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Medical Laboratory Technology: Volume 1
coverslip on top. Cells are counted under proper magnication over the specied grids of the
haemocytometer in the improved Neubauer counting chamber. The number of cells counted
in the volume of uid held in the chamber (counting area multiplied by the depth of the uid)
and the dilution made of the blood are then taken into consideration in reporting the number
of cells in the undiluted blood (cells per unit volume (μL) of undiluted blood).
Specimen
EDTA-anticoagulated venous blood is commonly used but other anticoagulants do not seem
to aect the results. In case of capillary blood from skin puncture, non-anticoagulated blood
is quickly diluted without leing the blood clot.
Equipment and supplies
• Microscope with 40× magnication of eye piece.
• Ruled counting chamber—preferably the improved Neubauer chamber.
• Cover slip for the haemocytometer. This is a standard cover slip that holds 0.1 mm
depth of uid.
• Blood Sahli pipee—20 and 50 μL.
• Graduated pipee, 1 mL.
• Pasteur pipee or capillary tube.
• Hand tally counter or bead counter.
• Rubber tubing for mouth suction (with mouth piece). Keep the mouthpiece always
dipped in 70% alcohol in a beaker or bole and avoid the common use of the mouth
piece between technicians (biohazard!).
• Surgical gauze.
• Erlenmeyer conical asks (25 mL) or test tubes in racks. These are used as containers
for diluting the blood.
Reagents
Diluting uids: Diluting uids are used to dilute the blood to facilitate counting under the
microscope. Apart from diluting they may ‘x’ cells in order to preserve or remove unwanted
cells When specic cells are counted.
White cell diluting uid: White cell diluting uid haemolyses the red cells due to acidity,
so that the counting of white cells becomes easy. Turk solution (2% acetic acid, v/v
aqueous):
Acetic acid, glacial 2 mL
Distilled water (q.s.) 100 mL
Aqueous methylene blue solution (0.3%> w/v) 10 drops*
Procedure (Figure 10.10)
1. Label an Erlenmeyer ask (25-mL) or a 10-mL test tube held in a rack. Take as many test
tubes (or asks) as the number of specimens.
2. Pipee 0.38 mL of diluting uid into the ask, using the 1 mL-graduated pipee
(0.01-mL graduation).
3. With the help of 20 μL Sahli pipee, draw well-mixed anticoagulated venous blood up
to the mark (mix the blood immediately before drawing into the pipee).
4. Hold the pipee in horizontal position and wipe the outside of the pipee with
absorbent paper or surgical gauze. Check the level of the blood specimen; it should still
be on the mark, otherwise, repeat Step 3.
* Note: Methylene blue solution is prepared by dissolving 0.3 g of methylene blue in 100 mL of distilled water. Filter
the methylene blue solution (0.3%) before adding to the acid solution. Gentian violet (1 mL of 1% solution in water,
w/v) can be used in place of methylene blue.
Routine Haematological Tests
269
Counting of white blood cells by haemocytometry: (a) Neubauer grid on the haemocytom-
eter showing white cell and red cell counting areas, (b) Filling of haemocytometer with
diluted blood, (c) Air bubbles should be excluded, (d) The depth of the uid column with
the use of standard cover slip is 0.1mm, (e) Eye movement pattern while counting cells
and the cells counted, (f) Screen the distribution under low-power objective and in case
of bad distribution; repeat the procedure
5. Blow the blood into the container with diluting uid. Rinse the blood pipee (Sahli,
TC*) three times and leave it inside the ask. Gently mix the blood well by swirling
and wait for 2 min for complete haemolysis. Dilution of the blood is (0.38 + 0.02)/
0.02 = 20.
6. Fill the counting chamber by capillary (Figure 10.10) and avoid overlling (Figure 10.11).
7. Place the counting chamber on the stage of the microscope, turn the objective to low
power and locate the grid.
* Additional Information TC pipees are “to-contain” pipees which are washed out. TD pipees, on the other
hand, are “to-deliver” pipees, like the serological pipees, which are not washed out. The drop of liquid that stays
at the tip of the pipee, after delivering the uid, is considered in measuring the volume. Sahli pipee is a TC pipee
and needs to be washed out with the solvent.
