Menelik Medical and Health science College

Clinical Laboratory Methods

for 1st year Post Basic Nursing students

02/13/2025 1
 Introduction to clinical laboratory

▫Laboratory – is a place that is equipped with different

instruments, equipments, chemicals etc for performing
either experimental works or research activities.
▫Clinical laboratory: A laboratory where tests are done
on clinical specimens in order to get information about
the health of a patient as pertaining to the diagnosis,
treatment, and prevention of disease. (Whole blood,
serum, plasma, urine, stool, sputum etc).

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 Function of clinical/medical laboratory in a
health care system
• Laboratory has an important role in:
A. Providing quality health care service
– Laboratory investigation increase the accuracy of disease
diagnosis.
B. Achieving efficiency and cost effectiveness
– The laboratory reduce the expenditure of money on drugs.
C. Achieving good health planning and management
– provide information on the health status of a community and
disease trend

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 Why laboratory tests are ordered?

• To confirm clinical diagnosis

• To screen for disease

• To monitor progress of disease, e.g. blood glucose test to

follow up patients with diabetes
• To monitor effectiveness of disease treatment

• To detect side-effects of drugs. e.g. aminotransferase

measurements in patients being treated with hepatotoxic
drugs.
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 Clinical laboratory units

 Hematology- deals with blood and its constituent

 Clinical chemistry – deals with measurement of various
bio-chemical changes in serum and body fluids
 Immunohematology- deals with blood banking and
transfusion medicine

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 Medical microbiology – deals with medically important
microorganisms.
o Bacteriology, Virology, Mycology, Parasitology,
Immunology

 Urinalysis - deals with the analysis of urine

 Serology- is the study of antigen and antibody and

their interaction in vitro.

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Selection of laboratory tests depends on:

 The value of information the test provide

 Cost of the test
 Availability of the necessary laboratory materials for the test
 Availability of well trained and experienced laboratory staff

 The ability of a diagnostic test to indicate a disease is present or

absent depends on its quality

 The quality is described in terms of:

i. Sensitivity
ii. Specificity
iii. Predictive values 7
i. Sensitivity
• The ability of a test to detect truly infected individuals

ii. Specificity
• The ability of a test to identify non infected individuals
correctly
• The ability of a method to identify all samples, which
do not contain the substance being detected
iii. Predictive value
• The ability of a test to predict the presence or absence
of disease from test result

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Clinical laboratory methods includes:
– Hematological tests
– Immunohematological tests
– Serological tests
– Urine and body fluid tests
– Clinical chemistry tests
– Microbiological tests
– Parasitological tests
– Molecular tests and etc. 9
 Basic hematological tests
 Haematological tests are used to diagnose
haematological disorders, anaemia, leukemia, infectious
and inflammatory disease.

 The frequently used haematological tests in most

clinical laboratories includes:-
Complete blood count (CBC)
Erythrocyte sedimentation rate (ESR)
Blood film (BF)
Coagulation test
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 Blood collection

 The objective of blood sample collection is to obtain

a representative sample of circulating blood
 Blood sources for hematological tests are:
 Venous blood
 Capillary or peripheral blood

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 A. Venous blood collection

 It is the procedure of collecting blood sample from veins

 Necessary for most test that requires anticoagulant or
large quantities of blood, plasma or serum

 Three main veins used for vein-puncture are cephalic,

median cubital and basilic veins

 Veins in the wrist or ankle may be used

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13
Anticoagulants
Anticoagulants are substances that prevent blood
from clotting
Anticoagulant may or may not be used depending on
the types of the test
The choice of anticoagulant depends on the test purpose
The common anticoagulants used in hematology are:
o Ethylene Diamine Tetra Acetic acid (EDTA)
o Sodium citrate
o Heparin and oxalates
The proportion of anticoagulant to blood must be
optimal
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 Procedure of venous blood collection

1. Apply the tourniquet

2. Clean the puncture site using 70% alcohol solution
3. Select vein and insert the needle
4. Release the tourniquet after appropriate blood has taken
5. Apply a ball of cotton to the puncture site and gently
withdraw the needle
6. Dispense the blood to the test tube which contain proper
anticoagulant
7. Instruct the patient to press on the cotton

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Advantage:
It allows various tests to be repeated for checking
Allows additional tests to be performed
Plasma or serum can be frozen for further reference
Reduce the possibility of error resulting from dilution
with interestial fluid

Disadvantage:
o Lengthy in procedure
o Technical difficulty in children and obese individual

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 B. Capillary (Skin puncture)

 Used when small amount of blood is required

For example for hemoglobin determination, WBC
count & blood smear preparations
 It is also used when vein puncture is impractical
• In neonates
• In case of sever burn
• In patients whose arm veins are being used for IV
medication

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 Sites of skin puncture

• Adults
lateral surface of either the tip of the ring or
middle finger

• Infants
lateral surface of big toe
lateral surface of the heel

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Advantages
 Can be obtained with easily
 Not relatively lengthy

Disadvantage
 Repeat testing is usually restricted because of smallness
of blood collected.

