Next Generation Sequencing for Blood Group Antigen Profiling

Abstract There are over 300 serologically defined red blood cell (RBC) antigens and 33 serologically defined platelet antigens, most of which have known genetic changes in 45 RBC or 6 platelet genes that correlate with antigen expression. Exposure to non-self RBC antigens during transfusion or pregnancy can lead to the development of alloantibodies, which on re-exposure can lead to clinically significant and even fatal complications. Therefore, it is vital to know which antigens are present on RBCs. However, traditional serologic phenotyping methods are labor intensive, costly, sometimes unreliable, and reagents are not always available. As such, routine antigen typing is only done for ABO and D antigens. A large percentage of blood is given for hematologic malignancies that will soon get routine whole genome sequencing (WGS). For a minor added cost this data could be used for RBC antigen prediction. However, there are no published reports of using WGS data to predict RBC antigens. This is likely for several reasons: (1) none of the existing WGS data sets have paired serologic RBC phenotypes, (2) there are no fully annotated and complete databases of genotypes to phenotypes, (3) all of the known alleles are defined using cDNAs numbered relative to the start codon without human genome coordinates, and (4) lack of software capable of automated RBC antigen prediction. We have created a fully interactive web site of all known blood group genotype to phenotype correlations, fully annotated with relevant information, and mapped to and visually overlaid to their corresponding human reference genome gene sequences, with algorithms to predict antigen phenotypes from inputted sequences. These predictions are part of the General Genome Reports for the 100 patients getting WGS as part of The MedSeq Project. In addition, each patient is undergoing an extensive antigen phenotypic workup using traditional blood bank serology and currently available SNP PCR based assays, which are being used to validate and improve our prediction strategies. As clinical WGS becomes pervasive we hope that comprehensive blood group prediction will be done on everyone, allowing for easy identification of rare donors and the prevention of alloantibody formation using extended upfront matching of antigens from sequenced recipients and donor. Disclosures No relevant conflicts of interest to declare.

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Non-Hemolytic Antibody Induced Loss of RBC Antigen during Transfusion of Crossmatch Incompatible Blood.

Blood ◽

10.1182/blood.v106.11.557.557 ◽

2005 ◽

Vol 106 (11) ◽

pp. 557-557 ◽

Cited By ~ 1

Author(s):

James C. Zimring ◽

Gregory A. Hair ◽

Traci E. Chadwick ◽

Seema S. Deshpande ◽

Kimberly M. Anderson ◽

...

Keyword(s):

Blood Group ◽

High Titer ◽

Surface Protein ◽

Biological Properties ◽

Target Antigen ◽

Blood Group Antigens ◽

A Cell ◽

Clinically Significant ◽

Antigen Loss ◽

A Minor

Abstract Background: Transfusion of red blood cells (RBC) into patients with anti-donor RBC antibodies (crossmatch incompatible transfusion) can result in antibody mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival. This phenomenon has now been reported for the major clinically significant blood group antigens, including Rh, Kell, Kidd and Duffy. Although this has been described multiple times in humans, no mechanistic elucidation has been accomplished. In an effort to investigate the mechanism of this process, we describe the first animal model of non-hemolytic antibody induced RBC antigen loss. Methods: mHEL mice express the model antigen Hen Egg Lysozyme (HEL) as a cell surface protein on RBC. Since mHEL mice are on a C57BL/6 background, the mHEL antigen represents a single antigenic difference between donor RBC and recipient mice. Immunizing C57BL/6 mice with HEL/CFA results in the generation of high titer IgG anti-HEL responses rendering the mice crossmatch incompatible with mHEL RBC. This system was utilized to study the effects of transfusing mHEL RBC into crossmatch incompatible recipients. Results: Similar to the antibody induced antigen loss observed in humans, transfusion of donor mHEL RBC into crossmatch incompatible mice results in selective loss of HEL antigen from donor RBC without affecting other blood group antigens or reducing the circulatory lifespan of the donor RBC. In addition, recovered RBC that have lost their antigen have normal morphology. This process is antigen specific and occurs in mice that have received passive injections of anti-HEL antisera. A spleen is not required for antigen loss to occur. However, antigen loss does not occur in animals with a targeted deletion of the FcγIII receptor. Although polyclonal anti-HEL antisera consistently causes antigen loss, and IgG1 and IgG2b are the predominant subclasses of anti-HEL IgG in the antisera, no antigen loss is observed in response to purified monoclonal anti-HEL antibodies of the IgG1 and IgG2b subclass. Conclusion: These studies demonstrate that antibody induced antigen loss is a process that involves interaction of RBC, anti-RBC IgG and FcγIII receptors, thus providing mechanistic insight into the phenomenon of antigen loss during incompatible transfusion. The lack of antigen loss in response to monoclonal anti-HEL IgG1 or IgG2b suggests that antigen loss occurs in response to a minor IgG subtype in antisera, depends upon biological properties of the antibody (such as affinity), or that additional serum cofactors are involved.

