G6PD deficient sample The test tube TR has a brown
(full expression): color matching with the
G6PD deficient sample The tube TR has intermediate
(intermediate females): color as compared to positive
reference tube PR and negative
on the degree of expression of
CLASS I Complete Chronic, congenital nonspherocytic,
anemia without drugs/oxidative stress
CLASS II 90% or more Acute hemolytic crisis induced by
CLASS III 40–90% Oxidative drugs/infection induces
self-limiting hemolysis without
CLASS IV Less than 40% Associated with milder clinical
conditions, depending on the variant
% G6PD deficiency of up to 20% as obtained by G-SIX test,
corresponds to the normal range of 4.5 – 13.5 U/g Hb activity.
1. Do not expose the reagents during storage or during
test to direct sunlight. Before performing the test, if the
reagent vials show any moisture or condensation on
the inner walls; they must be discarded, use another
2. The reagent vials should be used immediately after
3. Young red cells have a higher G6PD content than
the older ones, regardless of the genetic variant that
is present. If the enzymes have defective activity,
older cells are preferentially destroyed during mildto-moderate hemolytic phase. Since reticulocytes
released to replace lost cells have high enzyme levels,
false negative results may occur if blood is tested
immediately after a hemolytic episode.
4. The blood should be tested within an hour of collection
as recommended. Delay in testing may give rise to false
5. It is extremely important that the 5 mL test tubes used
for postincubation sample dilution are free from acids
or alkalies as this may interfere with end color stability.
6. Transfer of correct samples to the correctly labeled
reference tubes PR, TR and NR is extremely vital for
7. Vitamin C supplements or a large dietary intake of
vitamin C may interfere with the reaction.
8. The positive reference PR must be a brown color. The
negative reference must have a cherry pink to cherry
red color. These colors must be achieved to validate
test run and correct transfer of incubated samples to
correct and corresponding reference tubes.
9. If the positive and negative reference tubes (PR and
TR) have a different color than expected the test must
be re-run. It must be noted however, that the test
reference will show varying colors from red to brown
depending upon the degree of G6PD deficiency in the
1. A decreased level is associated with G6PD deficiency,
which is a sex-linked disorder. Affected males inherit
the abnormal gene from their mothers who are usually
asymptomatic carriers. In some cases of this disorder,
there is lifelong hemolysis; but more commonly,
the condition is asymptomatic and results only in
susceptibility to acute hemolytic episodes that may be
triggered by drugs such as primaquine, sulfonamides,
and antipyretics, by ingestion of fava beans, or by viral
2. The major types of G6PD deficiency are:
b. Mediterranean type, found in both
i. Caucasians and Orientals such as Greeks,
ii. Sardinians and Sephardic Jews.
c. Rare, congenital non-spherocytic anemia.
d. Nonimmunologic hemolytic disease of the
3. G6PD levels are increased in:
g. Other megaloblastic anemias.
Quantitative Estimation of G6PD
(Courtesy: Tulip Group of Companies)
Glucose-6-Phosphate-Dehydrogenase (G6PDH) deficiency
is one of the most common human enzyme deficiencies in
the world. During G6PD deficiency, the red cells are unable
to regenerate reduced Nicotinamide adenine dinucleotide
phosphate (NADPH), a reaction that is normally catalyzed
by the G6PD enzyme. Since the X chromosome carries the
gene for G6PD enzyme, this deficiency mostly affects the
The two major conditions associated with G6PD
deficiency are hemolytic anemias and neonatal jaundice,
which may result in neurological complications and death.
Screening and detection of G6PD deficiency helps in
reducing such episodes, through appropriate selection of
treatment, patient counseling and abstinence from disease
precipitating drugs such as antimalarials and other agents.
G6PDH in the RBCs is released by a lysing agent present in
the reagent. The G6PDH released catalyzes the oxidation
of glucose 6 phosphate with the reduction of NADP
to NADPH. The rate of reduction of NADP to NADPH
is measured as an increase in absorbance, which is
proportional to the G6PDH activity in the sample.
G-6-P + NADP Gluconate-6 - P +
G6PDH activity : 4.6 to 13.5 at 30°C/
(U/1012 RBCs) : 146 to 376 at 30°C/
It is recommended that each laboratory establish its
own normal range representing its patient population.
L1 : G6PDH Reagent 5 × 1 mL 5 × 5.5 mL
L2 : Starter Reagent 10 mL 50 mL
Contents are stable at 2-8°C till the expiry date mentioned
Reconstitute G6PDH reagent (L1) with distilled water as per
the volume mentioned on the label. This working reagent
is stable for 6 hours at RT and at least 3 days when stored at
The Starter Reagent (L2) is ready to use.
Fresh whole blood sample collected in EDTA, Heparin
or ACD. Red cell G6PDH in whole blood is reported to be
stable for 7 days at 2–8°C, but is unstable in hemolysates.
Mix well and incubate for 5–10 minutes at RT
Mix well and incubate for 5 minutes at 30/37°C and
read the initial absorbance A0 and repeat the absorbance
reading after every 1, 2 and 3 minutes. Calculate the mean
absorbance change per minute (DA/min).