Number of while cells counted
20
Medical Laboratory Technology: Volume 1
8. Focus on one of the ‘W’ marked areas (which cover the entire eld of view, as shown
in Figure 10.10a). Each ‘W’ marked square has 16 smaller squares to facilitate counting.
The area of the ‘W’ marked square is 1 sq. mm.
9. Scan all four ‘W’ marked corner squares in order to nd out the distribution of cells.
Cells are identied by their blue colour with a dark dot in the centre (nucleus); there
will be no red cells.
10. Focus on one of the corner squares and count the white cells systematically (Figure
10.10), starting from the upper left small square of each ‘W’ square. Apply the same
margin rule as for the red cell count, that is, count cells lying on two adjacent margins
and discard those on the other two margins.
11. Repeat the entire white cell count on the other side of the chamber (preferably a replicate
of the same specimen diluted separately). Make an average of the two sides (4 sq. mm
area).
12. Calculation
Number of white cells/μL or cu.mm of whole blood =
Where,
Area counted Depth of fluid
D×× iilution
Dilution = 20X
Area counted = 4 x 1 sq.mm = 4 sq.mm
Depth of uid = 0.1 mm (constant)
Factor=
40150.
Therefore, as long as the dilution and area counted remain constant, the above
formula can be simplied as follows:
WBC/μL or cu.mm = Number of cells counted × 50
Note In order to convert the above value to the Systemic International unit, which in
French known as Systeme International d’Unités (per litre), multiply by 106.
Filling of haemocytometer chamber: (a) Overlled, and (c) under-lled, chambers give
erroneous results. Correctly lled chamber (b) has a clean outline of the grid-platform.
Routine Haematological Tests
Additional Information In case of a high leukocyte count, dilution may have to be increased to
100 times or more, use 1.98 mL of diluting uid. The dilution will be (1.98 + 0.02)70.02 = 100 times.
Sources of error
• Diluting uid should not be contaminated with blood cells.
• Keep the counting chamber and cover glass free from dust, lint and dried blood.
• Improper volume measurement (specimen and diluent), improper lling of the count-
ing chamber, use of defective pipees, improper counting.
• Disturbance of the chamber during the switching of objective (do not allow the objective
to touch the cover slip).
• Failure to clean the blood sampling pipee.
• Failure to wash the TC pipee properly.
• Wrong calculation.
• Clerical mistake in recording.
Cleaning procedure
Use mild detergent (IX sodium bicarbonate) followed by washing with tap water and rinsing
with deionized water. Dry the haemocytometer by polishing with lens paper, tissue paper or
with soft cloth. Do not put the haemocytometer in dichromate solution.
miCrosCopiC stuDy of blooD smear
Examining a stained blood smear is a routine part of the complete blood count (CBC). A blood
smear enables the technologist to view the cellular components of the blood. The procedure
of studying the blood smear includes preparing a blood smear on a microscope slide,
which is dried, xed and stained. Cellular components of blood are then viewed under the
microscope. The smear is primarily used in reporting the dierential count and any abnormal
morphology of white cells and red cells. Study of the blood smear helps in the diagnosis of
various anaemia, leukaemia, infections including those of blood parasites, such as malaria.
Principle
A thin blood smear is prepared by spreading a small drop of blood evenly on a slide so that
there is only one layer of cells. The blood smear on the slide is then stained with a polychromie stain. The Romanowsky stain is widely used. The stain also contains a xative (metha-
nol) that helps in preserving the cellular morphology for an extended period. The smear is
used for the dierential count of white cells, detecting abnormal erythrocytes, identifying certain blood parasites and estimating the number of thrombocytes. In casé of Leishman stain,
the purple colour of cell nuclei is due to molecular interaction between eosin Y and an azure
B-DNA complex. The intensity of staining depends on the azure В content and on the ratio
azure B/eosin Y The pH of the solutions, xation and staining time can inuence the staining
result.
