Frequency of β-thalassemia trait and other hemoglobinopathies in northern and western India.

INTRODUCTION: India is an ethnically diverse country with an approximate population of 1.2 billion. The frequency of beta-thalassemia trait (βTT) has variously been reported from <1% to 17% and an average of 3.3%. Most of these studies have been carried out on small population groups and some have been based on hospital-based patients. There is also a variation in the prevalence of hemoglobinopathies in different regions and population groups in the country. A high frequency of Hb D has been reported from the North in the Punjabi population, Hb E in the eastern region of India and Hb S is mainly reported from populations of tribal origin from different parts of the country. OBJECTIVES: To study the gene frequency of βTT and other hemoglobinopathies in three regions East (Kolkata), West (Mumbai) and North (Delhi) in larghe population group (schoolchildren) for a more accurate assessment of gene frequency for planning of control programmes for haemoglobinopathies. MATERIALS AND METHODS: This study included 5408 children from 11 schools in Delhi, 5682 from 75 schools in Mumbai and 957 schoolchildren from Kolkata who were screened for βTT and haemoglobinopathies. These included 5684 children from 75 schools in Mumbai and 5408 children from 11 schools in Delhi. Children were 11-18 years of age of both sexes. The final report is, however, only on 11090 schoolchildren from Mumbai and Delhi as data from Kolkata was restricted both in numbers and objectives and could not be included for comparison. RESULTS: The overall gene frequency of βTT in Mumbai and Delhi was 4.05% being 2.68% and 5.47% in children of the two cities respectively. In Mumbai, the gene frequency was evenly distributed. Majority of the children with βTT from Mumbai were from Marathi (38.9%) and Gujarati (25%) speaking groups. Gene frequency was >5% in Bhatias, Khatris, Lohanas and Schedule Castes. In Delhi, a higher incidence was observed in schoolchildren of North and West Delhi (5.8-9.2%). The schoolchildren of North and West Delhi comprised predominantly of Punjabi origin compared to children in the South of the city (2.2%, 2.3%). When analyzed state-wise, the highest incidence was observed in children of Punjabi origin (7.6%) and was >4% from several other states. Majority of the traits from Mumbai were anemic (95.1% male and 85.6% in female). The prevalence of anemia was lower (62.7% male and 58.4% female) children with βTT from Delhi. This was a reflection of the higher prevalence of anemia in children without hemoglobinopathy in Mumbai than in Delhi. Nutritional deficiency was probably more severe and rampant in children Mumbai. Gene frequency of Hb D was greater in schoolchildren from Delhi (1.1%) than in Mumbai (0.7%). Hb S trait (0.2%) was observed exclusively in children from Mumbai. A low incidence of Hb E trait (0.04%) was seen in children in Mumbai. A higher incidence is reported from the East. The number of cases studied from the eastern region was small as the data from the East (Kolkata) could not be included in the analysis. CONCLUSION: This study comprises a larger number of children studied for the gene frequency of βTT and other hemoglobinopathies from India. Population groups with higher gene frequencies require screening programmes and facilities for antenatal diagnosis as well as increased awareness and educational programmes to control the birth of thalassemic homozygotes. The overall carrier frequency of βTT was 4.05% and reinforces the differential frequency of β-thalassemia trait in schoolchildren from Delhi and Mumbai and the higher incidence of hemoglobin D in Punjabis as reported previously. The birth incidence calculated thereof for homozygous thalassemics would be 11,316 per year which are added each year to the existing load of homozygous thalassemics. This is much higher than the previously reported number of births annually. Hence suitable control measures need to be undertaken urgently in India.

Utility of family studies in diagnosing abnormal hemoglobins/thalassemic states.

