Human Erythrocyte Antigen Research Articles

Molecular testing in a patient with multiple alloantibodies and warm autoimmune hemolytic anemia: A case study

Abstract Introduction/Objective We present a case of RBC alloimmunization and warm autoimmune hemolytic anemia (WAIHA) in a patient with a recent RBC transfusion. Serological testing followed by molecular testing revealed a rare D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/- predicted phenotype. Methods/Case Report A 75-year-old black male with secondary myelofibrosis presented with a sudden onset of chest pain, dyspnea and weakness during an RBC transfusion. The patient was subsequently admitted for further workup. Labs showed low hemoglobin, low haptoglobin, elevated LDH and a positive direct antiglobulin (DAT) test. The patient was started on intravenous immunoglobulin and steroids for suspected WAIHA. On immunohematology serologic testing, blood typed as B-positive and polyspecific DAT positive with anti-IgG positive and anti-C3 negative. Serum plasma studies showed probable warm autoimmune hemolytic anemia and allo anti-S, -E, and -C by previous history. Eluate studies showed panagglutinin and antibody with D-like specificity. Immucor PreciseTypeTM Human Erythrocyte Antigen (HEA) Molecular BeadChip test predicted a partial e antigen and potential hrB-. Genotyping for RHD variants was performed and revealed RHD*01 (homozygous or hemizygous). Targeted genomic testing did not detect variants associated with common partial or weak RhD antigens. Based on this testing, the patient was not predicted to be at risk of allo-anti-D. An RHCE genotype for C, c, E and e variants including hrB and hrS showed RHCE*ce48C,733G/RHCE*ce733G with predicated phenotype of D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/-. Based on the RHCE testing, the patient was predicted to be at risk for allo-anti-c, -e, -f (ce) and possibly anti-hrB. Results (if a Case Study enter NA) NA Conclusion In this case, serologic testing alone did not reveal a precise diagnosis. Molecular testing aided in the diagnosis of WAIHA and will help guide future transfusions. Pathologists must consider molecular testing when faced with a challenging serologic diagnosis.

A cross-sectional pilot study to estimate the frequency of minor blood group alleles and phenotypes in RhD-negative North Indian blood donors by DNA microarray analysis

Abstract: INTRODUCTION: There are scarce data on Indian blood donors with respect to blood group phenotypes using molecular diagnostic modalities. Hence, we planned to estimate frequencies of blood group alleles/phenotypes using DNA microarray analysis in the north Indian RhD-negative blood donor population. With this initial pilot study, we plan to expand it to our entire donor population. METHODOLOGY: The cross-sectional prospective study was conducted on 50 Indian blood donors (O RhD negative), to study the blood group genotype frequency. Genotyping for the most relevant red blood cell antigens (Rh, Kell, Duffy, Kidd, MNS, Lutheran, and Dombrock) was done using Bioarray Precise TypeHM Human Erythrocyte Antigen BeadChip kit containing probes directed to polymorphic sites. RESULTS: In the Rh system, the most common alleles were RHCE*e/RHCE*e (98%) and RHCE*c/RHCE*c (80%). Phenotype K-k+ (genotype- KEL*02/KEL*02) was seen in 98% of samples, Js(a-b+) (KEL*02.07/KEL*02.07) was detected in 98% (49/50) of the samples tested. Jk(a + b+) (JK*01/JK*02) was the most common phenotype (48%) in the Kidd blood group system. In MNSs system, M+N+ (GYPA*01/GYPA*02) 44% and S+s+U+ (GYPB*03/GYPB*04) 34% were the most common phenotypes detected. CONCLUSION: This pilot study shows the feasibility of genotyping a Northern Indian donor population. To the best of our knowledge, it is the first study on molecular blood grouping in Indian blood donors using the Bioarray platform.

