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BACKGROUND: ABO blood group iso-antibodies are naturally occurring antibodies found in serum and other body fluids. METHODS: Serum, saliva and milk samples from 5 mothers identified as "Bombay" phenotype were tested for ABH-iso-antibodies by routine serological techniques. RESULTS: All the five mothers showed presence of iso-antibodies in the samples tested. Higher titer values in milk than their serum were observed on subjects whose samples were collected in immediate post-partum phase as compared to those whose samples were collected after a lapse of a few months. CONCLUSION: High titer iso-agglutinins against ABH antigens were detected in milk samples besides their presence in saliva as well as serum.

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BACKGROUND & OBJECTIVE: The antigen H present on the surface of red cells in varying concentration, is maximum in O group red cells, but absent in Bombay phenotype individuals. This differentiation is generally detected by seed extracts of Ulex europaeus. The titre of such an extract is usually low and is subjected to batch variation. Hence, we carried out this study to raise potent murine monoclonal antibody against H antigen. METHODS: Spleen cells of female BALB/c mice immunized with O group red cells were fused in presence of polyethylene glycol (PEG) 1500 with a mouse myeloma cell line Sp2/0 Ag14 in hypoxanthine aminopterine thymidine (HAT) selective medium and incubated at 37 degrees, 5 per cent CO(2) and 95 per cent humidity for a week. RESULTS: The culture supernatants showing anti-H activity, were further subcloned and two clones 3E8A10 and 3E8A11 generated which showed a good potency, avidity and specificity. INTERPRETATION & CONCLUSION: The anti-H clones thus produced indigenously provided a potent reagent in distinguishing normal O group from Bombay phenotype individuals. The unlimited availability makes this reagent cost-effective to ensure a constant supply of hybrid clones with the similar specificities.

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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.

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While studying the serologic characteristics of certain monoclonal blood group antibodies, we observed a hybridoma clone (5A-11E10) with anti-N-like serologic specificity that was dependent on the presence of the bicarbonate anion. The diluted cell culture supernatant preferentially agglutinated M-N+ RBCs by immediate spin. This supernatant also agglutinated M-N+ RBCs that had been treated with trypsin or sialidase (to remove N-reactivity), suggesting anti-'N' activity. Anti-'N' specificity was confirmed by the supernatant's non-reactivity with N+ RBCs treated with papain (to remove 'N' reactivity) or with ('N'-negative) M+N-U- RBCs. The requirement for bicarbonate in the MoAb's formulation was not a function of pH. Both sodium and ammonium bicarbonate supported agglutination, but neither sulfate nor carbonate was effective.

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BACKGROUND: Anti-neutrophil cytoplasm antibodies (ANCA) play an important role as specific and sensitive markers for small vessel vasculitis and in some other systemic disorders. Indirect immunofluorescence test, known as the "Gold Standard" for screening of ANCA, can be further substantiated by ELISA for confirmation and for identifying sub-specificities like anti-Myeloperoxidase (anti-MPO), anti-Proteinase 3 (anti-PR3) and anti-Lactoferrin (anti-LF). AIMS: The present study was undertaken to investigate the incidence, specificities and strength of ANCA in suspected vasculitis cases and to correlate their presence with that of these auto-antibodies and with the disease. SUBJECTS AND METHODS: Sera from 130 clinically suspected vasculitis patients were studied. Indirect immunofluorescence microscopy (IIF) was used to identify cytoplasmic (c-ANCA), perinuclear (p-ANCA) and atypical (X-ANCA) patterns using ethanol and formalin fixed polymorphonuclear cells (PMN) and HL-60 cells from a human promyelocytic leukaemic cell line as substrates. ELISA was performed for identifying ANCA sub-specificities to anti-MPO and anti-PR3 and HEp-2 cells were used for detection of anti-nuclear antibodies (ANA). RESULTS: ANCA positivity was noted in 42.3% of these patients, wherein p-ANCA positivity rate was 34.6% and c-ANCA positivity was noted in 5.4% subjects. Three patients showed the unusual X-ANCA positivity. ELISA determined the sub-specificities: Out of 45 p-ANCA positive patients, 38 patients (84.4%) had anti-MPO and out of 7 c-ANCA positive patients, 5 patients (71.4%) had anti-PR3 antibodies. One patient with Class IV Lupus Nephritis, showed both anti-MPO and anti-PR3 antibodies and 17.8% p-ANCA positive patients had anti-Lactoferrin antibodies. CONCLUSIONS: Use of the Immunofluorescence method coupled with identification of ANCA sub-specificities by ELISA, is recommended for detection of ANCA in clinically suspected cases of small vessel and other systemic vasculitis.

