On Red Blood Cell Fragility and Reticulocyte Count In Hemolytic Anemic Patients with and Without G-6 PD Enzyme Deficiency

Background: Erythrocyte G-6PD enzyme deficiency is an important cause of Hemolytic anemia with consequent increase in reticulocyte count. Objective: To assess the osmotic fragility of RBC status and reticulocyte count in G-6PD enzyme deficient patients with hemolytic anemia in order to find their hemolytic status. Methods: The cross sectional study was carried out in the Department of Physiology, BSMMU, Dhaka from July 2002 to June 2003 to observe the osmotic fragility of RBC status and reticulocyte count in patients with hemolytic anemia. For this, total number of 50 hemolytic anemic patients(Group-B) with age ranged from 5 to 30 years of both sexes were studied. Among them, 25 were without G-6PD deficient hemolytic anemia ( group-B1 ) and 25 were hemolytic anemia with G-6PD enzyme deficiency (group-B2 ). Age & sex matched 30 apparently healthy subjects with normal blood G-6PD level were included to observe the baseline data ( Group-A ) and also for comparison. All the subjects were selected from Out Patient Department of Hematology, Bangabandhu Sheikh Mujib Medical University ( BSSMU ), Dhaka. Blood erythrocyte G-6PD enzyme level, osmotic fragility of RBC & reticulocyte count were measured by standard laboratory techniques. Analysis of data was done by unpaired Student ‘t’test. Result: Mean starting & completing points of hemolysis of RBC were significantly higher in Group B2 vs GroupA and also with Group B1 and similar higher levels of these values were also observed in Group B1 than those of GroupA, but the differences between them were not statistically significant. Reticulocyte count was significantly higher in Group-B2 vs Group B1 and also with GroupA and similar higher levels of this values were also observed in Group B1 vs GroupA which was also statistically significant. Conclusion: From this study, it may be concluded that, increased hemolysis of RBC with higher reticulocyte count occur in G-6PD deficient hemolytic anemic patients which may be due to membrane defect.

Various hematological changes occur in hemolytic anemia without and with G-6PD enzyme deficiency including osmotic fragility of RBC which depend on the integrity of its membrane 11 .Workers of different countries reported that osmotic fragility was increased in hemolytic anemia without and with G-6PD enzyme deficiency 12 .On the contrary, osmotic fragility of erythrocyte has been found to be decreased in drug induced hemolytic anemia associated with erythrocyte G-6PD enzyme deficiency 13 .Again, a normal osmotic fragility of erythrocyte has also been observed in similar group of patients 14.However, the common clinical consequences of this enzyme deficiency are neonatal jaundice and sporadic hemolytic crisis 9 .
Hemolysis of RBC is evidenced by increased reticulocyte count 12 .The reticulocyte count has been increased in erythrocyte G-6PD enzyme deficient hemolytic anemia associated with acute viral hepatitis 15 .On the other hand, the reticulocyte count has been decreased in drug induced hemolytic anemic patients 16 .On the contrary normal reticulocyte count has also been reported 17 .
In Bangladesh, many people are suffering from hemolytic anemia due to G-6PD deficiency.Unfortunately,most of them are treated without knowing the underlying cause.
In our country, there is lack of adequate information about deficiency of G-6PD enzyme in the hemolytic anemic patients and a few published data regarding the effects of G-6PD enzyme deficiency patients are available in our country 18,19 and also from different other countries 14,15 .
Therefore, the present study was undertaken to observe the osmotic fragility of RBC status along with reticulocyte count in hemolytic anemic patients without and with G-6PD enzyme deficiency.The outcome of the study may be helpful to create awareness among the clinicians about the presence of G-6PD enzyme deficiency and also to do the needful in avoiding various complications due to this deficiency in hemolytic anemia.

