College of Health Sciences
Department of Medical Laboratories
Second Year – Second Term
Hematology – 1
Practice NO (3)
Preparation of Haemolysates
EDTA is the most convenient anticoagulant because it is used for the
initial full blood count and film although samples taken into any
anticoagulant are satisfactory. Cells freed from clotted blood can also
be used.
Preparation of Haemolysate for the Quantification of Haemoglobins
and Stability Tests
Preparation of haemolysate for the quantification of haemoglobins and
stability tests can be used for qualitative electrophoresis and is
necessary for quantitation of Hb A2 and F or variant haemoglobins by
elution. It is also essential for reliable stability tests and globin
electrophoresis.
 Lyse 2 volumes of washed packed cells in 1 volume of distilled
water
 add 1 volume of carbon tetrachloride (CCl4).
 Alternatively, lyse by freezing and thawing, then add 2 volumes of
CCl4. Shake the tubes vigorously for approximately 1 min
 then centrifuge at 1200 g (3000 rev/min) for 30 min at 4°C.
 Transfer the supernatant to a clean sample container and adjust the
Hb to 100 ± 10 g/l with water. If an unstable Hb is suspected,
organic solvents should be avoided
Prepared By Dr Abdelrhman Elreshid 1
Practice NO (4)
QUANTITATION OF Hb F
Hb F may be estimated by several methods based on its resistance to
denaturation at alkaline pH, by HPLC, or by an immunological method.
Of the alkaline denaturation methods, that of Betke et al is reliable for
small amounts (<10–15%) of Hb F, whereas for levels of more than 50%,
and in cord blood, the method of Jonxis and Visser is preferable;
however, this method is not reliable at levels of less than 10%.
Immunological methods have been devised to measure Hb F by
immunodiffusion, for which commercial kits are available, and by
enzyme-linked immunoassay (ELISA).
Modified Betke Method for the Estimation of Hb F
Principle
To measure the percentage of Hb F in a mixture of haemoglobins,
sodium hydroxide is added to a lysate and, after a set time,
denaturation is stopped by adding saturated ammonium sulphate. The
ammonium sulphate lowers the pH and precipitates the denatured
haemoglobin. After filtration, the quantity of undenatured
(unprecipitated) haemoglobin is measured. The proportion of alkaliresistant (fetal) haemoglobin is then calculated as a percentage of the
total amount of haemoglobin present.
Equipment
Filter paper. Whatman No. 42.
Vortex mixer.
Glass tubes.
Prepared By Dr Abdelrhman Elreshid 2
Reagents
Cyanide solution. Potassium cyanide, 25 mg; potassium
ferricyanide, 100 mg. Dissolve in 500 ml distilled water. Store in a
dark bottle.
Saturated ammonium sulphate solution. Bring 1 litre of water to
the boil and add ammonium sulphate until the solution is
saturated. Cool and equilibrate at 20°C before use.
1.2 mol/l Sodium hydroxide. Sodium hydroxide 4.8 g; distilled
water to 100 ml. Prepare monthly. Equilibrate at 20°C before use.
Method
1. Prepare a lysate as described above. The lysate may be stored at
4°C for up to 1 week before use.
2. Add 0.25 ml lysate to 4.75 ml cyanide solution to make a solution
of haemiglobincyanide (HiCN).
3. Transfer 2.8 ml of the haemiglobincyanide solution to a glass test
tube and allow to equilibrate at 20°C.
4. Blow in 0.2 ml of 1.2 mol/l of NaOH and mix on a vortex mixer for
2–3 sec.
5. After exactly 2 min, blow in 2 ml saturated ammonium sulphate
solution and mix on a vortex mixer. Leave tubes to stand for 5–10
min at 20°C.
6. Filter twice through the same Whatman No. 42 filter paper, using
a clean test tube to collect the filtrate each time. If the filtrate is
not completely clear, filter again through the same paper. This
filtrate contains the alkali-resistant haemoglobin.
7. To measure the total haemoglobin, transfer 0.4 ml of the
haemiglobincyanide solution from step 2 into another tube and
add 13.9 ml of water.
8. Read the absorbance of the alkali-resistant and total haemoglobin
at 420 nm against a water blank.