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 Procedure for skin puncture

1. Rub the site with cotton with 70% alcohol solution

2. After the skin has dried, make a puncture 2-3mm
deep with a sterile lancet
3. Wipe the first drop
4. Take appropriate amount of blood
5. Stop the blood flow by applying slight pressure at
the site of puncture with cotton

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 1. Complete Blood Count (CBC)
 The Complete blood count consists of:
• Red blood cell data
– Total red blood cell count (RBC)
– Hemoglobin (Hgb) determination
– Hematocrit (Hct) or packed cell volume (PCV)
– RBC indices
• White blood cell data
– Total white blood cell (leukocyte) count (WBC)
– Differential WBC count

• Platelet count

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CBC can be done to:
 Detect anemia or determine severity of blood loss
 Used to investigate infections
 Diagnose leukemia
 Monitor the response to some types of drugs or radiation
treatment

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WBC count
• Measures the number of white cells in your blood.
• Total WBC count (total leukocytes count)
– Significance/Value of the test
• used to investigate infections & to monitor
treatments or to investigate probable blood cancer.
– Methods/techniques
• Manual total WBC count
• Automated electronic counting device or
hematological analyzers:
– Provides greater accuracy & precision than the
manual method.
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 Interpretation of WBC counts

• WBC normal range or reference range varies with: age,

gender, and race.
• It is increased in children
• WBC count is slightly higher in pregnancy.
• Total number of cells per mm3 or microliter (µL) is
reported usually.
• Normal range = 4000 to 11,000 per µL or 4 to 11x109/L
• Leucopenia= decrease in WBC
• Leukocytosis= increase in WBC count
• WBC count may increase, decrease or remains constant in
different types of diseases
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• Significance of the test
• Total WBC count is increased during infection,
inflammation, damage to the body tissue ( such as a
heart attack, sever physical or emotional stress, kidney
failure or disease such as cancer)

• A low WBC count may occur after cancer chemotherapy

or radiation therapy and bone marrow dysfunction.

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• Differential WBC count ( leukocyte cell type)
• Measures the amount of each type of white blood cell
– There are 5 major types of leukocyte cells
– These are:
• Neutrophils, Eosinophils, Basophils, Monocytes
& Lymphocytes

– The quantity of each leukocyte cell type provides

important information in diagnosing the patient’s
condition

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a. Neutrophil
– Normal range: 60-70%
– Neutrophilia= absolute increase in neutrophil
– Increase in bacterial infection (usually in acute
bacterial infection) & neutrophilic leukemia

– Neutropenia is a decrease in neutrophil

– Immature neutrophil is known as band neutrophil;
mature neutrophil is known as segmented neutrophil.
– Increased amount of band neutrophil suggests acute
inflammation
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b. Lymphocytes d. Eosinophils
• Lymphocytosis, an increase • Normal range: 1-3%
in the number of • Eosinophilia, an increase
Lymphocytes, occur in some
in eosinophil, occurs in
viral infection
parasitic infection &
• Normal range= 20-40%
allergic reaction.
• Infant & children have higher
lymphocytes than neutrophil

c. Monocytes e. Basophils
• Have kidney shaped nucleus • Normal range= 0-1 %
• Normal range: 2-10% • Basophilia, an increase in
• Increase in number non- basophil, occur in allergic
specifically reaction 29
Red blood cell (RBC) count: is done to determine whether
there is an adequate number of RBC in the circulation or not
Significance of the test
Investigate anemia
Anemia- is a general term that refers to a decrease in RBCs
Anemia can occur from either a decrease in the number of
RBCs, a decrease in the hemoglobin content or both.
A lower RBC count can result from a number of causes;
Massive RBC loss such as acute hemorrhage
Abnormal destruction of RBC
Lack of substances needed for RBC production
With hematocrit & hemoglobin it is used to calculate red
cell indices which are used to classify anemia 30
• Methods
• Manual and automation
– Manual red cell count is almost never done because
of errors inherited in the procedure

