COURSE TITLE

3
rd
Year Medicine & Medical Sciences Program

HAEMATOLOGY

(PRACTICAL MANUAL)

By

Haematology Department

Faculty of Medicine & Medical Sciences
Umm Al-Qura University
Makkah, K.S.A.




















2
Preface
Laboratory medicine is a key to the practice of clinical medicine. It would
be hard to imagine a situation where adequate medical care could be provided to
the patient in the absence of comprehensive and reliable laboratory services.
This manual provides a comprehensive yet short account of laboratory
procedures. The emphasis has been made on the practical aspects of various
routine tests, in the field of haematology regarding red cells, white cells and
platelets.






































3
References
1- Practical Haematology by Sir John Dacie, S.M. Lewis.
2- Clinical Haematology, Theory and Procedures by Mary Louise Thrgeon.
3- Clinical Haematology and Fundamentals of Haemostasis by Denise M.
Haemening.
4- Diagnostic Haematology by Bernadette F. Rodak.
5- Clinical Diagnosis and Management by Laboratory Methods by John
Bernard Henry MD.
6- Manual of Laboratory Medicine by Armed Forces Institute of Pathology,
Pakistan.
7- Technical Manual of American Association of Blood Bank (AABB)



































4
TABLE OF CONTENTS
SR.NO. PRACTICAL PAGE
1 Hazards in laboratory & safety precautions. 5
2 Light Microscopy. 8
3 Collection & handling of blood. 13
4 Methods of haemoglobin estimation. 18
5 Red blood cell count. 22
6 Methods of PCV determination. 26
7 ESR estimation. 30
8 Calculations of the absolute values (Red blood cell indices) 33
9 Preparation of blood smear and gross examination. 35
10 Reticulocyte count. 39
11 Demonstration of Heinz bodies. 41
12 Demonstration of haemoglobin H 43
13 Detection of haemoglobin S. 44
14 Estimation of haemoglobin F. 46
15 Haemoglobin electrophoresis. 48

16 Osmotic fragility of erythrocytes. 51



5




Practical Sheet: 1

Hazards in Laboratory and Safety Precautions

1- Introduction:
Laboratory is probably the most hazardous place in any medical set up.
Laboratory workers are exposed to a variety of hazards and hence must observe
certain precautions to protect themselves from these as much as possible.
Laboratory hazards mainly arise from:
a) Infected specimens.
b) Dangerous chemicals.
c) Faulty equipment.

2- Hazards from infected specimens:

Almost all specimens in laboratory are from patients. Many of them suffer
from known or unknown infectious diseases and tests are required either to
diagnose these or to follow the course of the disease. The specimens of blood,
urine, sputum, stool or aspiration fluids may all be infected. Infections, which can
be transmitted through these, may be parasitic, fungal, bacterial or viral.
Most dangerous transmitted infections include:
A- Blood:
1- AIDS.
2- Hepatitis.
3- Hemorrhagic fever.
B- Stool:
1- Typhoid bacilli (salmonella).
2- Dysentery organisms (shigella).
3- Cholera.
4- Parasitic ova and cyst.
C- Sputum:
1- Tuberculosis.
2- Other respiratory tract infections.

3- Hazards from chemicals:
A large number of chemicals are used in various laboratory tests. These
include:
a- Corrosives as strong acids and alkalies.
b- Poisons as potassium cyanide, benzene compounds, mercury and toxic
gases.
6
c- Carcinogens like benzidine, orthotoludine etc.
d- Explosives and inflammable chemicals like perchloric acid, ether etc.
With the exception of last group, which may destroy whole of the working
area, others are dangerous for working staff mainly. These may cause injury to the
laboratory workers by the following ways:
i- By direct contact with skin, mucous membranes or inadvertent
swallowing while pipetting.
ii- By inhalation of vapors or fine powder.
iii- By absorption from lungs or GIT after aspiration or inhalation.

4- Hazards from faulty apparatus:
Almost all apparatus used in laboratory is capable of giving rise to some kind
of injury to the person using it.
a- Glass apparatus may break while in use causing cuts and may even soil the
cut area with its contents which may be dangerous to body tissues.
b- Gas equipment may cause explosion, fire or gas poisoning.
c- Centrifuges can cause mechanical injury when they are unbalanced or
improperly closed.
d- Electrical apparatus is the most common source of electric shock or even
fire in laboratory. This may give rise to electric shock when insulation
inside the equipment gets defective. As apparatus itself is usually made of
metal, current may spread into it. In some cases switch is defective or its
plug is broken and while connecting equipment to main supply or
switching on, one may receive electric shock.

5- Safety precautions:

1- Specimen containers should be such that spilling does not occur. Care should
be taken in removing and replacing caps of containers.
2- Strict aseptic precautions should be observed when collecting blood or other
specimens. It is better to wear the gloves.
3- Soiling of request forms and other papers must be prevented.
4- If a patient is known to be suffering from hepatitis or AIDS or other dangerous
infectious diseases, the specimen containers must carry a red label. This will
alert all persons handling this specimen.
5- Bench workers must wear laboratory coats and gloves. In some cases use of
masks is recommended.
6- Washing hands with soap and water should be a frequent practice particularly
when leaving the work area.
7- Eating and drinking inside the laboratory should be strictly prohibited. Foods
and drinks should not be placed in laboratory refrigerators.
8- Use of carpets and cushions should be discouraged. These are extremely
difficult to clean or disinfect in case of spills. A smooth, polished floor is
easiest to clean.
7
9- Mouth pipetting should be avoided as for as possible. Instead use rubber teats.
However if it is to be done, it is better to plug upper end loosely with cotton
wool. All care must be taken to ensure that fluid is not sucked into the mouth.
10- Use of disposable laboratory ware is ideal but may not be possible. All
disposable ware should be collected in thick bags for later and proper disposal.
11- If re-useable glassware is used, it should be put in a strong disinfectant
solution. At the end of the day transfer these to a strong detergent. Next day
place these in boiling water for 10 to 20 minutes, cool and then wash with tap
water.
12- All potentially infected material like stool, urine, bacterial cultures etc. must
be incinerated after disinfections.
13- In case of spilling or breakage in centrifuge or on bench or floor, first collect
all glass pieces with forceps and then wash the area with strong disinfectant
followed by washing with strong detergent. Then wash with water and wipe to
dry.
14- All reagent containers must be of proper size and shape and properly
stoppered. These shall never be left open. Containers must be clearly labeled.
If these contain dangerous chemicals or infective material, a danger sign or
poison label must be pasted on the container.
15- If fuming chemicals are to be used it is advisable to use these in a cabinet or
hood made for such purpose.
16- Infected material should preferably be dealt with in a laminar flow cabinet.
17- Saline eye wash bottles must be supplied so that eyes can be immediately
washed in case of any contact with the eyes.
18- Electric and gas points should be checked from time to time for any leakage.
19- All water baths and centrifuges should be cleaned periodically.
20- Before leaving the work area laboratory coats and gloves should be removed
and hands should be washed.

6- Common disinfectants:

a- FORMALI NE 5 10 % solution for cleaning floors, surfaces and containers.
b- HYPOCHLORI TE with 10 % available chlorine e.g. Clorox. This is used for
overnight soaking of contaminated laboratory glassware, cleaning surfaces,
floors and equipment.
c- GLUTARALDEHYDE 2 % is good for cleaning of infected metallic objects.
d- PHENOLS as Lysol, sudol etc. for soaking of infected material.

7- Detergents:
A number of detergents are available in the market. Any one can be used.






8


Practical Sheet: 2

Light Microscopy

Objectives:

a- To recognize different parts of a compound light microscope.
b- To learn focus an object using appropriate objective lenses e.g., 10x for
focusing, 40x for wet preparations, 100x for oil immersion.
c- To adjust the light source to optimum depending on the preparation being
examined by using the condenser and iris diaphragm.
Introduction:

The use of microscopes in all their various forms is known as microscopy.
The microscope is used in the haematology laboratory to study various
blood cells and inclusion bodies. Using a system of lenses and illumination
sources, it makes a microscopic object visible.
Microscopes can magnify an object from 1001000 times of its original
size.

