Swati Kulkarni

Studies on the ABH-Iso-Agglutinins in serum, saliva and milk from mothers with "Bombay" (Oh) phenotype

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.

Production of Murine Monoclonal Antibody to Fetal Hemoglobin

Fetal hemoglobin (Hb F) is a major hemoglobin (Hb) component at birth. Hb F levels are markedly elevated in a number of inherited and acquired disorders. Measurement of Hb F levels is usually carried out by alkali denaturation which is not very accurate for low and high values. An accurate estimation of Hb F, and also of F cells, is desired in many hematological disorders like sickle cell disease, in monitoring the efficacy of hydroxyurea (HU) therapy, to assess feto‐maternal hemorrhage (FMH) during pregnancy and in the postpartum period. We have raised a murine monoclonal antibody to human Hb F, that accurately measures the number of F cells by flow cytometry. The antibody was found to be potent and specific for F cells.

Partial matching of blood group antigens to reduce alloimmunization in Western India

Red blood cell alloimmunization occurs due to the genetic disparity of red cell antigens between donor and recipient. In the present study, we report a spectrum of red cell alloantibodies characterized in patients with different clinical conditions in a reference center in India. Majority of the antibodies identified were against the blood group antigens c, D, E, M, N, S, s and Jka. Hence, apart from ABO and RhD, we recommend partial antigen matching between donor and patients for other Rh (C, c, E, e) and MNS blood group antigens to potentially reduce the risk of alloimmunization by 75%. Matching of Kell antigen is not recommended in Western India.

Noninvasive fetal RHD genotyping from maternal plasma

Alloimmunization to antigens of Rh blood group system is of clinical relevance during pregnancy. Despite the use of antenatal anti-D immunoglobulin prophylaxis, some proportion of RhD-negative pregnant women still become immunized. RhD-negative pregnant women with a heterozygous partner can be reassured and managed less intensively if RhD-negative status of the fetus was confirmed. The conventional techniques used for prenatal testing of fetal RhD status are mainly invasive such as chorionic villus sampling, amniocentesis and cordocentesis, and carry risk of transplacental hemorrhage and pregnancy loss. The discovery of cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new possibilities for noninvasive prenatal diagnosis. With the use of real time polymerase chain reaction technology, circulating fetal DNA has been detected robustly in the plasma of pregnant women, even in the first trimester of pregnancy. Various assays have been developed to confirm the fetal RHD status by targeting the cffDNA using a combination of multiple RHD exons. The commonly used exons are 4, 5, 7, and 10. Common causes leading to discordant results in fetal RHD typing are low fetal DNA concentration due to extraction inefficiency, lack of fetal DNA control, silent maternal RHD alleles, and manual error. False negative results can prove critical in case of alloimmunized pregnancies and hence use of appropriate controls and strict reporting criteria is important to increase the sensitivity of the assay. Owing to the wide genetic variation of the Rh blood group system, the development of fetal genotyping strategies according to ethnic origin of the patients would further increase the sensitivity. Fetal blood group genotyping by noninvasive method is safe, and numerous groups have reported fetal RHD genotyping in D-negative mothers with close to 100% accuracy. Noninvasive fetal RHD typing can be performed in RhD-negative alloimmunized and nonimmunized pregnancies to decide on clinical management and to restrict the anti-D immunoglobulin prophylaxis, respectively. Thus, the introduction of this test for screening all RHD-negative pregnant women is highly desirable.

Molecular genotyping of Indian blood group system antigens in Indian blood donors

BACKGROUND Haemagglutination has been the gold standard for defining the blood group status. However, these tests depend upon the availability of specific and reliable antisera. Potent antisera for extended phenotyping are very costly, weakly reacting or available in limited stocks and unavailable for some blood group systems like Indian, Dombrock, Coltan, Diego etc. The Indian blood group system consists of two antithetical antigens, Ina and Inb. The Ina /Inb polymorphism arises from 252C > G missense mutation in the CD44 gene. This knowledge has allowed the development of molecular methods for genotyping IN alleles. MATERIAL AND METHODS Blood samples were collected from 715 blood donors from Mumbai. DNA was extracted using phenol-chloroform method and genotyping for Indian (Ina/IN*01, Inb /IN*02) blood group alleles was done by Sequence Specific PCR. RESULTS Seventeen donors among 715 were heterozygous for Ina antigen i.e. In (a+b+). The Ina antigen positivity was confirmed serologically, using anti-Ina prepared in-house and the genotype-phenotype results were concordant. The frequency of Ina (2.37%) was higher than Caucasians and comparable to those reported among Indians of Bombay. CONCLUSION This is the first study reporting molecular screening of Indian blood group antigens in Indian population. The frequency of Ina and Inb antigens was found to be 2.37% and 100% respectively. Red cells of Ina positive donors can be used as in-house reagent red cells for screening and identification of corresponding antibodies. Thus, DNA based methods will help in large scale screening of donors to identify rare blood groups, when commercial antisera are unavailable.

