Clinical laboratory methods

 Introduction to medical laboratory science

 Basic Hematological tests
 Immunohematology
 Basic serological tests
 Clinical chemistry
 Urinalysis

By: Habtamu M. E.mail:habtamumolla601@gmail.com

Debre Markos , Ethiopia , 2022

12/21/2022 Habtamu M. 1
 UNIT-1.
Introduction to clinical
laboratory
Learning objectives
At the end of this session, you will able to:
 Define the term laboratory, medical laboratory.
 Describe the roles of laboratory in health care systems.
 Discuss on the criteria of selection of laboratory tests.
 Interpret laboratory tests.
 Do calculation sensitivity, specificity, predictive values ,
efficiency.

12/21/2022 Habtamu M. 2
 Cont’d…
 Laboratory – is a place that is equipped with different
instruments, equipment's, chemicals etc for performing
either experimental works or research activities.
 Medical laboratory is part of the laboratories that is
equipped with various biomedical instruments and
chemicals for performing different lab diagnostic activities by
using biological specimens (Whole blood, serum, plasma,
urine, stool, sputum etc). It is also called clinical laboratory
science.
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 Role of clinical laboratory in the health care system

1. Patient diagnosis, screening , treatment and follow up:

 To rule out (R/o)a disease and diagnosis
To screen for disease
To provide prognostic information

2. Public health aspects such as epidemic control,

surveillance and delivery of health information.
cause of the disease in the community.
prevalence and incidence rate of infectious disease

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 Out comes of clinical laboratory tests

• Reduce disease transmission

• Reduce health care costs

• Greater patient satisfaction

• Improvement in the quality of health system

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 Without clinical laboratory test support

• The etiology of disease may not be identified correctly

• Patients less likely to receive the best possible care

• Resistance to essential drugs will to continue to spread

• Epidemics and spread of major communicable diseases will

not be checked reliably.

• Valuable financial and human resource may not use

properly

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 Selection of laboratory tests

 Criteria :

 The value of information they provide

 Cost of the test

 Availability of the necessary laboratory materials

 Availability of well trained and experienced

laboratory staff

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 Types of diagnostic tests
1. Qualitative - positive or negative
 HIV test
 HBsAg test
 HCG test
2. Semi quantitative - +1, +2,+3 etc
 Urinalysis biochemical test
 Widal and weilflx test with titration
3. Quantitative – numerical amount with unit
 Biochemical analysis
 Hematological analysis

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 Interpretation of laboratory test results

 Reliable laboratory test results:

 provides information for clinicians to classifies peoples as
diseased or disease free based on certain cutoff value or
reference value
 Depends on quality of diagnostic test which is described in
terms of:
 Accuracy
 Precision
 Sensitivity
 Specificity
 Predictive value
 Test efficiency

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 Indicators of test reliability
1. Accuracy(Trueness) - The closeness of the
measured result to the true value
 A test method is said to be accurate when the test
value approaches the absolute “true” value of the
substance (analyte) being measured

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 Cont’d…
2. Precision(repeatability) - closeness of
repeated measurement to each other
A test method is said to be precise when
repeated analyses on the same sample give
similar results

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 Cont’d…
• Both accuracy and precision shows how well test
method performs
• For effective diagnosis and management of a
patient, a method with high precision and accuracy
must be used

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 Cont’d…
3. Sensitivity - is also called true positive rate
 can have two different meanings
 ability of diagnostic test to detect very small amount
of analytes
 ability of test to detect truly infected individuals
Sensitivity=total number of true positive X100%
total number of positive individual
= Tp X100% Tp
+FN
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 Cont’d…
4. Specificity :
 Ability of test method to identify all samples which do not
contain the substance being tested
 Ability of the test to detect non-infected individuals
correctly
Sp = total number of true negative X 100%
total number of non-diseased individuals
Sp = TN X100%
TN +Fp
 Sensitivity and specificity shows how well test method
is able to distinguish disease from non- diseased.

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 Cont’d…
5. Predictive values:- ability of a test to predict the presence
or absence of disease from test results.
 Positive predictive value- the probability that an individual
with a positive test result has the disease
 PPv = TP X 100%
TP+ FP
 Negative predictive value- probability that an individual
with negative test result does not have the disease
NPV = TN X100%
TN +FN
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 Cont’d…
6. Test efficiency- the over all ability of the test to correctly
identify positives from negatives and implies the
absence of false positives and false negatives
 combination of sensitivity & specificity

 determine total effectiveness of test method

TE = TP+TN X 100%
TP+TN +FP+FN
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 Cont’d…
To determine the sensitivity and specificity of a new test

Gold standard test is required.

This helps to know the correct disease status of an

individual.
uses a 2 x 2 table to compare the performance of the new

test to the gold standard test.

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 Cont’d…
A 2X2 table

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 Cont’d…
 example: A pap smear test was undertaken to screen women for cervical
cancer in debre markos referal hospital. Apathologist screen 2400 women
consisting of 200 women whose cervices are abnormal (to an extent
sufficient to justify concern with respect to possible cancer) and of those s
180 have cervical cancer and 2180 have not cervical cancer.
 A test was applied and results are tabulated as follows.

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 Calculate and Interpret from the above table

1. Sensitivity
2. Specificity
3. PPVT
4. NPVT and
5. Yield of the screening test
6.Eficiency
7. prevalance

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 Cont’d…
1. sensitivity

 sensitivity=(Tp/Tp+FN)*100
 =(180/180+20)*100
 =90%
2. specificity
• specifcity= (TN/TN+Fp)*100
• =(2180/2180+20)*100
• 99.09%
3. positive predictive value
• PPV=(TP/TP+FP)
• (180/180+20)*100
• =90%

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 Cont’d…

Negative predictive value

•NPV= (TN/TN+FN)*100
7. Prevalency
•=(2180/2180+20)*100 p=( TP+FN/Total)*100
•=99.09%
5. Yield
=(180+20)/2400)*100
Y= (TP/Total)*100 =8.33%
=(TP/TP+TN+FP+FN)*100
=(180/180+2180+20+20)*100
=7.5%
6. Eficency
E=(TP+TN/Total)*100
=(180+2180/2400)*100
=98.33%

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1. sensitivity

sensitivity=(Tp/Tp+FN)*100
=(180/180+20)*100
=90%
2. specificity
• specifcity= (TN/TN+Fp)*100
• =(2180/2180+20)*100
• 99.09%
3. positive predictive value
• PPV=(TP/TP+FP)
• (180/180+20)*100
• =90%

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 Cont’d…
• Exercise: In Debre markos referal hospital within one
weak 120 patients diagnosed for syphilis. Among them 68
were positive and 52 were negative by VDRL test. By
another confirmatory test, TPHAT 63 were positive and
45 were negative.
 Calculate sensitivity of the test?
 Calculate specificity of the test ?
 Calculate positive predictive value of the test?
 Calculate negative predictive value of the test?
 Calculate the efficiency of the test?

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 Chapter 4 :Basic hematological tests

Learning objectives:

At the end of the session, you are expected to:

-Describe the principle of basic hematological tests

-Interpret values of these tests

-Aware how blood sample is collected

-Perform some of these tests

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 Cont’d….
Definition of Haematology
 Greek term

 Haima means blood

 Logos means study

 Hence hematology is the science or study of blood

 It encompasses:

• The study of blood cells and coagulation

• Analyses of concentration, structure and function of cells in blood and their

precursors in the bone marrow

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 Cont’d…
• Hematology- deals with examination of the constituent
of blood
• Hematological tests are used to diagnose anemea,
leukemia, haematological disorders, infectious and
inflammatory disease.
• The frequently used hematological tests in most clinical
laboratories includes:-
• Complete blood count(CBC)/(FBC)
• Erythrocyte sedimentation rate (ESR)
• Blood morphology

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 The role of Hematology Laboratory

 Confirming a physician’s clinical impression of a

possible hematological disorder

 Establish a diagnosis or rule out a diagnosis

 Detect an unsuspected disorder

 Monitor the effect of radiation or chemotherapy

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 Composition & function of blood

Blood
 It is the only fluid tissue, more viscous than water.

 It constitutes 6-8% of the total body weight

 It consists of cells suspended in a fluid called plasma

 It is about 45% cells & 55% plasma

 Has a pH of 7.35-7.45

 Volume

 5-6 liters in adult male

 4-5 liters in adult female

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 Cont’d

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 Cont’d…
Plasma
 It is a complex solution of proteins, salts and numerous metabolic substances
 It acts as a transport medium carrying its constituents to specialized organs of the
body
 Consists of:
 About 91.5% water
 About 8.5% solutes of which about 7% are proteins

 Out of the 7% protein:

54% albumin
38% globulins
7% fibrinogen

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 Cont,d…
• Plasma is the liquid, cell-free part of blood, that has been treated with anticoagulants (e.g.

heparin, potassium oxalate).

 It will be collected from anti coagulated blood through centrifuge

• Serum is the liquid part of blood after coagulation, therefore devoid of clotting factors as

fibrinogen.
 Serum = plasma - fibrinogen

Formed Elements
• The three main blood cells/formed elements are:

 Red blood cells (erythrocytes)

 White blood cells (leukocytes)
 Platelets (thrombocytes)

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 Erythrocytes (Red Blood Cells)

They are the most numerous cells

in the blood

The normal RBC count is 4.5 to 6

million cells per microliter

Their primary function is gas

exchange (O2 and CO2)

They are non nucleated cells

The cytoplasm consists of Hgb.

12/21/2022 Life span 120 days
Habtamu M. 33
 Leukocytes (White Blood Cells)

Leukocytes are:
 A heterogeneous group of nucleated cells

 They are responsible for the body’s defenses

 They are transported by the blood to the various tissues where they exert

their physiologic role, e.g. phagocytosis

The normal WBC count is ~4,000 to 10,000/L (4–10 x 103/L)

Leukocytes are usually divided into:

 Granulocytes - which have specific granules, and

 Agranulocytes -which lack specific granules

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 Cont’d…
Are divided in two main groups.