Endothelial Expression of Duffy Antigens in Patients with Fyb-GATA Homozygosity and Suspected Anti-Fy3 Antibody Formation: A Case Series

BACKGROUND The Duffy blood group consists of 6 different antigens expressed on a glycoprotein with 7 transmembrane domains, and is carried on the surface of both erythroid and non-erythroid tissues. The majority individuals of African ethnicity do not express the Duffy glycoprotein on their erythroid cells but appear to be primarily susceptible to alloimmunization against only the FY1 (Fya) antigen. The accepted explanation for this finding is that these individuals are homozygotes for a variant in the FY2 (Fyb) antigen in which a GATA-box mutation in the promoter region specifically prevents erythroid expression; the uninhibited expression of the Fyb antigen on non-erythroid tissues protects against alloimmunization. Occasionally, however, patients with FYB-GATA homozygosity develop clinically significant antibodies against other Duffy antigens, such as FY3 (Fy3), despite the fact that these antigens do not have known antithetical alleles. A mechanistic explanation for this observation is lacking. METHODS From a single center, four adult sickle cell patients with anti-Fy3 were identified, with antibody specificity verified at a reference laboratory. All four individuals had previously undergone human erythrocyte antigen genotyping using the IDCORE XTTM flow bead PCR assay and were found to be FYB-GATA homozygotes, with a predicted RBC phenotype of Fy(a-b-). The development of apparent anti-Fy3 antibodies in the post-transfusion plasma of these individuals prompted the analysis of stored tissue that had been collected for other diagnostic purposes (gall bladder, liver, breast and bone marrow, respectively) for endothelial expression of Duffy antigens. This analysis was performed via IHC using the anti-Duffy monoclonal antibodies 2C3 and MIMA-29. The 2C3-mAb recognizes the Fy6 antigen, a linear epitope on the first extracellular domain region of the Duffy glycoprotein adjacent to the antithetical Fya/Fyb antigens, while the MIMA29-mAb binds to a region in extracellular domain 4 which is thought to carry the Fy3 antigen. IgG isotype antibodies were used as a negative control, and Duffy-antigen expressing RBCs were used as a positive control. RESULTS Both 2C3-mAb and MIMA29-mAb stained Duffy-positive RBCs. No staining was apparent on the fixed bone marrow biopsy with either antibody and may have reflected the paucity of endothelial tissue in the sample. On the gall bladder, liver, and breast biopsies, 2C3-mAb stained endothelium strongly while while MIMA-29 mAb staining was negative. No staining with IgG antibody negative controls was observed in any sample. CONCLUSION The three individuals in this series with interpretable results appeared to have only partial expression of Duffy antigens on non-erythroid tissue. While this expression included the extracellular domain that carries the antithetical Fya/Fyb antigens, it does not include the domain that carries the Fy3 antigen. As there is no known antithetical antigen to Fy3, the most likely explanation is that Fy3 is a conformational antigen that, possibly due to tissue-specific differences in tertiary structure, is only expressed on erythroid cells. It may therefore be concluded that at least some individuals with a Fy(a-b-) phenotype secondary to FYB-GATA homozygosity, while protected against sensitization to the Fyb antigen, remain susceptible to sensitization against the Fy3 antigen.

Prevalence of Rh, Kell, Kidd, Duffy, and MNS antigens in the Hispanic donor population of South Texas.

As population diversity in the United States expands, understanding antigen prevalence by ethnic group is essential. Differences in antigen prevalence among ethnicities have caused increased alloimmunization in chronically transfused patients. Recognizing these differences in patients and donors can reduce the risk of patients developing alloantibodies. Also, determining the antigen prevalence by ethnicity will improve the ability of blood centers to have compatible blood available. Thus far, there has not been significant published data on antigen prevalence of the U.S. Hispanic population. A retrospective cross-sectional study was performed to determine the prevalence of red blood cell (RBC) antigens, as determined by human erythrocyte antigen genotyping, in South Texas Hispanic blood donors. A total of 3455 donors, seen from 1 January 2015 to 31 May 2020, were included in the study. These donors met the inclusion criteria of self-selecting Hispanic ethnicity and successfully donating a RBC component. The antigen results for each included donor were entered into a data collection spreadsheet. The prevalence of each antigen was calculated. A binomial test was performed to determine if the observed results are statistically different as compared with the published prevalence of antigens in white and black populations. After statistical analysis, the p value for most antigens was statistically significant (p < 0.05). The Kidd blood group antigens were the only major antigens that did not show a significant difference. Cohen's h showed a large effect size for most antigens when compared with those of the black population and a small to medium effect size when compared with those of the white population. For most blood groups antigens, their prevalence in Hispanic donors was significantly different than that published for both white and black populations.