Light Microscope:

1- Principle:
To magnify an object the light microscope uses a system of lenses (objectives
and oculars) to manage the path of a light beam that travels between the object
being studied and the eye.
2-
Types of light microscopy:

a- Bright field microscopy:
Uses a light source, either a light bulb or daylight that illuminates the
entire specimen field. The light rays that strike an objective in the specimen
are bent and then refocused by the objective lens. This method is used to
examine stained preparations.

b- Dark field microscopy:
Uses a light microscope equipped with a special condenser and
objective to brightly illuminate the microorganism in the specimen against a dark
background. The dark field condenser directs the rays of light into the specimen
field at such an angle that only the rays striking an object in the field are bent or
refracted. This method is used for the examination of unstained living
organisms e.g. Treponema species.
9


c- Fluorescence microscopy:
A specimen is stained with a fluorescent dye that absorbs the energy of
short light waves like those in blue. The dye then releases or emits light of longer
wavelength such as green light (fluorescence). Commonly used fluorescent dyes
are acridine orange, auramine/rhodamine, and calcofluor white.

d- Phase contrast microscopy:
Uses a modified light microscope that permits greater contrast between
substances of different thickness or density. A special condenser and objective
controls the illumination, by causing light to travel different routes through
various parts of a cell. The result is an image of structures with differing degrees
of brightness or darkness, collectively called contrast. The denser materials appear
bright and the parts of the cell that have a density close to water will appear dark.

3- Materials:
1. Compound microscopes.
2. Lens cleaning paper/cloth.
3. Immersion oil.
4. Wet preparation.
5. Stained preparation.

4- Methods:

a- Different parts of a compound light microscope:
Study different parts of a compound light microscope by the picture and
the description mentioned as under (fig. 2.1):
1- Eye piece (ocular lens): A magnifying lens with magnification power of
10x.
2- Body tube: Contains mirrors and prisms that transmit the image from the
objective lens to the ocular lens.
3- Objective lenses: Primary lenses that magnify a specimen, low power
(10x), high power (40x), and oil immersion (100x).
4- Stage: Holds the slide in position.
5- Condenser: A lens system that condenses light before it passes through
the specimen.
6- Iris diaphragm: Controls the amount of light entering the condenser.
7- Coarse and fine adjustment knobs: Used for focusing the specimen.
Turning the knob changes the distance between the objective lens and
the specimen.
8- Light: Source of illumination, a bulb.




10


b- Focusing of an Object Using Different Objective Lenses:
1- Principle of Focusing:
a- Using objectives (10 xs and 40 xs):
The condenser lens focuses light on the specimen. Some of the light rays pass
directly into the objective lens, while other rays strike the specimen and are bent.
These are brought into focus by the objective lens to form an image of the object
being studied.
b- Using Oil Immersion Objective (100 xs):
When using the oil immersion objective, a drop of special oil is placed on the
slide. The bottom of the objective is immersed in the oil. The image is brought
into focus, with contact maintained between the oil and the front lens of the
objective. The oil helps to keep the light rays together as they pass between the
specimen and the objective lens.

2- Total Magnification Power of a Microscope:
The ocular lens enlarges the image formed by the objective. The total
magnification obtained with any one of the objective lenses is determined by
multiplying the power of the objective lens by the magnification of the eye piece
(e.g., using oil immersion objective (100 x) with a 10 x ocular lens, the total
magnification of the objective will be 100 x 10 = 1000).

3- Resolving Power of a Microscope:
The resolving power of any microscope is a measure of its ability to
discriminate between two adjacent objects. The absolute limit of the resolving
power is roughly the wavelength of the light used to illuminate the specimen. The
wavelength of visible light ranges from 400-800 nm.

4- Examination of a Wet Preparation Using 10 & 40x Objectives:
a- Place the slide of wet preparation onto the stage of a light microscope and
hold it with the help of moving clips attached to the stage.
b- Select the 10 x objective and focus the slide using coarse adjustment knob, by
moving it up or downward. When a specimen is in focus, make it clearly
visible using fine adjustment knob. When the image is clear in 10x
objective, turn to 40x objective and again focus (if out of focus) using fine
adjustment knob until object is clearly visible.
5- Examination of Stained Preparation Using 100x Objectives:
a- Place a drop of immersion oil on the stained preparation (smear) and place it
onto the stage of a microscope.
b- Focus the smear using 10 x objective in a similar fashion as described above.
When a smear is in focus in 10 x, turn to 100 x objective and touch it with
oil. Use the fine adjustment knob to make the viewing field visible.



11




6- Maintenance of Microscope:
1-The Microscope is delicate instrument which is must be :
a-Handled gently.
b-put in a clean environment away from chemicals, direct sunlight, evaporation,
heating source or moisture.
c-Clean immediately if the stage is contaminated with saline to avoid corrosion .
2- The temperate climate humidity and high temperature causes growth of the
fungus which can damage optical surfaces.


7- Cleaning the Microscope:
a- Optics:
Before and after using the low & high power lenses, it must be wiped(cleaned) by
lens paper tissue while the Oil immersion cleaned by lens paper tissue and (few
drops from xyelen) (if needed ).

b- Non-optics:
Using the soft camel-hair brush to remove the dust from the inside and outside of
the eyepieces.
Condenser and iris :
Cleaned by soft cloth or tissue moistened with toluene, and mirror if present with
a soft cloth moistened with 5%alcohole.
The iris diaphragm is very delicate and if damaged or badly corroded it is usually
beyond repair
The other parts cleaned with mild detergent and remove grease or oil with
petroleum ether, followed by 45% ethanol in water.( tissue paper or soft cloth
moistened with 5% alcohol )








12




Fig 2.1. Binocular light microscope.


13


Practical Sheet: 3

Collection and Handling of Blood

1- Introduction:
The collection of blood for different tests, its transport and storage until it
is tested, is the most important step for any laboratory investigations. Any
possible mistake at this level renders all the labor fruitless. The test report
becomes doubtful or insignificant. Therefore all necessary precautions must be
taken at this step.

2- Principle:
Patients request forms for tests should be read and checked properly. The
tests requested should be noted carefully. Before collecting specimen all
containers must be labeled properly giving full details of patients identification.
Blood can be collected from three different sources:
a- Venous blood.
b- Arterial blood.
c- Capillary blood.

a- Venous blood:
It is the most commonly required blood sample. The majority of routine
tests are performed on it. This is obtained directly from the vein. Best site for the
collection of venous blood is the deep veins of the ante-cubital fossa (fig 3.1). If it
is difficult to locate vein here, then veins in forearm, dorsum of hand, femoral
vein, scalp vein and jugular vein can be used. All sites other than the forearm
require extra caution and expertise for collection of blood.















Median
Vain
14






























(Fig 3.1. Collection of venous blood in the deep veins of the ante-cubital fossa).

1- Material:
Tourniquet.
Vacuetoiner (with butterfly or needle) or syringe.
Alcohol swab.
Bandage /medi-plast.

2- Procedure:
Appropriate syringes and needles should be selected.
If multiple specimens are to be collected then it is better to use a butterfly
needle.
Now visualize and palpate the vein.
After all the precautions have been made and the patient is comfortable, a
tourniquet should be applied on the upper arm.

Cephalic vain
Median Vain
Basalic vain

15
The area to be punctured should be sterilized with a spirit swab and
allowed to dry for a while.
Insert the needle into the vein in such a way that it traverses a little
distance under the skin and enter the vein from the lateral side of the vein.
Do not enter the vein directly and vertically, as chances of puncturing the
other side of the venous wall, are more in this way.
Now draw the blood according to your requirement. After this, first loosen
the tourniquet and place a clean spirit swab at the site of vein-puncture and
with draw the needle. Apply appropriate piece of band or medi-plast.
Put the blood into a suitable container, already labeled, and proceed
accordingly.

Table 3.1 blood collection tube type
Collection tube
color and additives
Used Department
Yellow
Sodium polyethylene
sulfonate (SPS)
Blood culture Microbiology
Light blue
(sodium citrate 1:9
ratio)
1. Coagulation studied:
PT, PTT and factor assay
2. Platelet function
Haematology
Lavender (EDTA)
1. CBC and most
haematology test
2. PCR (CD4 count)
3. HbA1C
Haematology
Molecular biology
Chemistry
Blood bank
Gray (potassium
oxalate and sodium
fluoride)
1. Glucose level
2. Glucose tolerance
level
3. Alcohol level
Chemistry
Black
(sodium citrate 1:4
ratio)
Westergren sedimentation
rate (ESR)
Haematology
Green
( lithium heparin)
1. Arterial blood gases.
2. Ammonia level.
3. Hormones.
4. Electrolyt.
Chemistry
Red
(plain no
anticoagulant)
Serum
1. Blood grouping.
2. antibodies testing.
Haematology
Blood bank
Serology
Chemistry






16
3- Precautions:
1- Vein puncture area must be thoroughly cleaned.
2- Tourniquet should not be left in place for a long time, to avoid prolonged
stasis of blood. Stasis may alter the concentration of certain constituents
particularly RBCs, haemoglobin, albumin and calcium.
3- Blind attempts to puncture the vein should not be made.
4- Subcutaneous manipulation of the needle to enter a vein should not be done
as it causes a lot of pain.
5- Once the needle is withdrawn, pressure should be maintained for 1 2
minutes, otherwise extravasation of blood will cause ecchymoses which is
not only painful but also ugly looking.
6- Blood should immediately be transferred to appropriate containers having
full information about the patient.
b- Arterial blood:
This sample is occasionally required particularly for the estimation of
blood gases. This is obtained directly from a superficial artery like radial,
brachial, temporal or femoral arteries.
c- Capillary blood:
This method is occasionally used to draw only a small amount of blood in
special microtube (fig 3.2) or strip as use in blood sugar. Therefore, only few tests
can be performed.

