MBChB YEAR 3, PRACTICAL MANUAL.

08/12/2023
PRACTICAL ONE.
BLOOD

1.5.1 Anticoagulants Used in Hematology Laboratory

Anticoagulants are defined as substances which prevent blood clotting/coagulation, and allow separation of
the blood into cellular and liquid (plasma) components. Generally plasma contains coagulation factors.
The three anticoagulants commonly used in hematology laboratory are:
1.5.1a Ethylene Di-Amine Tetra-Acetic Acid (EDTA):
EDTA can be found in three salt forms:
1. Tri-Potassium EDTA
2. Di-Sodium EDTA
3. Di-Lithium EDTA
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EDTA can be crystalline or liquid. Liquid EDTA tubes requires specific filling volume to avoid dilution
effect. So, blood: anticoagulant ratio must be maintained (this is applicable to all anticoagulants). EDTA acts
by chelating/removing ionized calcium (calcium is required for blood to clot, so when it is removed blood
will not clot). Generally tri-Potassium EDTA is better than di-Sodium EDTA and di-Lithium EDTA.
Always, be sure to mix blood with anticoagulant in a manner that guarantee proper complete mixing, by
gentle repeated inversion of the tube, in figure of 8 inversion for at least 20 times, do not shake or use
vigorous inversion, since this may cause hemolysis, and disintegration of cells, and the final effect will be
erroneous low results for cellular components of blood
EDTA is the most commonly used anticoagulant in the hematology laboratory, and is the anticoagulant of
choice for the CBC. Excess EDTA (i.e. more EDTA, you fill less blood volume, so EDTA is in excess),
causes shrinkage of RBC’s, causing falsely/erroneously reduced Hematocrit (HCT), and subsequent increase
in MCHC and decrease in MCV (MCV and MCHC are RBC indices that will be studied later). Platelets are
also affected, they will swell and subsequently disintegrate, causing erroneously high platelet count, since
platelets will be disintegrated into more than one fragment, each fragment will be counted as one platelet (for
example if one platelet will be disintegrated into 4 fragments, the 4 fragments will be counted as 4 platelets,
but actually they represent one platelet, causing erroneously high platelet count).

Sodium Citrate: Is the anticoagulant of choice for coagulation and platelet function tests. It acts by
precipitating calcium, thus it will not be available for clotting process. It comes in a liquid form, as 3.8% tri-
sodium citrate. For coagulation testing, the ratio of 9 volumes of blood to one volume of anticoagulant (9
volumes blood: 1 volume anticoagulant) is very critical, as variation from this ratio may cause errors.
See tube provided.

1.5.1b Heparin
Heparin is an acid mucopolysaccharide, it acts by complexing with anti-thrombin to prevent blood clotting
(antithrombin is one of the natural/physiological inhibitors of blood coagulation).
It is not suitable for blood films staining, since it gives too blue coloration to the background, when films are
stained with Romanovsky stains, also, heparin may cause leukocyte and platelet clumping, this is why
heparin is not suitable for routine hematology tests.
It is the preferred anticoagulant for osmotic fragility test.
Heparin also is used in capillary tubes for spun hematocrit (HCT) (heparin cover the entire capillary tube
glass), these capillary tubes are also called microhematocrit capillary tubes.
Heparin is also used for L.E. cell preparation (L.E. = Lupus Erythromatosus).
Heparin is found in basophil and mast cell granules. Heparin is used therapeutically as an in vivo
anticoagulant.

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1.5.1c Tri-sodium citrate
See tube provided

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ACTIVITY:
I. Identify any other vacutainer using their colour codes and give their uses.

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PRACTICAL TWO.

HAEMATOLOGY TESTS

1. Preparation of Blood Films

Principle: Blood film enables us to evaluate WBC, RBC, and PLT morphology
It also allows us to perform differential WBC count, furthermore estimation of WBC and platelets counts can
be done on blood films. Blood films are made on glass microscopic slides.

Sample:
Finger stick blood or EDTA anticoagulated venous whole blood may be used.
Films of peripheral blood must be made immediately.
Films may be made from EDTA anticoagulated blood as long as two to three hours after collection. All
specimens should be free of clots.

Fig 3.4a
Procedure:

1- Use clean standard size glass slides (3 inch x 1 inch = 7.5 cm x 2.5 cm), wiped from dust just immediately
before use.