• Normal range = 4-6 million/µL, but varies with age

Normal value adults - Women = 4.0 - 5.5 X106/mm3
- Men = 4.5 - 6 X 106/mm3

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• Hemoglobin (Hgb) determination
– Significance of the test
• To detect anemia & its severity
• To monitor anemic patients response to treatment
• To check Hgb level of a blood donor prior to donation
• Evaluate Polycythemia

– Methods :
• electronic devices (digital) and automated machines
– Hgb normal range
• Normal value New born = 14-20 g/dl
Women = 12-16 g/dl
Men = 13.5-17.5g/ dl 32
• Hematocrit (packed cell volume)
– Hematocrit is the ratio of the volume of erythrocytes to
that of the whole blood
– The measure of the ratio of the volume occupied by the
red cells to the volume of whole blood.
• Value of the test:
– To screen anemia
– To calculate red cell indices
– To diagnose polycythemia
– To monitor treatment of anemia or polycythemia

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High hematocrit value means:
the blood contains too many red blood cells (a
condition is called polycythaemia)
a lack of oxygen ( which can occur from living at high
altitude)
can also be caused by too little water in the body
(dehydration)

 Low hematocrit value indicates anemia

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Centrifuged blood (normal)

Plasma

Buffy coat (WBCs and Platelets)

Red blood cells

• Normal range:
– Adult male= 40-55%
– Adult female= 36-48%

• HCT value increase in polycythemia .

• HCT value decrease in anemia

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 Red blood cell indices

• Red blood cell indices are measurements that describe

the size and hemoglobin content of red blood cells.

• Useful in morphological characterization of anemia

• Red blood cell indices help to classify anemia

• Are calculated from the red cell count, hemoglobin

content & packed cell volume

– The red cell indices are MCV, MCH, & MCHC

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• Mean Cell Volume (MCV)
– Is the average volume of a single red blood cell i.e. it measure
average size of single RBC
• Calculated from hematocrit and red cell count
• MCV is expressed in femtoliters (fl).
• 1fl = 10-15ℓ
• MCV = Hct
RBC count /l
• Normal value for men and women: 80-100fl
• The MCV indicates whether RBCs appears Normocytic, Macrocytic or
Microcytic
– Interpretation:
• Low MCV value: is found in microcytic anemia’s particularly iron
deficiency, anemia of chronic disease & thalassemia (a type of
hereditary anemia)
• Raised MCV values: are found in macrocytic anemia, marked
reticulocytosis & chronic alcoholism. 38
Mean Cell Hemoglobin Concentration (MCHC)

• Is the average hemoglobin concentration per unit

volume of total red cells

• It is expressed in g/dl

• calculated from Hgb & HCT.

• MCHC (g/dl) = Hgb (g/dl)
HCT
Normal value =31.5-36g/dl
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 Mean Cell Hemoglobin (MCH)

• MCH is a measure of the average weight of Hgb

in a red blood cell expressed in pg. (picogram);
• 1pg= 10 -12 g
• Calculated from Hgb & RBC count
• MCH = Hgb (g/l)
RBC count/l
• Normal value men & women =27-33 pg

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 Platelet count

• Platelets are cytoplasmic fragments of megakaryocytic

mother cells & are produced in bone marrow
• They are always present in blood, but they are not
active unless damage has occurred
• They have life span of 7.5 days

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 Normal range = 150 - 400X 103/mm3

• Platelet count may be requested to investigate abnormal

bleeding which can occur when the platelet count is very
low
• It is also performed when patients are being treated with
cytotoxic drugs/other drugs that can cause
thrombocytopenia
– A decreased number of platelets =
• Thrombocytopenia
– An increased number of platelets =
• Thrombocytosis 42
 2. Erythrocyte Sedimentation Rate (ESR)

• Measures how quickly the red blood cells settle or sink to the
bottom of the tube.
• The rate at which the RBCs fall or settle out from the plasma
• Normally, red blood cells sink slowly. But inflammation makes
red blood cells stick together in clumps.
• These clumps of cells are heavier than single cells, so they sink
faster.
• If an ESR test shows that your red blood cells sink faster than
normal, it may mean you have a medical condition causing
inflammation.
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• The anticoagulated blood is allowed to stand undisturbed for a
specified period of time (usually 1 hour)
• When whole blood is placed in a vertical tube , red cells will tend
to fall toward the bottom
• The length of fall of erythrocyte in a given interval of time is
called erythrocyte sedimentation rate
• ESR is used to follow up patients, i.e. it has no diagnostic value
since it is non specific.
• Rise during inflammation

