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Medical Laboratory Technology: Volume 1
1. Collect the blood in a clean test tube without anticoagulant.
2. Allow the blood to clot at room temperature for an hour.
3. Separate the serum after an hour with the help of a Pasteur pipee.
PreParatiOn Of BlOOd films
Blood lms can be prepared immediately after collection or up to 1 h later if
ethylenediaminetetraacetic acid (EDTA)-blood is used and stored at 20°C (Figure 9.3).
Figure 9.3 Preparation of blood smear: (a) Making a spreader slide, (b) Cleaning the slide before
making smear, (c) Making blood smear (good smear and bad smear), (d) Making smear
from nger stick
Basic Laboratory Procedures in Haematology
Two types of permanent blood lms are used. Each lm provides dierent kinds of
information. The thick lm (Figure 9.4b) is used to increase the sensitivity of the test for
detecting parasites in the blood, while the morphology of blood cells and microbial agents
are best studied using a thin lm (Figure 9.4a).
Figure 9.4 Making (a) thin blood lm, and (b) thick blood lm
To ensure accurate examination of blood lms, clean grease-free slides must be used. Old
slides should rst be cleaned in detergent and washed with 70% alcohol. New slides should
be cleaned with alcohol before use.
Supplies
Coon balls, ethanol or isopropanol (70%), sterile lancet, cover slips for wet mount, normal
saline for wet mount, microscopic slides (clean, grease-free and scratch-free), slides with
perfectly smooth edges for spreaders (for thin lm only).
239
Procedure for fresh wet lm
1. Disinfect the tip of the third or fourth nger with alcohol and let the skin dry completely.
Prick the lateral side of the ball with the lancet, not too close to the nail bed.
2. Place a drop of blood, the size of a pinhead, on the centre of a slide. Be careful not to
touch the skin.
3. Invert the slide and add a small drop of saline. Mix the blood and saline with the
corner of a cover slip. Cover the preparation with the cover slip. Press the slide with a
thin stick or a match so that the centre of the blood lm is almost colourless. Examine
immediately.
Some microbial agents (Borrelia, trypanosomes and microlarieae) can be detected
(Figure 9.5b) in a fresh wet blood lm by their motility; however, for species identication a
permanent preparation may be necessary. Trypanosomes lyse and disappear within hours,
and therefore lms for this organism must be prepared and stained immediately.
Procedure for Preparing Thick Blood Film
1. Grasp the slide by its edge and from below bring the end of the slide into contact with a drop
of blood (Figure 9.3d). Be careful not to let the slide come into contact with the skin.
2. Invert the slide and place it on a at surface and hold it with the index nger and the
thumb of the left hand (Figure 9.4).
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Medical Laboratory Technology: Volume 1
3. Spread the blood evenly in a lm about 10 mm wide with the corner of the slide, or a
needle or a toothpick, or by slowly rotating the slide (Figure 9.5). The lm should be
spread quickly and have the correct thickness (one should be able to see hands of a
wristwatch but not gures through the smear).
4. Allow the lm to dry (on a sunny bench, under a lamp, an electric fan or in an incubator,
37°C), protected from dust and ies. The smear must be completely dry to make the
blood lm adhere to the slides. In humid climates, prolonged drying may be necessary.
A thick lm should not be xed.
Figure 9.5 In wet preparation, (a) Mix the drop of blood with saline and examine under low power of
the microscope, (b) Movement of trypanosomes may be seen
Procedure for Preparing Thin Blood Film
1. Make a spreader slide: Select a slide with perfectly smooth edges and make a diagonal
scratch across the two corners at one end with a le and snap o the two corners with a
pair of pliers (Figure 9.3a).
2. Take a drop of EDTA-anticoagulated blood of about 4 mm diameter to one end of the
slide. You may use a dipstick to transfer the drop of blood (Figure 9.3).
3. If the blood specimen is obtained from a nger stick (Figure 9.3d), grab the slide by its
edge and from below bring the end of the slide into contact with a small drop of blood,
being careful not to let the slide come into contact with the skin.
4. Invert the slide, place it on a at surface and steady it with the index nger and thumb
of the left hand (Figure 9.3c).
5. Place the end of the spreader at an angle of 45° on the slide slanting towards the drop
of blood. Draw the spreader back until it touches the drop of blood and wait until the
blood has spread along the entire edge of the spreader.