Granulocytes

- Neutrophil

- Eosinophil

- Basophiles

 Agranulocytes

-Lymphocyte

- Monocyte

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 Cont’d…

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 Neutrophils
Neutrophils are the most common type
They are the primary defense against
bacterial infection
Their size ranges from 10-12m in
diameter.
They are capable of amoeboid
movement.
There are 2-5 lobes to their nucleus
that stain purple violet.
The cytoplasm stains light pink with
pinkish dust like granules.
Normal range: 2.0-7.5 x 103/l
12/21/2022 Increased
Habtamu M. in acute bacterial infections
37
 Eosinophils
Have the same size as neutrophils or may be a bit
larger (12-14m)

The nucleus:

is often bilobed with a "spectacle" arrangement.

stains a little paler than that of neutrophils

Cytoplasm: contains many, large, round/oval

orange pink granules

Normal range: 40- 400/l

Eosinophilia is associated with allergic reactions

and helminthiasis

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 Basophils
Size: 10-12m in diameter.

are the least common type (≤1% )

Nucleus a kidney shaped often

obscured by a mass of large basophilic
granules

The granules contain:

 heparin (an anticoagulant)

 histamine (a fast vasodilator)

12/21/2022 Normal range: 20-200/l

Habtamu M. 39
 Lymphocytes
Small lymphocytes

are usually small (7-10m) or (12-14m)

has a dark round to oval nucleus, and
only a rim of pale blue staining cytoplasm
nucleus is about the same diameter as a
normal erythrocyte & occupies most of the
cell
Large lymphocyte
Nucleus:
 a little paler than small lymphocytes
 is usually eccentrically placed in the cell
12/21/2022
Cytoplasm:
Habtamu M.
It is more plentiful 40
 Monocytes
Largest white cells measuring 14-18 m
Normally comprise ~2 to 8% of leukocytes
Cytoplasm:
abundant staining light gray to light blue
& finely granular
Nucleus has very finely granular
chromatin and is often folded, bean
shaped, oval, or irregular
Monocytes have two functions:
Phagocytosis of microorganisms
Antigen processing and presentation.
Normal range: 700-1500/l
Monocytosis is seen in bacterial infections
(e.g. tuberculosis) and protozoan
12/21/2022
infections
Habtamu M. 41
 Platelets
They are small, non-nucleated,
round/oval cells/cell fragments

Their size ranges 1-4m in diameter

The cytoplasm stain pale blue and

contain many pink granules

They are produced in the bone marrow

by fragmentation of megakaryocytes

Their primary function is preventing

blood loss

Life span of ~10 days

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Normal
Habtamu M.
range: 150-400 x 103 /l 42
 Cont’d…

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 Blood specimen collection
• The objective of blood sample collection is to obtain a representative sample of
circulating blood.
• Blood sources/sample for hematological tests are
1. Capillary/micro blood

2. Venous blood

3. Arterial Blood
• Arterial blood is an ideal specimen for many analyses because its composition is consistent
throughout the body whereas venous blood varies relative to the metabolic needs of the areas of the
body it serves
• Not used for routine tests
• More invasive
• Technically difficult

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 Venous Blood Collection

• Collection of blood from the vein (venipuncture)

• Also referred to as phlebotomy

• Necessary for most tests that require larger quantities of blood

Sites of Puncture
• Veins of the forearm are preferred; wrist or ankle can also be used

• Veins in the antecubital fossa of the arm are the preferred sites

 They are larger than those in the wrist or ankle regions

 Hence are easily located and palpated in most people

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 The three main veins in the forearm

1. Medial Cubital vein

 First choice well anchored and

easy to penetrate

2. Cephalic vein
 On the outside surface

 Well anchored

3. Basilic vein
 Not well anchored, tends to roll,

painful and can cause nerve

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 Cont’d…
Advantage Venous Puncture :
• It allows various tests to be repeated for checking doubt
fullresults & allows additional tests to be performed.
• Plasma or serum can be frozen for further
reference.
• Reduce the possibility of error resulting from
dilution with interstial fluid.
Disadvantage Venous Puncture :
- Lengthy in procedure.
- Technical difficulty in children,
obeseindividual and patient in shock.

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 Cont’d…
Blood collection procedure by venipuncture
1. Assemble all the things required during blood collection
2. Read carefully the patients form, identify the patient and decide patient and
decide the total amount of blood needed for the entire test.
3. Select the blood collection container and label them with the patients
identification number
4. Introduce your self to the patient. Ask the patient to sit alongside the table
used for taking blood. Lay his arm on the table, palm upwards.
- The procedure of blood collection should be explained by the vein-
punctures' to the patient to minimize apprehension.
- NB: Never draw blood from standing patient or patient sitting on a high stool.

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 Cont’d…
5. Select the puncture site carefully after inspecting both arms.
6. Apply the tourniquet before drawing blood.
 The tourniquet should not be left in place unless the technician is
ready to proceed immediately with the vein-puncture
 Position the tourniquet 7.5 – 10 cm above the venipuncture site
with strip equal on both sides
7. Using the index figure of your left hand , feel for the vein where you
will introduce the needle.
8. Disinfect the site with a swab dipped in methanol or 70% alcohol.
Rub the venipuncture site thoroughly

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 Cont’d…
9. Remove the syringe from the protective warp or test tube used
during sterilization and the needle from the sterilized vial, assemble
them and see the needle is fixed tightly.
10.Puncture the vein, try to enter the skin first and then the vein , at a
30 to 40 degree angle. Continue with draw the position and fill the
syringe with the request amount of blood.
11. Release the tourniquet by pooling on the looped end.
12.Place a swab of cotton wool over the hidden point of the needle.
With draw the needle in one rapid movement from under the swab

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 Cont’d…
13. Ask the patient to firmly on the cotton wool swab for 3 to 5 minutes. This stops bleeding from the
wound. Do not bend the arm , this may cause hematoma.

14. Remove the needle from the syringe and gently expel the blood in to appropriate container.

15. Mix the blood immediately and thoroughly but gently with the anti coagulant. Label the bottle
clearly with the name of the patient, date, sex and registration number.

16. Immediately discard the syringe and the needle in appropriate waste disposal equipment.

17. Before the patient leaves , re-inspect the venipuncture site to ascertain that the bleeding has
stopped. If the bleeding has stopped , apply an adhesive tap over the cotton wool swab on the
wound , otherwise continue to apply pressure until the bleeding stops. Do not leave the patient
until the bleeding stops

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 Needle •

• Needle Size or Gauge depends on • Colored coded for size

the size and depth of the vein to be
punctured
• Gauge is the diameter of the needle

• The larger the gauge number the

smaller the needle

• Range from 16 –25

• Routine: 21 gauge

• Children: 23 gauge
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 Skin/ Capillary Puncture

 Used when small amount of blood is required for e.g.

Hemoglobin, WBC count & blood smear preparations.
 It is also used when vein puncture is impractical
• In neonates
• In case of sever burn
• In patients whose both arm veins are being
used for IV medication.
NB-Skin puncture specimen is preferred over venipuncture
specimen for the study of cell morphology because the
anticoagulant could affect the cell morphology

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 Cont’d…
Sites of Puncture
 Ring/middle finger - adults & children – half way b/n centre
& ball (vascular & fleshy).
 Heel - infant < 3 months – Side of heel.
 Big toe – older infants (> 3 months) – side of great toe.
 Ear lobe – today is not routinely used as a blood collection
site.

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 Cont’d…
Sites of Puncture

o Adults and children:

Palmer surface of the tip of the ring or middle finger or free

margin of the ear lobe

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 Cont’d…
• Infants:
• plantar surface of the big toe or the heel.
h
• The areas of the foot of a baby or infant that are
suitable for obtaining capillary blood

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 Cont’d
Considerations:
• Edematous, congested and cyanotic sites should not be

punctured
• Cold sites should not be punctured as samples collected

from cold sites give falsely high results of hemoglobin

and cell counts
• Cold site should be massaged until it is warm

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Capillary sample collection method
 Materials Required

• Cotton or gauze pads

• 70% alcohol or other skin antiseptic

• Sterile disposable lancet (automatic

lancet)

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 Cont’d…
Blood collection by finger puncture procedure
1. Assemble the necessary equipment, lancet alcohol pad,
dry surgical gauze, capillary tube, microscope, slide and
other supplies(glass, marking penile, lead panicle, etc).
2. Be sure that the patient is seated comfortably.
3. Find a spot on the middle or ring finger of the left hand.
The spot is located on the side of the figure, which is
less sensitive than the tip

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 Cont’d…
4.Clean the site with a sterile cotton wool swab dipped in 70% alcohol, then remove the
alcohol with a dry sterile cotton wool swab. This remove dirt, and epithlial debris. Warm up
the part chosen site for pricking, increase the blood circulation. And leave the area relatively
sterile.

5.Grasp the figure firmly and make a quick , firm puncture with a sterile lancet (sharp pointed
blade). The puncture should be 2-3 millimeter deep at the pre located spot on the side of the
figure in line with the figure print striations.

- If a good puncture has been made , the blood will flow freely. If it does not , use gentle
pressure to make the blood form a round drop.

NB.- Excessive squeezing will cause dilution of blood with tissue fluid.

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 Cont’d…
6. Wipe away the first drop of blood with sterile cotton wool. The
first drop of blood may be contaminated with tissue fluid and will
interfere with the laboratory result if used. The succeeding drops
are used for test.
7. Collect the specimen by holding a capillary tube to the blood drop
(for hematocrit determination), or by sucking in to
the Sahli pipette for the hemoglobin determination and for blood
count, or by touching the drop to the glass slide for preparing
smear

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 Arterial blood collection
 An ideal specimen for many analyses
 Because its composition is consistent throughout the body
 Whereas venous blood varies relative to the metabolic needs of the areas of
the body it serves
 Primarily for evaluation of arterial blood gases (ABGs)
 Is critical for the diagnosis and management of respiratory diseases
 Assessment of oxygenation problems in
 Pneumonia
 Pneumonitis

 Pulmonary embolism etc.

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 Cont’d…
 Not used for routine tests
 More invasive
 Technically difficult
 Lots of pre-analytical interferences:
 Exposure to air before testing
 Delay in transport
 Requires proper specimen collection
 E.g. In patients with metabolic diseases, it is difficult to differentiate whether it is venous
or arterial origin

 Pressure in arteries makes more difficult to stop bleeding – leads to hematoma

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 Anticoagulants
• Anticoagulant may or may not be used depending on the
types of the test.
• Anticoagulants are substances that prevent blood from
clotting.
• The common anticoagulants used in hematology are:
- Ethylene Diamine Tetra Acetic acid (EDTA)
- Sodium citrate
- Heparin and oxalates.
• The choice of anticoagulant depends on the test
purpose.
• The proportion of anticoagulant to the blood must
be optimal.
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 Complete Blood Count
 Provides important information about the kinds and number of
cells in the blood ; RBC WBC ,and platelets.
A CBC can be done to:
 Investigate the cause of certain symptoms like fatigue, weakness, fever,
bruising or weight loss.
 Detect anemia or determine severity of blood loss.
 Diagnose polycythemia, leukemia
 Monitor the response to some types of drugs or radiation
treatment.
 Investigate a history of abnormal bleeding

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 Cont’d…
 The complete blood count (CBC) is one of the most commonly ordered
clinical laboratory tests
 A CBC includes:
 The red blood cells (RBCs) count
 Hemoglobin determination ( Hgb)
 Hematocrit measurement (Hct)
 Red cell indices
 Mean cell volume (MCV)
 Mean cell hemoglobin(MCH)
 Mean cell hemoglobin concentration (MCHC)
 Red cell distribution width (RDW)
 Total WBCs count and differential count
 Total Platelet count and other parameters

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 Cont’d…
 Cell counts are nowadays performed by automated
procedures/instruments
 Depending on the type of automated instrumentation used,
some of these parameters are directly read from the
instrument and some are calculated
 Most automated instruments directly read the
 WBC count
 RBC count
 Platelet count
 Hemoglobin concentration (Hgb)
 Mean red cell volume (MCV)
 Mean Platelet volume (MPV)

12/21/2022 Habtamu M. 67
 Cont’d…
 Calculated parameters include:

Hematocrit (Hct)

Mean cell hemoglobin (MCH)