(fig 3.2. microtube for collection capillary blood)














17

In case of infants and young children, to perform one or two specific tests,
the blood is obtained from the heel pulp, finger pulp or ear lobule (fig 3.3).
Platelet count should never be performed on this sample.
























(Fig. 3.3. Various sites for the collection of capillary blood).

3- Interpretation:

a- Plasma:
It is obtained by removing all formed elements from anti-coagulated blood
at 5000 r.p.m for 3 5 minutes. This is required for, Coagulation profile,
Fibrinogen assay etc.

b- Serum:
It is the fluid left behind after blood has clotted. This is the most common
specimen required for chemical and serological tests.
A plain test tube is used for blood collection and blood is allowed to clot.
The tube is then gently centrifuged to obtain clear serum.




Capillary blood from the
pulp of finger
Capillary blood
collection from heel
of foot
18


Practical Sheet: 4

Methods of Haemoglobin Estimation

1- Introduction:
Whole blood haemoglobin concentration in g/dl, can be estimated by a
number of methods to diagnose and follow up the anaemia patients. Haemoglobin
is the oxygen carrying protein in red cells. Two most commonly used methods
are:
A) Cyanmethaemoglobin Method.
B) Acid Haematin Method.

2- Using to:
1- Screening test for health maintenance examination.
2- Screening test for patients taking anti-inflammatory drugs, which can
cause occult blood loss.
3- To follow the response to therapy for anaemia.
4- To follow the course of chronic anaemia.
5- To assess the degree of chronic anaemia such as thalassemia.
6- To assess the degree of blood loss following acute bleeding.

3- Procedures:
a- Sample:
- Both capillary & venous blood may be used for this test.


b- Methodology:

1- CYANMETHAEMOGLOBIN METHOD:
This method is the most accurate and most commonly used method. A
standardized method is recommended by ICSH i.e. (International Committee for
Standardization in Haematology).

a- Principle:
The blood is diluted in a solution containing Potassium Cyanide and
Potassium Ferricyanide (Drabkins Solution). It converts haemoglobin (Hb) and
methaemoglobin (Hi) to cyanmethaemoglobin (HiCN), which is a stable
compound. The absorbance of the solution is measured in photoelectric calori-
meter at a wavelength of 540nm or with a yellow green filter and compared with a
standard solution of HiCN.



19

b- Reagents and Equipment:
1- Diluent (Drabkins Solution).
2- 5 ml pipette.
3- Cuvettes.
4- Test Tubes.
5- 20 micro liter pipettes.

c- Procedure:
Venous blood or free flowing capillary blood can be used. Measurements
can be carried out on blood, which has been stored at 4
o
-C for several days,
provided it is free from infection and contamination.
20l blood is added to 4 ml of diluent and maixed well by inverting the tube
several times. It is allowed to stand at room temperature for 5-10 minutes so that
all haemoglobin is converted to HiCN.
The absorbance is then measured in the spectrophotometer at 540 nm and
the reading is noted. Absorbance of a known standard is also read in
spectrophotometer with each batch of tests and Hb is calculated by the following
formula:

Hb (g/dl) = Absorbance of test X Concentration of Standard
Absorbance of Standard

2- ACID HAEMATIN METHOD:

a- Principle:
Haemoglobin is converted into acid haematin by addition of 0.1 N HCl.
The resultant solution is then matched against a reference solution in a colorimeter
or colored strip (SAHLIS Haemoglobinometer).

b- Reagents and Equipment:
1- Sahlis haemoglobinometer.
2- Sahlis pipette.
3- 0.1 N HCl.
4- Dropping pipette.

c- Procedure:
1- Prepared plastic tube and labeled , Add 100 micro liter of N HCl by
micropipette .
2- Shake the sample 5 times gently , Aspirate 20 micro-litre by
micropipette and add to the plastic tube that contain N HCL and mixed .

3- Transfer the solution into the graduated tube of Sahlis
haemoglobinometer by plastic pipette.
4- Allow it to stand for 5 minutes, so that haemoglobin gets converted into
acid haematin.
20
5- Compare the color of the solution in the graduated tube with that of
reference strip on either side of haemoglobinometer.
6- If the color of the graduated tube is darker, add drop by drop either 0.1N
HCl or distilled water by the dropping pipette and mix with glass rod,
until the color matches with the reference strips.
7- Note the reading on the graduated tube. This is the haemoglobin level in
g/dl. Some tubes also give level in percentage. To convert into g/dl,
multiply the percentage with 0.146(Example:10% X 0.146=14.6 g/dL)
8- Estimation of haemoglobin by Sahlis method is an inaccurate method,
and should only be used when photoelectric calorimeter is not available.
4- Reference Values:







5- Precautions:
1- Before the sample is read the solution should be clear.
2- In case of high WBC count centrifuge the specimen and use the
sedimentation .
3- In case of haemoglobin S (HbS) or haemoglobin C (HbC) dilute the mixture
in 1:1 ratio with distilled water and then read in colorimeter and multiply the
reading by 2.
4- In case of abnormal globulins add 0.1 g of potassium carbonate to the
solution.

6- Interpretation:
Reference values for haemoglobin vary with the patients age and sex. The
values also vary with certain physiological conditions.

7- Clinical Significance:

As an indication of anaemia:
Reduction in haemoglobin concentration in comparison to normal value
means anemia. This level should be evaluated with RBCs count and indices .
Decreased levels of haemoglobin are found in;
1- Anaemias.
2- After severe hemorrhage.
3- Haemolysis due to transfusion of incompatible blood, reactions to
chemicals and drugs, bacteraemia, and artificial heart valves.
4- Variety of systemic diseases e.g. leukemia, lymphoma, uremia, cirrhosis,
hyperthyroidism, carcinomatosis and systemic lupus erythematosis.


Age group Hemoglobin (g/dL)
Adult Male 14.0 18.0
Adult Female 12.0 16.0
New borns(<1 week old) 14.0 22.0
New borns(6 month old) 11.0 14.0
Children (1-15 years old) 11.0 15.0
21
I ncreased levels of haemoglobin are found in:
1- Haemoconcentration states of blood e.g. sever burns
2- Polycythaemia .
3- Chronic obstructive pulmonary disease (COPD)
4- Congestive cardiac failure (CCF).
5- High altitudes.

8- Quality Control:
1- If the patients specimen running in automated machine there is three
levels controls running 3 times per day while if its running by manual
method send the patients specimen to the reference laboratory.
Perform duplicate testing in your own lab.
2- All personnel performing Hb should be checked for color
blindness(Sahlis methodology)
































22
Practical Sheet: 5

Red Blood Cell Count

1- Introduction:
The number of erythrocytes present in one liter of blood is the total red
cell count.
The main function of red blood cells is to carry oxygen from the lungs to body
tissues and to transfer carbon dioxide from the tissues to the lungs. This is
achieved by means of the haemoglobin present in the red cells that combines
easily with oxygen and carbon dioxide.

2- Uses :
1- Screening test for health maintenance examination.
2- Screening test for patients taking anti-inflammatory drugs, which can
cause occult blood loss.
3- To follow the response to therapy for anaemia.
4- To follow the course of chronic anaemia.
5- To assess the degree of chronic anaemia such as thalassemia.
6- ,gTo assess the degree of blood loss following acute bleeding.

RBCs can be counted using an electronic counter or by visual (manual)
methods. The manual method is more cumbersome and does not give accurate
results.

3- Principle:
For RBC count, the solution that is used is isotonic with erythrocytes. The
diluting fluid does not lyse the leukocytes. Normally the leukocytes are too few to
interfere with RBC count. In case of leukocytosis however, the leukocytes are
easily identified and are not counted.

