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248
Medical Laboratory Technology: Volume 1
Volume of standard—
15 g/dL (mL)
Volume of diluent (mL)
Drabkin’s solution
Total volume of mixture (mL) 5 5 5 5 5
% Standard in whole solution 0 40 60 80 100
Label on tube Blank S-l S-2 S-3 S-4
Final concentration of standard solutions (g/dL) 0 6 9 12 15
0 2 3 4 5
5 3 2 1 0
Now we have a blank (0) and four standards: 0 (Blank), 6, 9, 12 and 15 g/dL
These are referred as B, S-l, S-2, S-3 and S-4, respectively as shown in Table 10.1.
B. From reference blood specimen: If the reference laboratory supplies you with a single
blood specimen with known concentration of Hb (e.g., 15 g/dL), proceed as follows in order
to obtain dierent levels of standards:
1. Take four test tubes and label them—S-l, S-2, S-3 and S-4.
2. Pour varying amounts of Drabkin’s solution into the labelled tubes in the following
order, respectively: 12.5, 8.30, 6.25 and 5.0 mL.
3. To each add 20 μL of well-mixed reference blood specimen.
4. These respectively represent the same concentration as the above dilutions.
Labels on tube Blank S–l S–2 S–3 S–4
Concentration of standard (g/dL) 0 6 9 12 15
Step 2: Determine the absorbance reading for each and put in a tabular form as shown in
Table 10.1.
Tabulation of haemoglobin standards and their corresponding absorbance values
Serial No. Reference Concentration of standard (g/dL) Absorbance
1 Blank 0 0
2 S-l 6 0.18
3 S-2 9 0.27
4 S-3 12 0.36
5 S-4 15 0.45
6 Test ? 0.29
Step 3: Using a linear graph paper prepare the calibration curve (Figure 10.2).
Routine Haematological Tests
Calibration curve for haemoglobin determination
At the beginning of each day aend to the following:
• Clean the matched cuvees of the colorimeter/spectrophotometer.
• Fill one of the cleaned tubes with fresh Drabkin diluting uid (blank), which is used to
zero the colorimeter/spectrophotometer.
• Read a reference blood specimen (see above).
• If the value is beyond ±5%, check the standard curve and repeat the reference blood
specimen.
249
Alkaline Haematin D Method
The alkaline haematin D method oers several advantages over the haemoglobin cyanide
method.
• It is accurate and less expensive.
• The calibration procedure uses chlorhaemin, a stable crystalline compound that is
commercially available.
• The AHD reagent does not include potassium cyanide, which is highly toxic.
• All reagents are locally available.
Principle
When blood is mixed with an alkaline solution containing a non-ionic detergent, the
haemoglobin is converted to alkaline haematin, which is a stable coloured compound. The
absorbance of the alkaline haematin is measured using a haemoglobinometer or colorimeter.
The haemoglobinometer directly determines the Hb concentration of the blood sample,
whereas with a colorimeter/spectrophotometer, the Hb concentration of the blood sample is
obtained from the absorbance using a prepared calibration curve or table of values.
Equipment and supplies
• Spectrophotometer, haemoglobinometer or colorimeter.
• Test tubes, test tube racks, corks or rubber stoppers, cuvees, grease pencil, coon
wool or gauze.
• Alkaline haematin D standard (AHD), with known concentration of Hb. This can
be obtained commercially or from reference laboratories. Follow manufacturer’s
instructions to prepare the standard (16 g/dL).
Reagents
1. Alkaline haematin D (AHD) standard. This is commercially available or supplied by
the central laboratory.
2. Alkaline haematin D reagent
Sodium hydroxide (NaOH) 4 g
Triton X-100 (or equivalent) 25 g
Distilled water 1000 mL
(Use ltered rainwater if distilled water is not available)
Dissolve the sodium hydroxide in distilled water in a clean conical ask. Stir using a
glass rod until crystals have completely dissolved. Add the Triton X-100 (or equivalent) and
mix well. Filter the solution into a clean glass-stopper reagent bole using Whatman No.
1 (or equivalent) lter paper. Label the bole and write the date. Store the reagent at room
temperature. Alkaline haematin D (AHD) reagent can be kept for several months at room
temperature (20–25°C) and for 8 months in the refrigerator (4–8°C). If a precipitate forms
during storage, the reagent should be ltered before use.
Reagent quality control check
It is important to check the quality of the newly made AHD reagent. An alkaline haematin
D standard solution (AHD standard) is available commercially or may be obtained from the
reference laboratory for checking the quality of the new batch of AHD.