Mean cell hemoglobin concentration (MCHC)

Red cell distribution width (RDW)

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 Cont’d…
Basic Principles:

Cell counting and sizing (WBC,RBC,PLT)

 Electrical impedance method

 Light scatter and/or absorption (with or without cytochemistry)

Hemoglobin: spectrophotometric method

 Cyanmethaemoglobin

 Cyanide free Hemoglobin

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 Cont’d…
Electrical Impedance:
Counting and volumetric sizing based on the detection and

measurement of changes in electrical resistance produced by a particle

suspended in a conductive liquid as it is drawn through a small aperture
Resistance or change in current when cell passes between two

electrodes in NaCl solution

A blood sample is diluted in saline, which is a good conductor of

electrical current

12/21/2022 Habtamu M. 70
 Cont’d
 DC current is applied between the two
electrodes.
 Electrical resistance or impedance occurs
as the cells pass through the aperture
causing a change in voltage.
 Each cell momentarily increases the
electrical resistance between two
electrodes.
 The number of pulses is proportional to
the number of cells counted.
 The amplitude and size of the pulse
depends on the cell volume.
12/21/2022 Habtamu M. 71
 Cont’d…
FlowCytometry
Three main components
 Fluidics: controls cell detecting and cell flow
 Optical system that “questions” the cells while they cross a laser beam
 Electronics: controls the equipment, depicts and analyses data
Measuring cells in a flow system (suspension) instead of on
a static microscope
Uses lasers to measure both forward and side scatter

12/21/2022 Habtamu M. 72
 Cont’d…
 Correlation checks between the Hgb and Hct are a significant

part of quality assurance for the CBC and are known as the “rule
of three”
 The formulas for correlation checks/rule of three are as follows:

Hgb × 3=Hct ± 3 and RBC × 3= Hgb

 Additionally, each instrument presents a pictorial representation

of the hematological data registered as either a histogram or a

scatterplot,

12/21/2022 Habtamu M. 73
 Cont’d…
Total WBC count
 A white cell count is used to investigate infections and unexplained
fever and to monitor treatments which can cause leucopenia.
Normal values for WBC count
• Children at 1 year--------------------6.0-18X103/mm3
• Children 4-7 years ------------------5.0-15.0X103/mm3
• Adults………………………….4.0-10X103/mm3
• Pregnant women ---------------------up to 15X103/mm3
• Adults of African origin 2.6-8.3 x 109/L

• Ethiopian adult WBC Reference range: 3.0-10.2 x 109/L (Tsegaye A et

al Clin Diagn Lab Immunol 1999; p410-
28-Fe4b-212 4)

12/21/2022 Habtamu M. 74
 Cont’d…
Interpretation of WBC count
• A total WBC count, when used with a differential count it is used to
differentiate whether the infectious agent is bacteria or viral.
• Leukocytosis-increase number of WBC
– Acute infections (e.g. pneumonia, abscess, whooping cough,
tonsillitis, acute rheumatic fever, septicemia, gonorrhoea,
cholera, septic abortion, etc)
– Note: Acute infections in children can cause a sharp rise in WBC
count (than in adults to a corresponding infection).
– Metabolic disorders (e.g eclampsia, uremia, Diabetic coma,
acidosis)
– Inflammation and tissue necrosis (burns, gangrene, fractures and
trauma, arthritis, tumors, acute myocardial infarction

12/21/2022 Habtamu M. 75
 Cont’d…
• Poisoning (e.g. chemicals, drugs, snake venoms)
• Acute hemmorhage
• Leukemias and myeloproliferative disorders
• Stress, Menstruation, strenuous exercise
• Appendicitis
• Pregnancy
• Hemolytic disease of the new born

12/21/2022 Habtamu M. 76
 Cont’d…
Leukopenia
• The WBC may drop below normal
It occurred in:-
• Viral, bacterial, parasitic infections
• e.g. HIV/AIDS, viral hepatitis, measles, rubella, influenza, rickettsial
infections, overwhelming bacterial infections such as miliary tuberculosis,
relapsing fever, typhoid, paratyphoid, brucellosis, parasitic infections
including leishmaniasis and malaria.
• Drugs (e.g. Cloroamphenicol, phenlybutazone)
• Rheumatoid arthritis, cirrhosis of the liver, and lupus erythematosus

12/21/2022 Habtamu M. 77
 Cont’d…
• Radiation and certain drug therapy
cytotoxic) and reactions to chemicals.
• Hypersplenism.
• Production failure as in Aplastic anemia and
megaloblastic anemia (Folate and vitamin B12
deficiencies).
• Bone marrow infiltration (e.g. lymphomas,
myelofibrosis, myeloma).
• Anaphylactic shock.

12/21/2022 Habtamu M. 78
 Cont’d…
Differential WBC count
• There are 5 major kinds of WBC, namely neutrophil,
lymphocyte, monocytes, eosinophil and basophile .
• Immature neutrophils are called band neutrophil and
are included as part of the test.
• Each cell type plays a different role in
protecting the body.
• The differential WBC count is the enumeration of the
relative proportion of the various types of WBCs as
seenin stained examinations.
12/21/2022 Habtamu M. 79
 Cont’d
Neutrophilia/neutrophilic leucocytosis:
• an increase in the number of circulating neutrophils
above normal (>2.0-7.0 x 109/L)
• This is occur in:-
• Overwhelming infections
• Metabolic disorders: uremia, diabetic acidosis
• Drugs and chemicals: lead, mercury, potassium chlorate
• Physical and emotional stress
• Hematological disorders: myelogenous leukemia
• Tissue destruction or necrosis: burns, surgical operations

12/21/2022 Habtamu M. 80
 Cont’d…
Neutropenia:
a reduction of the absolute neutrophil count below 2.0 x
109/L
• Myeloid hypoplasia
• Drugs (chloramphenicol, phenylbutazone)
• Ionizing radiation
Hypergranular neutrophils (neutrophils with toxic
granules): these are
• neutrophils with coarse blue black or purple granules.
• indicative of severe infection or other toxic
conditions.

12/21/2022 Habtamu M. 81
 Cont’d…
Eosinophilia:
• An eosinophil count above 0.5 x 109/L
• Occurs during:
• Allergic diseases: bronchial asthma, seasonal rhinitis
• Intestinal parasitic infections: e.g. trichinosis,
taeniasis
• Skin disorders
• Chronic myelogenous leukemia

12/21/2022 Habtamu M. 82
 Cont’d…
Eosinopenia:
• An eosinophil count below 0.04 x 109/L.
• Occurs during:
 Acute stress due to secretion of adrenal glucocorticoid and
epinephrine
 Acute inflammatory state
Basophilia:
• Abasophil count above 0.2 x 109/L
• Rare condition
• Occurs during:
• Allergic reactions
• Chronic myelogenous leukemia
• Polycythemia vera

12/21/2022 Habtamu M. 83
 Cont’d…
Monocytosis:
• a monocyte count above 1.0 x 109/l
• Occurs during
• Recovery from acute infections
• Tuberculosis
• Monocytic leukemia
Monocytopenia:
• a monocyte count below 0.2 x 109/l
• Occurs during
• Treatment with prednisone
• Hairy cell leukemia

12/21/2022 Habtamu M. 84
 Cont’d…
Lymphocytosis:
• absolute lymphocyte count above 4.0 x 109/L in adults and
above 8.0 x 109/L in children.
• Seen during
• Infectious lymphocytosis associated with coxackie
virus
• Other viral infections: Epstein-Barr virus,
cytomegalovirus
• Acute and chronic lymphocytic leukemia
• Toxoplasmosis

12/21/2022 Habtamu M. 85
 Cont’d…
Lymphocytopenia:
• Lymphocyte count below 1.0 x 109/l in adults and below 3.0
x 109/l in children
• Seen in:
 Immune deficiency disorders: HIV/AIDS
 Drugs, radiation therapy

12/21/2022 Habtamu M. 86
 Cont’d…
• Red blood cell (RBC) count
 RBCs carry oxygen from lung to the tissue and also carry
carbon dioxide back to the lungs.
 Red cells can be counted manually or using electronic
devices
 Normal value adults
–Females 3.6 – 5.6 x 1012/L
---Males 4.2 – 6.0 x 1012/L

12/21/2022 Habtamu M. 87
 Cont’d…
Interpretation
• RBC count is increased in:
– Polycythemia vera
– Secondary polycythemia due to other causes such as
dehydration and the effect of altitude
• RBC counts below Normal in:
– anemia
– secondary to other disorders
• With hematocrit & hemoglobin it is used to calculate red cell
indices which are used to classify anemia

12/21/2022 Habtamu M. 88
 Cont’d…
 Platelet count
Is the total number of platelets per liter of whole blood
 Clinical significance of Platelet count
To investigate bleeding disorders.
 Capillary blood should not be used because there is a tendency

for the platelets to clump as the blood is being collected.

12/21/2022 Habtamu M. 89
 Cont’d…
Reference range
platelets per liter of blood
 150-400 x 109
Ethiopian: 98 – 337 x 109/L ((Tsegaye A et al Clin Diagn Lab
Immunol 1999; p410-414)
Thrombocytosis
Chronic myeloproliferative diseases:
Essential thrombocythemia
Polycythaemia vera
Chronic myelogenous leukemia
Myelofibrosis
Following splenoctomy

12/21/2022 Habtamu M. 90
 Cont’d…
Thrombocytopenia
Thrombocytopenia purpura
Aplastic anemia
Acute leukemia
Gaucher’s disease (an autosomal recessive disorder marked by a shortage of an
enzyme called glucocerebrosidase. develop progressive bone disease and an enlarged
spleen)
Infections, e.g. typhoid and other septicemias
Deficiency of folate or vitamin B12
Drugs (e.g. cytotoxic, quinine, aspirin), chemicals (e.g. benzene), some
herbal remedies
Hereditary thrombocytopenia (rare condition)
Following chemotherapy and radiation

12/21/2022 Habtamu M. 91
 Cont’d…

 High haematocrit value can also be caused by too little water in

thebody(dehydration).Low haematocrit value used to identify anaemia\
 Reference range varies between age, gender and altitude.
Reference values:
 Males- Normal: 40% to 54%
 Females- Normal: 36 to 46%
 A decrease Hct

 Anemia
• An increase Hct

 Hemoconcentration, polycythemia vera, or polycythemia secondary to chronic hypoxia

12/21/2022 Habtamu M. 93
 Methods of Hct measurement

Methods

Macro method
Wintrobe method

Micro methods
Microhematocrit method

 Electronic method
Calculated value

12/21/2022 Habtamu M. 94
 Hemoglobin determination
• Hemoglobin is the protein contained in RBCs that carries O 2 from the lungs to

the tissues and carries CO2 from tissues back to the lungs.