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Various autoantibodies like anti-nuclear antibodies (ANA), anti-double stranded DNA (anti-dsDNA), anti-histone antibodies (AHA), anti-neutrophil cytoplasmic antibodies (ANCA), anti-myeloperoxidase (anti-MPO), anti-proteinase3 (anti-PR3) and anti-lactoferrin (anti-LF) antibodies were studied in 173 acute hospitalised patients suffering from malaria of which 160 patients had P. falciparum and remaining 13 had P. vivax infection. Standard methods like indirect immunofluorescence (IIF) microscopy along with Confocal microscopy and ELISA were used for identifying and quantifying the autoantibodies and IIF patterns on PMN and HL-60 cells were studied for ANCA classification. Also HEp-2 cells were used for ANA detection, while estimation of anti-dsDNA, AHA, anti-MPO, anti-PR3 and anti-LF were tested using ELISA. Sera from malaria patients showed prominent immunofluorescence staining patterns where 23.8% cases had ANA in P. falciparum group as compared to 15.4% in P. vivax group and ANCA was found to be present in 20% in P. falciparum and 15.4% in P. vivax group. An interesting observation was that, of the total ANCA positives, 59% had p-ANCA, 5.9% had c-ANCA and 44.1% of the cases showed the 'atypical' or X-ANCA pattern. When p-ANCA positivity was compared with c-ANCA positivity among these patients, a good statistical correlation was noted with OR = 16, chi 2 = 16.43, EF = 0.46 and p-value = 5.037E 0.5. ELISA showed 31.2% anti-MPO and 6.2% anti-PR3 in P. falciparum cases while the two ANCA positive cases in P. vivax had anti-MPO. Anti-LF was found to be present in 40.6% cases. Neither the P. falciparum nor P. vivax contained autoantibodies with specificities similar to the characteristic lupus autoantibodies such as double stranded DNA (dsDNA). ANCA positivity develops in some types of malarial infection also with the presence of various autoantibodies which is important from a clinical point of view and should be carefully evaluated in those geographic areas where malaria is endemic. It also alerts us to the fact, whether in cases of repeated malarial infections in susceptible individuals, vasculitic disorders, which through ANCA pathways develop, could lead to renal and other complications.

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A 28 year old female patients presented with refractory anaemia since childhood and recurrent still-births at 28-30 weeks of gestation. One still-born child was hydropic at birth. Bone marrow showed characteristic morphological changes of congenital dyserythropoietic anaemia (CDA)-Type III. Electron microscopy showed disruption of the nuclear membrane, spongy appearance of nuclei, stacks of microtubules in intermediate normoblasts and myelin figures in erythroid cells. In vitro culture and karyotype data from the bone marrow of the patient is presented. Recurrent still-births in association with congenital dyserythropoietic anaemia has rarely been reported in the literature.

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BACKGROUND: Malaria caused by Plasmodium vivax and Plasmodium falciparum is common in the Indian subcontinent. Studies conducted elsewhere have suggested that malarial infection causes intense immunostimulation. We screened patients with malarial infection for autoantibodies and measured the immunoglobulin, circulating immune complex and complement levels to determine the extent of immunological alterations in these patients. METHODS: One hundred adults with acute malarial infection confirmed by examination of the peripheral blood smear and 25 age- and sex-matched controls were studied. An autoantibody screen and serum immunoglobulin complement (C3 and C4) and circulating immune complex levels were measured at the time of admission and 4 weeks after they became afebrile. A direct Coomb's test was also done. RESULTS: Anti-ssDNA, anti-dsDNA and rheumatoid factor were positive at the time of admission in 51, 30 and 38 patients respectively. None of the controls were positive for these autoantibodies except for one who was positive for rheumatoid factor. The IgM, IgG and IgA levels were raised in 16, 25 and 36 patients respectively. Circulating immune complex levels were raised in 32 patients and complement C3 and C4 were low in 8 and 31 patients. Follow up studies at 4 weeks in 19 patients showed that the autoantibodies were negative. However, the immunoglobulin, C4 and circulating immune complex levels remained elevated. Six per cent of patients had a positive direct Coomb's test with reticulocytosis at the time of presentation. CONCLUSION: Acute malarial infection can cause false-positive results for anti-ssDNA, anti-dsDNA and rheumatoid factor and may also cause a rise in the serum immunoglobulin, complement and circulating immune complex levels.

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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%).

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We report transfusion reactions due to acquired anti-IgA antibodies in a 32-year-old woman with acute lymphoblastic leukemia. Previously, such reactions have been reported only after several hundred transfusions. In our patient they commenced within the first few transfusions.

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55 cases of autoimmune haemolytic anaemia with positive direct Coombs' test were investigated for the nature of autoantibodies. The studies involved characterization of the autoantibodies on the basis of their thermal amplitude, serological specificity, nature of immunoglobulin and light chain specificity. The findings showed that the autoantibodies could be classified as cold autoantibody (18 cases), warm autoantibody (5 cases) and biphasic autoantibody which reacted at 37 degrees C as well as at 6 degrees C (32 cases). Further classification of these cases was made on the basis of antibody specificity to blood groups, immunoglobulin type in relation to the optimum temperature of their reaction.

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