Objective. To resolve all indeterminate cases on HPLC screening with the help of family studies and to further confirm the results by genetic analysis. Methods. In our 11 years experience with HPLC at Sir Ganga Ram Hospital, we solved many cases with the help of family studies on parental blood samples in which patient could have possibly been homozygous vs compound heterozygous. Genetic analysis was done on index case as well as on parental samples with ARMS-PCR technique to confirm the results. Results. In 100% of cases, we noted that the diagnosis obtained by family studies was commensurate with that obtained by DNA analysis. Conclusion. In centers, which do not have the facility for genetic analysis, family studies by HPLC can be equally useful.

Beta-thalassaemia mutations in northern India (Delhi).

The present study was undertaken to define beta-thalassaemia mutations prevalent in northern India (Delhi). Forty six children of beta-thalassaemia major and their families were investigated. DNA was extracted from leucocytes and screened for mutations prevalent in the Indian population. These mutations included 619bp deletion, IVS 1-1 (G-T), IVS 1-5 (G-C), frameshift mutations FS 8/9 (+G), FS 41/42 (-CTTT), Codon 16(-C), Codon 15 (G-A), codon 30 (G-C), IVS 1-110 (G-A) and -88 (C-T). 619 bp deletion mutation was detected directly by amplification of DNA by PCR followed by agarose gel electrophoresis. Other mutations were studied by DNA amplification and dot blot hybridization using synthetic normal and mutant oligonucleotide probes labelled at 5' end with gamma-32 P-ATP. Five mutations accounted for all the chromosomes in 46 patients. 619 bp deletion mutation was found to be the commonest mutation (34.8%) followed by IVS 1-5 (G-C) in 22.8 per cent, IVS 1-1 (G-T) in 19.6 per cent, FS 8/9 (+G) in 13 per cent and FS 41/42 (-CTTT) in 9.8 per cent. Nineteen (41.3%) patients were homozygous and 27 (58.7%) double heterozygous for different beta-thalassaemia mutations. This observation of limited number of mutations is significant and will be useful in planning strategies for prenatal diagnosis of beta-thalassaemia in northern India.

ESR and iron status in pregnancy.

ESR (Westergen) correlated significantly with the iron status (as measured by Hb concentration, haematocrit, red cell count, MCH, P/H ratio, serum iron, TIBC and percent saturation of transferrin) in a group of pregnant women (PW) at term. Serum ferritin correlated negatively with the ESR but the correlation was not statistically significant. Serum ferritin levels of < 50 micrograms/L were present in 9 (34.6%) PW with ESR > or = 50 mm 1st hour and 5 (19.2%) PW with ESR < 50 mm 1st hour. The mean ESR in PW was 55.7 (+/- 22.9) and was > or = 50 mm 1st hour in 50% and < 75 mm 1st hour in 82.7%. The difference in the mean ESR in anaemic and nonanaemic PW was highly significant (p < 0.001), 87.5% anaemic PW with serum ferritin > 50 micrograms/L had ESR > or = 50 mm 1st hour, suggesting the possible effect of chronic infection in raising ferritin levels in these PW.

Effect of maternal iron deficiency anaemia on foetal outcome.

One hundred and two pregnant women and their neonates were examined to evaluate the effect of maternal haemoglobin concentration (Hb. conc) and iron deficiency anaemia on the placental weight and the foetal outcome. Haematological and serum ferritin values were determined. It was observed that 34.3% of the pregnant women were anaemic. Maternal Hb conc. and serum ferritin showed a highly significant correlation (r = 0.40, p < 0.001) indicating that iron deficiency was the most important cause of anaemia amongst them. The maternal Hb conc. showed a significant correlation with placental weight (p < 0.05), birth weight (p < 0.01), Apgar score (p < 0.001) and birth asphyxia. Maternal serum ferritin also correlated positively with cord ferritin (p < 0.001). The study did not reveal any association between high Hb and adverse foetal outcome.

Erythrocyte T-activation: clinical importance & recognition by a simple routine technique.

