College of Health Sciences
Department of Medical Laboratories
Second Year – Second Term
Hematology – 1
Practice NO (5)
G6PD Assay
RED CELL ENZYME ASSAYS
Many methods are available for assaying each enzyme, and for this reason the
ICSH has produced simplified methods suitable for diagnostic purposes. These
methods are not necessarily the most appropriate for detailed study of the kinetic
properties of the variant enzymes, but they are relatively simple to set up and
allow comparison of results between different laboratories.
General Points of Technique
Collection of Blood Samples
Blood samples may be anticoagulated with heparin (10 iu/ml blood), EDTA (1.5
mg/ml blood), or ACD. In any of these anticoagulants, all normal enzymes are
stable for 6 days (and most for 20 days) at 4°C and for 24 hours at 25°C. However,
enzyme variants in samples from patients may be less stable. Therefore, we
recommend that ACD is used as anticoagulant and that the samples are tested
promptly. Ideally, samples of blood should be transferred to central laboratories
in tubes surrounded by wet ice at 4°C. Frozen samples are unsuitable because the
cells are lysed by freezing. Further details of enzyme stability were given by
Beutler. Approximately 1 ml of blood is required for each enzyme assay.
Washing the Red Cells
Centrifuge the anticoagulated blood at 1200-1500 g for 5 min and remove the
plasma together with the buffy coat layer.
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Resuspend the cells in 9 g/l NaCl (saline) and repeat the procedure three times.
This will remove about 80-90% of the leucocytes.
This simple method is adequate in most instances when more complicated
manoeuvres are impractical, but it has the disadvantage that some of the
reticulocytes and young red cells are lost together with the buffy coat.
Preparation of Haemolysate
Mix 1 volume of the washed or filtered suspension with 9 volumes of lysing
solution consisting of 2.7 mmol/l EDTA, pH 7.0, and 0.7 mmol/l 2mercaptoethanol (100 mg of EDTA disodium salt and 5 μl of 2-mercaptoethanol in
100 ml of water); adjust the pH to 7.0 with HCl or NaOH.
Ensure complete lysis by freeze-thawing. Rapid freezing is achieved using a dry-ice
acetone bath or methanol that has been cooled to -20°C. Thawing is achieved in a
waterbath at 25°C or simply in water at room temperature. Usually the
haemolysate is ready for use without further centrifugation, but a 1-min spin in a
microfuge is recommended to remove any turbidity (this may be unsuitable for
some red cell enzymes that are stroma-bound). Dilutions, when necessary, are
carried out in the lysing solution. The haemolysate should be prepared freshly for
each batch of enzyme assays. Most enzymes in haemolysates are stable for 8
hours at 0°C, but it is best to carry out assays immediately. G6PD is one of the
least stable enzymes in this haemolysate, and its assay should be conducted
within 1 or 2 hours of the lysate being prepared. The storing of frozen cells or
haemolysates is not recommended; it is preferable to store whole blood in ACD.
Control Samples
Control samples should always be assayed at the same time as the test samples
even when a normal range for the various enzymes has been established.
Take the control samples of blood at the same time as the test samples and treat
them in the same way. When receiving samples from outside sources, always ask
for a normal “shipment control” to be included.
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G6PD Assay
The activity of the enzyme is assayed by following the rate of production of
NADPH, which, unlike NADP, has a peak of UV light absorption at 340 nm.
Method
The assays are carried out at 30°C, the cuvettes containing the first four reagents
and water being incubated for 10 min before starting the reaction by adding the
substrate, as shown in Table below . Commercial kits are also available.[*]
Table Glucose-6-phosphate dehydrogenase assay
Reagents
Assay (ml) Blank (ml)
Tris-HCl EDTA buffer, pH 8.0
100
100
MgCl2, 100 mmol/l
100
100
NADP, 2 mmol/l
100
100
1:20 Haemolysate
20
20
Water
580
680
Start reaction by adding: G6P, 6 mmol/l 100
—
EDTA, ethylenediaminetetra-acetic acid; NADP, G6P, glucose-6-phosphate.
The change in absorbance following the addition of the substrate is measured
over the first 5 min of the reaction. The value of the blank is subtracted from the
test reaction, either automatically or by calculation.
Calculation of Enzyme Activity
The activities of the enzymes in the haemolysate are calculated from the initial
rate of change of NADPH accumulation:
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where 6.22 is the mmol extinction coefficient of NADPH at 340 nm and 103 is the
factor appropriate for the dilutions in the reaction mixture. Results are expressed
per 1010 red cells, per ml red cells, or per g Hb by reference to the respective
values obtained with the washed red cell suspension. However, the ICSH
recommendation is to express values per g Hb, and it is ideal to determine the Hb
concentration of the haemolysate directly. When doing this, use a haemolysate to
Drabkin's solution ratio of 1:25.
G6PD is very stable, and, with most variants, venous blood may be stored in ACD
for up to 3 weeks at 4°C without loss of activity.
Some enzyme-deficient variants lose activity more rapidly, and this will cause
deficiency to appear more severe than it is. Therefore, for diagnostic purposes, a
delay in assaying well-conserved samples should not be a deterrent.
Normal Values
The normal range for G6PD activity should be determined in each laboratory. If
the ICSH method is used, values should not differ widely from the given values.
