Accelerated RBC destruction by
Schistocytosis Presence of cell fragments in circulation Increased intravascular mechanical trauma
Acanthocytosis Irregularly spiculated surface Irreversibly abnormal membrane lipid content
Echinocytosis Regularly spiculated cell surface Reversible abnormalities of membrane lipid content
High plasma free fatty acids, bile acid abnormalities
Effects of barbiturates, salicylates, etc.
Elliptocytosis Oval cells Hereditary anomally, usually harmless
mark in 2 red cell pipettes. The first is diluted to the 101 mark
with Hayem’s (RBC diluting fluid) and the second with
0.45% sodium chloride solution. Both pipettes are shaken
for 2 minutes and counts made from both pipettes, the
percentage of cells hemolyzed in the 0.45% saline solution
is thus determined. Less than 30% of normal erythrocytes
are hemolyzed by this technique. An abnormal increase
in red cell fragility, as in congenital hemolytic icterus will
cause hemolysis of more than 70% of the cells.
Principle: Tubes containing solution of varying
concentration of saline buffered to pH 7.4 are used.
Heparinized or defibrinated blood is added to each tube
in a proportion of 1 to 100 and the degree of hemolysis in
each is noted using a photoelectric colorimeter. The result
may be reported as a graph or stating the concentration at
which hemolysis begins and that at which it is complete.
g, Na2 HPO4 27.31 g and NaH2 PO4 2H2O 4.86 g are dissolved
in distilled water and the final volume adjusted to 2 liters.
This solution will keep for months in a well-stoppered
bottle. In preparing solutions for use it is convenient to
make first a 1% solution from the 10% stock solution by
dilution with distilled water. Dilutions equivalent to 0.85,
0.75, 0.65, 0.60, 0.55, 0.45, 0.40, 0.35, 0.30, 0.20 and 0.10%
NaCl are convenient test concentrations. Intermediate
concentrations such as 0.475 and 0.525% are useful in
If the test is performed very occasionally, smaller
volumes of the solutions may be made up as given at the
Use: Heparinized or defibrinated blood, oxalated and
citrated blood may change the tonicity which is not
Add 0.05 mL of blood to each tube containing 5 mL
of the different concentrations of saline. Mix well and let
the tubes stand at room temperature for 30 minutes.
measure the amount of hemolysis in each tube in a
photoelectric colorimeter with green filter. The supernatant
from 0.85% NaCl is used as the blank because there is no
hemolysis in this concentration (normal) of saline. The
supernatant from the 0.1% NaCl is used to estimate 100%
lysis (the supernatant can easily be decanted into the
cuvette of the colorimeter). The depth of color should be
such that the reading on the colorimeter scale for complete
lysis does not exceed 50 (optical density 0.5). If necessary,
the supernatant may be diluted with an equal volume of
0.1% NaCl or the initial proportion of blood may be 1:200
instead of 1:100. With a good colorimeter, as little as 1%
The blood added should be exactly 0.05 mL. It can be
done by using capillary automatic pipettes. Alternatively
straight glass pipettes graduated till 0.05 mL may be used.
Less time consuming and far less accurate method is to
add one drop of blood to each tube.
Factors Affecting Osmotic Fragility Tests
In carrying out osmotic fragility tests by any method three
variables capable of markedly affecting the results must
be controlled, quite apart from the accuracy with which
the saline solutions have been made up. These are; (i) the
relative volumes of blood and saline, (ii) the final pH of
the blood-saline suspension, and (iii) the temperature at
which the tests are carried out.
Spherocytes, being already round are unable to swell very
much and therefore, rupture even when a small amount of
water has entered the cell. Hemolysis may thus commence
even at 0.75% and may be complete at 0.4% (a feature of
spherocytosis). On the other hand, target cells seen in
thalassemias and iron deficiency anemia cells can swell a
great deal before they rupture because they are relatively
flat. Fragility is, therefore, said to be decreased.
A. Increased fragility (> 0.5%) occurs in:
3. Autoimmune anemia (ABO and Rh) incompatibility
B. Decreased fragility (< 0.3%) occurs in:
Decreased fragility indicates that red cells are excessively
flat. Occurs in iron deficiency anemia, thalassemia, and
QUALITATIVE ASSESSMENT OF G6PD DEFICIENCY
1. Sodium nitrite 1.25 g in 100 mL distilled water.
2. Glucose 5 g in 100 mL distilled water.
3. Methylene blue 150 mg in 100 mL distilled water.
Withdraw 6 mL of blood and add to 1.2 mL of ACD solution.
Label three test tubes as A, B and C, add as follows:
0.1 mL sodium nitrite solution
0.1 mL methylene blue solution
0.1 mL sodium nitrite solution
c. To tube C, add 2 mL of blood only.
Mix well and keep the tubes A, B and C at 37°C for
3 hours. Mix again and aerate at 1, 2, and 3 hours
Take three test tubes each containing 10 mL of distilled
To one, add 0.1 mL of mixture of A.
To second, add 0.1 mL of mixture of B.
To third, add 0.1 mL of contents from C.
Test tube with distilled water and contents from C
Distilled water drop 18 17 16 15 14 13 12 11 10 9 8 7
1% NaCl 9 10 11 12 13 14 15 16 17 17 17 18
NaCl% 0.28 0.32 0.36 0.40 0.44 0.48 0.52 0.56 0.60 0.64 0.68 0.72
Test tube with distilled water and solution from B
Test tube with distilled water and solution from A.