Screening of blood donors for sickle cell trait using a DNA-based approach: Frequency in a multiethnic donor population.

Minority RBC donors are important to support the transfusion needs of patients with sickle cell disease. Testing of donors for sickle cell trait (SCT) is performed to avoid transfusion of hemoglobin S+ (HbS+) RBCs to specific patient groups and to investigate leukoreduction failures. A screening assay based on hemoglobin solubility is commonly used. The purpose of this study was to validate a DNA approach for HbS screening. Hemoglobin solubility screening (Pacific Hemostasis or SICKLEDEX) and PreciseType human erythrocyte antigen (HEA)-HbS (Immucor) targeting c.20A>T in the β-globin gene were performed according to manufacturer's directions. Resolution of differences in results included gene sequencing and high-performance liquid chromatography (HPLC). Initial validation of HEA-HbS performed by testing 60 known samples, 20 HbS/A, A/A, and S/S, gave expected results. However, in the subsequent parallel testing phase, 4/58 samples HbS+ by solubility assay tested negative by HEA-HbS; the negative results were confirmed by β-globin gene sequencing. Samples from donors self-identifying as White testing HbS+ by solubility assay (n=60) were retested by HEA-HbS and HPLC. The HEA-HbS assay was concordant with HPLC which is recognized as the gold standard for hemoglobin variation. A DNA-based approach is an alternative to screen donors for SCT, found in approximately 7% of Black and 1.7% of our random donors. HEA-HbS correlated with HPLC results in all samples tested, supporting the use of HEA-HbS as the test of record. The method allows higher throughput screening and testing at the donor center allows association of the screening result with the donor record to avoid repeat testing.

Concordance of two polymerase chain reaction–based blood group genotyping platforms for patients with sickle cell disease

In recent years, polymerase chain reaction-based genotyping platforms, which provide a predicted phenotype, have increased in both patient and high-throughput donor testing, especially in situations where serologic methods or reagents are limited. This study looks at the concordance rate between two platforms commercially available in the United States when used for testing samples from patients with sickle cell disease (SCD), a group particularly vulnerable to alloimmunization. DNA extracted from samples from 138 patients with SCD was tested by human erythrocyte antigen (HEA) BeadChip (Immucor, Norcross, GA) and by ID CORE XT (Progenika-Grifols, Barcelona, Spain). Predicted phenotype results were compared, and a concordance rate was calculated. Discrepancies were resolved by Sanger sequencing. All testing was done under an institutional review board-approved protocol. A concordance rate of 99.9 percent was obtained. Sanger sequencing was performed on four samples with discrepancies in the Rh blood group system. Three samples had a similar allelic variant detected by ID CORE XT. Two of the three discrepant samples were correctly identified as V+w, VS- by ID CORE XT but not by HEA BeadChip. The third sample, predicted to have a phenotype of V+, VS+ by sequencing, was called correctly by HEA BeadChip but not by ID CORE XT, which had predicted V+w, VS-. The fourth discrepancy was identified in a sample that ID CORE XT accurately identified as RHCE*ce[712G] and predicted a partial c phenotype. This result was confirmed by Sanger sequencing, whereas HEA BeadChip found no variants and predicted a c+ phenotype. The high concordance rate of the two methods, along with the known limitations of serology, warrant further discussion regarding the practice of serologic confirmation of extended phenotypes. Clinical significance of the identified discrepancies remains to be determined.