2- Place a small drop of well mixed anticoagulated whole blood, in the center line of the slide, about 1.5 to 2
cm from one end, with the aid of a capillary tube.
3- Immediately, without delay, with the aid of a second clean slide with uniform smooth edges (spreader
slide), with a 30 –40 degrees angle, move back so blood drop will spread along the edge of the spreader slide,
when this occurs, spread, or smear the film by a quick, unhesitating, uniform forward motion of the spreader.

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Fig 3.4b

Blood films are made by placing a drop of blood on one end of a slide, and using a spreader slide to disperse the
blood over the slide's length. The aim is to get a region, called a monolayer, where the cells are spaced far enough
apart to be counted and differentiated. The monolayer is found in the "feathered edge" created by the spreader slide
as it draws the blood forward. Fig 3.4c, Left smear is unstained, right smear is stained with Wright-Giemsa stain.

Fig 3.4c

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Notes:

1. Before preparing the films, you must check that blood samples are free from clots, and this is done
with two wooden applicator sticks. If clots are present the specimen is unsatisfactory.
2. Films can be labeled with patient’s name and /or Lab. No. on the thick end of the film itself, after
being dried, by using a pencil.
3. With anemia (low Hct, reduced viscosity), the spreading angle should be greater, to avoid running
off the slide.
4. With polycythemia (high Hct, increased viscosity), the spreading angle should be less, to avoid short,
too thick films.
5. With large blood drops, increase the spreading angle.
6. With small blood drops, decrease the spreading angle.
7. If the anemia is too severe, let the blood specimen settle, so that blood is divided into two layers,
plasma layer and red cell layer, then discard part of the plasma layer, then mix the blood specimen,
by doing this you have increased the viscosity of blood, by this you will be able to prepare a nice
blood film.

3.5 Staining Blood Films with Romanovsky Stains

Blood films are stained so that morphology of blood cells become more easily viewed, identified, and
evaluated. In addition, blood films may be examined for the presence of blood parasites (Malaria,
Trypanosoma, and Babesia). Furthermore, stained blood films can provide important information about a
patient’s health, they may lead to a diagnosis or verify a diagnosis, or they may rule out a diagnosis.
Evaluation of stained blood films also may lead to the decision of performing other hematology special blood
stain procedures in order to identify specific cell components.
As soon blood films are air dried, it is best to stain them as soon as possible. Blood films are stained with one
of the Romanovsky stains, which are universally used for staining blood films. There remarkable property is

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creating distinctions in shades of staining granules differentially and this is dependent on two staining
components: Azure B (the basic dye) and Eosin Y (the acidic dye). Other factors which affects the staining
results include: 1) Staining time, 2) Ratio of Azure B to Eosin Y, 3) pH of the staining solution.

•Azure B will stain the acidic cell Components (e.g. nucleus, because it contains nucleic acids; basophilic
granules also take the Azure B staining because they contain heparin, which is acidic in origin),
•while Eosin Y will stain the alkaline basic components (e.g. Eosinophilic granules in eosinophils, because
these granules contain spermine derivatives, which are basic in origin). Red cells have affinity for acidic
Eosin Y dye, because it contains hemoglobin which is basic in origin.
Romanovsky stains include:
Giemsa Stain
Wright’s Stain
Leishman Stain
May-Grünwald Stain

The widely and popular used Romanovsky stains are:

1. Wright's stain is composed of oxidized methylene blue and eosin azures. It is a simpler method .
2. Giemsa stain is thought to produce more delicate staining characteristics. It combines eosin Y with azure
B and methylene blue in methanol with glycerin added as a stabilizer.
3. Leishman's stain is similar to Wright's stain except for the method used to oxidize the methylene blue. It
is also a simple method, which is especially suitable when a stained blood film is required urgently or the
routine stain is not available (e.g. at night).
4. May-Gruwaldis a good method for routine work.
5. Field's stain is a rapid stain used primarily on thin films for malarial parasites.

3.5.1Leishman Stain Procedure:

1-Let the films be air dried.
2-Put the films on a staining trough rack.
3-Flood the slides with the stain.
4-After 2 minutes (or more, if the stain in newly prepared), add double volume of water, and blow to mix the
stain with water, until a shiny layer is seen.
5-After 5-7 minutes, wash with a stream of water.
6-Wipe the back of the slides with gauze.
7-Set the films in upright position on a filter paper to dry.