Equipment and supplies
1. Microscope (with facilities for high magnication)
2. Microscope slides (clean and grease-free); if necessary, clean with ethanol (70%) or
ether and polish with a piece of soft clean cloth. Making of a special spreader slide is
described in Chapter 9.
3. Spirit lamp or Bunsen burner
4. Measuring cylinder 5- or 100-mL
5. Beakers and boles containing clean tap water
6. Washed bole containing buered water
7. Timer
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Medical Laboratory Technology: Volume 1
8. Racks for staining and drying slides
9. Boles for holding stains
10. Methanol
11. 70% Ethanol or ether
12. Capillary tube or applicator stick for holding a drop of blood specimen.
Stains and reagents
Bueredwater
Disodium hydrogen phosphate
(Na2HPO4 · 2H2O) 3.8 g
Potassium dihydrogen phosphate (KH2PO4) anhydrous 2.1 g
Distilled water (q.s.) 1000 mL
Quantitatively transfer salts, after weighing, into a 1000-mL volumetric ask. Add su-
cient water to dissolve salts. Check the pH with narrow range pH paper. The pH should be
between 7.0 and 7.2. No adjustment is necessary if one uses freshly obtained distilled water.
If the pH is lower than 7.0, add the dry salt of disodium hydrogen phosphate (a small quantity at a time), with constant stirring. Continue the process until the pH comes between 7.0
and 7.2. Allow the newly added salt to dissolve, mix and stabilize before adding a new lot.
Conversely, add potassium dihydrogen phosphate if the pH is above 7.2 in order to bring it
down. Use of a pH meter for adjusting the pH is recommended. Finally make to 1000 mL vol-
ume (q.s.). Commercially available buer tablets are more convenient to use. Keep the buer
solution in a wash bole for easy dispensing.
Leishman stain
Stock Leishman stain:
Leishman powder 1.5 g
Acetone free methanol (CH3OH) (q.s.) 1000 mL
Rinse out a clean staining bole with acetone-free methanol. Add a few clean dry glass
beads. (This helps in mixing). Add the staining powder and methanol and mix well. Label
the bole and write the date. The stain is ready for use the following day. Filter, if necessary.
Note The stain improves on standing. Leishman stain solution is stable for ve years. The
boles must be kept closed. The advised storage temperature is 18–30°C. Used solutions
and solutions that are past their shelf-life must be disposed of according to local disposal
guidelines.
Diluted Leishman stain:
Use diluted Leishman stain for processing the smear. Prepare 1:3 dilutions using one part of
stock stain (described above) and three parts of buered water. Mix well. Do not store the
diluted stain. Prepare enough for one day.
Specimen
Skin puncture is ideal for the microscopic study of blood. EDTA-anticoagulated blood can
also be used. Other anticoagulants such as heparin alter the appearance of leukocytes and
thrombocytes and should not be used. The process of skin puncture has been described
earlier. Let the blood ow freely. First take samples for determining the leukocyte count and
then for making a smear. Do not take blood from the index nger, thumb, infected nger or
from the ear lobe.
Procedure
1. Preparation of smear: Properly spread lms are essential for the accurate identication of cells.
a. Specimen from skin puncture: If the blood specimen is obtained by skin puncture
disinfect the skin with 70% alcohol, dry the alcohol, prick the skin and let the blood
Routine Haematological Tests
273
ow freely; wipe o the rst drop and put the second drop on the clean grease-free
slide. Blood from the toe or heel can be taken from infants; however, blood from the
ear is not suitable as it has too many monocytes. Note: For disinfection, 70% alcohol is
more ecient than absolute alcohol. The alcohol used for disinfection is methylated
and generally tinged blue with the addition of copper sulphate. It is a strong poison
and should not be consumed.
b. Specimen from anticoagulated blood: Transfer a drop of well-mixed blood specimen
(anticoagulated venous blood) with a pair of applicator sticks (blood specimen is
held between sticks) or microhaematocrit capillary tube (non-heparinized) to a clean
grease-free slide. Place the drop of about 5 mm diameter, located approximately
1 cm from the end of the slide. Do not use a Pasteur pipee as it yields too big a drop
for making a good smear. Blood held between two disposable wooden sticks may be
used.
