Tests for Sickling Sickle cells are abnormal forms resulting from the presence of abnormal hemoglobin, which in the deoxidized state undergoes a hydrophobic bond dependent polymerization. Rigid threads or rods of S hemoglobin result, with

G6PD deficient sample The test tube TR has a brown

(full expression): color matching with the

 positive reference tube PR.

G6PD deficient sample The tube TR has intermediate

(intermediate females): color as compared to positive

reference tube PR and negative

reference tube NR depending

on the degree of expression of

the deficiency trait.

Quantitative Test

Class of

deficiency

% of G6PD

deficiency

Clinical relevance

CLASS I Complete Chronic, congenital nonspherocytic,

anemia without drugs/oxidative stress

CLASS II 90% or more Acute hemolytic crisis induced by

oxidative drugs

CLASS III 40–90% Oxidative drugs/infection induces

self-limiting hemolysis without

previous hematologic disorder

CLASS IV Less than 40% Associated with milder clinical

conditions, depending on the variant

involved

% 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.

Remarks

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

set for conducting the test.

Clinical Hematology 243

2. The reagent vials should be used immediately after

opening.

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

positives.

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

achieving correct results.

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

sample.

Clinical Implications

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

or bacterial infections.

2. The major types of G6PD deficiency are:

a. Type A, found in blacks.

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

newborn.

3. G6PD levels are increased in:

a. Pernicious anemia.

b. Werlhof’s disease.

c. Hepatic coma.

d. Hyperthyroidism.

e. Myocardial infarction.

f. Chronic blood loss.

g. Other megaloblastic anemias.

Quantitative Estimation of G6PD

(Courtesy: Tulip Group of Companies)

Summary

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

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.

Principle

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 - PDH

G-6-P + NADP Gluconate-6 - P +

 NADPH + H

Normal Reference Values

G6PDH activity : 4.6 to 13.5 at 30°C/

(U/g Hb.) 6.4 to 18.7 at 37°C

(U/1012 RBCs) : 146 to 376 at 30°C/

 202 to 522 at 37°c

It is recommended that each laboratory establish its

own normal range representing its patient population.

244 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contents 5 × 1 tests 5 × 5 tests

L1 : G6PDH Reagent 5 × 1 mL 5 × 5.5 mL

L2 : Starter Reagent 10 mL 50 mL

Storage/Stability

Contents are stable at 2-8°C till the expiry date mentioned

on the labels.

Reagent Preparation

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

2–8°C.

The Starter Reagent (L2) is ready to use.

Sample Material

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.

Freezing is not recommended.

Procedure

Wavelength/filter : 340 nm

Temperature : 30/37°C

Light path : 1 cm.

Addition S Sequence (mL)

G6PD working reagent (L1) 1.0

Whole blood 0.01

Mix well and incubate for 5–10 minutes at RT

and add substrate reagent

2.0

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)

ΔA /min = A2A1

5

Calculations

G6PDH Activity = ΔA × 47780

(U/1012RBC) RBC Count (/mm3

)

G6PDH Activity (U/gHb) = ΔA × × 4778

Hb (g/dL)

Temperature Conversion Factors

Assay Desired Reporting Temperature

Temperature 25°C 30°C 37°C

25°C 1.00 1.32 1.82

30°C 0.76 1.00 1.39

37°C 0.55 0.72 1.00

Notes

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

reported in Hb concentration.

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

essential.

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

episode.

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

Qualitative Method

Peripheral blood film staining method (Acid elution

technique).

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

a jar kept at 37°C

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

distilled water)

¾ In this mixture keep the slide for 30 minutes

¾ Wash with distilled water

¾ Stain with 1% eosin solution for 5 minutes.

Clinical Hematology 245

Interpretation

Normal red cells will appear as ghost cells. Fetal hemoglobin containing cells will appear as bright red cells.

Quantitative Method

STEP A

Preparation of hemolysate (Fig. 9.18):

1. Take 8 cc of EDTA blood, centrifuge it for 15 minutes,

remove the plasma.

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.

STEP B

Quantitative alkali denaturation method for estimation of

fetal hemoglobin

1. Take 9.5 mL of Drabkin’s solution in a test tube, add

0.5 mL of hemolysate. Cyanmethemoglobin (Hi CN)

is formed. Mix well.

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

filter paper number 3.

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).

OD of total 10

OD of total hemoglobin

HbF and HbS concentration as percentage of total Hb

concentration in various disorders is given below:

Disorder HbF% HbS%

Normal < 1% (in adults) not present

Sickle cell trait (AS) Normal 30–40%

Sickle cell anemia (SS) 1–20% 75–95%

HbS b thalassemia

[Sb+

 (some b chains present)] 2–10% 60–85%

[Sb° (no b chains present)] 5–30% 76–90%

HbS-C (SC) 1–5% 50–55%

HbS-D (SD) 1–5% 95% (S+D)

b Thalassemia major 10–98% —

a Thalassemia Reduced —

Normal Values

Adults 0–2% of total hemoglobin

Children

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.

Tests for Sickling

Sickle cells are abnormal forms resulting from the presence

of abnormal hemoglobin, which in the deoxidized state

undergoes a hydrophobic bond dependent polymerization. Rigid threads or rods of S hemoglobin result, with

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

Moist Cover Slip Preparation

Cover a small drop of fresh blood on a slide with a coverslip,

seal with petrolatum, paraffin or nail polish, keep at warm

room temperature and examine for sickling every 12 hours

up to 72 hours. To find percentage of circulating sickling