• Hemoglobin is normally present in red blood cells only

• Two primary structures: Hgb = heme + globin

 Globin = protein portion

 Heme (non-protein portion): Protoporphyrin & Iron

• Hgb molecule consists of four polypeptide chains:

 2α-chains, each with 141 amino acids and

 2β-chains, each with 146 amino acids

12/21/2022 Habtamu M. 95
 Cont’d…
 Hemoglobin, the main component of red blood is a conjugated protein that serves
as the vehicle for transportation of oxygen and carbon dioxide.
 Hgb determination is used to:
1. Screen disease associated to anemia
2. Determine the severity of anemia
3. Follow the response to treatment of anemia
4. Evaluate anemia

5. Helps in citing changes in Hgb concentration before & after operation

& blood transfusion

12/21/2022 Habtamu M. 96
 Cont’d…
There are different methods

(1) Spectrophotometric

a) Cyanmethemoglobin
b) Hemo-Cue
c) Oxyhemoglobin

(2) Visual comparative method

a) Sahli - Hellinge method
( 3) Cu SO4 specific gravity

12/21/2022 Habtamu M. 97
 Cont’d…
• Hemoglobin concentration is expressed in g/l,
g/100ml, or g/dl of blood.
• Expected value (normal value)
» Adult males:13-18g/dl
» Adult females: 11-16g/dl
» New borns: 14-23g/dl
• The formulas for correlation checks/rule of three are as part of
quality check of the procured is follows:

• Hgb× 3 =Hct ± 3 and RBC × 3=Hgb.

12/21/2022 Habtamu M. 98
 Cont’d…
Interpretation
• Each health institution should establish its own reference
ranges
• Decreased RBC, HGB and/or HCT values….Anemia
– Decreased production, increased loss/destruction

• Increased RBC, HGB and/or HCT values….Polycythemia

– Increased production

• Critical values: HGB <7.0 or >18.5 g/dL

12/21/2022 Habtamu M. 99
 Red cell induces
 Red cell indices are absolute values calculated from:

Hgb

PCV

RBC count
 Red cell indices are of considerable clinical importance in the
diagnosis and classification of anemias
 Red cell indices are dependent upon the accuracy of the various red

cell parameter estimations

12/21/2022 Habtamu M. 100

 Cont’d…
• It is the average volume of red cell expressed in femto litres (fL)

• Femtoliter is 10-15 of a liter

• MCV is obtained by dividing the PCV by red cell number

• The MCV indicates whether RBCs appears normocytic, macrocytic or microcytic

• MCV (fl) = PCV (l/l)

No. of RBC/l
Example: PCV = 0.45(l/l), RBC = 5  1012/l
MCV = 0.45 (l/l) = 90  10-15 = 90 fl

5  1012l

12/21/2022 Habtamu M. 101

 Cont’d…
Normal Values: Men and women: 80-100 fl
 MCV

Increased in macrocytic anemia

Decreased in microcytic anemia

12/21/2022 Habtamu M. 102

 Mean Cell Hemoglobin (MCH)

 It is the average amount of hemoglobin per individual red cell

expressed in picograms (pg = 10-12 )

 It is given by:

MCH (Pg) = Hgb (g/L)

RBC/L

Example: Hgb = 150g/L, RBC = 5  1012/L

MCH (pg) = 150g/L = 30  10-12 = 30pg

5  1012/L

12/21/2022 Habtamu M. 103

 Interpretation.
Normal Value:
 Men and women: 27-31 pg

MCH is increased in:

 Macrocytic anemia

MCH is decreased in:

 Microcytic anemia

 Iron deficiency anemia

12/21/2022 Habtamu M. 104

 Cont’d…
Mean Cell Hemoglobin Concentration (MCHC)
 It is the average hemoglobin per unit volume of red cells

MCHC (g/L) = Hgb (g/L)

PCV (L/L)
 Example: Hgb = 148g/L, PCV = 0.45 (L/L)

MCHC = 148g/L = 328g/L =32.8%

0.45(L/L)
12/21/2022 Habtamu M. 105
 Interpretation
Normal Values: Men and women: 32-36 % (320-360 g/L)

MCHC is increased in: Hereditary spherocytosis

MCHC is decreased in: Iron deficiency anemia

Exercise: A complete blood count was performed for a patient and the
following profiles were recorded:

WBC= 8,000/mm3, PCV = 50%, Hgb = 15g/dl

RBC count = 6 x 1012/L

 Calculate the MCV, MCH and MCHC values for the patient

12/21/2022 Habtamu M. 106

 Erythrocyte sedimentation rate(ESR)
• Erythrocyte sedimentation rate is the rate of fall (sedimentation) of red cells

when an anticoagulated blood is allowed to stand undisturbed for a specified

period of time, usually 1 hour.
• The rate is expressed in mm/hr

• It is a non specific test used as an index of the presence and extent of

inflammation (the so-called 'acute phase response') and its response to treatment,
e.g., tuberculosis, rheumatoid arthritis.
• Albumin which tends to counteract rouleaux formation diminishes in concentration

(hypoalbuminemia) in inflammatory processes further increasing the sedimentation

rate.

12/21/2022 Habtamu M. 107

 cont’d…
• 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 ESR is non-specific test.
• Rise during inflammation

12/21/2022 Habtamu M. 108

 Cont’d…
• Several factors affect ESR positively or negatively.
I. Plasma proteins
- Fibrinogen --------accelerate ESR
- Albumin -----------retard ESR
II. Red cell factors (shape and size)
- Anemia increase ESR
- Microcytes sediment slower than macrocytes
- Sickel cells and spherocytes have low sedimentation rate
III.Mechanical influences
 Vibration reduces ESR by retarding rouleaux formation
IV. Temperature
 High temperature causes falsely elevated ESR due to inhibition of zeta potential.

12/21/2022 Habtamu M. 109

 Cont’d…
• There are two methods for the determination of ESR.
1. Westergren
2. Wintrobe‘s
Westergren method
• The westergren ESR tube is a straight pipette 30cm long, 2.5mm
internal diameter and calibrated from 0-200.
• Reference value
Women……0-20 mm/ hr
Men……...0-15 mm/hr

12/21/2022 Habtamu M. 110

 Cont’d…
Significance of Measuring the ESR
• normal ESR can not be taken to exclude the presence of
organic disease
• majority of acute or chronic infections and most neoplastic
and degenerative diseases are associated with changes in the
plasma proteins which lead to an acceleration of the
sedimentation rate

12/21/2022 Habtamu M. 111

 Blood film preparation and staining
• Making And Staining Blood Films
• Stained blood film examination includes :
• Making blood film
• Fixing and staining blood film.
• The reliability of blood film examination depends heavily on
well made & well stained films that are systematically
examined .
• There are three methods of making blood films.
A. wedge method (glass slide)- most common
B. cover glass method

12/21/2022 Habtamu M. 112

 Cont’d…
• Blood stain dyes used in blood work are two general classes
1.Basic dyes (like methylene blue) - used to stain nuclei and certain
other structures.
2.Acidic dyes (like eosin) - used to stain acidophilic
structures
• Stained red cell examination is the best method of studying:-
– the variations and abnormalities in red cell size, shape,
structure, Hgb content & staining properties.
– It is also useful in diagnosis of blood disorders & blood parasites
(e.g Malaria, Trapnosmiasis, Borella).

12/21/2022 Habtamu M. 113

 Chapter 5 :Immunohaematological tests

Learning Objectives

At the end of this chapter, the student will be able to:

• Define Immunohematology

• Discuss antigen and antibody reaction

• Describe ABO and Rh blood grouping

• Perform Cross-matching test

• Discuss about blood and blood components

12/21/2022 Habtamu M. 114
 Cont’d…
• Definition: Immunohematology:
– is more commonly known as "blood banking“
– deals with the concepts and clinical techniques related to
modern transfusion therapy.
• It is the area of laboratory medicine dealing with the general procedures
involved in collecting, preparing, storing and transfusing blood
• Refers to immunologic reactions involving blood components
• An application of the principles of immunology to the study of red cell
antigens and their corresponding antibodies on blood for resolving the
problems of blood transfusions

12/21/2022 Habtamu M. 115

 Cont’d…
Blood group system
 The main importance of blood grouping:-
- safe blood transfusion and blood banking
- Understanding prevention of Rh immunization associated
with pregnancy.
- For organ transplantation
-To determine whether two people could be blood relatives.
- When a woman is planning to become pregnant or
pregnant woman.
- To help identify a person suspected of committing a crime

12/21/2022 Habtamu M. 116

 The ABO blood group system

Blood group/ phenotypes Plasma antibody Red cell antigen

A Anti-B A

B Anti-A B

O Anti -A and Anti-B Null

AB Null A and B

12/21/2022 Habtamu M. 117

 Blood Cell antigens

• A unique set of red blood cell Ag is determined through genetic inheritance.

• Antigens protrude from the surface of RBC in three dimensional

configurations.
• As a result, they are accessible to Ab molecules for agglutination reaction.

• In biochemical terms these antigens may take the form of:

 Proteins
 Glycoprotein
 Glycolipids

12/21/2022 Habtamu M. 118

 Blood group Abs

Blood group antibodies are classified into:

 Natural antibodies
 not provoked by previous RBC sensitization
 Mainly IgM type
 complete antibodies, cold agglutinins (25oC)
 Cannot cross the placenta
 Immune antibodies
 Produced due to previous antigenic stimulation either by transfusion
or pregnancy
 Mainly IgG type
 Incomplete antibodies, warm agglutinins (37oC)
 Cross the placental barrier

12/21/2022 Habtamu M. 119

 Antigen - Antibody interactions

• The binding of an antigen with an antibody

• When Ag and Ab combines, an immune complex is

produced
• The presence of in vitro antigen and antibody interaction
can be detected by:
 Hemolysis

 Agglutination (most commonly used)

 Precipitations
12/21/2022 Habtamu M. 120
 ABO blood group system

• Karl Landsteiner in 1900:

– The beginning of modern blood banking and transfusion

medicine
– Described the blood groups as A, B and O.

– Stated rule- Land steiner’s rule

“normal, healthy individuals possess ABO antibodies to the ABO

antigens lacking on their RBCs.”
• Von Decastello and Sturli - Described group AB

12/21/2022 Habtamu M. 121

 General characteristics of the ABO Ags

• ABO antigens are widely distributed and located

– on RBCs, lymphocytes, platelets, tissue cells, bone marrow, and organs such
as the kidneys.
• Soluble forms of the antigens can be
– synthesized and secreted by tissue cells
– found in association with cellular membranes and in all body fluids except,
CSF
– Are glycoproteins
• The antigens expressed on red blood cells determine an individual's blood group

12/21/2022 Habtamu M. 122

 Cont’d…
• ABO system Ags, which are intrinsic to the RBC membrane

– exist as either glycoprotein or glycolipid molecules

– are detectable as early as 5 to 6 weeks in utero

– are weaker on cord blood and result in weaker ABO
phenotype reaction
– develop slowly and reach the full expression of adult
levels at approximately 2 to 4 year of age

12/21/2022 Habtamu M. 123

 Cont’d…
Antibodies of ABO system
• They are naturally occurring
• They are present in reciprocal to the missing
ABO antigen.
• They are IgM –type in nature, react at room
temperature
• Can fix complement
ABO blood typing
• The main purpose of ABOblood typing is to
prevent transfusion reaction.
12/21/2022 Habtamu M. 124
 Routine ABO phenotyping

• There are two methods of ABO blood typing

1. Forward /cell typing/

2. Reverse /serum typing/

 Forward /cell typing :-

• Principle:- based on the presence or absence of the

corresponding antigens on the red cells surfaces of the
individual
12/21/2022 Habtamu M. 125
 Cont’d…

Forward/Direct (cell grouping)

 Testing of red blood cells for the presence of ABO antigens

(Unknown cell + known anti-sera)

Reverse /Indirect (serum grouping)

 Testing of serum or plasma for the expected ABO antibodies (Unknown

serum + known cell)
Clinical significance
• For safe blood transfusion
• Prevention of Hemolytic Disease of the Fetus and Newborn (HDFN)

12/21/2022 Habtamu M. 126

 Forward grouping

+ = reactions
NB: o = No reaction

12/21/2022 Habtamu M. 127

 Cont’d…
 Reverse /serum/ typing

 Principle:-To confirm ABO blood grouping based on the

presence or absence of antibodies in serum of a person.
• Serum + RBC A-Ag or B-Ag) ⤏(+/-)Agultination.
• Sample(Antibody) +Reagents(cells/ antigens)

12/21/2022 Habtamu M. 128

 Cont’d...
• Interpretation
• (+) agglutination antibodies specific for either
A or B antigen is present

• (0) agglutination- No antibody is present for Ags

12/21/2022 Habtamu M. 129

 Points to be considered in routine phenotyping

• Donor and recipient RBCs  must be tested using known anti- A and anti-B.