Human erythrocyte antigens in Brazilian Capuchin monkeys (Sapajus sp.).

Investigation of erythrocyte antigens in New World monkeys, especially in the Brazilian ones, is scant and incomplete. Determining the presence of 29 erythrocyte antigens from 11 human blood group systems (ABO, H, Rh, Kell, Duffy, Kidd, Lewis, P, MNS, Lutheran and Diego) on erythrocytes in nine Capuchin monkeys (Sapajus sp.). A majority (20 of 29) of human erythrocyte antigens were not found in this monkey genus. Erythrocyte phenotyping was very similar within this animal group, as five Capuchin monkeys differed from the other four in the ABO system only. The erythrocyte phenotype for this group of animals is less diversified than in humans. Some monkey erythrocyte antigens were similar in frequency, whereas others were different from those observed in human ethnicities.

Hemoglobin A clearance in children with sickle cell anemia on chronic transfusion therapy.

Chronic transfusion therapy for sickle cell anemia reduces disease complications by diluting sickle-erythrocytes with hemoglobin A (HbA)-containing erythrocytes and suppressing erythropoiesis. Minor antigen mismatches may result in alloimmunization, but it is unknown if antigen mismatches or recipient characteristics influence HbA clearance posttransfusion. Children with sickle cell anemia on chronic transfusion therapy were followed prospectively for 12 months. All patients received units serologically matched for C/c, E/e, and K; patients with prior red blood cell (RBC) antibodies had additional matching for Fya , Jkb , and any previous alloantibodies. Patients' RBC antigen genotypes, determined by multiplexed molecular assays (PreciseType Human Erythrocyte Antigen, and RHCE and RHD BeadChip, Immucor) were compared to genotypes of transfused RBC units to assess for antigen mismatches. Decline in hbA (ΔHbA) from posttransfusion to the next transfusion was calculated for each transfusion episode. Sixty patients received 789 transfusions, 740 with ΔHbA estimations, and 630 with donor Human Erythrocyte Antigen genotyping. In univariate mixed-model analysis, ΔHbA was higher in patients with past RBC antibodies or splenomegaly and lower in patients with splenectomy. RBC antigen mismatches were not associated with ΔHbA. In multivariate linear mixed-effects modeling, ΔHbA was associated with RBC antibodies (2.70 vs. 2.45 g/dL/28 d, p = 0.0028), splenomegaly (2.87 vs. 2.28 g/dL/28 d, p = 0.019), and negatively associated with splenectomy (2.46 vs. 2.70 g/dL/28 d, p = 0.011). HbA decline was increased among patients with sickle cell anemia with prior immunologic response to RBC antigens and decreased among those with prior splenectomy, demonstrating that recipient immunologic characteristics influenced the clearance of transfused RBCs.

Prevention of hemolytic disease of the fetus and newborn: what have we learned from animal models?

This review aims to highlight recent advances in our understanding of how anti-red blood cell (RBC) antibodies prevent erythrocyte immunization with an emphasis on new murine models. New murine models with clinically relevant human erythrocyte antigens have been used to understand the alloimmunization process and its inhibition. The search to elucidate the mechanism of action of IgG-mediated inhibition of erythrocyte alloimmunization has provided new evidence in support of a potential role for epitope masking, immune deviation and/or antigen modulation in this process. In addition, recent evidence suggests that blends of monoclonal antibodies targeting nonoverlapping epitopes on the RBC surface can improve the efficacy of monoclonal antibodies approaching that of polyclonal IgG. Animal models with defined alloantigens have helped to identify important mechanistic components that lead to alloimmunization and its inhibition by IgG. A better understanding of the underlying mechanisms leading to hemolytic disease of the fetus and newborn is required to develop the most effective prevention strategies for future patients.

Red blood cell minor antigen mismatches during chronic transfusion therapy for sickle cell anemia.