4- Procedure:
a- Reagents & Instruments:
1) Improved Neubauer chamber and cover-slip. It is also known as
Haemocytometer.
It is a thick glass slide with H shaped moats in it. Area between two lines
of H is 0.1 mm lower than the area on sides.
When a cover slip is fixed across these limbs, a depth of 0.1 mm is provided
in the center. Above and below the horizontal moat, is the ruled area. Moat
prevents the mixing of two samples on either side of the chamber. (fig.5.1)





23










2) Red cell diluting fluid:
3.2 g of Na-citrate and 1.0 ml of commercial formaldehyde solution made
up to 100 ml with distilled water.
b- Sample:
EDTA anticoagulated blood.

c- Method:
1- Prepare plastic tube and labeled ,the dilution of blood is (1:200).
2- Add 4ml of diulent to the tube.
3- Mix the sample gently 5times and aspirate 20 microliter to the tube and
mixed.
4- Place the cover slip on the Neubauer chamber.
5- Filled the chamber for RBC count by micropipette 10microliter in each
side.
6- Wait for 1 2 minutes to allow the RBC to settle.
7- Count in the central square of the chamber. Select five small squares (one
in each corner and one in the center). Count the cells lying on left and bottom
lines in addition to cells lying within the squares ( mention to the left square in
the bottom).

5- Precautions:
1- Diluent should be correct.
2- There should be no over flow in the moats.
3- No air bubbles should be present in the chamber area.
4- No debris should be present in the chamber.
5- No scratches should be present in the ruled area of the chamber.
6- Pipettes used must be clean and dry.




Areas for WBC
& RBC counting
(Fig.5.1. Haemocytometer or improved Neubauer
Chamber)

24

6- Calculations:

RBC (10
12
/L) = No. Of cells counted (N) x Dilution x 10
6

Volume counted (l).
7- Explanation:

Total ruled area, divided into 9 squares = 3mm.sq.
Area of central large square, divided into 25 small squares = 1mm sq.
Area of one small square = 1/ 25 = 0.04mm.sq.
Depth of the chamber = 0.1 mm. volume of one small square = 0.04 x 0.1 = 0.004
cu mm. = 0.004l.
Volume of five small squares = 0.004 x 5 = 0.02l
Therefore;
If 5 small squares are counted and the dilution is 1: 200 then,
Red cell count per liter = N x 200 x 10
6
/ 0.02
= N x 200 x 50 x 10
6

= N x 10,000 x 10
6

= N x 10 x 10
9

= N x 0.01 x 10
12

8- Reference Values:







9- Interpretation of the Results:
a- Decreased values are seen in:
1- Anaemias:
They can be due to:
a) Blood loss.
b) Decreased red cell production.
c) Increased red cell destruction.
d) Dietary insufficiency of iron and certain vitamins, particularly B-6, B-12 and
folic acid, which are essential for the production of erythrocytes.
2- Diseases which affect the bone marrow:
a) Leukemia.
b) Multiple Myeloma.
c) Hodgkins disease.
3 -Subacute bacterial endocarditis.
4 -Rheumatic fever.

Age group RBC count
Adult Male 4.7 6.1 x 10
12
/ L
Adult Female 4.2 5.4 x 10
12
/ L
New borns 4.4 5.8 x 10
12
/ L
Infnt/Children 3.8 5.5 x 10
12
/ L
25
b- I ncreased values are seen in:
1- Polycythaemia vera.
2- Secondary polycythaemia e.g, in tumors as hypernephroma and cancer of
liver.
3- Dehydration.
4- Acute poisoning.
5- Severe diarrhea.

10- Manual and automated methods:
The above-described method is the manual procedure. For automated
method Haematology cell counter, e.g. Coulter counter is used.


11- Quality Control:
1- If the patients specimen running in automated machine there is three
levels controls running 3 times per day while if its running by manual
method send the patients specimen to the reference laboratory.
Perform duplicate testing in your own lab.




























26
Practical Sheet: 6

Methods of Hematocrit (Hct/Ht) OR Packed Cell Volume (PCV)
Determination

1- Introduction:
When red blood cells in blood are separated by centrifugation in a tube,
the volume of the red cells per unit volume of blood is termed as packed cell
volume. OR
The percentage volume of red cells in a volume of whole blood. It is
expressed in liters of RBC per liter of blood.
It may also be expressed in percentage when it is called haematocrit.

2- Value of measurement of Hct:
1- Screening test for health maintenance examination.
2- Screening test for patients taking anti-inflammatory drugs, which can
cause occult blood loss.
3- To follow the response to therapy for anaemia.
4- To follow the course of chronic anaemia.
5- To assess the degree of chronic anaemia such as thalassaemia.
6- To assess the degree of blood loss following acute bleeding.

The packed cell volume can be estimated either by:
- Macro method (Wintrobes method)
- Micro method (centrifugal)


3- Principle:
Haematocrit is defined as the volume occupied by erythrocytes(RBCs) in a
given volume of blood and is usually expressed as a percentage of the volume of
the whole blood sample
The hematocrit is usually determined by spinning method using a blood
filled capillary tube in a centrifuge machine. The Coulter counter series of
analyzers provide an indirect measurement of haematocrit.

4-Procedure:
a-Reagents & Instruments:
1- Capillary tubes, plain (75 mm).
2- Micro hematocrit centrifuge.
3- Micro hematocrit reader.
4- Hematocrit tube sealant.
b-Sample:
capillary and venous blood in anticoaggulated EDTA tube.



27
c- Method:

1- Mixed the anti-coagulated venous or capillary blood gently 5 times.
2- Fill the capillary tube with blood. Preferably two tubes should be used for
each sample as breakage or leakage is common.
3- Seal one end of the tube with plasticine (sealing wax), and place these
tubes in the micro hematocrit centrifuge.
4- Centrifuge for 5 minutes at a speed of 10,000 r.p.m. This will separate
RBCs from plasma and leave a band of buffy coat at the interface
consisting of WBCs and Platelets.
5- Take out the tube and place it in the holder of micro-hematocrit scale in
such a way that the bottom margin of red cells layer is against the 0 mark
of the scale.
6- Similarly adjust the capillary tube so that the top margin of plasma layer
coincides with the slanting line of 100 mark.
7- Now adjust the sliding line so that it cuts between the red cell layer and
the buffy coat(fig.6.1).
8- Note the reading. This is the packed cell volume.








(Fig.6.1.Different layers of blood in micro-haematocrit tube after
centrifugation)








Plasma
Buffy coat
Red Blood Cells
28


d- Advantages of micro method:
1- Less amount of blood is used.
2- Capillary blood can be used.
3- It is a rapid method.
4- Plasma trapping is less.
e- Precautions:
1- Incomplete sealing of the capillary tubes will give falsely low results,
because in the process of spinning, RBCs and a small amount of plasma
will be forced out from the tubes.
2- Shortened spin time or slowed centrifugation speed may yield falsely
elevated results.
3- Micro-haematocrit centrifuge should never be forced to stop by applying
pressure to the metal cover plate. This will cause the RBC layer to sling
forward and results in a falsely elevated value.
4- The hematocrit is usually three times the haemoglobin.
f- Sources of error:
a- Incorrect anti-coagulant concentration.
b- Incorrect mixing.
c- Storage for 6 8 hours.
d- Incorrect centrifugation.
e- Haemolysis.
f- Clots in the blood sample.

5- Results:
1. Table of Reference Values:
Category Hematocrit (%) PCV (L/L)
Newborn 53 65 0.53 0.65
Infant/Child 30 43 0.30 0.43
Adult Male 42 52 0.42 0.52
Adult Female 37 - 47 0.37 0.47

2. Interpretation of the Results:
Variations in PCV:
PCV may be increased or decreased under various physiological as well as
pathological conditions.
Increased PCV is seen in:
1- Newborns.
2- High altitudes.
3- Dehydration.
4- Burns
Decreased PCV is seen in:
1- Anaemia (Macrocytic,thalassemia,spherocytosis,hypochromic anemia
,sickle anemia).
2- Pregnancy.

29


3. Quality Control:

1- Always check the working of machine every week by running a routine
patient sample and reading the results, then placing the capillary tubes
back in the centrifuge machine and spinning them for several more
minutes. The second microhaematocrit reading should agree with the first
set.
2- If the patients specimen running in automated machine there is three
levels controls running 3 times per day while if its running by manual
method send the patients specimen to the reference laboratory. Perform
duplicate testing in your own lab.

































30


Practical Sheet:7

Erythrocyte Sedimentation Rate

1- Introduction:
Anticoaggulated whole blood is made up of blood cells suspended in plasma.
When such blood stands for a period of time, the cells settle out. Various factors
affect the rate of settling. The ESR is a measure, under controlled conditions, of
the rate of red cell settling. Variety of procedures is available now days but they
are not reproducible. The modified Westergren sedimentation rate described here
can be performed easily. It is more reliable method than others.