• Donor and recipient serum or plasma must be tested for the expected ABO
antibodies using reagent A1 and   B red cells.

• Cord blood and samples from infants < 4 months old should be tested by
forward ABO grouping.
• The ABO phenotype is determined when the red blood cells are directly
tested for the presence or absence of either A or B antigen.
• Serum testing provides a control for red blood cells testing, since ABO
antibodies would reflect Landsteiner’s rule.

12/21/2022 Habtamu M. 130

 Cont’d…

The ABO phenotypes and their corresponding genotypes

• Physical expression of inherited traits, determined by reacting red cells
with known antisera (Phenotype)
• Actual genes inherited from each parent, family studies are required to

determine the actual genotype (Genotype).

Phenotypes Genotypes
A AO,AA
B BO,BB
O OO
AB AB

12/21/2022 Habtamu M. 131

 Table : ABO phenotyping Reaction

Phenotype Rbc reaction Serum/ plasma reaction

Anti-A Anti-B A -cell B-cell

A + 0 0 +

B 0 + + 0

AB + + 0 0

O 0 0 + +

+= agglutination, O= no agglutination

12/21/2022 Habtamu M. 132

 Forward and Reverse Grouping Reaction Patterns Of the ABO
groups

12/21/2022 Habtamu M. 133

 Cont’d…

12/21/2022 Habtamu M. 134

 RH Blood group system

• In 1940 Landsteiner & Wiener discovered a human blood factor, which they
called Rhesus factor
• They immunized guinea pigs and rabbits with blood from the Macacus rhesus
monkey
• The antiserum obtained agglutinated not only the red cells of the rhesus monkey
but also 85% of human.
• This discovery followed the detection of an antibody occurred in the serum of a
woman who delivered a stillborn fetus by Levine & Stetson in 1939
• They also postulated that the antibody had arisen as the result of immunization of
the mother by a fetal antigen which had been inherited from the father

12/21/2022 Habtamu M. 135

 Cont’d…
• In 1941, Levine & his coworkers showed that not only could
Rh negative mother become immunized to an Rh positive
fetus in utero but also that the antibody could then traverse the
placenta and give rise to erythroblastosis fetalis or Hemolytic
Disease of fetus or the New born (HDFN).

12/21/2022 Habtamu M. 136

 Antigens of the Rh system (D antigen)

• There are over 40 known Rh- Ags, five of which can cause post transfusion problems.
• Some individuals posses a weak form of D- Ags known as weak – D
or Du variants.

• D antigen is the most immunogenic antigen in the Rh system

• Other antigens C, E ,c, e …

• More than 80% of D-negative people receiving a D-positive red blood

cell transfusion produce an antibody with anti-D specificity
• For that reason, a D-negative patient should receive D- negative red
blood cells.

12/21/2022 Habtamu M. 137

 Rh- antibodies

• Rh- antibodies are usually made by exposure to Rh antigens through

transfusion or through pregnancy
• Most are IgG (IgG1) and bind the corresponding antigen at 370C
• It can cause a severe hemolytic transfusion reaction in a recipient if transfused
with blood possessing the offending antigen.
• Being IgG, are capable of crossing the placenta and are associated with
hemolytic disease of the new born (HDN).
• Anti-D is common cause of sever HDN (erythroblastosis faetalis)
• Anti-D is produced when Rh negative individuals receive blood from
Rh positive individual having D- Ag which is potent immunogen.

12/21/2022 Habtamu M. 138

 Methods of Rh Blood grouping Technique

– Direct slide and

– Direct tube

• Reverse grouping cannot be done due to the absence of naturally

occurring Rh antibodies in the serum of persons lacking the
corresponding Rh antigen.

Clinical significance
– For safe blood transfusion

– Prevention of Hemolytic Disease of the Fetus and Newborn (HDFN)

12/21/2022 Habtamu M. 139

 Cont’d…
 RBCs (D-Ag) + Anti-D(reagent) ⤏ Agglutination
Interpretation:
 + ve Agglutination ⤏ Rh postive
 -ve Agglutination Rh-negative or Du variant
Weak D (Du) typing
• Rh negative by direct (cell) Rh typing must be further tested for the presence
of Du- variants by indirect anti- globulin test (IAT)/ coomb’s test.
Results by IAT:-
• No agglutination – Negative for both D- Ag & Du- Ag.
• Agglutination – positive for Du- Ag.

12/21/2022 Habtamu M. 140

 Other minor blood group systems

• Kell Blood Group system

• Duffy Blood Group system

• Lutheran blood group system

• Lewis Blood group system

• P blood group system

• MNSs blood group system

• Kidd (JK) blood group system

12/21/2022 Habtamu M. 141

 Transfusion Practices

Includes:
• Selection of blood donor

• Pre-transfusion testing
• Transfusion therapy

Donor Selection
• Age: 18-60 yrs

• Hemoglobin: no less than 12.5 g/dl

• Hematocrit: male- no less than 41%, female–no less than 38%

12/21/2022 Habtamu M. 142

 cont.’d…

12/21/2022 Habtamu M. 143

 Cross matching test

 It is a test to determine the compatibility b/n recipient’s blood and donor’s blood
 The final test (element) of pre-transfusion testing
Purpose of cross-matching:
 Enables the patient to receive a blood transfusion with benefit and without
danger
 Prevents life threatening transfusion reaction

 It maximizes in vivo survival of transfused blood cells

 Serves as a double check of ABO errors

 Provides a second means of antibody detection and checks the results of antibody
screen

12/21/2022 Habtamu M. 144

 Pre-transfusion testing (PT)

PT protocols are based on a balance between:

• Patient safety
• Decrease unwanted transfusion reaction
• Selected methods
• The speed of the tests
 It includes:
• ABO and Rh typing of the donor and recipients
• Screening of unexpected Abs of donors and recipients
• Compatibility testing

12/21/2022 Habtamu M. 145

 Types of Cross- match

• There are two kinds of cross-match:

Major cross – match

Minor cross – match

 Major cross- match: involves testing the donor’s red
cells with recipient’s serum
 Minor cross-match: involves testing the donor’s serum
against recipient’s cells
12/21/2022 Habtamu M. 146
 Cont’d…
 major- cross match: Detects antibodies in the recipients’ serum that may
damage the cells of proposed serum.
– Recipient serum + Donor RBCs------+/- agglutnation
– Result: -Agglutination ------------- incompatible
-No agglutination ------------compatible
 Minor cross match: Detect antibodies in donor’s serum capable of
affecting the RBC of the recipient.
– It has minor importance.
– Donor serum + recipient RBC  +/- agglutination

12/21/2022 Habtamu M. 147

 Selection of blood for cross-matching

• Whenever possible blood of the patient’s own blood group

should be given.
• Group-A pt: Should receive group A blood, if not available
group O
• Group–B pt: Should receive group B blood if not available
group O
• Group–O pt: Should only receive group O blood

12/21/2022 Habtamu M. 148

 Cont’d…
• Group–AB pt: Should receive from group AB, if not
possible can receive blood from group A, B &O.
• Type AB blood is called the universal recipient (AB+ve)

• Type O blood is called the universal donor (O-ve)

12/21/2022 Habtamu M. 149

 Methods of Cross- matching

• When cross-matching is carried out, the patient’s serum is tested

against the donor’s cells.
• The patient’s serum should be fresh, not more than 48 hrs old

• Methods include:

 Saline cross-matching

 Protein cross-matching

 Anti-human globulin cross-matching

 The enzyme cross-matching

12/21/2022 Habtamu M. 150

 Hemolytic Disease of the New Born (HDN)

• Destruction of the RBCs of the fetus and newborn by

antibodies produced by the mother
• Only IgG antibodies are involved because it can cross the placenta (not IgA or
IgM).

Types of HDFN
• HDFN is often classified into three categories based on antibody
specificity:
– Rh
– ABO and
– Other (non-Rh) antibodies

12/21/2022 Habtamu M. 151

 Hemolytic Disease of Fetus and New born (HDFN), due to
Rh

• Originally known as erythroblastosis fetalis

• Initially observed in babies from D- negative women with D-positive

mates.
• HDFN is a condition in which the RBCs of a fetus or neonate are
destroyed by Immunoglobulin G (IgG) antibodies produced by the mother.
• Initial pregnancies were not usually affected

• Infants from subsequent pregnancies were often still born or severely

anemic and jaundiced

12/21/2022 Habtamu M. 152

 Cont’d…

12/21/2022 Habtamu M. 153

 Cont’d…
• The placental barrier limits the number of fetal RBCs entering the maternal
circulation during pregnancy and thus reduces chances of Ab production during
pregnancy.
• At the time of delivery, a fetal RBCs escape into the maternal circulation
(known as feto maternal hemorrhage)
• Factors that must be present for HDFN to occur:
1. The mother must lack the Ag, and following exposure to the Ag should produce
Abs of the IgG class.
2. The Fetus must possess the antigens
3. The Antigen must be well developed at birth

12/21/2022 Habtamu M. 154

 HDN due to ABO

• Occurs most frequently in group A or B babies born to group O

mothers.
• It is due to the increased incidence of IgG Abs in group O individuals
compared to other ABO groups.
• ABO incompatibility often affects the first pregnancy because of the
presence of non-RBC stimulated ABO Abs
• Red blood cell destruction by ABO Abs is more common than by anti-
D.
• Fortunately, most cases are subclinical and do not necessitate treatment.