Procedure
1. Fill a clean cuvee with distilled water. Place the cuvee in the cuvee chamber and
adjust the haemoglobinometer or colorimeter to read zero at 540 nm wavelength.
2. Replace the distilled water with the newly made AHD reagent. The haemoglobinometer
or colorimeter should read zero. If not, make a note of the absorbance value.
3. Pipee 20 μL of AHD standard into a test tube containing 3 mL of the freshly prepared
AHD reagent (1:150 dilutions). Usually the standard is equivalent to 16 g/dL Hb (or,
160 g/L, SIU). Follow manufacturer’s instructions.
Note SIU stands for Systemic International unit, which in French is known as
Système International d’Unités.
4. Note the absorbance reading of the standard in the new AHD solution and compare
this with the old solution, treated in the same manner. The dierence in the absorbance
reading should be within ±5%.
5. If the dierence is more than ±5%, discard the freshly prepared AHD reagent and
prepare a new batch, paying aention to accurate measurement of the constituents
and the cleanliness of the glassware.
Medical Laboratory Technology: Volume 1
Calibration of the haemoglobinometer
Haemoglobinometers comes under the general category of ‘Photometer’ where the energy
of light is used in the analytical process. Haemoglobinometers have a xed wavelength of
light and the instrument cannot be used for other photometric determinations. It is relatively
simple to use and is cheaper than spectrophotometers. The following steps are recommended
for the calibration of a haemoglobinometer:
1. The manufacturer provides the method of diluting the concentrated AHD standard
in order to make the ‘Reference standards’. Follow manufacturer’s instructions.
The following method is assuming that, after dilution, it yields 16 g/dL as the AHD
standard for reference (The method is taken from WHO Manual, 2007 with slight
modications).
Routine Haematological Tests
Absorbance of standard
2. Pipee 0.2 mL of the AHD primary stock standard in a 100-mL conical ask containing
30 mL of AHD reagent. Mix well (standard diluted 150x). The nal solution of this
diluted primary standard is equivalent to 16 g Hb/dL (or 160 g/L). This will be used as
the reference standard.
3. Take ve 10-mL test tubes and number them from 1–5. Place the test tubes in a test
tube rack.
4. Dilute the reference standard of Step 2 according to the following table. This yields
four standards and the blank for comparison.
Reagent Blank s-1 S-2 S-3 S-4 (Reference AHD)
Tube No. 1 2 3 4 5
AHD diluted reference (mL) 5 2 3 4 5
AHD diluting reagent (mL) 0 3 2 1 0
Total volume (mL) 5 5 5 5 5
Equivalent haemoglobin concentration 0 6.4 9.6 12.8 16.0
5. After dilution, stopper the test tubes using a clean cork or rubber stopper and mix by
inversion. Leave the tube to stand for 2–3 min.
6. Fill a clean cuvee with the undiluted AHD reagent (1, blank). Dry the outside of
the cuvee with coon wool or gauze and place it in the cuvee chamber. Adjust the
haemoglobinometer to read zero, the reading of the blank.
7. Replace the undiluted AHD reagent in the cuvee with the diluted AHD standards
tubes 2–5 in increasing order and take the absorbance reading of each.
Note If you are using a single cuvee for measuring absorbances of dierent
standards, it is a good practice to start with the lowest concentration rst in order to
avoid ‘carry over’ eects.
8. Prepare a calibration curve following the same instructions as given under
cyanmethaemoglobin method.
Note
• A standard curve should be linear.
• Prepare a standard every time you make a new reagent solution.
• It is a good practice to make a standard curve every day morning so that results are
reliable.
Determining haemoglobin concentration by formula
1. Set as many test tubes as there are number of test specimens (T1, T2, etc.)
2. Add 3 mL of AHD reagent in each.
3. Add 20 µL of the blood specimens in each T-marked test tube. Be careful not to mix-
up the patient’s accession number with the number on the test tube. Follow the same
sequence as recorded in the register.
4. Close the test tube, mix by inversion and wait for 2–3 min.
5. Take the absorbance readings of each test solution (TI, T2, etc.)
6. You can read the absorbance of the reference standard (Tube 5), for comparison, from
time to time.
7. Calculate the Hb concentration by the following formula.
Note The formula is applicable only when the calibration curve is linear.
Concentration of in test specimen =
Hb
Absorbance of test specim
een
Concentration of standard×
252
9. Carefully tally with the specimen number and then record results in your laboratory
register.