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CAUTION WITH LEISHMAN STAIN
INHALATION
Prolonged or repeated exposure may cause breathing difficulties and irritation.
INGESTION
May cause discomfort if swallowed.
SKIN CONTACT
Prolonged or repeated exposure may cause irritation.
EYE CONTACT
Prolonged contact may cause transient eye irritation.

8-Read the blood films microscopically.

If delay in staining blood films may occur, fix the films in absolute methanol, for 1-2 minutes, but do not
stain the slides until completely dried.

Giemsa staining Procedure

1. Prepare 10% Giemsa working solution, and place it in a small container.

2. Using a Pasteur pipette, fix the thin film by carefully dropping methanol onto the thin film.

3. Let the blood film dry in air on a drying rack or tray.

4. Place slides for staining blood films face down on a curved staining tray or face up on a staining rack.

5. Pour stain slowly on or under the slide until the blood films are covered.

6. Set the timer to 8-10 minutes for the staining.

7. Gently flush all the stain from the slides by dropping clean water over it.

8. Allow the slides to air-dry.

RED CELL SHAPES DIFFERENTIAL DIAGNOSIS

Irreversibly sickled Sickle cell syndromes (SS, SC, Sβthalassemia)

red cells
(drepanocytes)

Target cells Sickle cell disease, haemoglobin C trait, haemoglobin CC disease, thalassemias, iron

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 deficiency, Liver disease (cholestasis), asplenia,

Fragmented red cells Thrombotic micro-angiopathic haemolytic anaemias such as Disseminated intravascular
(schistocytes) coagulopathy (DIC), thrombotic thrombocytopenic purpura, haemolytic uraemic
syndrome.

Burr cells (echinocytes, In-vitro artifact following prolonged storage or slow drying of the smear due to high
crenated red cells) humidity, uraemia, Malnutrition

Spur cells Liver disease, Renal failure, Abetalipoproteinaemia, Spur cell anaemia, pyruvate kinase
(acanthocytes) deficiency

Tear drop cells Myelofibrosis, Myelophthisia (marrow infiltrations), Extramedullary haemopoiesis,

(dacrocytes) Hereditary elliptocytosis, Hereditary pyropoikilocytosis, Severe iron deficiency,
Megaloblastic anaemia, Thalassemias

Bite cells G6PD deficiency, Oxidative stress, unstable haemoglobins, congenital heinz body
anaemia

Pencil cells Iron deficiency

Stomatocytes Artifact( due to slow drying in humid environment), Liver disease, alcoholism, Rh-null
disease, Obstructive lung disease

Elliptocytes Hereditary Elliptocytosis (>25%)

Basket cells Oxidant damage, G6PD deficiency, Unstable haemoglobins Hereditary spherocytosis,
ABO incompatibility, Autoimmune hemolytic anemia (warm antibody type), Severe burns

Romanovsky Stain Blood Cell Characteristics

Cell Type Appropriate Appearance on Well-Stained Slide

RBCs Reddish pink.

Lymphocytes Dark purple nuclei with varying shades of blue cytoplasm

Neutrophils Dark purple nuclei with reddish, granular cytoplasm.

Monocyte Lighter purple nucleus with a gray-blue cytoplasm.

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Eosinophils Bright red/orange granules

Basophils Dark purple nuclei and granules.

Table 2.5.1
Sources of Errors in Staining
1- Stain Precipitate: May obscure cell details, and may cause confusion with inclusion bodies. Filter the stain
before use.
2- PH of the buffer or water:
• Too acidic pH causes too pinkish slides.
• Too basic pH causes too bluish slides.
3- Improper stain timing may result in faded staining or altered colors:
• Too long staining time causes too blue slides (overstaining).
• Too short staining time causes too red slides.
4- Forced drying may alter color intensities and/or distort cell morphology.
5- Non-stain related errors:
A. EDTA causes crenation of the cells after blood collection.
B. Severely anemic blood samples causes slower drying (before staining) due to excessive plasma.
C. Old blood specimens may cause disintegration in WBC’s and decrease in their numbers.
D. Collection of blood in heparin causes blue staining of

E. RBC’s with bluish background, which makes heparinunsatisfactory for routine hematology testing,
also heparininduces platelet aggregation and clumping, with subsequenterroneous platelet count with
automated counters.
Always filter the stain before each use, to eliminate stain precipitates.
Automatic stainers are available in the market, in which slides are moved automatically and they are stained
as they move.

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Fig 3.5a

Fig 3.5 b

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3.5.1 Variation in RBC morphology

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