12/21/2022 Habtamu M. 155

 Cont’d…
Prenatal investigations

• Tests on the mother

 Relevant laboratory tests should include:
♠ A full ABO grouping (forward and reverse)
♠ Rh typing
♠ Antibody screening
• Tests on the father
 Helpful in establishing the risk of immunization & in predicting the out
come of future pregnancies

12/21/2022 Habtamu M. 156

 Cont’d…
Postnatal investigations
• All newborns should be examined (even when HDN is not anticipated or suspected)
for:
 ABO blood type

 Rh0 (D) type

 Du (if Rh0 (D) negative)- weak D antigen, its detection requires the
indirect antiglobulin test (IAT)
• Anti-D immunoglobulin - immunoglobulin administered to Rh-negative mothers
after the birth of an Rh-positive baby, to prevent haemolytic disease of the newborn
in the next pregnancy

12/21/2022 Habtamu M. 157

 Cont’d…
Rho (D) Immunoglobulin (Human)- RhIG
– Administered to non immunized Rho- negative mothers

– Administered to who deliver Rho- positive babies

• It is used to prevent Rh- allo-immunization

• Candidates for this prophylaxis are:

– mothers who are Rh-negative and

– Du - negative, and

– have an Rh-positive or Du positive new born

• All Rh-negative women who have abortions are candidates unless the father or fetus
is known to be Rh-negative.

12/21/2022 Habtamu M. 158

 Cont’d…
• Fetal red cells in the maternal circulation might be destroyed
by administration of suitable quantity of IgG anti- D to
prevent Rh immunization of the mother, given to Rh-negative
women within 72 hours of delivery.
• This dramatically decreases the incidence of anti-D HDN.

• Combined prenatal-postnatal treatment is more effective than

postnatal treatment alone in suppressing Rh immunization.

12/21/2022 Habtamu M. 159

 Cont’d…
 The following are not RhIG candidates:

• Rho-negative women

– Who deliver Rh-negative babies

– Whose serum contains anti-Rho (D)
– Who are Rho- positive or Du- positive
• RhIG is a concentrate of IgG anti-D prepared from pools of human plasma
• It is given intramuscularly
– to non-sensitized D-negative women at 28 weeks of gestation (ante partum) and

– again within 72 hours of delivery (post partum) of a D positive infant

12/21/2022 Habtamu M. 160

 Determination of vials of RhIG
Acid Elution Stain Calculations

• # fetal cells/2000 adult cells x 100 = %of fetal cells

• % of fetal cells X 50 = number of mLs of fetal bleed
– FB/30 = # vials needed
– 300 ug RhIG will neutralize 30 mL of D+ whole blood
– If # calculated is <0.5 round down, > or =0.5 round up
• 1.4 round down to 1 and add 1 vial=give 2 vials
• 1.6 round up to 2 and add 1 vial – give 3 vial

12/21/2022 Habtamu M. 161

 Cont’d…
Treatment of HDN
• For infants who develop hyperbilirubinemia and/or anemia due to HDN,
exchange transfusion is usually carried out.
• Exchange transfusion: is a continuous removal of small amounts of blood from
neonate with simultaneous continuous infusion of donor blood until a one or two-
volume exchange is accomplished.
• By exchange transfusion the concentration of bilirubin and incomplete antibodies
decrease, simultaneously the infant is provided with compatible donor red cells.
• To give exchange transfusion for an infant clinical & laboratory findings must be
considered.
• Phototherapy . For mild HDFN and usually used for ABO HDN

12/21/2022 Habtamu M. 162

 Whole blood, blood components and derivatives

• Only human blood and its components are used for transfusion into humans to restore
blood volume, O2 carrying capacity, hemostasis, leukocyte function
• Transfusions are the introduction of either
o Whole blood
o Blood components (RBCs, Plts, plasma or WBCs ) or
o Blood derivatives (albumin, gamma globulin, F VII, VIII, VwF, or Ig etc)

• The use of component therapy:

o Gives a better form of treatment
o Often permits considerable economy in the use of blood
o Minimizes the risk of immune reactions

12/21/2022 Habtamu M. 163

 Whole Blood (w/b)

• It contains all cellular elements and coagulation factors

• Freshly drawn w/b maintains all its properties for a limited time

• Indicated for:

 To provide both O2 carrying capacity and blood volume expansion

 Used as a source material for blood component preparation

 For exchange transfusion in new born

 Whole blood less than 4-5 days old is often the component of
choice

12/21/2022 Habtamu M. 164

 Red cell preparation

• It is the product remaining after the removal of most of the plasma

from freshly drawn whole blood by centrifugation
• The commonly available RBC preparations is concentrated red cell
suspension
• Concentrated red cell is used to treat patients with anemia

• Transfusion of 1 unit RBC increases the Hct by 3% and the Hgb by

1 gm
• The final Hct=70-80% in 250-300 ml volume of red cell suspension.

12/21/2022 Habtamu M. 165

 Plasma products

• Plasma is a fluid portion of one unit of blood collected and separated in a

closed system and intended for intravenous use.
• The therapeutically useful plasma products are:
 Fresh Frozen plasma (FFP)- to replace coagulation factors
 Ordinary plasma (OP)/ single donor plasma- to volume and protein
replacement.
 Cryo depleted plasma only for protein replacement
• All plasma products have to be stored at -180C or colder

12/21/2022 Habtamu M. 166

 Platelet concentrates (PC)

 PC from random donations are the most frequently used form of platelet product
(“random” platelet)
 Each unit of PC: increases the platelet count by ˜ 5,000-10,000/ ml in an average
adult
 The normal adult dose is 6-10 units, and for child 1 unit/10kg

 Platelet should be stored at

• 1-60C with agitation for 72 hours if prepared in a close system
• 20-240C for 24 hrs if prepared in an open system
 Refrigerated PC don’t maintain function or viability as well as the PC stored at room
temperature

12/21/2022 Habtamu M. 167

 Chapter 6 : serological tets
 Learning objectives

 At the end of the session, you are expected to :

 Recognize the basic principle of serological tests
 Explain serological tests of syphilis, pregnancy, typhoid fever,
typhus , H. pylori and HIV,……….
 Interpret their results

12/21/2022 Habtamu M. 168

 Cont’d…
• Serology is the scientific study of blood serum

• In practice, the term usually refers to the diagnostic identification of

antibodies in the serum

• Such antibodies are typically formed in response to an infection

(against a given microorganism)

• Serology is a science of measuring antibody or antigen in body fluids

• The immune reaction is the production of antibody that protect the

body against antigen

12/21/2022 Habtamu M. 169

 Cont’d…
• Antigen tests- important for early diagnosis of an infectious disease

• Antibody tests- to diagnose microbial diseases, to screen donor blood, to monitor

effectiveness of treatment

Immunological techniques
• Three groups of techniques to measure antigen-antibody reaction

• Primary binding tests- directly measure the binding of antigen with antibody. eg.

ELISA, RIA, western blotting

• Secondary binding tests- measure secondary effects of antigen antibody interaction

e.g precipitation of soluble antigens, agglutination of antigens, neutralization of

bacteria

12/21/2022 Habtamu M. 170

 Cont’d…
• Tertiary binding tests- measure the consequence of

immune responses in vivo

• These tests are much more complex than primary and

secondary tests but their results reflect the practical

significance of the immune response.

E.g. tuberculin skin test.

12/21/2022 Habtamu M. 171

 Serology test for Syphilis

• It is a chronic systemic infectious disease

• It can be acquired sexually or congenitally

• It is caused by Treponema pallidum

• It can spread to all parts of the body through the blood stream

• Two types of antibodies:- reagin & specific antibodies

• Specimen:- serum, CSF, serous fluid from chancre/skin lesion

• Treponema Palladium is a cork screw shaped, motile organism

12/21/2022 Habtamu M. 172

 Stages Of Syphilis
 Primary syphilis – characterized by development of primary
inflammatory lesion called chancre.
-lesion appears at the point of contact, which is usually the
genitalia i. e penis, vagina or rectum
 This lesion is a firm, painless skin ulceration localized at the
point of initial exposure to the spirochete.
• Diagnosed by –
- Its characteristics chancre
- Positive serologic test
- Dark field microscopy

12/21/2022 Habtamu M. 173

 Cont’d…
• Secondary syphilis
- generalized rash ( any where in the body).
-a symmetrical reddish-pink non-itchy rash on the trunk and extremities.
-palms of the hands and the soles of the feet
-Diagnosed by - skin rash & Positive serologic test.
• Latent syphilis- non infectious stage diagnosis is only made by serology .
• Tertiary syphilis -soft, tumor-like balls of inflammation known as
granulomatous lesion in skin, bone liver, CNS etc.

12/21/2022 Habtamu M. 174

 Diagnosis of Syphilis

 Clinical findings (hard chancre, condolymata of vulva, skin rash…)

 Demonstration of spirochetes in clinical specimens

 Presence of antibodies in blood or CSF:-

 Treponemal tests
 Non-treponemal tests
1. Treponemal (specific) tests

A. The fluorescent treponemal antibody absorption test (FTA-ABS)

• Confirmation test, done on serum or CSF

B. The microhemagglutination treponema pallidum test (MMA-TP)

• Confrimation test, done only on serum

12/21/2022 Habtamu M. 175

 Cont’d….
. Non-treponemal (non-specific Regain antibody) tests
 VDRL- cardiolipin lechtin cholesterol antigen
• Heat inactivated serum is mixed with a buffered saline suspension of cardiolipin–lecithin–

cholesterol antigen.
• This serum-antigen mixture is microscopically examined for flocculation.

 RPR - cardiolipin choline chloride

• A cardiolopin lecithin-cholesterol antigen coated with carbon particle is mixed with patient’s

serum.
• Then flocculation reaction is observed macroscopically in the presence of cardiolipin antigen.

12/21/2022 Habtamu M. 176

 Cont’d…
Two types of antibodies are produced in response to treponema palladium infection.
1. Non treponemal antibody (regain antibody) is auto antibodies against tissue antigens.
-Are non specific because they are not directly against T. palliduim and are produced in
patients with other infectious disease as measles, hepatitis, leprosy, malaria &
tuberculosis.
-Are IgG & IgA class.
- Is identified by VDRL & RPR
2. Treponemal antibody (specific antibody) is antibodies directed against T. pallidium
antigens.
-Are IgM type and they are replaced by IgG.
-Are screened by FTA –Abs (Fluorescent Treponnema antibody Absorption
test)& MHA-TP (Microhemagglutination assay )

12/21/2022 Habtamu M. 177

 Cont’d…
Specimen
-Serous fluid from chancres & secondary skin lesion - to detect
motile T. pallidium.
-Blood - for antibody testing when syphilis is suspected but no
treponems detectedmicroscopically.
Serum/plasma must not be contaminated, clear free of cells.
-Cerebrospinal fluid - for serologic tests

12/21/2022 Habtamu M. 178

 Cont’d…
Veneral Disease Research Laboratory (VDRL)
Principle: Measured volume of heat inactivated serum is added to a measured
drop of buffered saline suspension of cardiolipin lecithin cholesterol antigen on
slide & after rotating the slide , the mixture is examined using 10X objective.

Aggregation of this particle in to large or small clumps is interpreted as

degree of reactivity.
• No clumping/ very slight roughness = non reactive

• Medium/ large clump = reactive.

