Varied distribution of RhD epitopes in the Indian population.

BACKGROUND: Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. METHODS: Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. RESULTS: No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. CONCLUSION: We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

Iron status of Hindu brahmin, Jain and Muslim communities in Surat, Gujarat.

AIM OF THE STUDY: To determine iron status of healthy, unrelated Brahmin, Jain and Muslim participants having different dietary habits. METHODOLOGY: Control participants other than above three communities, consumed vegetarian or non-vegetarian diet. Brahmin and Jain were strictly vegetarian but Jain did not consume roots or tubers. Muslims consumed non-vegetarian food. Standard techniques were used to measure hematological parameters, serum iron, total iron bindings capacity (TIBC), serum ferritin, transferrin and transferrin saturation. For statistical evaluation mean, standard deviation, pair t test, χ2 and ANOVA (F test) were employed. RESULTS: Study includes 565 male and 198 female children and adults. Among them 205 were children and remaining adults. All four categories i.e. control, Brahmin, Jain and Muslims showed higher incidence of anemia and iron deficiency in females compared to males. Mean values of hematological parameters did not vary significantly in four groups. Serum iron, TIBC, transferrin and ferritin levels indicated iron deficiency anemia more frequently in Jains and less frequently in Muslims (p<0.05). Iron status of Brahmin was comparable with controls (p<0.01). Majority of the participants had serum ferritin concentration >15 ng/mL. Except one male Jain child none of the participants had serum ferritin concentration <12 ng/mL. Jain subjects more frequently had serum iron concentration <60 µg/dL. CONCLUSION: Jain participants had higher incidence of iron deficiency anemia. Vegetarian diet consumed by Gujarati Hindu Brahmin community provided them with a sufficient iron to maintain their iron profile like Muslims consuming non-vegetarian diet.

Production of murine monoclonal anti-B.

BACKGROUND & OBJECTIVE: Monoclonal antibodies against red blood cell antigens used in research and as diagnostics in India are commercially procured from western countries. Indigenously generated potent clones are not available in India. Hence, the objective of the present study was to raise potent murine monoclonal antibodies against A, B and H blood group antigens indigenously and establish a stable clone of anti-B secreting cells. METHODS: Spleen cells of female BALB/c mice immunized with B group red blood cells were fused in presence of polyethylene glycol (PEG) 1500 with a mouse myeloma cell line Sp 2/0 Ag. 14 in hypoxanthine aminopterine thymidine (HAT) selective medium and incubated at 37 degrees C, 5 per cent CO(2) and 95 per cent humidity for a week. RESULTS: The culture supernatant of the wells showing anti-B activity, were further subcloned and a clone 2C4D5F10 was generated which showed a good potency, avidity and specificity. INTERPRETATION & CONCLUSION: The anti-B clones thus produced indigenously provided a useful reagent in blood group typing. The unlimited availability unlike polyclonal antisera makes this reagent more cost-effective. It also ensures a regular supply with the similar specificity.

Role of HLA antigens in Rh (D) alloimmunized pregnant women from Mumbai, Maharashtra, India.

Immunogenetic studies in various diseases provide potential genetic markers. We have studied the incidence of HLA A, B, C, DR and DQ loci antigen in Rh (D) antigen isoimmunized mothers compared to those nonimmunized isoimmunized Rh negative mothers. Seventy six mothers who were immunized to Rh (D) antigen due to pregnancy (responders) and fifty four mothers who did not develop Rh (D) isoimmunization despite positive pregnancies (nonresponders) were selected for the study. Standard methods of serological HLA typing, ABO and Rh (D) groups, and screening for Rh D antibodies were used. 392 unrelated individuals from the population were compared as controls. In addition 45 unrelated individuals from the same population were typed for HLA DRB and DQB gene using PCR-SSP kits. The genotype frequencies of HLA A2, A3, A28, B13, B17, B35, B52, B60, Cw2, Cw6, DR4, and DQ3 were significantly increased, while the frequencies of the HLA A11, A29, A31, B7, B37, B51, Cw1 and DR9 were decreased in the responder women when compared to the non-responder women. HLA A30 (19) split antigen was not identified in immunized women while HLA A23 (9) split antigen was not identified in non immunized women. HLA A3, B17, Cw2 and DR4 showed a significant relative risk among the immunized responder women. When compared with Rh immunized women (responders) reported from USA, England and Hungary the phenotype frequencies of HLA A11, A24, A28, B5, B17, B40, DR2 and DR5 were increased while HLA A23, B8, B18, and DR6 were decreased in the Indian Rh immunized women. Two locus haplotype frequency analysis observed among the responders women revealed that among the significant haplotypes expressed A2-B5, B7-Cw1, DR2-DQ1 were highly significant haplotypes in positive linkage, while A1-B5, and A1-B7 were in significant negative linkage disequilibrium. The haplotype frequencies were

Experience with intrauterine transfusions for severe Rh alloimmunization in a developing country.