Methods:
The present cross-sectional study was carried out in the Department of Physiology, BSMMU, Dhaka.For this,a total number of 80 subjects with age range from 5 to 30 years of both sexes were included.Among them, 50 patients with hemolytic anemia were included in Group B. On the basis of G-6PD enzyme level subjects of group B were further divided into GroupB 1, consisted of 25 patients without this enzyme deficiency and Group B 2, consisted of 25 patients with this enzyme deficiency(Group B 2 ) .Age & sex matched 30 apparently healthy subjects with normal blood G-6PD enzyme level were taken to observe the baseline data (Control) and also for comparison.All the G-6PD deficient & non deficient patients were selected from OPD of Hematology, BSSMU, Dhaka, and all the healthy subjects were selected from personal contact.Patients with acute hemolytic episode or receive blood transfusion in the last 2 months and the â thalassemia trait were excluded from the study.The objectives and benefit of the study were explained to all the subjects to ensure their voluntary participation and a written informed consent was taken from each subjects prior to the study.For all the subjects,G-6PD level, osmotic fragility of RBC and reticulocyte count were measured.Erythrocyte G-6PD enzyme level was determined by Spectrophotometeric method 20 ,osmotic fragility of RBC by Colorimetrically 21 and reticulocyte count by Brilliant cresyl blue method 22 .All of these tests were done in the Department of Hematology, BSSMU, Dhaka.Data were expressed as Mean ± SE.Statistical analysis of the results were done by unpaired Student 't' test by using SPSS program version 12.

Results:
Mean erythrocyte G-6PD enzyme level was significantly (P<0.001)lower in G-6PD enzyme deficient group(GroupB 2 ) than that of healthy control(GroupA) and hemolytic anemia without G-6PD enzyme deficiency(Group B 1 ).Again, this enzyme level was within normal range in Group B 1 and the difference of this value with healthy control was also statistically significant(P<0.001),(Table-I).
The mean starting & completing points of osmotic fragility of RBC were significantly (P<0.001) higher in G-6PD deficient (GroupB 2 ) and non deficient (GroupB 1) in comparison to those of healthy control(Group A).On the other hand, though starting and completing points of hemolysis were slightly higher in GroupB 1 than those of Group A but the differences were not statistically significant (Table-II).
The reticulocyte count was significantly(P<0.001) higher in hemolytic anemia with(Group B 1 ) and without(Group B 2 ) G-6PD enzyme deficiency compared to that of healthy control (Group A).Again,this count was significantly (P<0.001) higher in Group B 1 than that of Group B 2 (Table-III).

Discussion:
The patients with G-6PD deficiency had significantly higher osmotic fragility of RBC and reticulocyte count in comparison to those of healthy control.These findings are consistent with those of some investigators of different countries 23 .On the contrary, no remarkable change in osmotic fragility of RBC had also been reported by some other group investigators 24 .
Changes in red cell membrane integrity may be the possible cause of early destruction of RBC in G-6PD deficient in hemolytic anemia 25 .It has been suggested that abnormal degradation of hemoglobin may occur in G-6PD deficient hemolytic anemia 26 .Disturbance of intracellular metabolism may also be the another possible underlying cause in this type hemolytic anemia 16 .
The higher reticulocyte count in erythrocyte G-6PD enzyme deficiency may be due to shorter red cell life span 27 .In addition, it has also been suggested that lower level of reduced glutathione in erythrocyte of G-6PD deficiency limits their ability to resist oxidative stress and leads to premature destruction of RBC and there by leads to higher reticulocyte count [28][29] .
All the above mentioned suggestions may also be the underlying cause of excess hemolysis of RBC in the G-6PD deficient hemolytic anemic patients of present series and it is also supported by increased reticulocyte count in this series of patients.But it is difficult to comment on all the above mentioned factors as they were not studied.

Conclusion:
Therefore, this study concludes that in G-6PD enzyme deficiency, excess hemolysis of RBC occur possibly due to membrane defect.

Table - I
Erythrocyte