¾ If this is red—there is no G6PD deficiency
¾ If brown like B—full expression of deficiency of G6PD
¾ If between red and brown—intermittent expression of
Aging red cells are especially susceptible to oxidative
challenge by drugs, systemic infection, metabolic
acidosis and other stress. Oxidative stress induces rapid
intravascular destruction of susceptible cells, leading to
hemoglobinemia, hemoglobinuria and a sudden drop of
Young cells have higher G6PD content than the older
ones, regardless of the genetic variant that is present. If the
enzyme has defective activity, older cells are preferentially
destroyed during a mild to moderate hemolytic phase.
Reticulocytes released to replace lost cells have high
enzyme levels. False negative test results often occur if
blood is examined just after a hemolytic episode, because
the non-hemolyzed remaining cells are, by definition,
those with adequate enzyme levels. Newly generated
reticulocytes have still higher levels, and this can affect the
results for 3 to 10 days after the episode.
Drugs that hemolyze G6PD deficient cells are those
that either act as direct oxidants themselves or produce
peroxide activity. Primaquine, an antimalarial drug
is notable in this respect. Many sulfa drugs, quinine
derivatives, nitrofurans and antipyretic-analgesic
drugs can induce hemolysis in G6PD deficient patients.
Susceptibility seems to vary among different individuals.
The presence of coexisting fever, metabolic disease, or
hepatic or renal failure increases likelihood that symptoms
Commercially Available Kit for G6PD
(Courtesy: Tulip Group of Companies)♥
Glucose-6-Phosphate-Dehydrogenase (G6PD) deficiency
is one of the most common human enzyme deficiency in
the world. During G6PD deficiency, the red cells are unable
to regenerate reduced nicotine adenine dinucleotide
phosphate (NADPH), a reaction that is normally catalyzed
Since the X chromosome carries the gene for G6PD
enzyme, this deficiency mostly affects the males. 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,
G-SIX test is a ready to use, three-component reagent
system of the detection of G6PD deficiency in human
blood using the WHO recommended methemoglobin
reduction method. The test system contains three vials *P,
*T and *N predispensed with appropriate reagents along
with Quantitation graph paper.
Each batch of the reagent undergoes rigorous quality
control at various stages of manufacture for its sensitivity
a. Ideally, the product should be stored at 2–8°C. It may
also be stored between 20–25°C in a cool dark place
b. The shelf-life of the reagent system is as per the expiry
date mentioned on the G-SIX carton.
The G-SIX test is based on the principle of reduction of
methemoglobin by G6PD activity of the red cells under test.
The rate of reduction is proportional to the G6PD activity of
the red cells under test. During the test procedure, the test
sample is processed in triplicate so as to simultaneously
also derive positive and normal reference controls. During
screening method, the color of the test sample is compared
visually to the reference controls in order to arrive at the
diagnostic conclusion. Quantitation of the percentage of
G6PD deficiency can also be done spectrophotometrically.
Laboratory reagent for professional use only. Not for
Sample Collection and Preparation
1. The test requires minimum 3 mL of fresh whole blood
sample collected in EDTA or Heparin only. The samples
must be used within one hour of collection, since the
G6PD enzyme actively decreases on storage at 2–8°C.
stored up to 7 days at 2–8°C before performing the test.
3. No special preparation of the patient is required prior
to sample collection by approved techniques.
4. If the hematocrit of the sample is less than 30%, enough
plasma should be removed from the sample to bring
1. 5 mL capacity clean and clear glass test tubes of same
reference). Mark patients ID on the three vials. Use
2. Add 1 mL of the blood sample under test to each of the
vials *P, *T and *N and mix well by gentle inversion.
3. Recap the vials tightly using the screw cap (the plug
may be discarded) and place them vertically in an
incubator which has already been stabilized at 37°C.
4. Incubate undisturbed, at 37°C, for 3 hours.
5. Meanwhile set up three 5 mL test tubes on a test tube
stand and dispense 5 mL distilled/deionized water
number of samples are being run simultaneously set
up equivalent number of such distilled/deionised
7. Remove the vials after 3 hours incubation and mix
8. Uncap the incubated test vials*P,*T and *N and dispense exactly 50 μL (0.05 mL) of the well-mixed
incubated samples using different pipettes into the
corresponding appropriately labeled distilled water
reference tubes PR, TR and NR.
9. Mix evenly by gentle inversion.
10. Observe and compare the color of tube TR with PR and
NR against light to interpret the results.
11. The test results must be interpreted within 3 hours of the
preparation of tubes PR, TR and NR for screening test
and within 30 minutes for the quantitative procedure.
1. Set the spectrophotometer filter on 490 nm.
2. Dispense/aspirate required amount of NR as obtained
in point no. 9 of screening procedure into the cuvette.
3. Similarly read the OD of PR and place the corresponding
value on the G-SIX quantitation graph paper, which
equates to 100% deficiency on the Y-axis.
4. Make a straight line joining the blank value (0.00) and
5. Read the OD of TR and place it on the graph paper.
6. Find out the %G6PD deficiency, corresponding to the
OD value of TR on the Y-axis of the graph paper.
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