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 Wintrobe method
The tube is 11cm long with an internal diameter of
2.5mm

• Normal range -Women = 0-15 mm/hr

- Men = 0- 7mm/hr

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46
 3. Blood Film Examination

 A laboratory work-up that involves cytology of

peripheral blood cells smeared on a slide.
 The reliability of blood film examination depends
heavily on well made & well stained films that are
systematically examined

 Allows the evaluation of white blood cells (WBCs,

leucocytes), red blood cells (RBCs, erythrocytes), and
platelets (thrombocytes).

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 It may also be performed when a doctor suspects
blood disorders, such as an anemia, decreased or
abnormal production of cells in the bone marrow, or
increased cell destruction.

 Stained red cell examination is the best method of

studying the variations and abnormalities in red cell
size, shape, structure, Hgb content & staining
properties

 Also to investigate blood parasites

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 Blood film stains

• Dyes used in blood staining are:

• Romanosky stains:- are mixtures of acidic dye and basic
dyes that give a differential staining of the different
cellular components
1. Basic dyes (like methylene blue) - used to stain nuclei
and certain other structures.
2. Acidic dyes (like eosin) - used to stain acidophilic
structures
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Cont…

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 4. Coagulation test

 Bleeding time
• Measures how long it takes for a small cut on the skin to
stop bleeding.
• Normal bleeding time: 2-7 minutes
• Any clotting factor deficiency or platelet abnormality
will lead to increased bleeding time.

• Prolonged in:
– Thrombocytopenia
– Acute leukemia
– Aplastic anemia
– Liver diseases 51
 Prothrombin time (PT) test
• Prothrombin is another protein your liver produces.

• This test measures also how well and how long it takes
your blood to clot.
• It normally takes about 25 to 30 seconds.

• Abnormal results include: hemophilia, liver disease, mal-

absorption, disseminated intravascular coagulation,
Vitamin K deficiency

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 Immunohematological tests

• Immuno-hematology
– Deals with antigenic structures related to blood cells
& blood transfusion
• Blood type/group
– Blood type is identified by an antigen on the surface
of RBCs
– Major types of these antigens are:
ABO blood group antigens
Rh blood group antigens

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 1. ABO Blood grouping system
The main importance of blood grouping:-
 Safe blood transfusion and blood banking
 For organ transplantation
 To determine whether two people could be blood relatives
 When a woman is planning to become pregnant
 To help identify a person suspected of committing a
crime

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• In the ABO blood group system, we have two antigens
– A & B antigens
• Based on presence & absence of these antigens, we have
4 phenotypes
• Blood type A
• Blood type B
• Blood type O
• Blood type AB

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 Blood group Plasma Antibody Red cell Antigen

1. A ------------------ Anti-B ------------ A

2. B ------------------ Anti-A ------------- B

3. AB ----------------- Null ------------- A&B

4. O ------------------ Anti- A &B ------------ Null

The human red cell carries many antigens on

its surface 56
Antibodies of ABO system

– They are naturally occur

– They are present in reciprocal to the missing ABO
antigen
– They are IgM-type in nature
– Can fix complement

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There are two methods of ABO blood typing
A. Forward /direct grouping
• direct grouping: by using known anti-sera: anti-A, and anti- B.

B. Reverse /serum grouping

• Reverse or serum grouping: Determination of ABO antibodies in
serum

A. Forward /cell typing/direct :-

 Principle:- Based on the presence or absence of the
corresponding antigens on the red cells surfaces of the
individual
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 Forward/cell typing
RED CELLS TESTED BLOOD GROUP
WITH INTERPRETATION
ANTI- A ANTI- B

Positive Negative A
Negative Positive B
Positive Positive AB
Negative Negative O

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B. Reverse /serum/ typing

 Principle: To confirm ABO blood grouping based on the

presence or absence of antibodies in serum of a person
 Interpretation

• (+ve) agglutination: Antibodies specific for either A or B

antigen is present
• (-ve) agglutination: No antibody is present for Antigen

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 Reverse/serum typing
SERUM TESTED WITH BLOOD GROUP
INTERPRETATION
A cell B cell

Negative Positive A
Positive Negative B
Negative Negative AB
Positive Positive O

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 Rh-Blood group system
• Red blood cells may have the Rh antigen attached to
them, sometimes called Rh factor.
Rh Antigens
• There are over 40 known Rh Ags, five of which are
only expressed on red cells.
 These are D, C, E, c, & e
 The most common one is D antigen, it is highly
immunogenic than other Rh antigens.
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Rh Antibodies
 Rh- antibodies are not naturally occurring (Anti- D)
 Anti-D is produced when Rh negative individuals
receive blood from Rh positive individual having D-Ag
which is potent immunogen
 IgG type - they can pass through placenta
 Anti-D is common cause of sever HDN
(Erythroblastosis faetalis)

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• Rh factor is determined by the Rh test.
– Rh positive (Rh+) means the Rh antigen is
present on the RBCs.