Erythrocyte T-activation is reported in association with bacterial infections. Although it is an unfrequent phenomenon, it has been reported in cases of septicaemia and necrotizing enterocolitis. It is important to recognize these cases as, transfusion of blood & blood products and lead to haemolytic--transfusion reactions. Here we report a case of T-activation detected during routine immunohaematological procedure in the blood transfusion laboratory. This also emphasizes the role of a routine, cost-effective test to diagnose cases of T-activation.

Computer based application software for histopathological reporting system.

For better follow up of patient and the immediate retrieval of records, we have developed a computer based application software for histopathological reporting system (HIPRIS). With its help, among others, we can (i) retrieve the biopsy report of a patient from the accession number of the specimen; (ii) find out the number of cases for a particular period as well as can analyse cases by any relevant referral parameter like department, specialty and disease and (iii) find out the time gap between receiving the specimen and reporting of result. Our experience suggests that this system greatly improves the efficiency of the histopathological laboratory.

Effect of leukaemic sera & cell-extracts on splenic colony counts (CFU-S).

Sera and leukaemic cell extracts from patients of acute leukaemia were evaluated for their effect on the repopulating ability of the pluripotent stem cells and erythroid differentiation by an in vivo splenic colony count (CFU-S) technique. Normal donor marrow cells of mice were treated with sera and cell extracts from patients of acute leukaemic and healthy controls and injected in the recipient mice. The CFU-S performed on the seventh day to assess repopulating ability of the stem cell showed consistently lower CFU-S counts in the test groups, with leukaemic sera (P less than 0.01) as well as leukaemic cell-extracts (P less than 0.001). The erythroid differentiation assessed by 59Fe uptake by the spleens also showed significantly reduced counts in the two test groups (P less than 0.01 and less than 0.001 respectively). The results indicate that both leukaemic sera and cell-extracts exert a significant suppressive effect on the repopulating ability of the stem cells and on their erythroid differentiation.

Light & electron microscopy of proliferating oval cells during early chemical hepatocarcinogenesis.

Early hepatic changes were studied in male albino rats (70) of Sprague Dawley strain fed a choline devoid diet containing 0.05 per cent w/w AAF (2-acetylaminofluorene) for 12 days. Proliferating periductal and ductal cells appeared in the portal area on days 1 and 3 respectively in the experimental group. On day 7, these cells infiltrated within the sinusoids of adjacent lobules up to the first one or two layers of hepatocytes. Subsequently, these cells extended up to the midzonal region on day 21 and by day 24 the entire lobule was infiltrated. Formation of duct like structures by the proliferating cells was seen on day 21. Ultrastructurally both periductal and ductal cells showed only a few organelles. Periductal and ductal cells are the earliest cells to appear in the portal area in chemically induced hepatocarcinogenesis. Its undifferentiated ultrastructure, may suggest the stem cell nature of these cells.

The effect of serum from patients of chronic renal failure on colony forming units (CFU-S) in mice.

Repopulating ability of mouse bone marrow stem cells, treated with pre-dialysis, post-dialysis and control sera was assessed by colony forming units (CFU-S). Significant lower colony counts were observed in pre-dialysis group as compared to control group. There was an improvement in the colony counts when the cells were treated with post-dialysis sera. The study suggests the presence of inhibitor/s of CFU-S in the uraemic sera which is/are partially removed by haemodialysis.

Glucose-6-phosphate dehydrogenase deficiency & neonatal hyperbilirubinaemia.

Seventy four neonates with hyperbilirubinaemia and 47 non-jaundiced (control) neonates were studied for evidence of G6PD deficiency by spectrophotometric assays and 3 screening tests viz., ascorbate cyanide test, methaemoglobin reduction test and fluorescent spot test. The incidence of G6PD deficiency was significantly higher (P less than 0.001) in the hyperbilirubinaemic neonates (35.1%) as compared to non-jaundiced neonates (6.4%). The G6PD levels in the hyperbilirubinaemic neonates were significantly lower than in the non-jaundiced neonates (P less than 0.05). In 9(12.2%) hyperbilirubinaemic neonates G6PD deficiency was present, without evidence of any other factor known to cause hyperbilirubinaemia. Results of screening tests were essentially similar. However, even with all the screening tests, 11 of 29 neonates (37.9%) with mild G6PD deficiency were not detected showing the limitation of these tests.