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Urgent Transfusion in a New Patient with Rare Blood Type

Blood ◽

10.1182/blood-2020-141478 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-28

Author(s):

Catherine Dubé ◽

Scherzinger Kritikopoulos ◽

Marsha Downie ◽

Elizabeth Krok ◽

Katerina Pavenski

Keyword(s):

B Cells ◽

Blood Group ◽

Research Funding ◽

Antigen Expression ◽

Cord Compression ◽

Positive Control ◽

Blood Type ◽

Patient Specimen ◽

Field Reaction ◽

Clinically Significant

Background/Case Studies: Bombay is a rare blood group characterized by the absence of H substance at the surface of RBCs leading to naturally occurring anti-H antibodies. Anti-H presents the risk of severe hemolytic transfusion reactions in these patients. The case presented is of a 32-year-old female of Middle Eastern origin, who presented with a traumatic vertebral fracture with spinal cord compression and required urgent neurosurgery. Her presenting hemoglobin was 84 g/L. She had no previous group and screen on record, had never been transfused and had a history of a remote miscarriage. Study Design/Methods: Forward blood group with an automated gel-method instrument revealed the following reactions: negative with anti-A, unable to interpret (?) with anti-B, 4+ with anti-D. Reverse grouping revealed the following reactions: 4+ with A1-cells and an unexpected 1+ with B-cells. The antibody screen and 11 cell panel in gel (Micro Typing Systems) 2+; the panel, with enhancement reacted 3+ in Ficin. The auto control was negative. A second panel and pre-warm panel produced the same findings. An antibody reacting at 37C against a high-frequency antigen was suspected. Patient specimen was sent for investigation to the reference lab (RL), which performed blood group by manual tube test, antibody identification with panels by manual tube PEG-IAT method; RL also sent a sample for ABH sequencing (Sanger). Results/Findings: A thawed frozen plasma sample from a previous Bombay patient 12 years prior showed no reactivity against the patient's RBCs; positive control included. A frozen Bombay RBC unit was ordered urgently from the blood supplier and was crossmatch compatible. The patient underwent surgery and was transfused with a single unit of RBC for peri-operative bleeding. She was treated with erythropoietin and IV iron post-operatively and did not require any further transfusions. The investigation at the RL showed mixed field reaction on forward blood typing with anti-B and anti-A,B and negative reaction with anti-A commercial reagents. The RL reverse grouping showed 4+ with all O H+ and A1 red cells, but 2+ with B cells. The autocontrol and group O H- cells did not react, confirming anti-H and suggesting Para-Bombay group. ABH sequencing revealed a normal B allele (ABO*B.01) while genotyping of FUT1 revealed a null allele (FUT1*01N.12) and weak H allele (FUT1*01W.23). FUT2 genotyping (FUT2*01N.02) predicted a nonsecretor (sese) phenotype. Conclusions: This patient with non-secretor status, variant H production, clinically significant anti-H, greatly reduced B antigen expression, should be treated as a Bombay (Oh) for transfusion purposes. She was counselled and provided with an antibody card and a letter. This case illustrates the importance of timely communication with the clinical team about the risks and benefits of transfusion pending antibody identification, as it could have proved fatal in this case. Figure Disclosures Pavenski: Bioverativ:Research Funding;Shire/Takeda:Honoraria;Octapharma:Research Funding;Alexion:Honoraria, Research Funding;Sanofi:Research Funding;Ablynx/Sanofi:Honoraria, Research Funding.

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A Minor Genetic Variation In the Granzyme B Gene Predicts Relapse After HLA-Fully-Matched Unrelated Bone Marrow Transplantation for Hematologic Malignancies

Blood ◽

10.1182/blood.v116.21.681.681 ◽

2010 ◽

Vol 116 (21) ◽

pp. 681-681

Author(s):

Luis J. Espinoza ◽

Akiyoshi Takami ◽

Kayoko Yamada ◽

Mayu Yamada ◽

Yuki Motohashi ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Conflicts Of Interest ◽

Granzyme B ◽

Hematologic Malignancies ◽

Effector Cells ◽

Transplant Outcomes ◽

A Minor ◽

The Impact

Abstract Abstract 681 Granzyme B (GZMB) is a serine protease with important roles mediating target-cell apoptosis induced by natural killer cells and cytotoxic CD8+ T cells. The polymorphism rs8192917 (A55G) in the GZMB gene has been reportedly associated with the ability of effector cells to secrete GZMB. In this study we analyzed the impact of GZMB polymorphism on transplant outcomes in patients undergoing unrelated HLA-fully-matched myeloablative bone marrow transplantation (BMT) through the Japan Marrow Donation Program. The GZMB genotypes were retrospectively analyzed in a cohort of 360 pairs of patients with hematologic malignancies and their unrelated donors. The presence of the G/G genotype in the donor side was associated with a significantly higher incidence of relapse (62% vs. 32%, P=0.04; Fig 1). Multivariate analysis revealed that the donor G/G genotype, which is expected to produce lower levels of GZMB after stimulation, (Girnita et al, Transplantation 2009) was an independent risk factor for relapse (hazard ratio, 4.17; 95% confidence interval, 1.63 to 10.70; P=0.003). The host GZMB genotypes did not significantly influence the transplant outcomes. These results suggest an association of low GZMB secretors with disease relapse. These could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