This study reports our experience with 67 intrauterine transfusions (IUTs) carried out for 27 cases of severe Rh alloimmunization, which could be useful to other developing countries with similar situations. Most of the mothers were from sections of India other than Mumbai, their socioeconomic status was low, and they were referred during the second or third trimester. The mean gestation age at first IUT was 27+/-2.9 weeks and maternal anti-D titer ranged from 1:32 to 1:512. Ultrasonography (USG) was normal in eight cases, but showed minimal or gross ascites in 8 and 11 cases, respectively. The mean +/- SD hematocrit (HCT) in three groups defined by USG was 23.5+/-1.7, 15.9+/-4, and 12+/-5.9, respectively. Amniotic fluid analysis, which proved to be an important investigation, indicated IUT in eight cases having normal USG. Six cases were severely anemic (Hb deficit >7 g/dl). By fetal cell staining, the percentage of the donor's red cells in the fetal circulation was determined. Besides Hb, blood group, direct antiglobulin test, and mean cell volume, this parameter was also useful in assessing efficacy of IUT and the need for an exchange transfusion after birth. Of 11 fetuses having gross ascites, eight and one each from the remaining two groups, were stillborn. One death may be procedure related. Two neonates died due to hemorrhagic disorder and prematurity. The overall survival rate was 55.6%. Late referral, severe Rh alloimmunization, volume overload, delay in IUT because of nonavailability of blood and use of nonirradiated blood could be the reasons for the poor outcome. Strategies for improving results are discussed.

Viral infections in newborns through exchange transfusion.

Preprocedure sera of thirty one neonates requiring exchange transfusion were tested for serological markers of HBV, HCV, CMV, HIV and LFT. All the babies were investigated for these parameters one week and two months after transfusion to evaluate the risk of transmission of viral infection. Serological markers for these viral infections were also studied in the mothers and donors' blood to establish the route of infection. Donors' blood used for transfusion was pretested for HBsAg, VDRL and anti-HIV. HBsAg was detected one week post exchange in one baby and two months post exchange in two babies. Exchange transfusion was implicated in two of them, where one donor had HBsAg and the other anti-HBc. Vertical transmission accounted for the remaining one. Out of these HbsAg positive cases, one showed evidence of recently acquired CMV infection. Vertical transmission of anti-HCV was observed in one case. None of the neonates, mothers and donors were positive for anti-HIV. In view of probable serious consequences of HBV and HCV infections, blood used for exchange transfusion ought to be screened for anti-HBc and anti-HCV, besides routine HBsAg, VDRL and anti-HIV screening.

Role of C1q in Rhesus haemolytic disease of the newborn.

Present study attempts to find out if maternal anti-D can bind the first complement component C1q and its impact on phagocytosis and severity of Rh haemolytic disease of the newborn (HDN). One hundred Rh immunised women were enrolled, however six having Rh(D) negative infants were excluded. Immunometric assay revealed that of 94 sera, 18 (19.1%) were able to bind C1q but failed to bind C3. Six mothers had anti-C (two bound C1q) and one had anti-E (C1q nonbinding) in addition to anti-D. Various characteristics of anti-D like titre, concentration, IgG subtypes and phagocytic activity showed comparable results (P > 0.3) in C1q binding and nonbinding groups. No significant difference in the severity of Rhesus haemolytic disease of the newborn (Rh HDN), judged by the outcome of pregnancy, cord blood haemoglobin, peak and pre exchange transfusion (ET), indirect serum bilirubin and requirement of ET, was observed in these two groups. Hence this study suggests that though some anti-D sera can bind C1q, there is no further activation of complement pathway and the severity of Rh HDN is not influenced by this phenomenon.

Antibody dependent cell mediated cytotoxicity and erythrophagocytosis assays in Rh haemolytic disease of the newborn.