– Rh negative (Rh-) means the Rh antigen is not

present on the RBCs.

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• In clinical set up, a patient’s blood type is described as a
combination of blood group antigen and Rh antigen
• Blood type letter (s) followed by a plus (+) or minus (-) signs
are used
• The signs indicate if the Rh antigen is present or absent.

• Example:
 A+, A-, B+, AB-, AB+, B- etc.

 A- Means an individual has ‘A’ antigen but doesn’t have

the Rh antigen attached to the RBCs
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 Determination of Rh type

The need for Rh typing

 Avoiding transfusion reaction

 To prevent Hemolytic disease of the fetus and new born

(HDFN)
• Detection of Rh antigen on RBCs by using Rh antiserum
(anti-D).
• Only forward grouping is used
RBCs (D-Ag) + Anti-D (reagent) ⤏ Agglutination
Interpretation:
 +ve Agglutination ⤏ Rh positive
 -ve Agglutination ⤏ Rh-negative
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Hemolytic Disease of the Fetus and New Born (HDFN)

• Result when antibodies of maternal origin bind with

fetal red cell
• The antibodies are mainly IgG type and they are anti
D alone or with others

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 Assessment of HDFN
1. Prenatal testing:
 It is recommended that all pregnant women at their first
attendance at a clinic need to have
 ABO grouping
 Rh typing
2. Postpartum testing : The following tests are performed
by taking samples from infant blood
 Rh & ABO typing
 Hgb & HCT determination
 Billirubin determination
 Peripheral blood film morphology study
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 3. Coomb’s test
• is also known as antiglobulin test
• Coomb’s test is an investigation done for identification
of anti-D antibody or Rh antibody

• Can be used to detect red cells sensitized with IgG

autoantibodies or complements.

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 Types

• Two types
• Direct Coombs Test (Direct Antiglobulin Test- DAT) and
Indirect Coombs Test (Indirect Antiglobulin Test- IAT)

• Direct Coombs Test (Direct Antiglobulin Test- DAT)

• is used to detect antibodies that are bound to the surface
of red blood cells
• DAT is performed in:
– HDN/Erythroblastosis
– Transfusion reaction
– Drug induced sensitized RBC
– Autoimmune hemolytic anemia
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71
• Indirect Coombs Test (Indirect Antiglobulin Test-
IAT)
• The indirect Coombs test looks for free-flowing
antibodies against certain red blood cells.
• Used to detect the presence of Rh antibody in patient
serum.

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 Pre transfusion testing
 A process which donor's blood components are
checked for transfusion to recipients
ABO & Rh grouping of the patient
Antibody screening of the patient
Donor RBC selection
Cross match

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Cross- matching
• Also known as compatibility testing

• Is a kind of double checking of blood type

compatibility.
• Is done for ABO & Rh compatible blood

 Cross-matching is the last phase of pre-transfusion testing

 It is done to avoid incompatibility of blood for recipient
and donor
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• Is a lab test used to detect antibodies in patient
(recipients) serum that can lyse donor cells (mainly) or
antibodies in donor blood that can lyses patient’s RBCs

• Types of cross match

• Major and Minor cross match

• Value of the test
• Used to prevent life threatening transfusion reactions.

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A. major- cross match
 Detects antibodies in the recipients’ serum that may
damage the cells of the donor
 Recipient serum + Donor RBCs ⤏ +/- agglutination
 Result: Agglutination ------------- incompatible

No agglutination ------------ compatible

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B. Minor cross match
 Detect antibodies in donor’s serum capable of affecting
the RBC of the recipient

 Donor serum + recipient RBC  +/- agglutination

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ABO compatibility between recipient & donor
• If possible, transfuse the patient with the same type of
blood
• Concept of universal donor or receiver or choices of
other alternatives should be made in absence of the same
kind.