6. With a rm, fast motion, push the spreader along the slide maintaining an angle of 45°
(Figure 9.4). If the original drop of blood is small enough, the blood is drawn after the
spreader in a thin smear which ends in a drawn-out tail well before reaching the end of
the rst slide. A comparison of a good smear and bad smear is illustrated (Figure 9.3c).
7. Wave the slide so it dries quickly. In humid climate, the drying of the lm can be
speeded up by waving the slide 5 cm away from the ame of a spirit lamp or Bunsen
burner (Figure 9.6).
8. With a lead pencil, mark the thick part of the lm with the patient’s name or number.
Basic Laboratory Procedures in Haematology
Figure 9.6 The blood smear is xed (or dried) on a ame using a (a) spirit lamp, or (b) Bunsen beerner
Common mistakes in preparing thin blood lms
• If the end of the lm is lost—the drop of blood was too big.
• If the lm ends in a thick line—the spreader was lifted up too early.
• If the end of the lm is ragged—the edge of the spreader was uneven.
• If there are lines along the lm—blood was cloing.
• If there are lines across the lm—the spreader was pushed forward jerkily.
• If there are holes in the lm—greasy slide.
241
Cleaning Of laBOratOry glassware in HaematOlOgy
Glassware for haematological work must be free from detergent. Presence of even minute
concentrations of detergent will lyse the red cells. Hence clean the glassware as described
earlier in Chapter 4 and make sure that the nal wash with tap water and rinsing with
deionized or distilled water are done properly.
review QuestiOns
1. What is the most commonly used anticoagulant for haematological studies? Rationalize
your answer.
2. What is a buy coat? Which one of the formed elements of blood will be predominant
in the buy coat?
3. When would you recommend skin puncture in place of venepuncture?
4. How would you clean up microscope slides and cover slips? How would you store the
cover slips?
5. What is a spreader slide? How is it made? What is its role in making the blood smear?
6. At the time of making a blood smear from nger stick, you are instructed not to touch
the skin. Why?
7. Name some of the reasons for geing bad smears.
8. Which diseases are transmied through contact with body uids?
9. Would you x a wet mount? How do you make a wet mount?
Routine Haematological Tests
Anuradha Chakravarthy and Volkmar Dierolf
Chapter Outline
• Determination of Haemoglobin Concentration
▪ Cyanmethaemoglobin method
▪ Alkaline haematin D method
• Determination of Haematocrit
▪ Macrohaematocrit (wintrobe) method
▪ Microhaematocrit (capillary tube) method
• Red Blood Cell Indices
▪ Mean Cell Volume (MCV)
▪ Mean Cell Haemoglobin (MCH)
▪ Mean Cellular (corpuscular) Haemoglobin Concentration (MCHC)
• Interpretation of Abnormal Findings
• Erythrocyte Sedimentation Rate (ESR)
▪ Westergren method
▪ Wintrobe method
▪ Other methods
• Enumeration of Formed Elements
▪ Total leukocyte count by haemocytometry
• Microscopic Study of Blood Smear
▪ Dierential count
▪ Study of abnormal cell morphology
▪ Estimation of platelet count
▪ Other stains for blood smears
• Automated Systems in Haematology
▪ Electrical impedance cell counting
▪ Light-scaer cell counting
▪ Current progress in automation
• Reticulocyte Count
• Absolute Platelet Count
▪ Estimation of platelet count from stained blood smear
• Review Questions
10
Routine Haematological Tests
243
Determination of Haemoglobin ConCentration
Haemoglobin (Hb) is the red pigment contained in erythrocytes. It consists of protein
chains and ironcontaining molecules. Various methods were used in the past to determine
haemoglobin of which the cyanmethaemoglobin method is most reliable and widely used.
Specic gravity method is used by the blood bank before collecting blood in the eld.
This is described in Chapter 17 of the book. Methods that use discrete auto analyzers
are described with the automated system. Here we will focus on cyanmethaemoglobin
or hemiglobinocyanide method. Another method that will be described is the alkaline
haematin method.
Clinical significance
A decrease in Hb concentration in blood below normal values is a sign of anaemia. The Hb
concentration is lower in adult women as compared to adult males. Haemoglobin values
further drop during pregnancy due to haemodilution (a proportionately greater increase in
plasma volume as compared to the increase in red cell mass). Children also have values
lower than those seen in adult males. An increase in Hb concentration can occur due to
haemoconcentration (loss of body uid, e.g., in severe diarrhoea, vomiting), reduced oxygen
supply (e.g., congenital heart disease, emphysema) and in a rare condition, polycythaemia
vera. A decrease or increase in Hb concentration must be reported, as it is a sign of disease,
requiring further investigations.