Red blood cell (RBC) alloimmunization occurs at a high frequency in sickle cell anemia (SCA) despite serologic matching for Rh (C/c, E/e) and K antigens. RBC minor antigen genotyping allows for prediction of antigens and RH variants that may lead to alloimmunization. RBC antigen genotyping was performed on chronically transfused pediatric SCA patients, using PreciseType human erythrocyte antigen (HEA), RHCE, and RHD BeadChip arrays. All patients received C/c, E/e, and K serologically matched units (Category 1); patients with prior RBC antibodies were also matched for Fya , Jkb , and any antibodies (Category 2). The RBC genotypes of all leukoreduced (LR) units transfused over a 12-month period were determined by the prototype HEA-LR BeadChip assay. There were 2320 RBC units transfused to 90 patients in 1135 transfusion episodes. Thirty-five (38.9%) patients had homozygous or compound heterozygous RH variants. Seven new alloantibodies were detected, with alloantibody incidence of 0.706 in 100 units for Category 2 transfusions and 0.068 in 100 units for Category 1 (p = 0.02). Three patients on Category 2 transfusions formed new anti-Jsa and had a higher rate of exposure to Jsa than those who did not form anti-Jsa (20.4 vs. 8.33 exposures/100 units, p = 0.02). The most frequent mismatches were S (43.9%), Doa (43.9%), Fya (29.2%), M (28.4%), and Jkb (28.1%). Alloimmunization incidence was higher in those with prior RBC antibodies, suggesting that past immunologic responders are at higher risk for future alloimmunization and therefore may benefit from more extensive antigen matching beyond C/c, E/e, K, Fya , and Jkb .

The role of molecular typing and perfect match transfusion in sickle cell disease and thalassaemia: An innovative transfusion strategy

Chronic red blood cell transfusions remain an essential part of supportive treatment in patients with thalassaemia and sickle cell disease (SCD).Red blood cell (RBC) transfusions expose patients to the risk of developing antibodies: RBC alloimmunization occurs when the immune system meets foreign antigens.We created a register of extensively genotyped donors to achieve a better matched transfusion in order to reduce transfusion alloimmunization.Extended RBC antigen typing was determined and confirmed by molecular biology techniques using Human Erythrocyte Antigen (HEA) BeadChip (BioArray Solutions Ltd., Warren, NJ) in periodic blood donors and in patients with thalassaemia and SCD.During 3 years, we typed extensively 1220 periodic blood donors, 898 male and 322 female.We also studied 10 hematologic patients affected by thalassaemia and sickle cell disease referred to our institution as candidate to periodic transfusions.Our patients (8 females and 2 males with a median age of 48 years, range 24–76 years), extensively typed using molecular techniques and screened for RBC alloantibodies, were transfused with a median of 33.5 RBC units.After three years of molecular typing, the “perfect match” transfusion strategy avoided new alloantibodies development in all studied patients.

Hemoglobin Α Clearance Is Associated with RBC Antibodies in Chronically Transfused Children with Sickle Cell Anemia

Hemoglobin Α Clearance Is Associated with RBC Antibodies in Chronically Transfused Children with Sickle Cell Anemia

Genotyping of 19 red cell antigens, including RHD, using liquid bead arrays

BackgroundTraditionally, the determination of blood group has been performed using serological techniques. Due to the drawbacks of using antisera, molecular typing of blood groups has been introduced. However, the commonly used genotyping assays in blood banks have limitations because the panels for these were based on genetic variations in Caucasians. MethodsWe developed a multiplex human erythrocyte antigen genotyping assay using allele-specific primer extensions and hybridization with bead microarrays. Single nucleotide polymorphisms (SNP) were genotyped for the 18 red cell antigens and wild-type RHD, DEL, and complete deletion were examined for RHD. The cut-off of median fluorescence intensity (MFI) for each allele was optimized. In the evaluation stage, the results of 87 samples were compared with those from other genotyping methods, and 26 serologic results were also compared. ResultsThe cut-off values were determined using −1 and −2 standard deviation (SD) of the minimum adjusted MFI for SNP detection. Complete deletion was determined with raw MFI+2SD. In comparison with the other methods, the kappa values were 0.984 overall. Compared with serologic methods, our assay showed discrepancy in 2 S/s, 3 C/c, and 3 Dia/Dib antigens. ConclusionsOur method reliably predicts the presence or the absence of the 19 antigens.