9. Calculate the percentage alkali-resistant haemoglobin as follows:
Prepared By Dr Abdelrhman Elreshid 3
% Alkali resistant
haemoglobin
= A420 alkali
- resistant Hb
X 100
A420 total Hb X 20
Method of Jonxis and Visser
Principle
The increased resistance of Hb F to denaturation by alkali is detected by
recording the change in absorption at 576 nm in each min, caused by
the addition of ammonium hydroxide. At this wavelength, the
absorption of oxyhaemoglobin differs from that of the alkali
haemochromogen that is formed on denaturation.
When the logarithm of the percentage of haemoglobin remaining
undenatured is plotted against time, a straight line is obtained. By
extrapolation to time zero, the percentage of Hb F in the original
sample can be calculated.
Reagents
Ammonium hydroxide solution. NH4OH, 100 g, water to 1 litre.
Sodium hydroxide solution 0.06 mol/l. Sodium hydroxide, 2.4 g,
water to 1 litre.
Method
1. All reagents should be allowed to reach room temperature before
use. Add 0.1 ml of blood or lysate (100 g/l) to 10 ml of water and
mix.
2. Add 2 drops of ammonium hydroxide solution and mix.
3. Measure the absorbance in a spectrophotometer at 576 nm (AB).
4. Add 0.1 ml of the same blood or lysate to 10 ml of sodium
hydroxide solution; then add 2 drops of ammonium hydroxide
solution and mix thoroughly.
5. Measure the absorbance in a spectrometer at 576 nm at every
Prepared By Dr Abdelrhman Elreshid 4
min for 15 min (AT); then incubate the solution at 37°C for 15 min,
cool to room temperature, and measure the absorbance (AE). The
ratio AB:AE should be constant.
6. Calculate the percentage of undenatured haemoglobin at each
min as follows:
AT 576 - AE 576
X 100
AB 576 - AE 576
Plot the percentage on the logarithmic scale of semilogarithmic
paper against time. This should produce a straight line from which the
original amount of Hb F at time zero can be found by extrapolation.
Interpretation and Comments
The Jonxis and Visser method requires an accurate spectrometer
because the maximum absorption peak at 576 nm is very narrow and
the difference in extinction between oxyhaemoglobin and alkali
haemochromogen is relatively small.
Radial Immunodiffusion
The radial immunodiffusion procedure can be used for the quantitation
of Hb F. The principle is based on an antibody–antigen reaction; the
anti-Hb F is incorporated into the gel support medium resulting in the
formation of a visible opaque precipitin ring. The square of the
diameter of this ring is directly proportional to the concentration of Hb
F. A standard curve must be prepared from samples containing known
levels of Hb F plotted against their haemoglobin concentrations.
Interpretation of Hb F values
Prepared By Dr Abdelrhman Elreshid 5
Hb F
Range
(%)
Interpretation
0.2–1.0
Normal results
1.0–5.0
In approximately 30% of β thalassaemia traits
Some heterozygotes for a variant haemoglobin
Some homozygotes for a variant haemoglobin
Some compound heterozygotes for a variant haemoglobin
and β thalassaemia
Some individuals with haematological disorders (aplastic
anaemia, myelodysplastic syndromes, juvenile
myelomonocytic leukaemia)
Some pregnant women (second trimester)
Sporadically in the general population, particularly in AfroCaribbeans (representing heterozygosity for nondeletional
HPFH)
5.0–20.0 Occasional cases of β thalassaemia trait
Some homozygotes for a variant haemoglobin
Some compound heterozygotes for a variant haemoglobin
and β thalassaemia
Some types of heterozygous HPFH δβ thalassaemia
15.0–
45.0
Heterozygous HPFH African type (usually more than 20%)
Some cases of β thalassaemia intermedia
>45.0
β thalassaemia major
Some cases of β thalassaemia intermedia
Neonates
>95.0
Homozygous African-type (deletional) HPFH
Prepared By Dr Abdelrhman Elreshid 6
Hb F
Range
(%)
Interpretation
Some neonates (particularly if premature)
Prepared By Dr Abdelrhman Elreshid 7