Serum & cerebrospinal fluid ferritin levels in children with acute leukaemia.

Serum and CSF ferritin were estimated in 35 consecutive patients of acute leukaemia at the time of admission and on induction of remission. Serum ferritin levels were significantly raised in 94 per cent patients of acute leukaemia. The mean (+/- SD) serum ferritin (314.36 +/- 158.4 micrograms/1) was significantly higher when compared with control values (P less than 0.001). Remission induction resulted in significant fall in serum ferritin values to a mean of 149 (+/- 98.7) micrograms/l (P less than 0.05). Serum ferritin is thus of value in assessing the state of remission and is a sensitive indicator of the leukaemic cell mass and the state of activity of the disease. CSF ferritin levels in acute leukaemia were comparable to normal control values. CSF ferritin did not reflect CNS involvement in acute leukaemia and therefore its value as a tumour marker of CNS infiltration is doubtful.

Synergistic promoter effect of diethylstilbesterol & phenobarbitone in diethylnitrosamine induced hepatic neoplasia in rats.

An experimental model was designed to study the role of both diethylstilbesterol (DES) and phenobarbitone (PB) singly or in combination, in diethylnitrosamine (DEN) induced hepatic neoplasia. Experimental and control rats were injected DEN (200 mg/kg) or saline, ip. Acute morphological changes were studied at days 1, 2 and 3; and at weekly intervals for 3 wk. Four weeks after DEN pretreatment the experimental and control rats were randomized into various groups and fed DES (T1), PB (T2) or a combination of both DES and PB (T3). Five rats from each experimental group were sacrificed at 10, 20 and 30 wk. Group T3 showed gross nodules with a mean nodule score of 20.5 mm at 20 wk. Nodule score in T1, T2 and T3 at 30 wk were 7, 9 and 34.5 mm respectively. The sequential morphological lesions encountered were clear cell and acidophilic foci; acidophilic, basophilic and mixed nodules. Haemorrhage within the nodules was frequent when DES was administered either alone or in combination with PB. Oval cell proliferation and cholangiocellular lesions were produced in all experimental groups. Foci and nodules generally showed loss of glucose-6 phosphatase, adenosine triphosphatase and invariable presence of gamma-glutamyl transpeptidase and glycogen. A combination of DES and PB as promoter yielded earliest and highest nodule score. This suggests that DES and PB acted synergestically as promoters or that PB caused enzyme induction thereby enhanced the promotive effect of DES.

Supplementary iron dose in pregnancy anemia prophylaxis.

This study was conducted to determine the optimum dose of supplemental iron for prophylaxis against pregnancy anemia. One hundred and ten pregnant women were randomly allocated to three groups: Group A receiving equivalent of 60 mg, group B 120 mg and Group C 240 mg, elemental iron as ferrous sulphate daily; the content of folic acid was constant in all the three groups (0.5 mg). These women had at least consumed 90 tablets in 100 +/- 10 days. Blood was drawn at the beginning and at the end of the treatment. Fifty percent were anemic (less than 11 g/100 ml). The hemoglobin levels rose similarly in all groups and the differences were statistically not significant. Fifty-six percent had depleted iron stores (serum ferritin value less than 12 micrograms/l) at the beginning of the study. Following therapy a statistically significant increase in iron stores was observed in group B and C as compared to group A. The difference between group B and C was not significant. The side effects increased with increasing doses of iron; 32.4%, 40.3% and 72% in group A, B and C respectively. Based on these findings, the authors advocate that optimum dose of iron should be 120 mg instead of 60 mg as is currently being used in the National Nutritional Anemia Prophylaxis Programme.