Normal values
Haemoglobin concentration is commonly reported in g/dL.
Men: 14–17 g/dL; Women: 13–15 g/dL
Specimen
EDTA-anticoagulated venous blood is commonly used but the use of other anticoagulants,
for example, heparin, double oxalate, also yields the same result. Capillary blood can be used
directly.
Cyanmethaemoglobin Method
This is one of the oldest methods of Hb determination, Its reagents, however, should be
carefully handled, as they are highly toxic.
Principle
Cyanmethaemoglobin method is a photometric procedure for determining Hb concentration.
An aliquot of well-mixed whole blood is taken and reacted with a solution of potassium
cyanide and potassium ferricyanide (called Drabkin’s solution). The chemical reaction yields
a product of stable colour—cyanmethaemoglobin. The intensity of the colour is proportional
to the Hb concentration and obeys Beer’s Law (Chapter 31).
Equipment and supplies
• Photometer with 540 nm lter
• Cuvees
• Blood pipee (Sahli) of 0.2 mL (20 μL) capacity
• Automatic dispenser (5 mL)
• Surgical gauze, test tubes
• Test tube rack and other glassware
Reagents
Drabkin reagent (Poison): This is commonly known as Drabkin diluting uid (From Manual
Basic Techniques for Health Laboratories, 2nd Ed., WHO, 2003).
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Medical Laboratory Technology: Volume 1
Drabkin solution can be prepared from commercially available reagent tablets. The
manufacturer supplies instructions for its preparation. For laboratories equipped with an
accurate balance, it can be prepared as follows:
Potassium cyanide (KCN) 100 mg
Potassium ferricyanide (K3FeCN6) 400 mg
Potassium dihydrogen phosphate (KH2PO4) 280 mg
Distilled water (q.s.) 2000 mL
Note q.s. = quantum sucit, or make it to the volume after adding the ingredients
Dissolve the rst three chemicals in distilled water and mix. Add the detergent and mix
gently. The reagent should be clear and pale yellow in colour. When measured against water
as blank in a colorimeter/spectrophotometer, at a wavelength of 540 nm, the absorbance
should be zero. Transfer the uid to a brown bole. Label the bole ‘Drabkin Diluting
Fluid’ (POISON) with the date. It is stable for several months. Do not freeze, as this can
result in decolourization with reduction of the ferricyanide. If the reagent appears cloudy,
discard.
Warning Potassium cyanide is a highly poisonous chemical and should be used only by
experienced chemists. When not in use, it should be kept in a locked cupboard. After using
the chemical, wash your hands thoroughly. Never discard Drabkin’s solution in the sink
without washing it out thoroughly. If the sink has an acid solution, the poisonous gas HCN
(cyanide) will be released by acidication. Flush the sink with ample water and then discard
Drabkin’s solution in the sink and continue to ush water for some time.
Cyanmethaemoglobin standard
• Cyanmethaemoglobin is used to calibrate the instrument.
• Cyanmethaemoglobin standard is commercially available or may be obtained from a
reference laboratory.
• A reference solution previously calibrated against the cyanmethaemoglobin standard
can also be used.
• A blood sample of known Hb concentration, which is subjected to the cyanmethaemo-
globin procedure, is also used as a reference solution.
A calibration curve must be prepared before the colorimeter/spectrophotometer which can
be used for Hb estimation. From such a curve, a graph can be prepared and a table made for
the Hb values. This is described later in this section.
Procedure (Figures 10.1 and 10.2)
1. Label the test tubes (15-mL) as В (blank) and Τ (test).
Note Increase the number of ‘T-tubes’ (T1, T2 etc.) if there are more than one specimen.
2. Pipee 5 mL of Drabkin’s solution into the marked test tubes. Correct volume is not
important in case of the blank (B). Hence, while seing the automated dispenser
(recommended), use the blank for removing air bubbles.
Warning Drabkin’s solution is highly poisonous. Do not pipee by mouth.
3. Mix the blood specimen by gentle inversion or swirling.
4. Open the rubber cap and place it on the bench in the inverted position (the surface of the
cap with blood should face upwards).
