part of stool
– Formalin does not preserve trophozoites.

• PVA: Proportion 3 parts of PVA in 1 parts of stool.

• Sodium acetate formalin (SAF):used for protozoan cysts &

trophozoites, helminthes eggs & larva, and intestinal coccidian
(such as cryptosporidium parvum).

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 Processing of specimens
– Macroscopic observations
– Microscopic examination
– Serological diagnosis
1) Macroscopic Observations
Consistence Color Elements Stage Parasite contain

1 Hard Black Pulp & fiber nearly pure All stage except trophozoite

2 Formed Brown Conspicuously fibrous All stage except trophozoite

3 Soft Yellow Feces with scanty mucus All stage except trophozoite

4 loose Green Feces with much mucus All stage

5 Diarrheic Clay Mucus with much feces All stage except Cyst

others Others (blood, barium, etc

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 2) Microscopic examination
• Divided in to three steps:
– Direct wet mount
– Concentration technique
– Permanent stain smear
Direct wet mount preparations
• The primary purpose to detect protozoan trophozoites & their
characteristics motility.
• Prepared by mixing a small amount of specimen (approx.
2mg) in a drop of 0.85% saline (physiological saline) on clean,
dry microscope slide.
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 Direct wet Mount
– Saline method (Eosin Method)
– Iodine method

Concentration
• Need for fecal concentration technique:
– When the number of parasite in the sample is too few to be detected by
direct microscopy.
– To check whether treatment has been successful.
– To quantify (enumerate) the parasite.
• Concentration techniques can be:
– Sedimentation technique
– Floatation Technique
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a) Sedimentation Technique(formol-ether)
– is one of the most widely used techniques.
b) Floatation Technique(Zinc_sulphate)

Permanent Stains
• Used for the identification of some of the smaller protozoans by
their detailed cytological morphology.
• Three permanent stains widely used routinely:
– Iron hexatoxyline

– Wheatlev’s trichrome stain

– Modified acid fast stain

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 Preparation of Blood Smears for Parasite examination
• Blood is the specimen of choice for plasmodium species,
Babesia species, trypanosome species, Leishmania species &
Microfilaria (except O. volvulus).
• Blood sample may be obtained from the finger tips, earlobe, or
vein-puncture.
• it may be anticoagulated or non-anticoagulated.
• Two kinds of smears may be made.
– Thin &
– Thick.

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Types of Blood Film
• Thin blood smears are suggested for the observation of
morphologic detail & species identification.
• The thin blood film is made in the same manner as blood smear
used for a differential count in hematology.
• Thick smears are typically used for screening purpose,
particularly in cases where malaria or babesiasis is suspected.
Staining of Blood Film
• Generally, there are two permanent stains that are commonly
used in the clinical laboratory to stain parasite.
1. Wright’s &
2. Giemsa stains.
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 Question, comments &suggestion!!

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