RHD -Positive Alleles among D- C/E+ Individuals from India

Background: Molecular bases of blood group systems, including Rh blood group, have been poorly studied in the Indian population so far, while specificities of Europeans, East Asians and Africans have been well known for years. In order to gain insights into the molecular bases of this population, we sought to characterize the RHD allele in D- Indian donors expressing C and/or E antigen(s). Methods:RHD gene was analyzed in 171 serologically D-, C/E+ samples by standard molecular methods such as quantitative, multiplex PCR of short fluorescent fragments (QMPSF) and direct sequencing when necessary. Results:RHD whole gene deletion at the homozygous state was found to be the most common genotype associated with D- phenotype (118/171, 69.0%). Nonfunctional, negative hybrid genes with reported molecular backgrounds were observed in approximately one-third of the samples, while only four samples carry single-nucleotide variations, including one novel nonsense (RHD(Y243X)), one novel frameshift (RHD(c.701delG)), and two missense (RHD(T148R) and RHD(T148R, T195M)) alleles. Conclusion: Overall we report for the first time the molecular bases of D antigen negativity in the D-, C/E+ Indian population, which appears to be qualitatively similar to other populations, but with a population-specific, quantitative distribution of D-- alleles.

Molecular basis of weak D expression in the Indian population and report of a novel, predominant variant RHD allele: MOLECULAR BASIS OF WEAK D EXPRESSION IN INDIANS

The Rh blood group system is the most polymorphic system and is implicated in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. Molecular genetics of the RH genes have been extensively studied in Caucasians, Africans, and East Asians and the variant alleles giving rise to weak and partial D phenotypes have been reported. However, limited genetic studies have been carried out in the large Indian population, even though the variability of Rh expression has been documented.

Screening for DEL phenotype in RhD negative Indians

DEL phenotype represents a very weak form of D variant detected only by adsorption and elution technique. DEL phenotype individuals mistyped as RhD‐negative can lead to alloimmunization after transfusion or pregnancy. Molecular techniques have now been used to identify DEL variants. They are commonly encountered in the East Asian population with RHD(K409K) being the most frequent allele. RHD(M295I) is the most common DEL allele in Caucasians. As there is a paucity of data on DEL phenotype in the Indian population, the study aims to screen RhD negative individuals for two most common DEL mutations.

First report of Rhnull individuals in the Indian population and characterization of the underlying molecular mechanisms

Rhnull phenotype is an extremely rare condition characterized by no expression of Rh antigens at the surface of red blood cells. Although rare, genetic bases of this phenotype are well known and include mutations within either the RH (RHD and RHCE) genes or the RHAG gene. So far Rhnull has been reported in individuals of Caucasian, African, and Asian origin. Here, we report individuals from two families of Indian origin representing such a rare phenotype.

Antigen negative red blood cell inventory of Indian blood donors

BACKGROUND Screening the donor population for clinically important antigens and creating a database of phenotyped donors will eliminate the tedious task of large scale screening for antigen negative units. The aim of the present study is to identify donors lacking common antigens and a combination of common antigens to establish an antigen negative inventory. MATERIALS AND METHODS Blood samples of 1221 regular blood donors were phenotyped for the clinically important common antigens of the Rh, Duffy, Kell, Kidd and MNS blood group systems using standard tube technique. RESULTS Out of 1221 total donors tested, we observed that 261 donors lacked a combination of clinically important common antigens (C, D, e, Fya, Jka, s). After excluding the RhD negative donors in this study 15.56% lacked a combination of two or three common antigens. Of all donors, 3.2% lacked Fya and Jka antigens, 1.96% Fya and s, 1.88% Jka and s antigens and 0.57% lacked three common antigens. DISCUSSION An antigen negative inventory of donors who lack a single common antigen or a combination of common antigens was prepared from regular donors which will prove useful for efficient management of transfusion therapy in patients with multiple antibodies against common antigens.