2- Objectives:
The ESR is a non-specific test but even then it is used in wide range of diseases.
1- The ESR is dramatically affected by the red cell shape (e.g. sickle cells,
burr cells etc).
3- Principle:
When anti coagulated blood is allowed to stand un-disturbed, the RBCs
will normally settle down to the bottom of the tube. This principle is the basis for
ESR.
4- Procedure:
a- Reagents & Instruments:
1- Westergren Disposable sedimentation pipettes.
2- Westergren sedimentation rack.
3- Plastic disposable Pasteur pipettes.
4- Timer.
b- Sample:
1- Venous blood in sodium citrate or EDTA anticoagulant tubes (The ratio is
4 volume of blood to 1 volume of sodium citrate).
2- Hemolysed specimens should not be used.
c- Method:
1- Mix the blood thoroughly.
2- Fill the Westergren tubes according to the instructions of the
manufacturer.(up to the zero mm)
3- Place the tubes exactly vertical in the sedimentation rack, free from
vibrations, and sun light, for 60 minutes.
4- Read the length of the column after one hour. This measurement in mm is
the reading for ESR.
d- Reference Values:
1- Children: 0-10 mm/hour.
2- Males under 50 years: 0-15 mm/hour.
3- Males between 50 & 65 years: 0-20 mm/hour.
4- Males over 65 years: 0-38 mm/hour.
5- Females under 50 years: 0-20 mm/hour.
31
6- Females between 50 & 65 years: 0-30 mm/hour.
7- Females over 65 years: 0-53 mm/hour.
5- Results:
a- Interpretation:
The ESR is directly proportional to the weight of the cell aggregate and
inversely proportional to the surface area. Microcytes sediment more slowly than
macrocytes. Erythrocytes with abnormal or irregular shapes, such as sickle cells
or spherocytes, hinder rouleaux formation have a lower the ESR.

Factors that effect ESR are:
1- Plasma factors (viscosity of plasma).
2- Red cell factors (shape, Rouleaux formation and size).
3- Anti-coagulants (Na & K oxalates cause shrinkage of RBCs).
4- Mechanical factors (various methods give different values of ESR owing
to variations in the caliber of the tube and height of the column of the
blood).
6- Clinical Significance:
a- Markedly elevated ESR:
1- Malignant tumors.
2- Multiple Myeloma.
3- Lymphoma.
4- Leukemias.
5- Infections (bacterial as well as viral)
6- Collagen disease.
7- Ulcerative colitis.
8- Aplastic anaemia.
9- Pregnancy.
b- Moderately elevated ESR:
1- Viral hepatitis.
2- Tuberculosis.
3- Rheumatoid arthritis.
4- Lead poisoning.
5- Myocardial infarction.

7- Sources of Error:
a- Falsely elevated ESR:
1- Tilting of ESR tube.
2- Vibrations.
3- Elevation of room temperature.
4- Anisocytosis of RBCs.
b- Falsely lowered ESR:
1- Microcytosis.
2- Polycythaemia.
3- Old sample.
4- Sickle cells.

32


8- Quality Control:
1- Be sure that the rack is leveled, out of sun light and not affected by
vibrations.
2- Duplicate samples can be run. This can serve as an internal quality control.
3- Because anisocytosis and poikilocytosis affect the values for ESR, so it is
recommended that abnormal values must be checked by measuring
hematocrit and examining peripheral smears.





































33


Practical Sheet: 8

Absolute Values (Red Cell Indices).

1- Introduction:
The values obtained from the erythrocyte count, hematocrit and
haemoglobin concentration can be further used to calculate RBC indices or
absolute values, which define the size and haemoglobin content of the average
RBC in a given specimen of blood.

2- Values of use:
1- Screening test for health maintenance examination.
2- Screening test for patients taking anti-inflammatory drugs, which can
cause occult blood loss.
3- To follow the response to therapy for anaemia.
4- To follow the course of chronic anaemia.
5- To assess the degree of chronic anaemia such as thalassemia.
6- To assess the degree of blood loss following acute bleeding.

3- Principle:
The significance of these values lies in the fact that these form the basis
for the morphological classification of anaemia.
The three most commonly used RBC indices are:
i. Mean corpuscular volume (MCV).
ii. Mean corpuscular haemoglobin (MCH).
iii. Mean corpuscular haemoglobin concentration (MCHC).

i. Mean corpuscular volume (MCV):
This is the average volume of RBC, in cubic micron or femto liters (fl).
This can be calculated by using the following formula, if the PCV and the total
RBC count are known:
MCV (fl) = PCV (L/L) / RBC (x 10
12
/ L)
The value is reported in femto liters (fl)
Normal range for adults = 86 10 fl.
e.g. of calculation; Hct (44%) and RBC count 4.2 X 10
12
/ L ,the MCV=105fL

1- Interpretation:
Results below 80 fl. Indicates a smaller size cells that is microcytes,
similarly an MCV greater than 100 fl indicates that cells are of larger size i.e.
macrocyte.


34
2- CLINICAL SIGNIFICANCE:
a) Decreased values are obtained in:
1) Iron deficiency anaemia.
2) Thalasaemia
3) Sideroblastic anaemia.
4) Anaemia of chronic blood loss.

b) Increased values are obtained in:
1) Liver diseases.
2) Alcoholism..
3) Deficiency of vitamin B-12 or folate (Megaloblastic anaemia).

ii- Mean corpuscular haemoglobin (MCH):
This is the average weight of haemoglobin, in absolute units, in the RBC.
The results give the average contents of haemoglobin per erythrocyte in
pico-grams (pg) or micro- micro grams (g).
MCH (pg) = Haemoglobin (g/L) / RBC (x 10
12
/ L)
The normal MCH in adults = 29.5 2.5 pg.
e.g. of calculation; Hgb (14g/dl) and RBC count 4.2 X 10
12
/ L ,the MCH=33pg
The value is frequently higher in newborns and infants, because their
MCV is higher than in adults.

CLINICAL SIGNIFICANCE:
1- An increase in the MCH is associated with macrocytic anaemia.
2- A decrease in the MCH is associated with hypochromic anemia.

iii- Mean corpuscular haemoglobin concentration(MCHC):
This is the average concentration of haemoglobin in each individual RBC.
It is a ratio of the weight of haemoglobin to the volume of the RBC.
MCHC (g/dl) = Haemoglobin (g/dL) X 100/ Hematocrit (%)
The normal MCHC value = 32.5 2.5 g/dl.
e.g. of calculation; Hgb (14g/dl) and Hct (42%) ,the MCHC=33g/dl

Based on MCHC, erythrocytes may be classified as normochromic or
hypochromic.

4- Classification of Anemia:

MCV(fL) MCHC(g/dL) Classification
80-100 32-36 Normocytic,normochromic
>80 >32 Microcytic,hypochromic
<100 32-36 Macrocytic,normochromic




35

Practical Sheet: 9

Preparation of Blood Smear and Gross Examination

Introduction & Objectives:
Examination of a well-prepared and well-stained blood film is an important
part of haematological examination.
It is carried out for various reasons:
1- Differential leukocyte count.
2- Morphology of red blood cells.
3- RBC inclusion bodies.
4- Blood parasites detection, etc.
All these points make it essential that a blood smear must be prepared
correctly and stained properly to provide the physician / haematologist with an
accurate interpretation.
There are different methods used to prepare the blood smears, e.g.
- Slide-to-slide method.
- Coverslip-to-coverslip method.
- Coverslip-to-slide method.
- Automatic evaluation.
Blood smears are prepared with EDTA-anticoagulant blood to minimize
degenerative changes in the blood cells.
To ensure good preservation of cellular morphology, differential smears
should be made as soon as possible and it should be no later than 3 hours after
collection.
1- SLIDE - TO - SLIDE METHOD:

a- Principle:
A small drop of blood is placed near the one end of a clean glass slide. A
second slide is used as a spreader. The blood is streaked in a thin film over the
slide (fig.9.1). The slide is allowed to dry (air), fixed and then stained for
examination under the microscope.

b- Reagents and Equipment:
1- Glass slide (3 x 1 inches).
2- EDTA blood.
3- Leishmans Stain.
4- Leishmans buffer.
5- Distilled water.
6- Staining rack.
7- Microscope.




36

c- Making a blood smear:
1- Place a small drop of blood, 1.0 cm from one end of the slide (fig. 9.2).
2- Place spreader margin in front of it on glass slide at an angle of 45
degree.
3- Bring the spreader back to touch the drop of blood, which should spread
along its margin.
4- Push the spreader forward by rapid, uniform and smooth movement.
5- It should not reach the other end of the slide.



