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. NR serves as blank. 3. Similarly read the OD of PR and place the corresponding

 

Accelerated RBC destruction by

reticuloendothelial system

Schistocytosis Presence of cell fragments in circulation Increased intravascular mechanical trauma

Microangiopathic hemolysis

Acanthocytosis Irregularly spiculated surface Irreversibly abnormal membrane lipid content

Liver disease

Abetalipoproteinemia

Echinocytosis Regularly spiculated cell surface Reversible abnormalities of membrane lipid content

High plasma free fatty acids, bile acid abnormalities

Effects of barbiturates, salicylates, etc.

Stomatocytosis Elongated, slit-like zone of central pallor Hereditary defect in membrane sodium metabolism

Severe liver disease

Elliptocytosis Oval cells Hereditary anomally, usually harmless

Red Cell Fragility Test

Screening Test

One needs 0.45% sodium chloride solution and hemocytometer for counting red cells. Blood is drawn to the 0.5

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.

Quantitative Test

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.

Contd...

Reagents

A stock solution of buffered sodium chloride (AR) osmotically equivalent to 10% NaCl, is made up as follows. NaCl 180

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

critical work.

If the test is performed very occasionally, smaller

volumes of the solutions may be made up as given at the

next page.

Method

Use: Heparinized or defibrinated blood, oxalated and

citrated blood may change the tonicity which is not

desirable.

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.

240 Concise Book of Medical Laboratory Technology: Methods and Interpretations Remix and centrifuge for 5 minutes at 2000 rpm and

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%

hemolysis may be detected.

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.

Interpretation

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.

Clinical Implications

A. Increased fragility (> 0.5%) occurs in:

1. Hereditary spherocytosis

2. Hemolytic jaundice

3. Autoimmune anemia (ABO and Rh) incompatibility

4. Chemical poisons

5. Burns.

B. Decreased fragility (< 0.3%) occurs in:

1. Obstructive jaundice

2. Thalassemia

3. Sickle cell anemia

4. Iron-deficiency anemia

5. Polycythemia vera

6. Liver disease

7. Splenectomy (following).

Decreased fragility indicates that red cells are excessively

flat. Occurs in iron deficiency anemia, thalassemia, and

sickle cell disease.

QUALITATIVE ASSESSMENT OF G6PD DEFICIENCY

Methemoglobin Reduction Test

Reagents

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.

Method

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:

a. To tube A, add:

 0.1 mL sodium nitrite solution

 0.1 mL glucose solution

 0.1 mL methylene blue solution

 2 mL blood.

b. To tube B, add:

 0.1 mL sodium nitrite solution

 0.1 mL glucose solution

 2 mL blood.

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

(hourly intervals).

Take three test tubes each containing 10 mL of distilled

water.

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.

Wait for 10 minutes.

Test tube with distilled water and contents from C

should always be red.

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

Clinical Hematology 241

Test tube with distilled water and solution from B

should always be brown.

Interpretation

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

G6PD deficiency.

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

hematocrit.

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

will emerge.

Commercially Available Kit for G6PD

Assessment (Qualitative)

(Courtesy: Tulip Group of Companies)♥

Summary

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

by the G6PD enzyme.

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,

patient counseling and abstinence from disease precipitating drugs such as antimalarials and other agents.

Reagents

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

and performance.

Storage and Stability

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

away from light and moisture.

b. The shelf-life of the reagent system is as per the expiry

date mentioned on the G-SIX carton.

Principle

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.

Note

Laboratory reagent for professional use only. Not for

medicinal use.

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.

242 Concise Book of Medical Laboratory Technology: Methods and Interpretations 2. Blood samples may be collected in ACD and can be

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

back the PCV to 40 ± 5%.

Additional Material Required

1. 5 mL capacity clean and clear glass test tubes of same

diameter and height.

2. Incubator at 37°C.

3. 0.05 mL, 1 mL, 5 mL clean precision pipettes, spectrophotometer with 490 nm filter or colorimeter with blue

green filter.

4. Timer.

5. Test tube stand.

6. Distilled/Deionized water.

Screening Test Procedure

1. Open the pack of the reagent vials *P (for test reference),*T (for test reference) and *N (for normal

reference). Mark patients ID on the three vials. Use

immediately upon opening.

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

into each of these tubes.

6. Label these reference tubes as PR, TR and NR respectively and mark patient ID on each tube, (if more

number of samples are being run simultaneously set

up equivalent number of such distilled/deionised

water tube sets).

7. Remove the vials after 3 hours incubation and mix

gently.

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.

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.

NR serves as blank.

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

the OD of PR.

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.

Interpretation of Results

Screening Test

Normal sample: Tube TR has a clear red color,

matching with the normal

reference tube NR.