• Small clumping = weakly reactive

12/21/2022 Habtamu M. 179

 Cont’d…
Rapid plasma regain (RPR) card test
Principle- antigen used is a modified cardiolipin antigen in which micro
particulate carbon particles are suspended, in addition it contains a
balanced quantity of cholesterol and lecithin. In the presence of reagin,
clumps or flocculation appears which can be visualized as black
clumps against white back ground. The result is read macroscopically.

Reporting results
- Small to large clumps - reactive

- No clumping or slight roughness – non reactive

12/21/2022 Habtamu M. 180

 Cont’d…
• Treponemal (specific)test
1. Fluorescent treponemal antibody absorption test ( FTA Abs)
-It is used to confirm syphilis infection after positive screening
test.
 Principle patient serum is added on a slide coated with
T.pallidim antigen followed by addition of fluorescent
tagged anti human globulin & examine under fluorescent
microscope.
• Specimen heat inactivated serum ( at 56oc for 30 min.)
• Fluorescence indicates the presence of specific antibodies

12/21/2022 Habtamu M. 181

 Cont’d…
2.Microhaemagglutination (MHA-TP) Test
- Used to confirm syphilis infection after screening test result indicate syphilis.
-Detects antibodies to the bacteria and can be used to detect syphilis in all stages .
Interpretation of the serological test for syphilis
 Positive result
 It could be due to biological false positive or the patient has syphilis .
 Weakly reactive
- Very early infection
- Effect of treatment
- Biological false positive
 Negative reaction
 Patient doesn’t have syphilis
 Infection is too recent
 Effect of treatment
 Disease is inactive
12/21/2022 Habtamu M. 182
 HCG and Pregnancy test

• Human chorionic gonadotrophin (HCG) is a hormone secreted by trophoblastic

tissue in the placenta during pregnancy.

• Its production stimulates secretion of progesterone by the ovary.

• Adequate levels of progesterone are necessary for successful implantation and

prevent any further release of egg from the ovary.

• Used to confirm pregnancy and also to diagnose ectopic pregnancy and trophoblastic

tumor (serum sample is better in this case)

 Clinical applications of the test

• Confirmation of pregnancy early in the first trimester

HCG increase rapidly with time

12/21/2022 Habtamu M. 183

 Cont’d…
• Diagnosis of ectopic pregnancy with lower abdominal pain,

trophoblastic tumor
 HCG levels typically increase at slower rate
• Evaluation of threatened abortion during the 1st trimester

 HCG levels first rise then begin to decline

• A positive result may not always indicate pregnancy

Diseases, drugs and interfering substances (cancer in male)

12/21/2022 Habtamu M. 184

 Cont’d…
Direct agglutination test
• Reagent coated with anti HCG mixed directly with urine

•The reaction is based on the reaction between HCG in urine and the latex particles

coated with anti HCG

• In positive result agglutination will be observed

2. Indirect method
• If HCG present combine with anti-HCG antibody leave free the latex HCG, no

agglutination for positive tests.

•Urine specimen is first treated with anti-HCG and then reacted with the

latex suspension

12/21/2022 Habtamu M. 185

 Cont’d…
• If urine contains HCG, the anti HCG will be neutralized and then the latter will not be

available to HCG coated latex particles for bringing about agglutination.

3. Haemagglutination inhibition test (tube test)
• It is more sensitive than slide test
Principle: similar with latex slide test except the HCG is coated on red cells, not on
polystyrene particles.
• If the urine contains no HCG, the anti HCG antibody will react with the HCG on the red
cells and cause their agglutination
Factors that affect pregnancy test
• HCG high for up to four weeks after completed miscarriage

• An injection of HCG to treat infertility make results falsely high for several days after
injection.
• Blood in the urine or soap in the sample container can interfere the test results

• Diuretics and medication can also interfere the test result

12/21/2022 Habtamu M. 186

 Pregnancy test(strip test)
• Pregnancy tests are methods that detect the presence of Human choriono
gonadothrophin(HCG) hormone which is produced by the trophoblastic
tissue in the placenta, used not only to confirm pregnancy but also diagnose
ectopic pregnancy and trophoblastic tumors .
• HCG is a glycoprotein hormone with two non identical, non-
covalently
bound glycoprotein sub units  &
• 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 residuesHabtamu
12/21/2022 in theM.container 187
 Cont’d…
Test Principle: the test strip is based on the reaction between the
HCG in the urine and gold coated antibody on the strip.

A positive control is also impregnated on the strip

so that interpretation will be very easy.

12/21/2022 Habtamu M. 188

 Serological test for typhoid fever
 The etiologic agent is salmonella typhi. It occurs only in humans and
some
times termed as enteric fever since they colonize the intestine
• The ingested salmonella reach the small intestine From which
they enter the lymphatics Then go to blood stream From
this to many organs including intestine The organism multiply in
intestinal lymphoid tissue And excreta stool

12/21/2022 Habtamu M. 189

 Cont’d…
Widal test
• Widal test is serological test widely used to diagnose typhoid fever, measure
agglutinating antibodies to O& H antigens of salmonella typhi.
• Salmonella species have three distinct antigenic structures:
– O –antigen (somatic),
– H- antigen (flagellar), and
– capsular antigen(K/vi- antigen) .
• The patient serum is tested for O& H antibodies.
• In acute typhoid fever, 0 agglutinins can usually be detected 6–8 days after the
onset of fever and H agglutinins after 10–12 days

12/21/2022 Habtamu M. 190

 Cont’d…
• Salmonella antigen suspensions are commercially available. The
suspension must be allowed to warm to room temperature and be
well mixed.
• Two types of test are available to diagnose typhoid fever
-Rapid slide (screening) test
-Tube agglutination test.
Rapid slide test on clean slide, a drop of serum is placed (which is
not hemolyzed) and a drop of antigen suspension is added to
the mixture and added on it. Then mix, look for agglutination

12/21/2022 Habtamu M. 191

 Cont’d…
• Tube agglutination test serial dilution of patient serum is made in
physiologic saline. An antigen suspension is added to the mixture &
incubated for certain period of time. Finally the highest dilution (titer)
giving agglutination reaction is determined.
• Widal-test is reported by giving the titer for both O&H antibodies.

12/21/2022 Habtamu M. 192

 Limitation of the Widal test

• False positive reactions

- Due to past infection
- Cross reaction (malaria, relapsing fever, non
typhoid salmonella)
- Improper technique
• False negative reaction
- Early typhoid cases
- Poor antigen preparation
- Poor transport of reagents & storage.
12/21/2022 Habtamu M. 193
 Serological tests for typhus
• Typhus is a disease caused by bacteria called Rickettsia
through bite of infected tick.
• Rickettsia resemble viruses in that they are mostly obligate
parasites and
• Are unable to survive as free living organisms.
• They are about the size of the largest viruses and can just be
seen with the light microscope

12/21/2022 Habtamu M. 194

 Cont’d…
Weil-Felix reaction test
• Weil-Felix is an agglutination test for various rickettsial
infections (as typhus and tsutsugamushi disease) using
particular strains of bacteria of the genus Proteus that
have antigens in common with the rickettsiae to be
identified,
• It is developed by Weil, Edmund (1880-1922), and
Felix, Arthur (1887-1956), Austrian bacteriologists

12/21/2022 Habtamu M. 195

 Cont’d…
 The Weil-Felix reaction test, developed by Weil and Felix (1916), is based on
the fact that certain strains of Proteus vulgaris and P. mirabilis share
antigens with several of the rickettsia species that produce febrile disease,
such as typhus.
 Three strains of Proteus species have been found to be useful in diagnosing
rickettsial diseases; these have been labeled OX-2, OX- 19 and OX-K.
 Weil-Felix reaction is an agglutination test based on the cross- reactions,
which occur between antibodies, produced in acute rickettsial infections
and the OX-19 and X-2 strains of Proteus vulgaris and the OX-K strains of
Proteus mirabilis.

12/21/2022 Habtamu M. 196

 Cont’d…
Limitation of test
 The Weil-Felix reactions are non- specific and cannot be fully relied on to
diagnose acute rickettsial infections.
 False positive Weil-Felix reactions are may also occurs in:-
 Proteus infections,
 R. tsutsugamushi infections,
 Relapsing fever,
 Brucellosis,
 Rat bite fever,
 Infectious mononucleosis, and other acute febrile illness

12/21/2022 Habtamu M. 197

 HIV/AIDS

• HIV-1 is responsible for the main AIDS epidemic and common in

Ethiopia.

Serologic tests for HIV/AIDS

• There are a number of HIV tests with different specificity and sensitivity.

• For example- ELISA and rapid tests for screening test (ab)

- Western blot assay method for confirmatory test (ag)

• A ‘window’ period of seronegativity exists from the time of initial

infection to 6 or 12 weeks or longer

12/21/2022 Habtamu M. 198

 ELISA

• Rely on a primary antigen to antibody reaction by using

an enzyme which produces color and measuring the color

photometrically
• Require well trained and skilled laboratory personnel

• Done in well equipped laboratory

• Important to process many samples at once (96 samples at

once)
12/21/2022 Habtamu M. 199
 Western blot

• It is a technique in which proteins are separated

elecrophoretically, transferred to membranes and

identified through the use of labelled antibodies specific
for protein of interest.
• It is a confirmatory test for HIV

12/21/2022 Habtamu M. 200

 Rapid tests

• Interest in the development of HIV antibody tests that provide

results within short period of time

• Not require additional reagents or equipment not contained in

the kit
• Useful for small laboratories without electricity or equipment

• Done by adding serum/plasma to the test kit then see the

result at the test and control lines

12/21/2022 Habtamu M. 201

 Cont’d…
 Principle: An antigen is coated on the strip with positive control, up
on addition of serum /plasma or whole blood depending on the test
procedure, there will be reaction between antigen and antibody if
present in the sample.
• Reactive results: two colored bars (one for the control & the other
for the patient)
• Non- reactive: single colored bar (positive control only)
• Invalid result: without having any line and when single color bar
at
test area)
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 HIV test algorithm

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 Cont’d…
Stat Pack test procedure
1. Remove the chembio HIV1/2 STAT PACK test device from it’s pouch and
place it on a flat surface.
2. Label the test device with patient name or ID
3. Touch the 5µl sample loop provided to the specimen, allowing the
opening of the loop to fill with the liquid.
4. Holding the sample loop vertically, touch it to the sample pad in the
center of the sample (s) well of the device to dispense 5µl of sample
(serum, plasma or whole blood) onto the sample pad.
5. Add 3 drops (105 µl) of buffer slowly, drop wise, into the sample well.
6. Allow 15 min. from the time of wash reagent addition for the reaction to
occur, the result should be read immediately after the end of 15 minutes
incubation time. Don’t read the result after 20 minutes

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 Cont’d…
ABON test procedure
1. Remove the test device from the foil pouch and use it as
soon as possible (within one hour).
2. For serum/plasma specimen: Hold the dropper vertically
and transfer 1 drop of serum/plasma ~25 µl to the specimen
well of the test
3. Add one drop (~40µl) of assay diluents to the same area
and start a timer
4. For venipuncture whole blood specimens: Hold the dropper
vertically and transfer 2 drops of whole blood( ~50µl) to the
specimen well(S) of the test device then add 2 drops of
buffer(~80µl) and start the timer

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 Cont’d…
SD BIOLINE test procedure
1. Remove the test device and sample pipette from the foil
pouch and place it on a clean, flat and dry surface
2. Label the test device with patient name or identification
number
3. Add 20µl of drawn blood specimen with a 20 capillary pipette
into sample well or using micropipette add 10µl of plasma or
serum into the sample well
4. Add 4 drops (about 120µl) of the assay diluents vertically in
to the sample well .if you don’t hold the bottle vertically ,it can
lead to in accurate result.
5. Observe for development of colored bands on the result
window and Interpret test result at 10-20 minutes. Do not
interpret the test results after 20 minutes.
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 Serology test for H. pylori

• H pylori infection is very strongly associated with peptic ulcer disease (duodenal

and gastric) and chronic active gastritis.