Rh compatibility between recipient & donor

• Rh- patient should receive from Rh- donor
• Rh+ patient can receive from either Rh+ or Rh- donor.

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79
 Basic Clinical Chemistry Tests

1. Liver Function Tests (LFT)

• Liver functions:
• Synthetic Function
– Plasma proteins (albumin, globulins), cholesterol,
triglycerides and lipoproteins
• Detoxification and excretion
– Ammonia to urea (urea cycle), bilirubin, cholesterol, drug
metabolites
• Storage Function
– Vitamins A, D, E, K and B12
• Production of bile salts
– Helps in digestion 80
• Liver Function Tests
– Noninvasive methods for screening of liver
dysfunction

– Help in identifying general types of liver disorder

– Used for treatment follow up

81
Tests of liver function can be classified :

A. Based on substances produced by the liver

e.g. Albumin, coagulation factor
B. Based on substances metabolized by the
liver
e.g. Drug, billirubin, cholesterol
82
C. Based on substances released from the
damaged liver tissue
 Endogenous substances released by damaged
hepatocytes

e.g. enzymes (Alanine Aminotransferase -ALT,

Aspartate Aminotransferase -AST)

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 A. Hepatic Excretion Function:

 Billirubin:
• A byproduct of red blood cell breakdown

• A bilirubin test measures the level of bilirubin in

your blood.
• If liver is damaged bilirubin can leak out of liver
into blood and can cause jaundice (Yellowing of
skin and eyes)
• It is the yellowish pigment observed in jaundice

• High bilirubin levels are observed in:

– Gallstones, acute and chronic hepatitis 84
Normal value
– Conjugated billirubin = 0-0.2 mg/dl
– Unconjugated = 0.2-0.8 mg/dl
– Total billirubin = 0.2-1mg/dl

85
 B. Measuring hepatic synthetic ability

Used to asses liver diseases & quantifying the severity, but

not very sensitive to minimal liver disease.
Plasma proteins
 Most of the plasma proteins such as albumin, globulin
and plasma coagulation factors are synthesized in the
liver, therefore is a measure of hepatic function

86
 Serum albumin
• The most abundant protein synthesized by the
liver

• Normal serum levels: 3.5 – 5 g/dL

• Its levels decrease in all chronic liver diseases

87
 C. Substances released from damaged liver tissue

The most important enzymes measured in liver disease are:

A. Aspartate amino transferase (AST):

 Found in hepatocytes and myocytes (skeletal and
cardiac muscle)
 In liver disease the permeability of the hepatocytes
membrane increase & cystollic enzymes spill in to the
blood
 Normal value: 6 – 25 Units/l
88
Significance of the test
 help to diagnose liver disease (it is increased in
intracellular liver disease)
 monitor treatment of liver disease.
B. Alanine amino transferase (ALT)
Only found in hepatocytes
Like AST, increased permeability of the hepatocytes
increases concentration in plasma
Normal value 3 – 30 U/l
 The test is used to evaluate the liver disorder
More liver-specific than AST

89
C. Alkaline phosphatase (ALP)
 This enzyme is present in high concentration in the
liver, bone, placenta & intestine
 Serum ALP is commonly elevated in malignant diseases;
may be bone or hepatic origin
 Normal value: 20 – 150 U/l
D. Gamma glutamyl transferases (GGT)
 It is present in numerous tissues, including kidney ,liver,
pancreas & prostate
 GGT helps to determine whether an elevated ALP is
coming from bone or liver.

90
 2. Blood glucose test

 Glucose is the main source of energy used by the

body
 Measures the amount of glucose in blood.
 Insulin is the hormone which is produced /
secreted by -cell of the pancreas and released
into blood when the amount of glucose in the
blood rises
91
• Blood glucose tests may be done at different
times
Fasting blood glucose
2- hour post prandial blood sugar
Random blood sugar

• Significance of the test

– To diagnose diabetes
– To monitor treatment of diabetes
92
 There are different methods for the
determination of glucose.
 Example: determination of glucose with
glucometer

93
 3. Renal function tests
• Used to assess overall renal function
• waste products formed in the body as a result of
metabolism of nucleic acid, amino acid & protein
• Excretion of these compounds is important function of
the kidney
• The three non-protein nitrogenous substances are Urea,
Creatinine & Uric acid

94
 Urea
 Major excretory product of protein metabolism.
– Urea is formed in the liver as the end product of protein
metabolism and is transported to the kidneys for excretion.
– Nearly all renal diseases can cause an inadequate
excretion of urea, which causes the blood concentration to
rise above normal.
 Azothemia is increase level of urea.