If the G6PDH activity is very low, the absorbance change
per minute will also be very low. In such cases read the
initial absorbance A1 and read another absorbance A2
exactly 5 minutes later. Calculate the mean absorbance
change per minute (ΔA/minutes)
G6PDH Activity (U/gHb) = ΔA × × 4778
Temperature Conversion Factors
Assay Desired Reporting Temperature
Since the activity of G6PDH is reported in Hb concentration
or RBC count the same should be determined before
performing the assay. RBCs are well preserved when
collected in ACD and such samples give an accurate
count, for samples collected in Heparin counts become
unreliable after 2 days and in such cases results are best
Copper and sulfate ions inhibit the G6PDH activity;
hence use of good quality deionized or distilled water for
reconstitution of L1 and properly cleaned glassware is
Young red cells have a higher G6PD content then
the older ones, regardless of the genetic variant that is
present. If the enzymes have defective activity, older cells
are preferentially destroyed during mild to moderate
hemolytic phase. Since reticulocytes released to replace
lost cells have high enzyme levels, falsely elevated results
may occur if blood is tested immediately after a hemolytic
Normally the activity contributed by WBC, platelets
or serum is very small. In cases of severe anemia,
leukocytosis, or very low G6PDH levels, the use of a sample
after removing the Buffy Coat is recommended.
EXAMINATION OF FETAL HEMOGLOBIN
Peripheral blood film staining method (Acid elution
Always use freshly prepared smears
¾ Fix in 80% ethanol for 10 minutes
¾ Take 37.7 mL of citric acid solution prepared (21 g of
citric acid by dissolving, in 100 cc of distilled water) in
¾ To this, add 12.2 mL of Na2H PO4 solution (prepared
by dissolving 57.6 g of Na2H PO4.12 H2O (in 1000 cc of
¾ In this mixture keep the slide for 30 minutes
¾ Stain with 1% eosin solution for 5 minutes.
Preparation of hemolysate (Fig. 9.18):
1. Take 8 cc of EDTA blood, centrifuge it for 15 minutes,
2. To the RBCs sediment, add normal saline and fill the
centrifuge tube to 4/5th of it. Mix thoroughly and
centrifuge for 20 minutes. Remove the saline and
repeat the said process at least twice more.
3. To the packed RBCs, add equal quantity of distilled
water and half the quantity of toluene. Mix them
thoroughly and keep in the deep freezer for one hour.
4. Remove from the freezer chest. Thaw it and centrifuge
it for 30 minutes. Now there will be 3 zones in the tube.
5. Pass a pipette to the hemolysate zone through the
side of the test tube without disturbing the upper two
zones, suck up the hemolysate.
6. Check the hemoglobin of the hemolysate and adjust
to 8–10 g%. This hemolysate can be stored at –20°C
for 6 months and can be used for quantitative (alkali
denaturation method) estimation of fetal hemoglobin
or for hemoglobin electrophoresis.
Quantitative alkali denaturation method for estimation of
1. Take 9.5 mL of Drabkin’s solution in a test tube, add
0.5 mL of hemolysate. Cyanmethemoglobin (Hi CN)
2. Transfer 2.8 mL of HiCN solution in a tube and keep
at 20°C. To this, add 0.2 mL of 1.2 NNaOH solution.
Mix rapidly—incubate for 2 minutes at 20°C. Add 2 cc
of saturated solution of ammonium sulfate. Mix again
and let stand for 5-10 minutes. Filter through Whatman
3. Prepare the total hemoglobin by adding 0.4 mL of the
original HiCN solution to 6.5 mL of distilled water.
4. Using Drabkin’s solution as blank at 540 nm, read the
total hemoglobin and filtrate.
5. The optical density should fall between 0.05 and 0.5.
If it is beyond 0.5, dilute the hemolysate with distilled
water and repeat the said procedure.
6. Calculate as hemoglobin F (percentage of HbF).
HbF and HbS concentration as percentage of total Hb
concentration in various disorders is given below:
Normal < 1% (in adults) not present
Sickle cell trait (AS) Normal 30–40%
Sickle cell anemia (SS) 1–20% 75–95%
(some b chains present)] 2–10% 60–85%
[Sb° (no b chains present)] 5–30% 76–90%
Adults 0–2% of total hemoglobin
Newborn < 60–90% of total hemoglobin
1-5 months < 75% of total hemoglobin
6-12 months < 5% of total hemoglobin
1-20 years < 2 % of total hemoglobin.
Sickle cells are abnormal forms resulting from the presence
of abnormal hemoglobin, which in the deoxidized state
consequent production of the rigid sickle cells. Sickling
occurs in 10% of blacks, but only a few of these have
anemia. FIG. 9.18: Preparation of hemolysate
246 Concise Book of Medical Laboratory Technology: Methods and Interpretations Methods
Cover a small drop of fresh blood on a slide with a coverslip,
seal with petrolatum, paraffin or nail polish, keep at warm
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