с. Makingofthebloodlm: (Figure 10.12)
The general procedure of making a blood smear is described in Chapter 9. Here we
shall focus on making a very thin blood smear for microscopic examination under high
power.
• Place the slide on a at table top with the drop of blood on the right. With the thumb
and index nger of the left hand, hold the two left edges of the slide. With the right
hand hold the spreader slide and place the edge of the spreader just in front of the
drop of blood. There should be an approximate angle of 30°-45° between the two
slides. Draw the spreader back until it touches the drop of blood. Let the blood
run along the edge of the spreader. Push the spreader to the end of the slide with
a smooth quick movement. All the blood should be used up before you reach the
end. Keep the angle of the spreader constant (30° to 45°) throughout the process.
Prepare at least two smears for each specimen. A good slide preparation should be
thick at one end and thin at the other and should occupy the central portion of the
slide with clear margins on all sides from the edge of the slide. One can use special
‘spreader slides’ for making the smear (see Chapter 9 for details).
• Making smear at the bed side Blood smears can also be made even when a table is
not available at the patient’s bedside (Figure 10.13). This can be performed at the
bedside and after nger stick. The blood smear is made in the hand without the use
of a table. Slides are held in one hands and the longer side of the spreader slide is
moved over the smear slide using the other hand.
• Checking the blood smear (Figure 10.14)
Proceed to the next step if it is satisfactory for microscopic examination:
▪ There should be no lines extending across or down through the lm.
▪ The lm must be smooth at the end, not ragged and lined.
▪ The lm must not be too long or too thick.
▪ The lm must not contain holes often caused by a greasy slide.
• The blood smear should be dried quickly by waving it rapidly in the air. This
prevents distortion of the red cells. When the moisture is too high (in the rainy
season), dry the lm by waving it rapidly about 5 cm from the ame of a spirit
lamp (see Chapter 5 for details); to the side and slightly above the ame. Never put
the slide directly over the ame. Adequate drying is, however, essential to preserve
the quality of the lm. Hence, before staining the slide, make sure that the lm is
dry.
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Medical Laboratory Technology: Volume 1
Common causes of a poor blood smear (Figure 10.14)
The glass slide must be scrupulously cleaned. Dirty slides do not give an even smear. Use
an appropriate size of blood drop. Make the smear immediately after puing the drop of
blood on the slide; a delay causes uneven distribution of white cells on the lm. The spreader
slide must be moved steadily and condently; jerky movement of the spreader slide and loss
of contact between the spreader slide and the smear slide yields poor smears. The angle of
holding the spreader slide determines the thickness of the lm. Increasing the angle above 25°
yields a thicker smear, whereas a smaller angle results in a thin smear. The spreader slide must
be pushed completely across the smear slide in order to have an even distribution of the smear.
Preparation of blood smear: (a) Place a clean slide on a table; transfer a drop of antico-
agulated blood near the edge of the longer side, (b) Place the spreader slide at an angle
of 30 to 35°, (c) Pull back the spreader slide until it touches the drop of blood, and (d)
then push forward; air dry the smear Note Use of specically made spreader slide is
recommended (See Chapter 9).
Identification-marking on slides
Write the specimen identication number on the slide with any glass-marking pencil. Use
of paper labels is not recommended. Make sure that the markings do not wash away during
staining.
Routine Haematological Tests
Preparation of smear on hand
275
Comparison of (a) good smear against (b and с) bad smears
2. Fixing and staining of blood smear: It is advisable to stain the slide soon after making the
smear. Methanol (acetone free) present in the stain xes the slide in the staining process.
If staining is to be done later, the blood lm must be xed with methanol for 2–3 min.
Fixation prevents distortion of cells and smears can be kept for a long time and stained when
convenient. Staining with Leishman stain is described here (Figure 10.15). Use of other stains
will be described in the following pages.
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Medical Laboratory Technology: Volume 1
• Place slides on the staining rack with the blood smear (dull side) facing up. If the
staining rack is not available, place two glass rods over a sink to hold the slide.
