This hospital based cross sectional and comparative study was conducted in total of 100 patients attending the hospital with clinical as well as laboratory suspicion like fever and shortness of breath, hyperbilirubinemia, jaundice after antimalarial drug therapy, peripheral smear showing bite cells, Heinz bodies and other hemolytic blood picture were included. Patients of age more than 60 years of age were excluded from the study.
Study population and sample size was determined by using the formula: n = z2PQ
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D2
n = (1.96)2 ×0.07 × 0.93/ (0.0025 = 100 where, n = sample size, z = critical value = 1.96, P = prevalence of disease = 7%, Q = without disease (1-P), D = allowance error (5%).
The hematological parameters including hemoglobin concentration, Red blood corpuscular (RBC) count, Packed Cell Volume (PCV), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC) of the patient were obtained from Hematology analyzer 5 parts (Beckman coulter, DxH 520).
Spectrophotometric assay of G6PD
G6PD in RBCs is released by lysing agent present in the reagent. The G6PD released from the red cells catalyzes the Glucose-6-phosphate with reduction of NADP to NADPH. The rate of reduction of NADP to NADPH is measured as an increase in absorbance at 340 nm produced in the reaction catalyzed by the enzyme which is proportional to the G6PD activity in the sample. [13] Coral G6PD assay kit (Clinical System, Bambolim complex, Goa, India) was used and the procedure provided in the manual with the kit was followed. The absorbance was taken 2 min after reaction mixture was added with blood and final value was calculated by multiplying absorbance (∆A) with factor 4778 in Human Semi-automated analyzer divided by hemoglobin concentration of patients. The normal value of the G6PD activity at 37 °C is 6.4– 18.7 IU/g Hb. At low, medium and high G6PD, intra assay coefficient of variation (CV) were 5.91 %, 4.98 % and 4.83 % and inter-assay CV were 7.85%, 8.43% and 6.35%.
Methemoglobin reduction test
The action of nitrite on red cells results in formation of an oxidized form, methemoglobin, and in the presence of methylene blue, methemoglobin is reduced to hemoglobin through the oxidative pathway. The absence of glucose 6 phosphate dehydrogenase is determined by unchanged brown colored methemoglobin after addition of methylene blue and incubation. [8] 2.0 ml of blood was taken in the test tubes marked with ‘positive control’, ‘negative control’ and ‘test’. 0.1 ml of sodium-nitrite-glucose solution and 0.1 ml of methylene blue solution was added in test tubes marked ‘positive control’ and ‘test’ and mixed. No reagent was added in the negative control tube. After 3 hours of incubation, 0.1 ml of solutions from each tube was transferred to clean and labelled new test tubes. The volume was made up to 10 ml and observed for the comparison of ‘test’ with ‘positive control’ and ‘negative control’. If the ‘test’ was clear red similar to negative control, it was interpreted as negative and if it was brown, similar to positive control, it was interpreted as positive for G6PD deficiency. The controls were prepared from the same blood samples that were being tested or the samples from healthy individuals with hemoglobin concentrations not differing more than ± 0.5 g/dl than that of the samples being tested.
Statistical analysis was done using SPSS IBM ver. 22, New York. The parametric data were expressed in mean ± SD. Categorical data were expressed as frequencies with corresponding percentages and evaluated using Chi-Square test with level of significance set to 0.05 for all tests and P-value <0.05 was considered significant.