Clinical Utility of Genotyping Human Erythrocyte Antigens.

Traditional serological methods, which have been used for decades to evaluate the human erythrocyte antigen (HEA) composition of recipient and donor specimens, have some serious limitations. Specific reagent antisera are not available for all clinically relevant antigens (eg, V antigen). Reagent antisera are expensive, and serological testing is labor intensive. The results of serological testing are subjective and semiquantitative (eg, microscopic, weak, 1+, 2+, 3+, 4+), and may vary from one technologist to another. Further, in many clinical situations, serological testing may be difficult or impossible.Recent developments in nucleic acid-based testing (molecular diagnostics) have made it possible to genotype HEA in the clinical laboratory. Most allelic variations occur due to single nucleotide polymorphisms (SNPs), which can be detected and from which the phenotypes can be predicted. HEA genotyping offers several technical and clinical advantages compared with serological testing. The Immucor PreciseType HEA Test is the first and currently the only platform for clinical testing approved by the United States Food and Drug Administration (FDA), to our knowledge.

A Comparative Study of Solubility Based Sickle Cell Screening Versus Genomic Method in Blood Donors

Background:Sickle cell trait (SCT) is more common in minority African-Americans (AA) and Hispanic blood donors. Screening donors for Sickle Hemoglobin (HbS) carrier status (SCT) can be useful, especially in managing the transfusion needs of neonates and patients with sickle cell disease. Currently, the Sickledex test, based on the principle of relative insolubility of HbS in phosphate buffers, is routinely used for HbS donor screening. However, there are several limitations including the inability to perform high-throughput testing by automation as donor centers target larger numbers of minority blood donors. False positives results due to hyperlipidemia, dysglobulinemia and higher peripheral blood hematologic counts have also been reported. Hence alternative methods to identify SCT donors are highly desirable in blood centers. The purpose of this study was to validate testing, and to identify rates of False Positive (FP) and False Negative (FN) sickledex screening in blood donors by verifying the presence or absence of the most common mutation seen with HbS production using molecular methods. The comparison could offer evidence to use molecular methods as a test of record to manage HbS carriers at blood centers.Methods and ResultsBetween Jan 2012 till Dec 2014, 200 sickledex screening tests were done daily, totaling approximately 90,000 donors self-identifying as Caucasians and 60,000 donors identifying as African American or Hispanic tested during the study period. Amongst Caucasians tested, 154 had a positive sickledex screen result. Since the prevalence of SCT is less common in the Caucasian population, we hypothesized several of these Caucasian donors could have a false positive screening test. All 154 sickledex positive Caucasian donor samples were prospectively tested for the HbS mutation detection with molecular methods. BioArray Human Erythrocyte Antigen (HEA) Bead Chip DNA assay with PCR multiplex based technology was used to detect the HbS mutation (β-globin change -173 A>T, more commonly designated 20A>T). Of the 154 sickledex + Caucasians, 108 (70%) donors tested negative (FP Screen) and 46 (30%) tested positive for the HbS mutation. A retrospective review of sickledex results in our electronic database for minority donors (n-3431) who also had HEA BeadChip assay was performed to determine concordance. Of 3431 minority donors tested, only 2 (0.0006%) had a positive sickledex screening and were negative by HEA, with the remaining negative by both the sickledex screen and HEA tests. Among minority donors (n= 254) testing + for HbS by HEA, 3% (8/254) were negative by sickledex screening. To establish concordance of HEA assay with high performance liquid chromatographic (HPLC) assay, two separate cohorts were evaluated. Initially 12 sickle screen and HEA positive donors was subject to HPLC analysis, which confirmed SCT status in all 12 samples. Subsequently sickledex screen positive but HEA negative samples (n= 40), were subject to HPLC. All 40 showed absence of SCT by HPLC. Finally, 58 random sickledex + samples had gene sequencing and HEA performed in parallel. Out of 58 samples, 54 showed a positive HbS mutation by HEA with A>T (SCT) nucleotide change noted with gene sequencing. In the remaining 4 samples, HEA was negative with gene sequencing showing (3 T>T and in 1C>T) change at nucleotide 163 associated with Hemoglobin Okayama.Conclusion:The HEA bead chip assay testing for the HbS mutation showed 100% concordance with HPLC and gene sequencing in samples studied. Molecular methods can process multiple samples in a single setting to detect SCT. FP sickledex screening results in Caucasians needs confirmatory testing for the HbS mutation to avoid unnecessary donor notification and deferral. The association, if any, of false positive sickledex screening in Caucasian donors with the Hgb Okayama mutation is under further investigation. DisclosuresNo relevant conflicts of interest to declare.