For adults, these values are 8.83 ± 1.59 eu/g Hb at 30°C. However, newborns and
infants may have enzyme activity that deviates appreciably from the adult value.]
In one study, the newborn mean activity was about 150% of the adult mean.[37]
Interpretation of Results
In assessing the clinical relevance of a G6PD assay, three important facts must be
kept in mind:
1. The gene for G6PD is on the X-chromosome, and therefore males, having
only one G6PD gene, can be only either normal or deficient hemizygotes. By
contrast, females, who have two allelic genes, can be either normal
homozygotes or heterozygotes with “intermediate” enzyme activity or
deficient homozygotes.
2. Red cells are likely to haemolyse on account of G6PD deficiency only if they
have less than about 20% of the normal enzyme activity.
3. G6PD activity falls off markedly as red cells age. Therefore, whenever a
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blood sample has a young red cell population, G6PD activity will be higher
than normal, sometimes to the extent that a genetically deficient sample
may yield a value within the normal range. This usually, but not always, will
be associated with a high reticulocytosis.
In practice, the following notes may be useful:
1. In males, diagnosis does not present difficulties in most cases because the
demarcation between normal and deficient subjects is sharp. There are very
few acquired situations in which G6PD activity is decreased (one is pure red
cell aplasia where there is reticulocytopenia), whereas an increased G6PD
activity is found in all acute and chronic haemolytic states with
reticulocytosis.
2. In females, all the same criteria apply, with the added consideration that
heterozygosity can neverbe rigorously ruled out by a G6PD assay; for this
purpose, the cytochemical test is more useful than a spectrometric assay,
and a counsel of perfection is to use the two in conjunction with each other
and with family studies.
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Methaemoglobin reduction test
Screening for G6PD deficiency
Value of test: The methaemoglobin reduction test
is one of the simpler and less expensive tests to
screen for G6PD deficiency.
reduced G6PD activity in red cells can cause
acute intravascular haemolysis following exposure to
oxidant agents or fava beans (favism), neonatal
jaundice and less commonly, chronic haemolytic
anaemia. The severity of clinical symptoms is mainly
dependent on the variant of defective G6PD gene
inherited. For the main laboratory findings associated
with a haemolytic crisis
Principle of test
Haemoglobin is oxidized to methaemoglobin (Hi) by sodium
nitrite. The redox dye, methylene blue activates the pentose
phosphate pathway, resulting in the enzymatic conversion of
Hi back to haemoglobin in those red cells with normal G6PD
activity. In G6PD deficient cells there is no enzymatic reconversion
to haemoglobin.
Reagents
● Methylene blue, 0.4 mmol/l
● Sodium nitrite-glucose reagent*
*The reagent must be prepared fresh on the day
of use. To make 40 ml:
Sodium nitrite . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 g
Dissolve the chemicals in 40 ml of distilled
(deionized) water.
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Preparation of reagents in tubes for long term storage
To store the reagents in dried ready to use form:
– Mix equal volumes of methylene blue reagent with
sodium nitrite-glucose reagent.
– Dispense in 0.2 ml amounts into small glass tubes.
– Dry the contents of the tubes at room temperature.
– Stopper the tubes and store in the dark at room temperature.
Note: In dried form the reagents are stable for up to 6 months.
Blood sample: EDTA anticoagulated venous blood
is suitable. It should not be collected during a
haemolytic crisis but when the patient has recovered
and reticulocyte numbers have fallen back to normal
levels. This is because reticulocytes contain higher
levels of G6PD and may mask low G6PD activity in
mature red cells. The blood must be tested within 8
hours of being collected.When the patient is anaemic,
use a plasma reduced blood sample (remove sufficient
plasma until the PCV is about 0.40).
Test method
1 Take 3 small glass tubes and label Test, Normal,
Deficient.
2 Pipette into each tube as follows:
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Tube
Sodium
nitrite-glucose
reagent (fresh)
Methylene
blue reagent
Patient’s blood
Test
Normal
Control
0.1 ml
––––––
Deficient
control
0.1 ml
0.1 ml
––––––
––––––
2 ml
2 ml
2 ml
3 Stopper the tubes and mix well (gentle mixing).
Incubate all three samples at 35–37 _C for 90
minutes.
4 Take 3 large tubes (15 ml capacity) and label as
described in step 1. Pipette 10 ml of distilled
(deionized) water into each tube.
5 Transfer 0.1 ml of well mixed sample from the
Test, Normal, and Deficient tubes to the large
tubes. Mix the contents of each tube.
6 Examine the colour of the solution in each tube.
Interpretation of test results
Colour of test solution . . . . . Normal G6PD activity
is similar to the
red colour of the
Normal tube
Colour of test solution . . . . . Reduced G6PD activity
is similar to the (G6PD deficiency in
brown colour of the homozygote)
Deficient tube
Note: Results from a heterozygote are midway
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between normal G6PD activity and G6PD deficiency
in the homozygote.
Quality control
Follow the technique exactly.
The main sources of error when performing the
methaemoglobin reduction test are:
● Testing blood which has a high reticulocyte count
or too low a haemoglobin concentration (see
previous text).
● Not using freshly made sodium nitrite-glucose
reagent.
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