• Fix the thin blood lm with methanol for 2–3 min. This may not be necessary if the
staining can be done immediately.
• Staining with Leishman stain requires a lile experimentation. Dierences exist
between various lots of the stain as well as with the length of storage of the stain. Thus,
the length of time for which the stain is to be left on the slide has to be standardized
when a new batch of stain is received or made in the laboratory.
Method 1: Using undiluted leishman stain
1. Cover the slide with undiluted Leishman’s stain for 2 min (this also allows xation of
the smear).
2. Add on the slide buered water of about double the volume of stain. Allow the staining
to continue for 5–7 min. A metallic sheen should form on top of this mixture. Staining
time may have to be adjusted according to the reaction of the stain. Reduce the time if
overstained; increase the time if poorly stained.
Staining of blood smear with Leisman stain: (a) Take a drop of blood on the microscope
slide, (b) Prepare the smear, and (c) Air dry, (d and e) Add stain followed by buer, (f)
Mix the buer and stain by blowing, (g) Wash with distilled water and air dry
Routine Haematological Tests
3. Wash the stain o in a stream of buered water until it has acquired a pinkish tinge.
Do not tip the stain o; this leaves a deposit of stain on the blood lm and hampers
microscopic examination.
Method 2: Using diluted leishman stain
1. Fix the smear with methanol (2–3 min).
2. Cover the smear with diluted Leishman stain for 7–10 min.
3. Wash the stain o with buered water. Do not tip the stain o.
4. Finally add fresh-buered water for 2–3 min to dierentiate the lm. The time taken
for dierentiation depends on the stain and the pH of the water used.
5. Shake o all water adhering to the slide and set the slide in an upright position in a
drying rack. Keep the smeared surfaces of the slide facing down. This avoids picking
up dust.
Additional Information
• The pH of the water is of vital importance in dierentiating the dierent types of
leukocyte with Leishman stain. It should be between 6.8 and 7.2 and preferably
between 7.0 and 7.2.
• Care is required to avoid formation of deposits of stain, which appear on the lm as
masses of lile black dots.
• Always use clean glassware. Do not use acid in cleaning. Remove stain deposits with
methanol.
• Always use neutral water for washing (buered, if possible). Acid water produces a
lm that is too red and alkaline water results in one that is too blue. Neutral water
must be freshly prepared, as it becomes acid when exposed to air.
277
Differential Count
Dierential count is the percent distribution of various white cells in the peripheral blood
as determined from a blood smear stained with a polychromatic stain (e.g., Leishman stain).
Various types of white cells found in circulation cannot be recognized in the wet mount while
performing white cell count by haemocytometry. In addition to the relative WBC count,
microscopic study of a blood smear also helps to get an overall picture of the blood.
Complete blood count (CBC) includes six parameters of red cells (RBC, Hb, Hct, MCV,
MCH and MCHC), total leukocyte count (TLC) and the dierential count (often abbreviated as ‘di’). Dierential count is done with a stained blood smear under the microscope,
using the oil-immersion objective (total magnication 1000×). The blood smear used for the
dierential count is also used for reporting abnormalities in the RBC morphology and in the
estimation of the platelet population. As the blood picture is related to haematological ndings, the dierential count of the CBC should be done at the end. In this way, examination of
the smear can double-check the reports of cell population and content of RBC along with its
morphological variations.
Clinical significance
Dierential count is vital for the diagnosis of a number of blood-related disorders involving
either red cells or white cells. Its primary use is to identify changes in the distribution of
white cells, which may be related to specic types of disorders including infection (bacterial,
viral or parasitic) or leukaemias (myelogenous, lymphocytic, monocytic, etc.). Clinical terms
like the rise in specic white cells (neutrophilia, eosinophilia, lymphocytosis, monocytosis)
or their fall (neutropenia, eosinopenia, etc.) are based on the dierential count. On the other
hand, the terms leukocytosis or leukopoenia are based on the total leukocyte count (TLC). In
addition, the blood smear study reveals morphological abnormalities in blood cells, as well