Sources of error in haemoglobin estimation
• Error in blood collection and mixing with the anticoagulant.
• Volume measurement of sample and reagents.
• Trapped air bubbles in volumetric equipment and in cuvees.
• Use of dirty or defective equipment or glassware.
• Unacceptable reagent quality.
• Defective technique—volume measurement, mixing, inappropriate use of equip-
ment—wavelengths, cuvee orientation, scale adjustments etc.
Note Check the operation of the equipment everyday.
Medical Laboratory Technology: Volume 1
Determination of HaematoCrit
Haematocrit (Hct) or packed cell volume (PCV) is the amount of packed red blood cells
(RBCs), following centrifugation, expressed as a percentage of the total blood volume. It can
also be expressed as a fraction of one (proposed International Unit) or as percent of whole
blood (%). The numerical gures remain the same except that the decimal is shifted two digits
to the right in order to express Out of 100’ to convert it in percentage.
Two methods are available for determining Hct – macrohaematocrit and microhaematocrit
method. The microhaematocrit method needs smaller amounts of blood and takes less time
to report. Hence the microhaematocrit method has largely replaced the macrohaematocrit
method. It, however, requires disposable capillaries which is not always available in peripheral laboratories of developing countries.
Clinical significance
Haematocrit is an index of the red cell population. As in the case of Hb concentration, a
decrease in Hct value is a measurement for detection of anaemia (Figure 10.3). Erythrocyte
volume fraction, Hct or packed cell volume, convey the same idea, that is, reporting the red
cell volume in whole blood after centrifugation.
Relation between haematocrit value (%) and haemoglobin concentration (g/dL)
Routine Haematological Tests
Normally there is a linear relationship between Hb concentration, number of red cells in
circulation and the erythrocyte volume fraction. But the relation may be altered in certain
pathologic states. A decrease in Hct may also result from hydraemia (excessive uid in the
blood as occurs in pregnancy). An increase in Hct value indicates an increase in red cell pro-
duction, which may occur as a result of a decrease of oxygen supply (congenital heart disease,
emphysema), an abnormality in red cells (polycythaemia) or from dehydration. Some laboratories express percent values as a fraction of 1 (in line with the IU of expression–SI unit).
To convert percent to a fraction of 1, divide the value by 100 or shift the decimal two digits to
the left (for example, 47% = 0.47). The value of Hct is used, along with Hb concentration and
red cell count, for the calculation of mean cell volume (MCV), mean cell haemoglobin (MCH)
and mean corpuscular haemoglobin concentration (MCHC), the three important RBC indices
used in the diagnosis of various types of anaemia.
Note Some additional information may be available following Hct determination. If the
buy coat is thick, an increase in white cell count may be suspected. This occurs in case of
leukaemia.
Normal values
Traditional expression of Hct value is in percent (%). The SI unit is in fraction, that is, out of
1, is given in parenthesis. The gures given here are taken from WHO publications and may
vary by race.
Adult Male: 40–50% (0.40–0.50)
Adult Female: 37–43% (0.37–0.43)
Newborn infant: 50–58% (0.50–0.58)
Infant (3 months): 35–40% (0.35–0.40)
Children (5 years): 38–44% (0.38–0.44)
Principle
Erythrocytes are heavier than other cellular components of blood (white cells and platelets).
Hence, when whole blood is centrifuged, erythrocytes sele down to the boom of the
centrifuge tube. This seled red cell column is called haematocrit or packed red cell volume
(PCV), which is expressed as fraction of the whole blood (level of plasma). Two methods
are applied for the determination of haematocrit—macrohaematocrit and microhaematocrit
method.
Specimen
Only anticoagulated blood is used in determining haematocrit. Ethylenediamine-tetraacetate
(EDTA) is most commonly used as an anticoagulant. In case of capillary blood, obtained by
skin puncture, heparinized capillary tubes are used.
253
Macrohaematocrit (Wintrobe) Method
A larger volume of blood is needed in this procedure and hence, only venous blood can be
used (Figure 10.4). This method can give values of both tests—ESR and haematocrit. First the
ESR reading is taken for one hour (described in detail later) followed by spinning which yields
the Hct value. Thus reporting cannot be done before one hour. The tubes, unlike capillaries,
are reusable after proper washing.
Principle
Anticoagulated blood is taken in a Wintrobe tube, lled to the graduation mark and then
spun for the desired length of time. The volume of packed cells is read directly from the
graduation mark on the Wintrobe tube.
