Phenotyping and Genotyping of HNA: Prevalence, Risk of Alloimmunization, and HNA Incompatibilities in Indians.

Background: Antibodies to human neutrophil alloantigens (HNA) are involved in the pathophysiology of several clinical conditions including transfusion-related acute lung injury (TRALI), alloimmune and autoimmune neutropenia, and febrile nonhemolytic transfusion reactions leading to neutropenia. The cognate antigens are polymorphic structures expressed on several glycoproteins on the neutrophils, i.e., antigens HNA-1a, -1b, -1c, and -1d on Fc-γ-receptor IIIb; HNA-2 on CD177; HNA-3a and -3b on choline transporter-like protein 2; HNA-4a and -4b on CD11b/αM subunit of the αMß2-integrin (CD11b/CD18, Mac-1, CR3); and HNA-5a and -5b on αL-subunit (CD11a) of the αLß2 integrin (CD11a/CD18), leukocyte function associated molecule (LFA)-1. Currently, there is a lacuna of diagnostic methods for detection of HNA in India. This study aimed to determine the HNA frequencies in Indians, estimate the risk of alloimmunization, and prepare typed neutrophil panels, which can be used to detect HNA antibodies in neutropenia cases. Material and Methods: EDTA blood samples were collected from random 1,054 blood donors. HNA-2 was phenotyped on fresh EDTA samples using FITC labelled monoclonal anti-CD177 by flowcytometry. HNA-1 (FCGR3B) genotyping was carried out by DNA sequencing and PCR-RFLP. Antigens of HNA-3 (SLC44A2) and HNA-5 (ITGAL) were genotyped by PCR-RFLP using TaqαI and Bsp1286I restriction enzymes, respectively, while HNA-4 (ITGAM) was genotyped by PCR-SSP. Results: Allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were found to be 0.433, 0.444, and 0.087, respectively. FCGR3B*01+*02+*03- was the most common genotype (33.78%). Ten individuals showed deficiency of FCGR3B individuals, while 23 showed hyperexpression, i.e., FCGR3B*01+*02+*03+. FCGR3B*04and *05 occurred with a frequency of 0.002 and 0.024. HNA-2 was found to be a high frequency antigen occurring in 98.8% population. Four percent individuals showed atypical expression of CD177 on their neutrophils. Allele frequencies of SLC44A2*01 and SLC44A2*02were 0.812 and 0.188, respectively, and that of ITGAM*01, ITGAM*02, ITGAL*01, and ITGAL*02 were 0.9546, 0.0454, 0.2372, and 0.7628, respectively. Conclusion: This is the first study in India to report the frequencies of HNA among blood donors. Typed neutrophil panels identified in the present study will enable us to investigate suspected cases of immune neutropenia in future.

Red cell antigen phenotypes in blood donors & thalassaemia patients for creation of red cell antigen-matched inventory.

Background & objectives: Patients with thalasssaemia are at a risk of alloimmunization and the presence of RBC alloantibodies further complicates transfusion therapy. Matching for the critical antigens of Rh, Kell, Kidd and Duffy blood group systems has been shown to minimize alloimmunization. The aim of the present study was to create a database of extensively typed donors for clinically significant and common blood group antigens of Rh, Kidd, Kell and Duffy systems for transfusion therapy of multitransfused thalassaemic patients. Methods: Five hundred O group regular blood donors were phenotyped for Rh, Kell, Duffy and Kidd blood group antigens using haemagglutination technique. Eighty four non-alloimmunized and 15 alloimmunized thalassaemia major patients with known antigenic profiles (determined by polymerase chain reaction with sequence-specific primers) were selected for this study. Results: By analyzing antigen profiles of 500 O group regular donors, a database of 193 donors matching perfectly for Rh, Duffy, Kell and Kidd antigens was prepared for 15 alloimmunized patients. For non-alloimmunized 84 thalassaemic patients, a database of 405 donors was created. Interpretation & conclusions: A database of 500 regular blood donors phenotyped for common antigens of Rh, Duffy, Kell and Kidd blood group systems was created, which would be useful in providing extended antigen-matched RBCs for thalassaemia patients. This will improve the quality and effectiveness of transfusion therapy.

Molecular basis of weak D expression in the Indian population and report of a novel, predominant variant RHD allele.

BACKGROUND: 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. STUDY DESIGN AND METHODS: In this study we sought to characterize the molecular bases of weak D expression in Indians. RHD gene in samples presenting with a weak D phenotype by serologic analyses (n = 223) was genotyped by conventional molecular approaches. RESULTS: In addition to referenced and novel single-nucleotide variations, a novel approximately 12-kb duplication event, including Exon 3, was identified predominantly in variant D samples (130/223, 58.3%) and characterized at the nucleotide sequence level. Functional analyses suggested that this genetic variation quantitatively affects the expression of the normal transcript and then subsequently the expression of the normal RhD protein. CONCLUSION: We describe a major novel, variant RHD allele in Indians that can be easily identified routinely by implementing a simple genotyping assay. Although we may consider this variation as a weak partial D variant, further studies and observations are needed to confirm the same. These findings may contribute to improve significantly Rh blood group diagnostics in more than one billion Indians.

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.

Molecular genotyping of clinically important blood group antigens in patients with thalassaemia.