(Fig.9.1. Peripheral blood smear on a glass slide)
(Fig.9.2. various steps in the preparation of smear)

3
15-30
o



37
d- Spin method:
By which 1 or 2 drops of blood, placed in the center of a glass slide are
briefly spun at high speed in a special centrifuge (e.g. Cytospin); the blood
spreads on the slide in a mono-layer.
By this method leucocytes and platelets are distributed uniformly and free
of distortion. The red cells show a tendency to become distorted, but this can be
overcome by diluting 2 volumes of blood with 1 volume of 9 g/l NaCl
immediately prior to putting the blood on the slide.
e- Labeling blood films:
A recommended method is to write the name of the patient and the date or
a reference number in pencil (graphite) on the slide/film itself. A paper label
should be affixed to the slide later.

f- Staining Procedure:

Romanowsky Stains; Preparation & Uses:

These stains are widely used for staining the peripheral blood smears and bone
marrow aspiration smears. These include the following stains:
1. Leishmans stain
2. May-Grunwald- Giemsa stain
3. Jenners stain.
4. Wrights stain.
1- Principle:
The distinct staining reaction depends upon the components of the stain,
which are:
- Azure B.
- Eosin Y.
The acidic elements in the cell, like nucleic acids and proteins of the cell
nucleus, bind the basic dye i.e. Azure B and stain blue.
The basic elements like haemoglobin, stains red with acidic component i.e.
Eosin Y.
Alkaline pH accentuates the basic staining. Therefore an optimum pH is to
be sought. A pH of 6.8 is recommended for optimal staining for all components.
Leishmans stain and May Grunwald Giemsa stain, are the most frequently
used stains.

2- Staining of blood films by Leishmans Stain Method:
a- Requirements:
1- Leishmans stain.
2- Buffer solution.
3- Staining rack.


38
Procedure:
1- Prepare a smear of peripheral blood by placing a small drop of blood on
a glass slide, 1cm from one end of the slide. Place spreader margin in
front of it on the glass slide at an angle of 45
0
. Bring it back to touch the
drop of blood, which should spread along the margin of the spreader.
Push the spreader forward by rapid, uniform and smooth movement. It
should not reach the other end of the slide.
2- Place the air-dried blood film in a jar-containing Methanol for 3-5
minutes, to fix the smear on the slide.
3- Keep it on a staining rack and put few drops of Leishmans stain on it,
just to cover the smear.
4- Leave it for 2 minutes.
5- Pour on it freshly prepared working buffer, about twice the amount of
stain and cover the whole slide with it.
6- Mix the buffer with the stain by blowing gently through a pipette.
7- Leave it for 8-10 minutes.
8- Pour off the stain-buffer mixture, wash it with buffer or distilled water
very gently, and clean/dry the under surface of the slide with tissue
paper.
9- Place the slide vertically in a slide stand and let it dry in air.
10- Place the dried slide on the microscope and scan it with the help of low
power (10x) objective, and check for cell distribution, clumping, and
abnormal cells.
11- Choose a specific area, add a drop of immersion oil, and switch to the
(100x) oil immersion objective.
12- It is important to examine cellular morphology and to count leukocytes
in areas that are neither too thick nor too thin.
13- The best area is in between the body and the tail of the blood smear.



39

Practical Sheet: 10
Reticulocyte Count

1- Introduction & Objectives:
Reticulocytes are immature RBCs that contain remnants of cytoplasmic
RNA and organelles such as mitochondria and ribosomes. It is done to assess the
activity of bone marrow.
2- Principle:
Reticulocytes are visualized by staining with supra vital dyes e.g., Brilliant
cresyl blue or New methylene blue that precipitate the RNA and other organelles,
forming a filamentous net work of reticulum. The reticulocyte count is ment for
assessing erythrocytic activity of the bone marrow.
3- Procedure:

a- Reagents, Chemicals and Equipment:

A. Reticulocyte stain;
Take 1.0 gram brilliant cresyl blue and dissolve in 100 ml of citrate saline
solution (0.049 grams of tri-sodium citrate dissolved in 100 ml of normal saline).
Filter the mixture and it is ready for use.
OR:
Dissolve 0.5 grams of new methylene blue and 1.6 grams of potassium
oxalate and dissolve it in distilled water to make the contents 100 ml in volume.
B. Anti-coagulated blood EDTA
C. Dropper.
D. Test tubes.
E. Glass slides for smear and microscopy.

b- Steps for the Procedure:
1- Mix equal amounts of Retic stain and blood in a test tube.
2- This mixture is kept at 37
0
c in a water bath or oven for 1520 minutes.
3- Prepare a thin smear of this blood-dye mixture using one small drop. Air-
dries it. Do not fix or counter stain the slides.
4- Count the reticulocytes under oil immersion lens.
5- Reticulocytes would show strands of bluish material in the cytoplasm.
c- Counting Reticulocytes:
1- An area of the film is chosen where the cells are not overlapping.
2- Count both RBCs and Reticulocytes, until at least 100 reticulocytes are
counted.
4- Calculations:
The calculation of reticulocyte percentage is: e.g.
Total number of retics seen in 50 fields = 100
Total RBCs present in these 50 fields = 2500
Therefore:
Reticulocyte percentage (%) = 100 /2500 x 100
40
Reticulocyte %age = 4

a- Absolute Reticulocyte Count:
Absolute reticulocyte count expresses the number of reticulocytes in 1 cu.
mm of whole blood. It is not a percentage of RBCs.
Absolute Retic count = Retic % x RBC x10
12
/l / 100
The normal value is 60,000 / cu.mm (Average).

b- Corrected Reticulocyte Count:
It is important to adjust the retculocyte count according to the degree of
anaemia. This is known as adjusted reticulocyte count.


Corrected Retic count = Calculated retic % X Pts PCV
Average optimum PCV (0.45)
5- Reference Range:

Adults and Children= 0.52.5 % (50 100 x10
9
/L)
Infants (full-term, cord blood) = 2 6%
(Mean: 150x 10
9
/L)

6- Precautions:
1- Reticulocyte count should be done on fresh blood, because if blood is stored the
reticulocytes will mature leading to falsely low count.
2- Reticulocytes should not be confused with Hb-H inclusion bodies in Hb-H
disease. Hb-H inclusions stains paler, are dot like and occur in most of the red
cells. If there is any doubt, the retic count should be re-performed after
incubating the red cells and brilliant cresyl blue solution for another 24 hours.
If Hb-H inclusions are present, the count should not be decreased.
3- Heinz bodies appear as small dots present near the cell membrane, and should
not be confused with reticulocytes.
7- Clinical Significance:
a- Elevated reticulocyte count:
i- Haemolytic anaemias.
ii- Acute and chronic haemorrhagic conditions.
iii- Following treatment of iron deficiency anaemia and megaloblastic
anaemia.
b- Decreased reticulocyte count:
i- Aplastic anaemia.
ii- Aplastic crises of haemolytic anaemia.
iii- Ineffective erythropoisis, as seen in:
1. Thalassemia.
2. Pernicious anaemia.
3. Sideroblastic anaemia


41

Practical Sheet: 11

Demonstration of Heinz Bodies

1- Introduction:
Heinz bodies are precipitated globin chains, which may be seen as red cell
inclusions.

2- Principle:
Heinz bodies can be demonstrated by cytochemical staining. They may also
be seen in unstained preparations as refractile objects by lowering the microscope
condenser and subduing the light or by dark ground illumination or phase contrast
microscopy.

3- Procedure:
a- Sample:
venous blood in anticoaggulated EDTA tube.
b- Reagents and Instruments:
1- Methyl violet solution is prepared by dissolving 0.5 g of methyl violet in
100 ml of 9 g/dl NaCl and filtered.
Or brilliant cresyl blue
2- Glass slides & Microscopes.
c- Steps of the Procedure:
1- Mix 1 drop of blood and 4 drops of methyl violet solution in a test tube.
2- Allow standing for 10 minutes at room temperature.
3- Prepare the films and allow drying.
4- See under oil immersion lens. Methyl violet stains the Heinz bodies
excellently as intense purple inclusions in RBC. Their size varies from 1
3m.
OR
1- Mix equal amounts of brilliant cresyl blue stain and blood in a test tube.
2This mixture is kept at 37
0
c in a water bath or oven for 1520 minutes.
3-Prepare a thin smear of this blood-dye mixture using one small drop.
Air-dries it.
4- See under oil immersion brilliant cresyl blue stains the Heinz bodies
excellently as intense blue inclusions in RBC. Their size varies from 1
3m
4- Reporting Results for Heinz Bodies:
Heinz bodies appear as blue, refractile, intracytoplasmic inclusions. They
are irregularly shaped bodies of varying sizes, up to 2m in diameter, and are
found close to the cellular membrane. There may be more than one Heinz body
present in an erythrocyte. The test detects the in vivo precipitated Heinz bodies.
The presence of Heinz bodies in the blood is a sign of chemical poisoning,
drug intoxication, G6PD deficiency or the presence of anunstable haemoglobin.
42
They may also be produced by the action on red cells of some aromatic
and amino compounds such as inorganic oxidizing agents.