254
Medical Laboratory Technology: Volume 1
Equipment
• Wintrobe haematocrit tube: The Wintrobe tube is a 110 mm long narrow test tube (Fig-
ure 10.4) with a 3 mm internal bore, graduated from 0 to 100 mm with the graduations
both in ascending and descending order on the two sides of the graduation. Thus at the
top, 0 and 10 cm (100 mm) coincide. The scale with markings in ascending order from
the top is used in ESR determination, while the scale with descending order is used for
Hct determination. It holds about 1 mL of whole blood.
Determination of haematocrit and ESR (erythrocyte sedimentation rate) by Wintrobe
macro-method: (a) Fill the Wintrobe tube, (b) with the help of a syringe, (c) While lling
the tube, keep it slanted and ll from the bottom, (d) While the tube is being lled, pull out
the syringe slowly; if you have to report for the erythrocyte sedimentation rate (ESR), put
the tube in a special stand (Figure 10.9) and read the fall of RBC column after one hour;
this reads 0.8 mm in the insert, (e) After taking the ESR reading, centrifuge the Wintrobe
tube, (f) and take the reading of the haematocrit. Details of ESR measurement will be
presented later.
• Transfer pipee: A long (22 cm), ne capillary Pasteur pipee or special syringe sup-
plied by the manufacturer to ll the Wintrobe tube with the blood specimen.
• Centrifuge: A centrifuge with an arrangement on the head to hold Wintrobe tubes. Some
manufacturers provide special soft-cushion-holders for the Wintrobe tube along with the
centrifuge. The laboratory can also make such holders from foam. The centrifuge should
be capable of producing a force of 2300 G. Calculate the speed (rpm) for your centrifuge in
order to meet the specication of 2300 G. A force of less than 2300 G gives a false high Hct
reading, and conversely, excessive force may lead to falsely low values. Increased time
or decreased time of centrifugation gives false-low or false-high values, respectively, for
haematocrit. The centrifuge can be standardized for speed and time by taking a reference
blood sample and determining the time and speed necessary to obtain the reference value.
Routine Haematological Tests
Procedure (Figure 10.4)
1. Carefully mix the blood specimen by repeated inversion.
2. Label the required number of Wintrobe tubes that corresponds to the specimens. Fill
the Wintrobe tube with the help of the Pasteur pipee (or syringe supplied by the
manufacturer) to the 10 cm mark which represents 100%. If the level of blood crosses
the mark, note the error from the extra divisions given at the top.
3. Filling of the Wintrobe tube requires special care in order to avoid trapping of an air
bubble and mechanical damage to the erythrocytes. To ensure this, place the tip of
the pipee at the boom of the Wintrobe tube and ll from the boom, gradually
withdrawing the pipee as the blood goes in. Try to keep the tip of the pipee under
the rising column of blood to avoid foaming.
4. Fill a second tube in the same way with the blood specimen, which gives a second
reading and the tube will be able to balance the centrifuge. If the amount of specimen is
limited, use water to ll the second Wintrobe tube with which to balance the centrifuge.
5. Place the Wintrobe tubes in the opposite cups of the centrifuge. Check the rubber
cushions at the boom of the centrifuge cups before inserting the Wintrobe tubes.
6. Turn the centrifuge on to slow speed, increase the speed gradually and nally bring up
to the required speed.
7. Centrifuge for 30 min at 3000 rpm for the bucket head centrifuge. Use the alarm clock
to time the process if the centrifuge is not provided with a timer.
8. After 30 min switch o the centrifuge and allow it to stop by itself. Do not use the brake.
9. Take out the Wintrobe tubes after the centrifuge has stopped and read the PCV directly
o the graduation given on the tube. For example, if the red cell column is two divisions
above the graduation mark of 4, the reading is 42, or the Hct is 42%. If the reading is to
be expressed in ‘erythrocyte volume fraction’, it will be 0.42.
Note Do not include the buy coat while reading the height of the red cell column. The
buy coat is the thin greyish-white layer of white cells at the top of the red cell column.
255
Microhaematocrit (Capillary Tube) Method
This method requires only a small volume of specimen and hence it is ideal for skin puncture
(Figure 10.5) and for a limited amount of specimen (paediatric patient, buins patient). It
requires disposable capillary tubes, a special centrifuge and a reading device. The chances of
error are high in case the reading is not taken properly. There are also a number of advantages
with this method, which make it most popular in all advanced laboratories. It takes less time,
capillaries are easy to ll, replicates are easily obtainable, and a large number of specimens
can be handled simultaneously.