A model for integrating molecular-based testing in transfusion services.

Molecular-based laboratory tests can predict blood group antigens and supplement serological methods, adding a unique technology to assist in resolving discrepant or incomplete blood group typing or antibody identification. Hospital transfusion services have options for integrating molecular-based methods in their routine operations. We describe here the model of a hospital-reference laboratory partnership. Blood samples for compatibility testing were obtained from patients in a 609-bed hospital serving an urban multiethnic and multiracial population. When results of blood group phenotyping by serological methods were inconclusive, samples were referred for molecular-based testing. The reference laboratory used several methods for genotyping, including polymerase chain reaction followed by restriction enzyme-linked polymorphism analysis, sequence-specific primer polymerase chain reaction and array-based approaches. Human erythrocyte antigen, RHCE and RHD single nucleotide polymorphism arrays were integrated into the laboratory as they became commercially available. The hospital-reference laboratory model made it possible to integrate blood group genotyping promptly by current technology without the expense of new laboratory equipment or adding personnel with technical expertise. We describe ten cases that illustrate the categories of serological problems that were resolved by molecular methods. In-hospital molecular testing for transfusion services has logistical advantages, but is financially impractical for most hospitals. Our model demonstrates the advantages of a hospital-reference laboratory partnership. In conclusion, hospital transfusion services can integrate molecular-based testing in their routine services without delay by establishing a partnership with a molecular blood group reference laboratory. The hospital reference-laboratory model promotes genomic medicine without the expense of new equipment and skilled personnel, while supporting the economy of centralised large-scale laboratory operations.

Red blood cell antigen genotype analysis for 9087 Asian, Asian American, and Native American blood donors

There has yet to be a comprehensive analysis of blood group antigen prevalence in Asian Americans and Native Americans. There may be ethnic differences in blood group frequencies that would result in clinically important mismatches through transfusion. Blood donors who self-identified as Asian or Native American were tested using a single-nucleotide polymorphism (SNP) DNA array (HEA BeadChip kit, Bioarray Solutions Ltd) that predicts expression of 38 human erythrocyte antigens (HEAs) and by serology for ABO, D, C, M, N, Jk(a) , and Jk(b) . The prevalence of blood group antigens was compared to published European prevalence. Discrepancies between SNP-predicted and serology-detected antigens were tallied. A total of 9087 blood donors were tested from nine Asian and Native American heritages. The predicted prevalence of selected antigens in the RHCE, JK, FY, MNS, LU, CO, and DO blood group systems were variable between Asian populations, but overall not significantly different than Europeans. Compared to European frequencies, Kell blood group allele frequencies were significantly different in the Chinese, Native American, Hawaiian/Pacific Islander, South Asian, and Southeast Asian heritage blood donors; Diego antigens Di(a) and Di(b) were different in donors of Native American and South Asian ancestries (p < 0.05). Of the donors tested, 4.5% showed a SNP-serology discrepancy that segregated within specific ethnic groups. This study provides HEA allele frequency and antigen prevalence data in a cohort of Asian and Native Americans donors. Several ethnic groups exhibited differences in HEA frequencies compared to Europeans. Genotype-serotype discrepancies were detected in all systems studied.

