Human Erythrocyte Antigen BeadChip Research Articles

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.

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.

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.

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Comments on: Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

Potential Benefits of Extended Genotype Matching to Chronically Transfused Patients with Sickle Cell Disease

Abstract▪2144▪This icon denotes a clinically relevant abstract Background:Management of RBC alloimmunization in Sickle Cell Disease (SCD) patients has been the subject of much debate, and currently there is no standard approach. Many programs transfuse SCD patients with RBCs that are phenotype-matched for D, C, c, E, e and K. Although these approaches reduce the incidence of alloantibody production, patients still become alloimmunized. Based on this we aimed to identify the rates of alloimmunization in chronically transfused SCD patients and compare the phenotyping with genotyping methods to find a better way to match RBC units to those patients. Methods: We selected 45 SCD patients (homozygous for hemoglobin S) with multiple transfusions, previously phenotyped for ABO, Rh (D, C, c, E, e) and K1. Phenotypes were determined by hemagglutination using gel cards (Diamed® ). Genotypes were determined by a DNA array using the Human Erythrocyte Antigen BeadChip (“HEA”) from Bioarray Solutions. All SCD patients included in this study were in chronical transfusion program; receiving multiple transfusions. The median age was 24y; there were 28(62%) females and 17(37.8%) males. The median of transfusions were 53 (5–78) and 40 (88.9%) patients received more than 20 phenotype-matched units for Rh (D, C, c, E, e) and K1. Results: Of the 45 SCD patients selected, 11 (24.4%) had alloantibodies. The antibody specificities found in these patients were anti-D, -C, -CW, -E, -Jka, -Jkb, -Fya, -Dia, -s. Although the patients were receiving Rh and K phenotype-matched units 8 (17%) of them became alloimmunized to Rh antigens and on those patients we found discrepancies between the previous phenotype and genotype-derived phenotype. Our results showed that the risk of immunization increases in patients over 40 years old (p= 0.05) and with the number of transfusion events. Patients with more than 20 RBC transfusions have a tendency for alloimmunization (p=0.65). We also observed that genotyping was more effective than hemagglutination in determining patient’s correct phenotype. Conclusion: Our data show that even with the implementation of Rh and K phenotype-matching in chronically transfused patients with SCD, they still become alloimmunized to other antigens with high immunization risk and also to Rh antigens due to the limitations of the hemagglutination. The relevance of genotype determination of blood groups for the management of multiple transfused patients with SCD has been demonstrated by allowing the determination of the true blood group genotype, by assisting in the identification of suspected alloantibodies and in the selection of antigen-negative. As donor genotyping for the most clinically relevant blood group antigens by automated DNA techniques are becoming available, extended genotype matching should be considered in this group of patients. Disclosures:No relevant conflicts of interest to declare.