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Use of Genomics for Transfusion Therapy Management in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood.v118.21.2324.2324 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2324-2324

Author(s):

Connie M Westhoff ◽

Stella T Chou ◽

Kim Smith-Whitley ◽

David Friedman

Keyword(s):

Sickle Cell Disease ◽

Blood Group ◽

Sickle Cell ◽

Conflicts Of Interest ◽

Hla Typing ◽

Blood Group Antigens ◽

Dna Testing ◽

Cell Disease ◽

Transfusion Therapy ◽

Clinically Significant

Abstract Abstract 2324 A genomic approach to blood group typing is now possible and high-throughput automated platforms have been developed to test for numerous blood group antigens in a single assay. These methods are reproducible and highly correlated with RBC serologic phenotype. We routinely perform a complete RBC phenotype for clinically significant minor red cell antigens on pre-transfusion samples from patients with sickle cell disease, and we antigen match patients for C, E, and K for transfusion. In this study we compared the historic serologic typing with that predicted from DNA testing for clinically significant antigens in 114 samples from chronically transfused patients with SCD to determine concordance and to evaluate the clinical utility of genotyping for the management of transfusion therapy. Serologic typing was performed by standard methods with licensed commercial reagents. DNA was isolated from WBCs, and minor antigen genotyping was performed with HEA (human erythrocyte antigen) BeadChip (BioArray, Inc). RH genotyping was by a combination of methods including PCR-RFLP, AS-PCR, exon-specific amplification and sequencing, and, for some, Rh-cDNA amplification and sequencing. Comparison of serologic typing with DNA-based testing for thirteen blood group antigens, CcEe, Fya/b, K, Jka/b, MN and Ss, in 114 samples found 8 discrepancies in 1,482 antigens analyzed, for 99.5 % concordance. Discrepancies were in several systems (C, Fy, Ss, and M), and at least one has been confirmed to be a serologic recording error. All are under investigation. DNA-based testing for RH found 54 of 114 patients inherited variant RHD alleles; many also had conventional RHD in trans. Sixteen patients had made anti-D, despite typing as D+. Ten of 35 patients (∼30%) whose RBCs typed as C+ had a hybrid allele encoding variant C antigen. Five had made anti-C, which prompted us to change our protocol so patients with variant C by DNA testing are transfused on a C- protocol. DNA testing found a large amount of diversity in ce-alleles in this population. Seventy-two of 114 patients carried at least one of nine different variant ce-alleles. Ten patients had made anti-e, despite typing as e+, and were homozygous for variant ce-alleles. In total, 49/114 patients with SCD were homozygous for variant RH alleles and were not truly Rh matched for D, C and e antigens by serology. Similar to the way in which HLA typing by DNA has revolutionized bone marrow transplantation by providing a superior alternative to serological testing, we find that minor blood group antigen typing by DNA improves efficiency, reduces cost, and expands antigen matching, especially in the Rh system. Continuing studies are needed to identify more precisely which variant alleles are associated with clinically significant antibody production to improve antigen matching for patients with sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

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AB0 Blood Groups and Coronary Heart Disease (CHD)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650035 ◽

1980 ◽

Vol 43 (02) ◽

pp. 137-140 ◽

Cited By ~ 22

Author(s):

Jan Erikssen ◽

Erik Thaulow ◽

Helge Stormorken ◽

Ole Brendemoen ◽

Arvid Hellem

Keyword(s):

Coronary Artery ◽

Blood Group ◽

Artery Bypass ◽

Laboratory Data ◽

Epidemiological Data ◽

Consecutive Series ◽

Related Factors ◽

Ab0 Blood Groups ◽

Clinically Significant ◽

Artery Disease

SummaryThe view based on epidemiological and laboratory data that blood group A subjects (=A) have clinically significant higher thrombotic potential than blood group 0 subjects (= O), is supported by the present finding of a significantly higher platelet retention in A than 0.The completely normal ABO distribution found among 71 cases of proven latent CHD, and the disproportionate excess of 0 vs. A in a consecutive series of 191 coronary artery bypass candidates apparently conflict with epidemiological data indicating a higher risk of achieving CHD in A than 0. The conflict may be solved by suggestinga) that the »thrombotic proneness« in A compared with 0 causes a poorer prognosis in CHD among the former, leaving a disproportionate excess of 0 among longterm CHD survivors, and b) that AB0-related factors have had an insignificant, independent impact on the evolution of preclinical coronary artery disease in our 71 men with latent CHD.

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