5. Draw an aliquot (20 μL) of well-mixed EDTA-anticoagulated blood specimen into the
blood pipee. Use the automated pipeing device. Do not use your mouth. Carefully
bring the level of blood to the graduation mark. Try not to cross the graduation mark as
Routine Haematological Tests
Absorbance of thhe test solution
Absorbance of the standard
Absorbance
245
blood will be left on the wall when brought back to the graduation mark. This adds to
the volume error.
6. Wipe the outside of the pipee with wet gauze while holding the pipee in the
horizontal position. If the outside of the pipee is not properly cleaned, the result may
show a falsely high value. After cleaning the pipee, check that the blood is still up to
the mark and there are no air bubbles.
7. Blow the blood sample into the Drabkin’s solution in the tube marked ‘T’. Rinse the
pipee 3–5 times with the Drabkin’s solution (diluting uid), by drawing up and
blowing out the uid into the tube of the pipee. Make sure that the blood sample is
completely washed out and inside of the pipee is clean.
8. Mix the contents of the tube by tapping (or vortex) and wait for 10 min.
9. Turn on the colorimeter and allow it to warm up for 10 min; set the wavelength selector
to 540 nm or use a suitable lter in this range. Zero the instrument without any cuvee
using the rst control knob.
(Note No light passes through when the cuvee is not placed in the instrument
illustrated). The colorimeter reading should be 0% Τ or ‘∞’ (sign of innity) absorbance
which is close to the value of 2.
10. Transfer both the solutions, i.e., blank (B) and test (T), to two matched cuvees. The
matched cuvees should give the same absorbance reading when lled with water.
11. Re-check the ‘instrument zero’ as in Step 9. Insert the cuvee with blank solution (B);
the needle of the colorimeter will swing to the opposite side. Adjust the needle to 100%
Τ or 0 absorbance with the help of second control knob.
Note 0% Τ represents no light received by the detector and 100% Τ represents full
light passing through the solution placed in the cuvee and received by the detector.
12. After seing the instrument at 0% Τ and 100% T, you are ready to take the absorbance
reading of the test solution.
13. Insert the cuvee with test solution (T) into the socket of the photoelectric colorimeter
and record the absorbance reading. Do not touch any adjustment knob while taking
the absorbance reading of the test solution. If the instrument is giving only %T reading,
convert it to the corresponding absorbance reading. If there is more than one specimen,
continue taking readings of other unknowns in the same way, replacing the test
solution (T) in the second cuvee. Leave the cuvee with the blank for re-checking.
During re-checking, use appropriate control knobs to reset 0%T and 100%T (Step 11).
Note The test specimens should be placed back in their original test tubes so that, if
necessary, they are available for a re-check.
14. Refer to the calibration curve to nd out the concentration of the unknown. Alternatively,
this can be simplied by taking the absorbance of the standard equivalent to 15 g/dL
and applying the following formula:
Hb concentration of test solution (g/dL) =
Note In this formula, concentration of the standard (15 g/L) and its absorbance
remain constant. Hence, a factor can be calculated as follows:
15
Factor=
This formula can then be rewrien as:
Hb concentration (g/dL) = Absorbance of test solution × Factor
× 15
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Medical Laboratory Technology: Volume 1
(Contd.)
Routine Haematological Tests
247
Determination of haemoglobin: (a) Use the Sahli pipette for taking whole blood specimen,
(b) Draw well-mixed blood into the pipette, (c) Wipe o the tip, (d) Dispense the blood
specimen into a tube with 5 mL of Drabkin's solution, or cyanide solution, (e) Wash out
the inside of the pipette repeatedly, (e) Mix and (f) Wait for 5 min, (g) Take the solution for
absorbance, (h) Set the 0% transmittance (without cuvette) with the help of a left control
knob, (i) Insert the cuvette with blank solution (B) and set the 100% transmittance with
the control knob on the right; nally, insert the standard solution (S) and test solutions
(T1, T2, etc.) and read their respective absorbances on the scale
Determining haemoglobin from calibration curve (Figure 10.2)
Step 1: Preparation of standard solutions of haemoglobin
A. From commercially available haemoglobin stock solution: Prepare dilutions of the stock
solution according to the directions from the supplier. You can also make your own dilutions
of the stock solution in order to obtain a range of standards.
Suppose you have received a reference stock solution, equivalent to 15 g/dL. You can make
a series of standards according to the following table.