• H. pylori infection is also an independent risk factor for gastric cancer and
primary malignant lymphoma of the stomach.
• Diagnose H pylori infection: 

1. H. pylori antibody testing

2.  Stool antigen detection -This method detects H pylori antigen in stool specimens
and can be used for diagnosis, therapeutic monitoring, and proof of eradication
post treatment.

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 Cont’d…
H. pylori antibody testing
• Detects antibodies to the bacteria and will not distinguish previous

infection from a current one.

• This test is not recommended for routine diagnosis or for evaluation of

treatment effectiveness.
• If test is negative, then it is unlikely that a person has had an H. pylori

infection.
• If ordered and positive, results should be confirmed using stool antigen or

urea breath test.

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 Chapter -7 Parasitological tests
 Learning objectives
 At the end of this chapter you will able to:
 Discuss the process, preservation and transportation of parasitological specimens.
 Describe the laboratory methods of parasitological infections.
 Describe the preparation of wet mount and iodine preparation.
 List the advantage and dis advantage of thin and thick blood film preparation.
 Describe concentration methods for the diagnosis of parasitic infections.

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 cont’d…
 Medical parasitology is the science that deals with organisms living in the
human body (the host) and the medical significance of this host- parasite
relationship.
Medical parasitology-The study of the parasites of man and
their medical consequences . 
It is a subject that researches:
 the biological features of human parasites,
 the relationship between the human being and the parasites,
the prevention and treatment of the parasitic diseases. 

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 General laboratory methods
1. Parasitic (morphological) diagnosis
 Macroscopic
 Microscopic
2. Immunological diagnosis
 Antibody detection
 Antigen detection
3. Molecular diagnosis
4. Culture
5. Animal inoculation
6. Xenodiagnosis

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 cont’d..
 Importance Of Immuno Diagnosis

1. If parasites live in the internal organs and difficult on gate

specimens E.g E. granulosis
2. When the ova of parasite may not shed in body fluids.
3. Parasites may not be seen on specimens either acute or chronic
stage.

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 Types of parasitological specimens

1. Blood: examination of blood films ( Dx of

hemoparasites
2. Stool: examination of the stool forms an important part
in the diagnosis of intestinal parasitic infections
3. Urine: when the parasite localizes in the urinary tract,
examination of the urine will be of help in establishing the
parasitological diagnosis. For example in urinary
Schistosomiasis
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 Cont’d…
4. Sputum: examination of sputum sometimes used In amoebic
abscess of lung or in the case of amoebic liver abscess
bursting into the lungs, the trophozoites of E. histolytica are
detected in the sputum
5. Urethral or
vaginaldischarge:forTrichomonas vaginalis.

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 Stool examination for parasitological diagnosis

Macroscopic examination Stool color.

 Normal:
– Adult: brown
– New born infants:- Black(meconium)
– Breast feed infants:- scrambled egg
– Infant feed on animal milk:- “ curd like”

 Abnormal:
 Clay or white:
• Absence of bile pigment (bile obstruction)

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 Cont’d…
 Black or tarry:
• Drug (e.g., iron),
• bleeding from upper gastrointestinal tract (e.g., small intestine),
• diet high in red meat and
• dark green vegetables (e.g., spinach)

 Red:
• Bleeding from lower gastrointestinal tract (e.g., rectum),
• hemorrhoids
• some foods
– red gelatin,
– tomato juice or soup
– large amounts beets
 Pale:
• Malabsorption of fats,
• diet high in milk and milk products and low in meat
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 Cont’d…
Stool consistency
• Normal: Formed, soft, semisolid or mushy
• Abnormal:
– Hard, dry, constipated stool
• Dehydration, decreased intestinal motility resulting from lack of
fiber in diet, lack of exercise, emotional upset, laxative abuse
– Diarrhea
• Increased intestinal motility (e.g., irritation of the colon by
bacteria)
– Cleary watery, loose mixed with mucus and blood

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 Cont’d…
Stool frequency
• HOW OFTEN DOES THE AVERAGE PERSON POOP?
• Normal
– three times a day to once every three days.
– average person poops about once a day.
• Abnormal
– four times a day or more and
• the stool has a liquid consistency – diarrhea
– less than two or three days a week and
• the stool is hard, dry, and difficult to pass-constipation

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 Cont’d…
Stool odor
Normal: Aromatic, affected by ingested food and person’s own bacterial flora

Abnormal:Pungent
– Infection, blood
Stool constituents Abnormal:
– Mucus:inflammatory condition
– Parasites
– Blood:gastrointestinal bleeding
– Large quantities of fat: malabsorption
– Foreign objects: accidental ingestion

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 Cont’d…
Normal:
water (about 75%).
Rest constitute:
 dead bacteria that helped us digest our food, living bacteria,
 undigested food residue (known as fiber),
 cellular linings, sloughed epithelial cells

 substances released from the intestines (such as mucus) and the liver.
 fat, protein, dried constituents of digestive juices (e.g., bile
pigments),
 inorganic matter (e.g., calcium, phosphates)

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 Cont’d…
Microscopic examination Materials:
• Microscope slides
• Cover slips
• solution bottle with pipette
• Wooden stick
• Fresh stool
• Gloves

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 Microscopic examination procedure

1.Used cleaned microscopic slide

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 2. Place a drop of normal saline on the slide

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 3. Take a small amount of stool with wooden applicator stick

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 Cont’d…
4. Mix stool with normal saline

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 5. Place cover slip- Avoid air bubbles

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 Cont’d...
6. Observe under microscopy as follow
NB. Ova, cysts, trophozoites and larva's can be identified as per
their characteristic features, in addition pus cells and RBCs may
be seen and should be reported.

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 Cont’d…

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 Preservation
Required if not delivered to lab immediately
Preserve protozoan morphology
Prevents development of worm eggs/larvae
Commercial kits - vials for PVA & formalin
3 parts fixative to 1 part stool
Record time collected and placed in preservative

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 Cont’d…
 Fixatives are required
-for preserving the morphology of parasite
-to arrest developmental stage of helmenthic ova
-Preserving cysts and eggs
1.formalin- it is all purposed preservative for ova, larvae,
- 5%-cyst of protozoa
-10%- helmenthic ova, larvae, &adult

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 Cont’d…
2. 10%v/v formol saline--cyst, egg, larvae and adult of some
helmenthes.
3. Refrigerator-3-50c preserve trophoziote for several days and
cysts for more than one month.
4. Merthiolate iodine formaldehyde(MIF)- -contain iodine&
eosin –fixative& staining
-good for most parasites found in feaces.
- used field survey

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 Concentration method of parasite detection

- Stool contains a number of debris that have non diagnostic

value in addition to different stages of parasites . Because of this,
a technique that helps us to separates debris from parasites is
developed .
- Concentration works by utilizing the difference in the density
and it enables us to examine great quantity of stool by separating
diagnostic stage of parasite from fecal specimen.

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 Cont’d…
Advantages
- To detect parasites when they are not seen in direct stool
examination but symptom of patient continues.
-To detect eggs of parasite which are few in
number(schistosomia spp, Teania spp)
-To check treatment successfulness
Disadvantages
- the motility of parasites lost during concentration and it
takes time.

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 Cont’d…
Types of concentration methods
A. Sedimentation techniques:- Sedimentation can be achieved by
different methods.
1.Formol- ether centrifugal sedimentation technique
 The best and most popular method than all concentration
techniques .
 It concentrates the egg and larvae of helminthes and cyst of
protozoa .
 It is most useful to detect the eggs of schistosomes in feces.
 Formalin-is used for fixation and preservation of parasites
morphology.
 Ether-dissolves fecal debris.

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 Cont’d…
Other sedimentation techniques includes:-
Formol-detergent sedimentation
 Acid - ether sedimentation
 Water- ether sedimentation
Sedimentation is super than flotation that:-
 It concentrates all type of parasites
 It is faster when centrifugation used
 Organisms are preserved & more abundant than flotation

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 Cont’d…
B. The flotation concentration techniques
 They are not recommended as sedimentation because
they concentrate small range of parasites --This is due
to all organisms will sediments if appropriate
techniques but all do not floats .
E.g. operculated egg of treamatodes not float in Nacl
solution and Zn so4 solution destroys ova of schistosoma
species and rupturing of operculated eggs .
Also NaCl solution causes shrinkage of protozoan cysts.

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 Perianal specimen

Cellophane (cellulose acetate ) tape preparation

 Purpose to collect the eggs of pinworm ,Entherobious
vermicularis as female deposits the eggs at night on the
perianal folds .
 The pinworm egg attach itself to the cellophane tape laced
against the perianal folds.
 The tape is fixed to a microscope slide and examine
microscopically.

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 Blood film preparation of hemoparasites

Preparation of thick films

 Slides must be clean, free from dirt, grease and fingerprints
 Mix the bloodUsing an applicator stick, apply 4 drops
(6microliter) of blood on to a microscope slide
 Spread the blood without excessive stirring to form a smear
approximately a 2 cm, through which newspaper print can be
red.
 This should be approximately 5 red blood cells thick.
 Allow to air dry horizontally (without using heat).
 Label slides

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 Cont’d…
Preparation of Thin Films
1. The microscope slides must be clean as for thick films otherwise the
blood will not adhere and the smear will be irregular.
2. Apply 1 drop ( 2 micro liter) of blood to the end of the slide, pace
another slide at an angle of 45o and bring it towards the drop of blood.
3. As soon as it touches it, the blood will disperse along the width of the
slide.
4. Before it reaches the edges, pull the drop along the length of the slide.
5. the correct amount of blood in the drop the film will form a good tail
before the end of the slide. The film here should be 1 RBC thick. Make 2
slides for each test.
6. Air dry, Label slides

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 Cont’d…
Thick blood film
 Good for rapid detection malaria parasites , particularly when they are few.
 In P. malariae parasitaemia is normally low
 About 30 times more sensitive (detecting about 20 parasites/µl)
 In a thick film the blood is not fixed red cells not found.
Thin films
 To confirm the plasmodium species enabling the parasites to be seen in the red
cells .
 It needs fixative ( absolute methanol) that allows specious identifications.
 Greatly assists in the identification of mixed infection
 Value in assessing whether a patient with falciparum malaria is
responding to treatment .
 Gives the opportunity to investigate anaemia and white cell abnormalities, red cell
morphologies.

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 End of the course
There should be no blind medicine!!! Thank you!!!!

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