 Uremia is increase blood level of urea accompanied by renal

failure
 Urea concentration of blood is often expressed in terms of
blood urea nitrogen (BUN) 95
 Clinical Significance:

• Increased urea (BUN): either Azotemia or uremia can

be resulted from
Increased protein intake
Decreased kidney function
Dehydration
• Decreased urea (BUN):
Decreased protein intake
Decreased liver function
• Not a good test for GFR: Influenced by diet and liver
function

• Normal value: 6 - 8mg/dl 96

 Creatinine
• Creatinine is a catabolic product of creatine
phosphate used in skeletal muscle contraction.
• Waste product of muscle metabolism

• Best test for overall kidney function; not affected by

diet or hormone levels
• creatinine rises when kidney function is impaired
• Sample – serum

Normal value Men = 0.7 - 1.2 mg/dl

Women = 0.6 - 1.1 mg/dl
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 Serological Tests
• Definition
– Serology, previously defined as” the study of sera.”
– But nowadays serology is not restricted to serum
– It uses range of clinical specimens including plasma,
whole blood, CSF and any other body fluids.
• Serological tests are applied for those diseases that
are difficult to diagnose or screen by other methods
– Example: syphilis serology
• Serology tests can be of antigen detection or antibody
detection

98
• Antigen detection
– To detect an antigen of a particular agent in the client
clinical specimen
– One has to use commercially produced known
antibody
– It is better than antibody detection but expensive
• Antibody detection
– To detect antibody produced by the host immune
system (against foreign agent)
– One has to use commercially prepared known antigen
– It is relatively cheap
99
1. Syphilis serology
• Example: RPR tests

2. Serological test for typhoid fever

• Widal test is serological test widely used to diagnose typhoid fever by
measuring antibodies to O & H antigens of salmonella typhi

3. The pregnancy test or HCG test

• Pregnancy tests are methods that detect the presence of HCG hormone
which is produced by the trophoblastic tissue in the placenta
 Serum HCG first reaches detectable level within 24 hour after
implantation
 Normally HCG is negative in blood & urine
 The sample of choice for HCG is early morning urine which is not
contaminated with soap residues in the container
100
• Interpretation of HCG test results
• POSITIVE: Two distinct red lines appear. One line
should be in the control region (C) and another line
should be in the test region (T).
• NEGATIVE: One red line appears in the control region
(C). No apparent red or pink line appears in the test
region.
• INVALID: Control line fails to appear. Insufficient
specimen volume or incorrect procedural techniques are
the most likely reasons for control line failure.
• Review the procedure and repeat the test with anew test
strip.
• If the problem persists, discontinue using the test kit
immediately and contact your local distributor. 101
• NOTE:
• The intensity of the red color in the test line region (T)
will vary depending on the concentration of hCG present
in the specimen.
• Therefore, any shade of red in the test region (T)
should be considered positive.

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 103
Source: Monica Cheesbrough 2009
104
• HIV serology ….. READING
ASSIGNMENT

105
 Urinalysis/urine analysis
• Urinalysis is a group of tests that detect and semi
quantitatively measure various compounds that are
eliminated in the urine
– includes the byproducts of normal and abnormal
metabolism as well as cells, and cellular fragments.
• Why urinalysis is done?
– As a general evaluation of health
– To screen for a disease or infection of the urinary tract.
– To monitor the treatment of certain conditions such as
kidney stones, a urinary tract infection (UTI), or some
types of kidney or liver disease.
– Diagnosis of some metabolic and endocrine disturbances
in the body such as diabetes. 106
 Urinalysis consists of the following measurements:
 Macroscopic exam or physical examination
 Chemical exam
 Microscopic exam of the sediment

 Physical examination of urine

Urine volume
Appearance- Clarity or transparency
Urine color

107
Urine chemical examination

N.B. There are different methods for urine chemical

examination, but here we discuss Reagent Strip Tests
• A urine dipstick interprets the presence of various
substances in urine indicating a color change on the strip
Procedures
1. Dip the test areas of the strip in the urine specimen (fresh, well
mixed and un-centrifuged).
2. Remove excess of the urine by tapping the edge of the strip against
the container.
3. Compare the test areas closely with corresponding color charts on
the bottle at the times specified.
Note: The strip should be compared with the corresponding color
charts in reasonably good light. 108
Chemical Exam

• When the test strip is dipped in

urine, the reagents are activated
and a chemical reaction occurs.
• The chemical reaction results in a
specific color change.