Procedure Notes:
Heinz bodies are detectable by using supravital stains as brilliant cresyl
blue, new methylene blue N, methyl violet, and crystal violet. Heinz bodies are
not seen when stained with Wright or Wright-Giemsa stain.
5- Clinical Applications:
H.bodies are formed when glycolytic enzymes in the erythrocytes are
unable to prevent the oxidation of Hb. As a result, the Hb is eventually denatured
and precipitated to form H.bodies. Erythrocytic enzyme systems decrease as the
cell ages; therefore, occasional H.bodies may be observed in normal blood.
Increased number of H.bodies represents unstable forms of Hb that are
present in a number of hemolytic disorders.
H.bodies occur in disorders such as, G6PD or, secondary to the action of
certain oxidant drugs and in cases of unstable Hb such HbH.






























43
Practical Sheet: 12

Demonstration of Haemglobin H

1-Introduction:
This mainly feature of hemoglobin H disease but small numbers of similar cells
may be seen in -thalassemia trait, particulary ,but not only , thalassemia triat.

2-Principle:
Patient with -thalassemia, how form hemoglobin H(4),have red cells in which
multiple blue-green spherical inclusion develop on exposure to billernt crysle blue
or new methylene blue as in reticulcyte preparation..

3- Procedure:
a-Sample:
venous blood in anticoaggulated EDTA tube.

b-Reagents, Chemicals and Equipment:
stain;
Take 1.0 gram brilliant cresyl blue and dissolve in 100 ml of citrate saline
solution (0.049 grams of tri-sodium citrate dissolved in 100 ml of normal saline).
Filter the mixture and it is ready for use.
OR:
Dissolve 0.5 grams of new methylene blue and 1.6 grams of potassium
oxalate and dissolve it in distilled water to make the contents 100 ml in volume.
B) Anti-coagulated blood EDTA
C) Dropper.
D) Test tubes.
E) Glass slides for smear and microscopy.


c-Method :
1- Mix equal amounts of brilliant cresyl blue
Stain and blood in a test tube.
2This mixture is kept at 37
0
c in a water bath or oven for 1-3hour.
3-Prepare a thin smear of this blood-dye mixture using one small drop. Air-
dries it.
4- See under oil immersion, the hemoglobin H precipitate multiple blue-
green spherical inclusion varying in size develop on exposure to brilliant
crysle blue
4-Reportin the result :
The number of the cell contains inclusions varies according to the type of -
thalassemia.
In -thalassemia triat only 0.01-1% of the red cells contain inclusions.
In hemoglobin H disease at least 10%of the cells develop inclusions.

44
Practical Sheet: 13

Detection of Haemoglobin S (HbS)

1- Introduction & Objectives:
HbS is found in the sickle cell disease. In this abnormal Hb, valine is
substituted for glutamic acid at the sixth position of the beta globin chain.
One of the properties of HbS, which is responsible for the clinical
symptoms, is its conversion into an insoluble gel form when exposed to low
oxygen tensions. Tubular filaments are produced and the red cells become sickle
shaped.
HbS can be detected by the qualitative Solubility Test, Sickling Test, and
Haemoglobin Electrophoresis.

Sickle Screening Tests

a- Qualitative solubility test

1- Principle:
The test is based on the principle that the HbS is relatively insoluble in
concentrated phosphate buffer in the presence of reducing substances.
Virtually all other haemoglobins, apart from HbS are totally soluble in the
buffer solution. HbS on the other hand will precipitate out and the solution will
remain turbid.

2-Sample:
Venous blood in anticoaggulated EDTA tube.

3-Reagents:
1. Reaction vials prefilled with sodium hydrosulfite
2. Phosphate buffer
3. Sickle screen control set
4. Deionized water
5. Tube reading rack

4-Procedure:
1-Bring all reagents to room temperature
2-Label one reagent tube for each patient and control
3-Place tubes in reading rack and add phosphate buffer to each tube until it
reaches the "Fill to this level" mark
4-Add 50 L of patient sample and controls to respective tubes.
5-Incubate at room temperature for 10-20 minutes.
6-note the appearance of the solution:
- HbA is soluble in concentrated phosphate buffer; hence it gives a
uniform red color without any precipitate.
45
- If whole of the haemoglobin is HbS, then only a red precipitate will be
formed, whereas the rest of the fluid will be clear.
- In cases of sickle cell trait, both homogeneous red solution of HbA as
well as a precipitate of HbS will be seen

5- Results:
The presence of HbS is indicated by a turbid solution.


6- Interpretation of the Results:
Negative:
If HbS is not present, the solution will be clear to slightly cloudy and the lines of
the tubes reading rack will be visible the tubes.
Positive:
If the HbS is present the solution will be turbid and the dark lines of the reading
rack will not be visible when the viewing through the tubes.
Whenever a solubility test is positive, haemoglobin electrophoresis must
be performed before a definite diagnosis is made.

7-Limitaions:
1-If the Hemoglobin level is 8g/dL or less, a false negative result may occur.
2-patient with multiple myeloma or other plasma cell dyscrasias may yield false
positive results.
3-Elevated HbF levels my yield false-negative results and therefore this test
should be performed on infants 6 month of age or younger.

b- Sickling test.

1- Principle:
This test is based on the decreased solubility of HbS at low oxygen
tension. Red cell which contain HbS, sickle when the oxygen tension is lowered.
2- Reagent:
Na-metabisulphite (2g dissolved in 100ml distilled water).
3- Materials:
- Glass slides and cover slips.
- White petroleum jelly or wax or DPX.
4- Procedure:
1. Mix 1 drop of whole blood with 2 drops of reagent.
2. Place 1 drop of the mixture on to a glass slide and cover with cover slip.
3. Seal the edges of cover slip with DPX.
4. Put the preparation in the 37C incubator for 2 hours and examine for
sickled cell.
5- Interpretation:
- Immediate sickling indicates HbS disease.
- Sickling after 1-2 hours and some times after 12 hours is suggestive of HbS
trait.
46
Practical sheet: 14

Estimation of Haemoglobin F

Acid Elution Technique (Kleihauer Test)

1- Introduction & Objectives:
Hb F can be identified in RBCs by the acid elution technique or estimated
by the alkali denaturation method.
2- Principle:
The test is based on the fact that HbF resists acid elution to a greater extent
than HbA. It is performed on smears of blood made on a glass slide. Cells, which
contain HbA, are cleared of their haemoglobin, (Ghost cells) whereas those cells,
which contain HbF, retain the haemoglobin and hence stain pink.
3- Requirements:
1) 80 % ethyl alcohol as a fixative.
2) Elution solution.
3) Eosin solution as a counter stain.
4) Slides and Microscope.
4- Procedure:
1- Prepare peripheral blood smear and dry it in air.
2- Fix the slides in 80 % ethanol for 5 minutes.
3- Remove the slides from fixative and wash in running tap water.
4- Dry the slides and place in elution solution for 20 seconds.
5- Rinse immediately in tap water, and dry the slides.
6- Counter stain with aqueous eosin for 5 minutes.
7- Dry the slides after rinsing in tap water.
8- See under oil immersion lens. Cells containing HbF stain pink whereas
which contain HbA appear as clear ghost cells (fig. 13.1).
Microscopic Appearance Of A Peripheral Smear In Kleihauer Test:

(Fig.13.1.Kleihauer test for fetal red cells

47
A deeply eosin staining cell containing fetal haemoglobin has been
eluted from the other red cells by an incubation at acid pH and these appear as
colorless ghosts.
5- Explanation of the Test:
It is a sensitive procedure, which identifies individual cells containing Hb
F even when few are present, and their detection in the maternal circulation has
provided valuable information on the pathogenesis of haemolytic disease of the
new born.

1- Reporting the Results for Hb.F:

a- Calculations:
The %age of Hb-F containing cells can be determined by counting the
number of dense-staining cells and the number of ghost cells per field. Using the
high power dry objective, count 500 ghost cells and record the Hb-F containing
cells seen during the count.
b- Results:
An adult specimen should have approx. the same number of dense Hb-F
containing cells as the normal adult blood. These cells should appear rarely. The
results are expressed as a %age.
Normal adults have less than 1% Hb-F containing cells. Infants values are
higher, with newborn infants having 70-90% Hb-F containing cells.

7- Sources of Error:
1- False positive result may be obtained if the EDTA blood is allowed to
stand overnight.
2- If patient has a very high %age of reticulocytes.
3- In haemoglobinopathies, e.g; sickle cell disease, the amount of HbF
varies, producing inconsistent staining results. Cells having small amount
of Hb F stain lightly.

8- Clinical Significance:
Increased amount of Hb F is found in various hemoglobinopathies such as:
1- Hereditary persistence of fetal Hb (HPFH).
2- Sickle cell anemia.
3- Thalasaemia.

9- Limitations:
The procedure is a semi quantitative method.





48

Practical Sheet: 15

Haemoglobin Electrophoresis

1- Introduction:
Haemoglobin is composed of four polypeptide chains called globin chains
and a heme group called iron protoporphyrin heme.