OBJECTIVE: To evaluate the role of antibody dependent cell mediated cytotoxicity (ADCC) and erythrophagocytosis in comparison to IgG subtypes and concentration of anti-D in haemolytic disease of the newborn (HDN). DESIGN, SETTING AND PATIENTS: One hundred Rh (D) immunised women attending an antenatal clinic of Nowrosjee Wadia Maternity Hospital at 32 to 34 weeks of gestation. Results of 90 women having Rh (D) positive infants were correlated with severity based on outcome of pregnancy, cord blood Hb and treatment given to infant. MEASUREMENTS: Immunoradiometric assay (IRMA) was used for anti-D quantitation. In ADCC assay 51Cr release in the culture supernatant was measured, and in the phagocytosis assay lytic activity was measured on spectrophotometer. Cord blood monocytes were used as effector cells in both the assays. RESULTS: Good correlation (P < 0.01) was observed between ADCC vs IRMA and ADCC vs phagocytosis. ADCC% specific lysis was significantly higher and still-birth rate was increased when mothers had IgG1 + IgG3 type of anti-D. Though all variables showed significant correlation (P < 0.01) with severity, ADCC assay was the most predictive. Cord blood haemoglobin showed a significant inverse correlation with ADCC and phagocytosis assays. CONCLUSION: In the absence of a cordocentesis facility severity of Rh HDN could be reliably judged by ADCC assay. If an isotope laboratory is not available, then instead of ADCC, phagocytosis assay may be employed.

Role of antibody dependent cell mediated cytotoxicity in ABO hemolytic disease of the newborn.

Study includes fifty O blood group mothers delivering A or B group infants suffering from jaundice and a control group consisting of thirty one O group mothers of non-jaundiced infants. Lytic ability of maternal IgG anti-A/anti-B was determined by 51Cr ADCC assay in which cord blood monocytes were used as effector cells. In control series mean% specific lysis (SL) was 18 +/- 3.1 for IgG anti-A and 17.9 +/- 3.1 for IgG anti-B. In jaundiced series IgG anti-A was more lytic than IgG anti-B. However, the increase in ADCC lysis was statistically insignificant. Even though > 1:32 titre was more often associated with > 35% SL, in general the immune A/B antibody titre showed poor correlation with ADCC lysis. Majority of the severe ABO-HDN cases had > 35% ADCC lysis.

Fetal outcome following intrauterine intravascular transfusion in rhesus alloimmunization.

The outcome of 14 pregnancies with severe rhesus alloimmunization was analyzed over a period of 16 months. Group A consisted of 7 cases who received ultrasound guided intravascular intrauterine packed red blood cell transfusions via the umbilical vein after determining fetal blood group and hematocrit. The outcome of these cases was compared with another 7 cases (Group B), who did not require intrauterine transfusions. The 7 cases in Group A received a total of 25 intrauterine transfusions between 25 to 33 weeks gestation. Procedure related complications encountered were transient fetal bradycardia on 4 occasions, difficulty in cord cannulation due to fetal movements in 2 cases and transient bleeding at puncture site in 2 cases. These complications were not associated with any maternal or fetal consequences. There was no procedure related mortality. Mean cord hemoglobin in Group A (12.52 g/dl) was significantly higher (p < 0.05) than in Group B (8.5 g/dl), and mean cord indirect serum bilirubin was significantly lower (p < 0.1) in Group A (2.5 mg/dl) than in Group B (5.8 mg/dl). Three neonates in Group A required one exchange transfusion each, as compared to all 7 in Group B who required a total of 12 exchange transfusions. All neonates in Group B survived, whereas 2 expired in Group A, one of severe intravascular coagulopathy and the other due to prematurity and hyaline membrane disease. Percutaneous ultrasound guided umbilical blood transfusions directly into the vascular system appears to be safe in experienced hands and has the potential to improve the prognosis of the severely alloimmunized fetus.

Incidence of Rh immunization between 1981 and 1992.