109
110
Chemical Reaction Chart

111
Microscopic examination of urine
 Carried out from urine sediments

 Used to detect:
 RBC’s
WBC’s
Epithelial cells
Casts
Crystals
Bacteria's
Yeasts
T.vaginalis, W.bancrofti, E.vermicularis, S. heamatobium
112
 Body Fluid Analysis
– CSF analysis
– Synovial fluid analysis
– Serous fluid analysis

113
 Examination of Cerebrospinal Fluid (CSF)

• CSF is contained in the cavity that surrounds the brain in

the skull and the spinal cord in the spinal column
• It supplies nutrients to the tissues of the central nervous
system and helps to protect the brain and spinal cord
from injury.
• The volume of the CSF in adults is 100–150 ml.
• The volume is less in children and varies according to
the body length.

114
Common reasons for investigation of CSF
• The most common reasons for investigating CSF are to
exclude:
— meningitis
— bleeding into the central nervous system
— certain cancers.
• Meningitis is an inflammation of the meninges, the
membranes lining the skull and covering the brain and
spinal column. It is often caused by infection
• Note: Immediate laboratory investigation of the CSF
may be life-saving if meningitis is suspected. 115
 Synovial fluid analysis

• Synovial fluid, often referred to as “joint fluid,” is a

viscous liquid found in the cavities of the movable joints

• Synovial fluid reduce friction between the bones during

joint movement.
• Examination of the synovial fluid is essential to
distinguish infectious from noninfectious arthritis

116
 Serous fluid analysis
• The closed cavities of the body—namely, the pleural,
pericardial, and peritoneal cavities—are each lined by
two membranes referred to as the serous membranes.
• One membrane lines the cavity wall (parietal
membrane), and the other covers the organs within the
cavity (visceral membrane)
• The fluid between the membranes is called serous fluid,
and it provides lubrication between the parietal and
visceral membranes.

117
• An effusion is fluid which collects in a body cavity or
joint.

• Fluid which collects due to an inflammatory process is

referred to as an exudate and that which forms due to a
non-inflammatory condition is referred to as a transudate.
• When the effusion is an exudate, it is important to
investigate whether the inflammatory process is an
infective one (septic) or caused by a non-infective process,
e.g. malignancy.
• When the fluid is a transudate, no further microbiological
testing is required.
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Parasitological tests

119
Specimens for parasitic detection
A. Stool Specimen
 Stool is the best specimen to diagnose intestinal
parasite and other parasites in which their diagnostic
stage is found in stool
B. Perianal specimen
 Cellophane tape preparation
 Purpose to collect the eggs of pinworm ( E.
vermicularis) as female deposits the eggs at night on the
Perianal folds
 The tape is fixed to a microscope slide and examine
microscopically

120
C. Urogenital specimen
 T. vaginalis is identified in a wet preparation of vaginal
and urethral discharge
 S. heamatobium is also identified by analyzing urinary
sediments
D. Examination of Sputum
 Ova of paragonimus westermani may be found in the
sputum
 The specimen should be collected in early morning and
examined as a saline/iodine wet mount.
E. Blood sample
 To detect plasmodium species

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 Major Techniques to Diagnose Parasitic
Infection
1. Macroscopic examination of stool

Presence of worm: for adult worm or segments

Consistency (degree of moisture): formed, semi-

formed, diarrhea
 Color: any abnormal color

Abnormal features seen: mucus, blood and fat

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2. Microscopic examination
The majority of intestinal, bloods, urinary and skin parasites
are usually detected microscopically either directly or
following concentration
2.1 Direct microscopy
Helps to detect and identify the stage of some parasites
Helps to study the motility of the organism
Cysts of protozoa and helminthes eggs can be detected
The direct microscopic methods can be two types
stained (e.g. eosin and iodine)
unstained (e.g. using saline wet mount)
2.2. Concentration
Helps us to separate debris from parasites
It enables us to examine great quantity of stool by separating
diagnostic stage of parasite from fecal specimen 123
2.2.1: Sedimentation concentration techniques

 Formol ether sedimentation technique

It concentrate the egg and larvae of helminthes and
cyst of protozoa.
It is most useful to detect the eggs of Schistosomes in
stool
Formalin - for fixation and preservation of parasites
morphology
Ether - dissolves fecal debris

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 Microbiological tests

• Staining
• Bacterial culture

Already discussed in microbiology

section
02/13/2025 125
ANY QUESTIONS?

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