HEA BeadChip™ technology in immunohematology

Abstract Classic methods to determine human red blood cell (RBC) antigens are based on serologic testing. Thanks to increased knowledge of the molecular basis associated with many blood group antigens, it is currently possible to predict their presence or absence on the red cell membrane. Several molecular techniques have been developed to detect the most important allelic variations attributable to single nucleotide polymorphisms. The human erythrocyte antigen (HEA) BeadChip™ system manufactured by BioArray Solutions (Immucor, Warren, NJ) is one of the commercial DNA array platforms currently available to predict HEAs by DNA analysis. This technology provides a useful tool to increase the inventory of antigen-negative RBC units and prevent immunization of patients who require chronic transfusion by providing compatible RBC units based on matching by DNA testing. Immunohematology 2015;31:81–90.

Blood group genotype analysis of Australian reagent red blood cell donors across three genotyping platforms: consistent detection of 7·0% phenotype genotype nonconcordance

Background and ObjectivesStandards mandate reagent red blood cells (RBCs) must be homozygous for certain antigens. Genotyping provides an enhanced approach to characterizing RBCs as mutations encoding weakened antigen expression may be detected. This study aimed to genotype reagent RBC donors and compare outcomes with serological phenotype. Genotyping was undertaken using three commercially available platforms.Materials and MethodsBlood samples were collected from donors (n = 300) who contribute to reagent RBC panels. Genotyping was performed on at least one of three commercial platforms; BLOODchip Reference v4.1 (Progenika); human erythrocyte antigen (HEA) and RHD BeadChip (BioArray Solutions Ltd); Hemo ID Blood Group Genotyping Panel (Agena Bioscience).ResultsGenotype and phenotype were concordant for 279 of 300 donors (21 of 300; 7·0% nonconcordance). Five donors carried RH mutations and fifteen donors carried FY mutations, including two donors who had an incorrect phenotype reported. One donor carried RH and FY mutations.ConclusionDiscrepancies between genotype and phenotype were detected in 7·0% of reagent RBC donors tested in clinically significant blood group systems. Genotyping of reagent RBC donors can improve transfusion safety by optimizing sensitivity of antibody screening and identification panels.

Molecular genotyping platforms for blood group antigen prediction

<h3>Aim</h3> This study investigated the advantages/disadvantages of commercial red blood cell (RBC) genotyping platforms. <h3>Methods</h3> DNA samples (<i>n</i> = 196) from extensively phenotyped red blood cell (RBC) donors were genotyped utilising three genotyping platforms: BLOODchip Reference v4.1 (Progenika); Human Erythrocyte Antigen (HEA) BeadChip v1.2 (BioArray Solutions Ltd); and HemoCarta Blood Group Typing Panel pre-released vl.O (Sequenom Inc). <h3>Results</h3> BLOODchip Reference is a low-throughput platform which genotypes 138 polymorphisms, encoding 33 RBC antigens and 12 human platelet antigens (HPA). HEA BeadChip is medium-throughput and genotypes 24 polymorphisms, encoding 34 RBC antigens. HemoCarta Blood Typing Panel is medium to high-throughput and genotypes 107 polymorphisms, encoding 78 RBC and 23 HPA and neutrophil antigens. Flexibility is offered by the BeadChip platform with the availability of different test chips and the HemoCarta platform by customisation into smaller modules. In this study a phenotype genotype discrepancy rate of 10.4% was detected and genotyping outcomes between platforms were 100% concordant where comparison was possible. <h3>Discussion</h3> All genotyping platforms worked well as an adjunct to serology with sensitivity and specificity of 100% on the sample set tested. Factors to consider when comparing platforms are the polymorphisms detected, depth of coverage (replicates), sample through-put, cost per sample and cost per requested result.