BACKGROUND: The Institute of Immunohaematology has had an Rh clinic at the Nowrosjee Wadia Maternity Hospital since 1957. Between then and 1977, 5% Rh-negative women produced Rh antibodies. Between 1978 and 1980, the rate of immunization was reduced to 3.3%. In the present study we determined the incidence of Rh immunization between 1981 and 1992. METHODS: ABO and Rh grouping was carried out by standard methods on 139,635 samples collected from antenatal women. Six thousand nine hundred and fourteen (5%) Rh-negative women were screened for Rh antibodies using an enzyme technique. Analysis was carried out by dividing the data into four groups, each of three years duration. RESULTS: The number of women attending the antenatal outpatients increased steadily but the frequency of Rh-negativity remained at approximately 5%. The rate of Rh immunization among Rh-negative women declined from 3.1% during 1981-83 to 1.7% during 1990-92. The rate among pregnant women dropped from 0.16% to 0.09% and this was statistically significant (p < 0.05). The incidence of Rh immunization among Rh-negative women increased from 0.4% in the first pregnancy to 17.5% after four or more pregnancies (p < 0.01). Only 15 out of 167 Rh immunized women had received anti-D immunoglobulin previously. CONCLUSIONS: The incidence of Rh immunization has declined during the last 12 years possibly due to family planning and extensive use of anti-D immunoglobulin. However, complete eradication of Rh haemolytic disease of the newborn will be possible only if a comprehensive Rh prophylaxis programme is instituted.

Distribution of IgG subtypes in maternal anti-D sera and their prognostic value in Rh haemolytic disease of the new-born.

In 107 Rh(D)-immunized women having Rh(D)-positive pregnancy screening for IgG subtypes was carried out between the 34th and 36th week of gestation. Using polyclonal IgG subtype-specific reagents, all four IgG subclasses were detected in anti-D sera though IgG1 and IgG3 were the most predominant classes. IgG3 anti-D was usually low titre. At the same level of Rh(D) antibody titre, haemolytic disease of the new-born was more severe when anti-D was IgG1 type than IgG3 type. When the IgG1 and IgG3 anti-D subtypes existed together, the risk of having a stillborn child was very high compared to other subtypes (IgG1 + IgG3 34.8%, IgG1 19.2% and IgG3 15.4%).

Quantitation of HLA antigens on red cells by immunoradiometric assay.

Immunoradiometric assay was employed to quantitate HLA antigens on red cells. Using this technique HLA-B7, HLA-B17 and HLA-A28 were detected on the red cells of all individuals studied irrespective of the serological status of the Bennett-Goodspeed (Bg) antigens. However, HLA antigenic sites for serologically Bg positive red cells were significantly more than that for Bg negative red cells (P less than 0.001). Bga positive red cells possessed maximum number of antigenic sites as compared to Bgb and Bgc positive red cells.

Quantitation of Ina blood group antigens.

Using indirect immunoradiometric assay the mean (+/- SD) number of Ina antigenic sites per erythrocyte was estimated to be 73857 +/- 18285 in normal adults and 20142 +/- 8571 in newborn infants. Pregnant women showed temporary reduction in Ina antigens (28144 +/- 13857), which returned to normal levels, 3-6 months after delivery. Ina antigens were shown to be denatured by papain treatment of red cells, proving it to be of glycoprotein nature.

Clinical significance of serum and cerebro spinal fluid bilirubin indices in neonatal jaundice.

To assess the value of unbound bilirubin (UB) and saturation index (SI) in serum and CSF as indicators of Kernicterus, we studied 50 icteric neonates (serum indirect bilibrubin (IB) greater than or equal to 7 mg/dl) and 20 controls (IB less than 7 mg/dl) during the first week of life. Serum and CSF were obtained simultaneously in all neonates. Of 36 neonates with IB greater than 12 mg/dl 19 had evidence of kernicterus. UB was estimated by Sephadex gel filtration and SI by salicylate displacement technique. Positive correlation (r = +0.85) was obtained between serum and CSF UB levels. There was a significant difference (p less than 0.05) between mean serum and CSF UB levels in kernicterus and non-kernicterus neonates (kernicterus serum UB = 0.71 +/- 0.22) mg/dl, CSF UB = 0.16 +/- 0.06 mg/dl: non-kernicteric serum UB = 0.40 +/- 0.10 mg/dl, CSF UB = 0.10 +/- 0.03 mg/dl). A critical serum UB level 0.5 mg/dl and a danger zone of CSF UB (0.1 to 0.15 mg/dl) was observed in presence of kernicterus. Neonates with kernicterus and 30% non-kernicteric had serum SI greater than or equal to 8. Mean values of serum and CSF SI were comparable in all neonates. The serum and CSF UB and SI, and the mean percentage cross over of UB from serum to CSF when statistically compared were not significantly influenced by risk factors.