2- Objectives:
Haemoglobin electrophoresis is the most useful method for the
demonstration of abnormal haemoglobins.

3- Principle:
Small sample of hemolysates prepared form packed cells are automatically
applied to the TITAN alkaline hemoglobin GEL.
The hemoglobins in the sample are separated by electrophoresis using alkaline
buffer and stained with acid blue stain. The patterns are scanned on a densitometer
and the relative percentage of each band is determined. Proteins, including
haemoglobin, are negatively charged and hence they move towards the positively
charged electrode.

4- Various Gel Applications:

a- Cellulose Acetate Gel(PH 8.4)
Medium used for initial haemoglobin electrophoresis is cellulose acetate.
It is a smooth, homogenous strip of membrane on which separation of different
types of haemoglobin is excellent.

b- Citrate Agar(PH6.0-6.2)
For more precise results, polyacrylamide gel, starch gel and agar gel are
used.
5- Reagents & Equipments:
1- Titan HB-Alkaline or Acid plates.
2- Lysing agent.
3- Stain (Blue Acid).
4- Stain Solution.
5- Haemoglobin Buffer.
6- Electrophoresis unit (haemoglobin separator, coloring unit and
densitometer).
6- Procedure:
a- Specimen Collection, Handling, and Preparation of Haemolysate:
Fresh EDTA anti-coagulated whole blood sample.
Samples can be stored refrigerated at 2-8
0
C for up to 1 week.
For the preparation of haemolysate:
49
1-centrifuge anticoagulated blood for 10 minutes to separate cells from
plasma.
2-Wash Packed cells three times by resuspending in 5 to 10 volumes of
normal saline solution (0.85% NaCl).
3-prepare the sample by mixing 10L of sample with 100 L of
hemolysate reagent.
4-vortex and incubation 10 minutes.


b- Method:
According to the Kit provided as, KIT TITAN by Helena- France.

1- Preparation of Haemoglobin separation chamber:
a- Pour 30 ml of Acid or Alkali buffer in the chamber according to the
procedure or the gel to be used.
b- Put haemolysates in the sample wells of the chamber along with different
controls.
c- Put the specific gel and prepare the chamber according to the
specifications of the kit.
d- Place the chamber in the machine and press the start button. The
electrophoresis or the separation of the haemoglobin will start
automatically.
e- Remove carefully, the gel from the machine, at the end of a cycle.
2- Poly Stain machine:
Place the wet gel, removed from the electrophoresis chamber, into an
applicator, carefully, and insert it in the machine for drying---- coloring----drying-
---destaining and finally drying of the gel.
At the end of this step, a dry gel with blue colored lines of the separated
haemoglobin in the given sample of blood.
3- Densitometer:
Place this dried, colored gel in the densitometer to read the different
percentages of various haemoglobins in the given blood sample.

7- Interpretation of Results:
The possible identity of the haemoglobin types present in the samples can
be determined by visual evaluation of the completed gel by comparing it with the
mobility of the controls .
a- Limitations:
Some abnormal haemoglobins have similar electrophoretic mobilities and
must be differentiated by other methods.
b- Reference Values:

Hb A Hb F Hb A2
Structure 2 2 2 2 2 2
Normal (%) > 95 % < 2% 1.5-2.5 %

50
c- Clinical Significance:
Haemoglobin electrophoresis is useful as part of haematology profile in
the diagnosis of haemoglobinopathies such as sickle cell disease and hereditary
persistence of fetal haemoglobin and thalassaemias.

Table: Results of laboratory investigations in interactions of Hb S and
or thalassaemia in adults.

MCV %S %A %A2 %F
A/S N 35-38 62-65 <3.5 <1
SS N 88-93 0 <3.5 5-10
S/ L 88-93 0 >3.5 5-10
S/ L 50-93 3-30 >3.5 1-10
S/HPFH N 65-80 0 <3.5 20-35
AS/+ Thalassaemia N/L 28-35 62-70 <3.5 <1
AS/Thalassaemia L 20-30 68-78 <3.5 <1
SS/Thalassaemia N/L 88-93 0 <3.5 1-10

N=NORMAL L=LOW

















51
Practical Sheet: 16

Osmotic Fragility of Erythrocytes
(Dacies Method)

1- Objectives & Principle:
In Osmotic Fragility Test (OFT), whole blood is added to varying
concentrations of sodium chloride solution and allowed to incubate at room
temperature. The amount of hemolysis is then determined by examining the
supernatant fluid either visually or with spectrophotometer.
If erythrocytes are placed in isotonic solution (0.85%) of sodium chloride,
water molecules will pass in and out of the membrane in equal amounts. In
hypotonic solutions, erythrocytes will hemolyse because more water molecules
enter into the cell than leave. This net influx of water molecules eventually
ruptures the cell membrane.
The main factor in this procedure is the shape of the erythrocyte, which is
dependent on the volume, surface area, and functional state of the erythrocytic
membrane. A spherocytic erythrocyte ruptures more quickly than normal
erythrocytes or erythrocyte that has large surface area per volume, such as target
or sickle cells.
The fragility of erythrocytes is increased when the rate of hemolysis is
increased. If the rate of hemolysis is decreased, the erythrocytic fragility is
considered to be decreased. The clinical value of the procedure is in
differentiating various types of anemia.

2- Specimen:
Fresh defibrinated or heparinized whole blood may be used.
3- Reagents, Chemicals & Equipment:
1- Stock buffered sodium chloride solution: Prepare by weighing 90 grams
of sodium chloride, 13.65 gm of sodium dibasic phosphate (Na
2
HPO4),
and 2.34 gm of monobasic sodium phosphate (NaH
2
PO
4
.2H
2
O). Add 1.0
L of deionized water.
2- 1% buffered sodium chloride: Prepare by mixing 20 ml of stock buffered
sodium chloride with 180 ml of distilled water.
3- Test tubes (14 in number).
4- Pipette, graduated 1 ml and 10 ml.
5- Centrifuge.
6- Spectrophotometer and cuvettes.
4- Quality Control:
A normal control sample should be tested simultaneously.
5- Procedure:
1- Prepare dilutions of buffered sodium chloride and place in labeled test
tubes (as shown in the following Table).
2- Cover the tubes and mix by inversion.
3- Add 0.02ml (20 l) of blood to each of 14 test tubes. Mix gently by
inversions.
52
4- Allow tubes to stand at room temperature for 30 minutes.
5- Remix gently and centrifuge at 2500 rpm for 5 minutes.
6- Transfer supernatant to cuvettes and read on spectrophotometer at 550
nm. OR
7- Examine the haemolysis macroscopically, ie visual reading.

Osmotic Fragility of erythrocytes (Dacies Method):
Preparation of Dilutions

Test Tube 1% NaCl(ml) Dist. H
2
O (ml) Final Conc.(%)
1 10 0.0 1.00
2 8.5 1.5 0.85
3 7.5 2.5 0.75
4 6.5 3.5 0.65
5 6.0 4.0 0.60
6 5.5 4.5 0.55
7 5.0 5.0 0.50
8 4.5 5.5 0.45
9 4.0 6.0 0.40
10 3.5 6.5 0.35
11 3.0 7.0 0.30
12 2.0 8.0 0.20
13 1.0 9.0 0.10
14 0.0 10.0 0.00

6- Visual Readings:
Note the tubes in which hemolysis is complete, partial or absent.


7- Calculations:

% Hemolysis for each tube = OD of Supernatant X 100
OD of tube with 100% Hemolysis.

Reporting Results:
In normal blood, hemolysis should end at 0.30% sodium chloride and
beginning of hemolysis should not occur in a concentration over 0.45%. Patient
and normal control results may be reported in graphic form (fig.14.1).








53
Normal Ranges for Osmotic Fragility:
Test Tube Conc. of NaCl (%) Haemolysis (%)
1 1.00 0
2 0.85 0
3 0.75 0
4 0.65 0
5 0.60 0
6 0.55 0
7 0.50 0-5
8 0.45 0-45
9 0.40 50-90
10 0.35 90-99
11 0.30 97-100
12 0.20 100
13 0.10 100
14 0.00 100






















(Fig.1
4.1.
Osmot
ic
fragili
ty
curve)

54


9- Clinical Applications:
The osmotic fragility test is of value in the diagnosis of different types of anemia
and conditions in which physical properties of the erythrocytic membrane are
altered.

Erythrocytic Fragility in Selected Disorders:

Decreased Fragility Increased Fragility
HbC disease Acquired Auto Immune Hemolytic .Anemia.
IDA Burns
Sickle Cell anemia Chemical Poisons.
Thalasaemia Haemolytic Disorder .of the new born (HDN)
Polycythemia Vera Hereditary Spherocytosis