Principle
Anticoagulated blood is centrifuged in a sealed capillary tube and the volume of packed
red cells and percentage of the whole blood (level of plasma) are determined by a special
haematocrit reader. In case of anticoagulated venous blood obtained from venepuncture, use
plain capillary tubes which are cheaper; while for the capillary blood obtained from skin
puncture, use heparinized capillary tubes.
Equipment and supplies
1. Microhaematocrit centrifuge (Figure 10.6): This is a special centrifuge that runs at
high speed and is capable of producing relative centrifugal force (RCF) of 12000 G
and runs at a speed of about 15000 rpm. The head is at with grooves to hold the
capillary tubes and has a head cover to hold the capillary tubes inside the groove.
Never run the centrifuge without puing on the head cover and the centrifuge cover.
256
Medical Laboratory Technology: Volume 1
The centrifuge also has a timer, which is usually set at 5 min; however, the time should
be standardized against a reference blood specimen.
2. Haematocrit reader: There are several types of readers used for reading the haemato-
crit. The simplest one is the card reader which can be made by hand (Figure 10.6b).
3. Capillary haematocrit tubes: The capillary Hct tubes (or capillaries) are approximately
75 mm in length and have an internal diameter of approximately 1 mm. If anticoagulated
whole blood is used, plain capillary tubes (usually marked with a blue line at the neck)
can be employed. With the blood collected by skin puncture, heparinized tubes must be
used. The marking of a red line at the neck identies these heparinized capillary tubes.
4. Modelling clay: This is used to seal the end of the Hct tube.
Blood specimen from nger stick. Specimen from nger stick is collected in heparinized
capillaries for haematocrit determination. For cell count, transfer the blood promptly to the
diluent before it clots.
Procedure
1. Draw the blood sample into an appropriate capillary tube by capillary action (Figure 10.6).
Use a plain tube for anticoagulated whole blood and a heparinized tube for skin puncture.
In case of skin puncture, the blood should ow freely or with very lile pressure in the
area; wipe away the rst drop with gauze and then collect the blood specimen. Fill the
tube to about 3/4 length. Two capillary tubes can be lled from the same blood specimen
so that they can work as replicates and provide two readings to be averaged.
2. Seal the capillary tubes with soft wax or modelling clay plugged to a height of about
2 cm at the boom. The clay plug should form a straight edge across the boom of the
blood sample. Alternatively, seal the clean end of the tube by heating it carefully over
a spirit lamp or microburner.
3. The sealed tubes can be kept in a vertical position, keeping the sealed end at the
boom, until all tubes are sealed. Commercially available plastic modelling clay for
the sealing of Hct capillary tubes is provided with numbered grooves at the edge for
keeping the lled capillary tubes in the upright position. The grooves are in pairs for
keeping duplicates of the same specimen. It is not important that capillaries be lled
to the same volume of blood.
4. The sealed capillaries are now ready to be spanned in a special Hct centrifuge. While
placing the capillary tubes in the centrifuge, follow the sequence of increasing number
Routine Haematological Tests
257
and check carefully between the patient accession number (that provides the identity
of the patient) and the groove number or slot number chosen. Make a record of groove
number against specimen number in order to avoid confusion. This is important, as
numbers cannot be wrien on the capillary tubes. Remember A wrong report is worse
than no report.
Haematocrit determination by capillary method: (a) Fill the capillary tube with anticoag-
ulated blood from nger stick or anticoagulated blood, (b and c) Seal and centrifuge the
capillary, (d and e) Read the haematocrit value on a reader; do not include buy coat, (f)
Sealant plate with numbers on the margin to identify the capillary tubes, (g) Follow the
same number on the haematocrit centrifuge. Microhaematocrit card reader is convenient
to use for determining haematocrit value. Line up the top level of the plasma on 100%
line and the bottom of the red cell column on 0% line and then read the haematocrit from
the line separating plasma from the red blood cells
5. Always keep the sealed ends facing outwards (towards the periphery of the centrifuge
head). It is a good practice to keep the duplicate capillaries of the same sample on
opposite radial grooves of the centrifuge head. You can use empty capillaries to
balance the centrifuge head when necessary.
6. Screw in the head cover and then close the centrifuge cover.
7. Centrifuge at high speed (13000 rpm + 2000 rpm) for 5 min.
6. When the centrifuge has stopped spinning, open the centrifuge cover and then unscrew
the head cover for taking out the capillary tubes.
7. Remove the capillary tubes in sequence, place them back in the same vertical position
as in Step 2, on the clay model holder. The capillary tube shows three layers: at the top