BACKGROUND & OBJECTIVES: In multitransfused thalassaemic patients, haemagglutination fails to phenotype the patient's blood group antigens due to the presence of donor-derived erythrocytes. DNA-based methods can overcome the limitations of haemagglutination and can be used to determine the correct antigen profile of these patients. This will facilitate the procurement of antigen-matched blood for transfusion to multitransfused patients. Thus, the aim of this study was to compare the serological phenotyping of common and clinically important antigens of Rh, Duffy, Kell, Kidd and MNS blood group systems with molecular genotyping amongst multitransfused thalassaemic patients. METHODS: Blood samples from 200 patients with thalassaemia and 100 'O' group regular blood donors were tested using standard serological techniques and polymerase chain reaction-based methods for common antigens/alleles (C, c, D, E, e, Fya, Fyb, Jka, Jkb, K, k, M, N, S, s). RESULTS: Genotyping and phenotyping results were discordant in 77 per cent of thalassaemic patients for five pairs of antithetical antigens of Rh, Duffy, Kell and Kidd blood group systems. In the MNS blood group system, 59.1 per cent of patients showed discrepancy. The rate of alloimmunization among thalassaemics was 7.5 per cent. INTERPRETATION & CONCLUSIONS: Molecular genotyping enabled the determination of the actual antigen profile in multitransfused thalassaemia patients. This would help reduce the problem of alloimmunization in such patients and would also aid in the better management of transfusion therapy.

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.

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: Serologic analysis was carried out by testing with anti-D, -C, -c, -E, and -e in Rhnull individuals and their family members. RH genes were analyzed by standard molecular approaches, including Sanger sequencing and quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments. RHAG gene was investigated by exon-specific PCR amplification and Sanger sequencing. RESULTS: In one family, RHAG gene was found to be deleted at the homozygous state in the propositus, suggesting Rhnull of the regulator type. In the other family, a novel splice site variant in RHCE in cis with whole RHD gene deletion was identified at the homozygous state. Further functional analysis by minigene splicing assay showed that this variation, that is, c.801 + 1G>A, completely impairs normal splicing, then inactivating the expression of RhCE protein. Contrary to the former case, these data suggest Rhnull of the amorph type. CONCLUSION: Overall, we report for the first time the molecular mechanisms responsible for Rhnull phenotype in individuals of Indian origin. This study contributes to extend the molecular spectrum of variations in Rhnull individuals.

Evolution of technology for molecular genotyping in blood group systems.

The molecular basis of the blood group antigens was identified first in the 1980s and 1990s. Since then the importance of molecular biology in transfusion medicine has been described extensively by several investigators. Molecular genotyping of blood group antigens is one of the important aspects and is successfully making its way into transfusion medicine. Low-, medium- and high-throughput techniques have been developed for this purpose. Depending on the requirement of the centre like screening for high- or low-prevalence antigens where antisera are not available, correct typing of multiple transfused patients, screening for antigen-negative donor units to reduce the rate of alloimmunization, etc. a suitable technique can be selected. The present review discusses the evolution of different techniques to detect molecular genotypes of blood group systems and how these approaches can be used in transfusion medicine where haemagglutination is of limited value. Currently, this technology is being used in only a few blood banks in India. Hence, there is a need for understanding this technology with all its variations.

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.

Detection of a rare subgroup of A phenotype while resolving ABO discrepancy.

Weaker subgroups of ABO blood group system give rise to discrepancies between forward and reverse grouping and cause diagnostic difficulties in routine blood banking. Weaker subgroups of A blood group that have been reported so far include A3, Aend, Ax, Am, Ay, and Ael. We report a case of a 54-year-old patient whose red cells showed a discrepancy between cell and serum grouping on initial testing. Serological investigation included absorption elution tests and saliva testing after performing initial blood grouping. Molecular genotyping of the ABO gene was performed by DNA sequencing of exons 6 and 7 of the ABO gene. The serological characteristics of the patient's red cells were similar to Ax subtype. The patient was a secretor and only H substance was present in the saliva. Serum did not show the presence of anti-A1. Molecular genotyping confirmed the ABO status as Aw06/O13. The weak A phenotype identified in the propositus had serological characteristics similar to Ax and showed the ABO genotype Aw06/O13. Although Aw06 allele has been previously reported in the Indian population, this is the first study to report O13 allele in the Indian population.

Heterogeneity of O blood group in India: Peeping through the window of molecular biology.

BACKGROUND: Molecular genotyping of ABO blood group system has identified more than 60 "O" group alleles based on the single-nucleotide polymorphisms present in the ABO gene. Heterogeneity of O group alleles has been observed in various countries from South America, Europe, Middle East, and Asia. India is a vast country with more than 1300 million population which is divided into various ethnic and tribal groups. However, very little is known about the heterogeneity of O alleles in Indians. MATERIALS AND METHODS: A total of 116 O group individuals from the mixed population of Mumbai, India, were enrolled in the present study. DNA was extracted using the standard phenol-chloroform method. The exons 6 and 7 of the ABO gene were genotyped by polymerase chain reaction-single-strand conformation polymorphism and/or DNA sequencing. The genotyping results were compared with our earlier findings. RESULTS AND DISCUSSION: Overall, ten different genotypes were identified. Three rare alleles, namely, O05, O11, and O26 were seen in the mixed group category. These results suggest that there is an internal heterogeneity in the mixed group while Dhodias and Parsis, the groups which were screened earlier, seem to be more homogenous groups. An important piece of information emerges out from this study, that is, O01O02 genotype is expressing some selective force in population groups screened in India as well as many other groups worldwide. CONCLUSION: In the future, molecular genotyping of the ABO blood group system among different ethnic and tribal Indian groups would help in generating data to fill up the gaps in the molecular ABO map of the world.