NAC Statement on RBC Genotyping

Alloimmunization – an immune response to foreign antigens after exposure to genetically different cells or tissue – in this context referring to foreign antigens on red cells to which a person is exposed through transfusion or pregnancy.

Alloantibody – an antibody directed against red cell antigen different from those in the recipient/patient.

Antisera – reagents containing antibodies used in serological testing to determine the presence or absence of specific red cell antigens (or the red cell antigen phenotype).

Autoadsoprtion – advanced serological methodology to differentiate between multiple antibodies (allo and autoantibodies) in patient/recipient plasma using patient/recipient’s own cells.

Autoantibody – an antibody directed against a red cell antigen present on the recipient/patient red blood cells.

Chronic transfusion therapy – the requirement for ongoing red cell transfusion support at regular intervals over a prolonged period.

Genotyping assays – laboratory tests that detect differences in DNA that can lead to changes in phenotype. These tests may be used in pretransfusion testing to predict red cell antigen types.

Hemoglobinopathy – a group of inherited disorders in which there is abnormal production or structure of the hemoglobin molecule resulting in chronic anemia.

High prevalence antigens – red cell antigens present on the red cells of >99% of individuals.

Low prevalence antigens – red cell antigens present on the red cells of <1% of individuals.

Next generation sequencing – DNA sequencing method that sequences multiple fragments in parallel, allowing for higher volume sequencing.

Non-ABO antigens – RBC antigens from one or more blood group systems other than the ABO system (e.g. Rh, Kell, Kidd or Duffy blood group antigens are non – ABO antigens).

Pan-reactive – antibodies in patient/recipient plasma that react broadly to all reagent red cells tested.

Private antibodies – antibodies arising in a prenatal individual and directed against low prevalence, paternally inherited antigens on a fetus or neonate. These antibodies are not detected on a standard antibody screen as the antigen targets are rarely present (low prevalence). The particular antigen antibody combination is private to the unique maternal paternal and fetal unit, is generally suspected based on a positive neonatal direct antiglobuin test with anemia.

RBC genotype – the DNA code associated with blood group antigens.

RBC phenotype – the antigens expressed on the RBC surface. These may be detected using serological tests or predicted using genotyping assays.

Sanger sequencing – DNA sequencing method that only sequences a single DNA fragment at a time. Gold standard methodology for DNA sequencing.

Serological discrepancies – situations where different antisera result in different interpretations of antigen presence or absence.

Serological test – a method of laboratory testing based on antigen antibody reactions and their detection. These tests may be used to identify red cell antigens (phenotype) or antibodies in pre transfusion testing.

Alloimmunization to red blood cell (RBC) antigens is a clinically meaningful outcome of transfusion or pregnancy that is not uncommon. Alloantibodies are seen in up to 3% of generalized hospital patients in one United States study,¹ and in up to 80% of chronically transfused hemoglobinopathy patients, most notably Sickle Cell Disease (SCD).²

Reduction of alloimmunization can be achieved by prophylactic matching of non-ABO antigens between donor and recipient for chronically transfused populations. Additionally, in patients who are already alloimmunized, therapeutic matching of non-ABO antigens is necessary to prevent the hemolytic consequences of transfusion and to facilitate appropriate transfusion care. Serologic methods of blood group determination for patients are inherently limited by insensitivity to minor structural antigen variations that are clinically meaningful, lack of commercially available reagents for rare antigens, and challenges in distinguishing between donor and recipient cells in recently transfused individuals.³

Genotyping assays overcome these limitations and allow for effective blood group antigen determination. Current advances in the field demonstrate a high concordance rate between genotyping assays and serologic methods, allowing for more widespread adoption of this methodology.⁴ However, limitations including cost and availability make it difficult to ensure equitable access for all patients who may benefit, and there is no single published guideline to identify the patients who would most benefit from this testing.

This document serves to describe clinical scenarios in which genotyping could be considered to predict phenotypes facilitating optimal RBC matching to avoid alloimmunization, or to select the safest RBC units for transfusion in patients who are already alloimmunized. Genotyping results should always be interpreted in conjunction with the patient’s clinical context, serologic phenotype, and alloimmunization history. Discordances should be reported to the National Immunohematology Reference Laboratory for further investigation and resolution.

Current State in Canada

Red cell genotyping for non-ABO and RH antigens is offered at Canadian Blood Services (CBS) and The Ottawa Hospital. The RBC Genotyping Subcommittee is unaware of any other provider of genotyping for red cell antigen genes in Canada outside of the province of Québec.

DNA arrays for extended RBC typing do not routinely include ABO antigens and genotyping to predict ABO antigens for transfusion purposes is not currently offered in Canada. When required, this testing is available at international reference laboratories using validated laboratory developed tests. This approach requires assessment of multiple alleles and currently employs Sanger sequencing or next generation sequencing (NGS) targeted to the ABO genomic locus.

ABO testing is increasingly being performed outside of transfusion medicine laboratories, by university or clinical HLA laboratories as part of organ transplantation programs. This testing has not yet been validated in the transfusion medicine setting. Ambiguity for ABO genotyping must be interpreted with care and ABO genotyping for transfusion purposes requires expert interpretation. Importantly, serologic testing for ABO remains the standard of care and ABO discrepancies in pre-transfusion patients should be managed by provision of group O blood.

Canadian Blood Services

CBS offers extended red cell antigen genotyping through the Health Canada licensed IDCOREXT assay. This assay is based on probe hybridization of targeted PCR products coupled to flow cytometry, and it predicts the expression of 37 blood group antigens in 10 blood group systems. In addition, CBS offers RHD and RHCE genotyping through the Health Canada licensed Immucor BeadChip Kit. The turn around time for these three genotyping tests is up to 14 days. For complex samples that cannot be resolved with these targeted genotyping platforms, CBS offers blood group gene sequencing (Sanger or NGS) through an international referral. These services are available to all Canadians outside of Québec.

The Ottawa Hospital

Genotyping for non-ABO and RHD is offered at The Ottawa Hospital using the HEA BeadChip and RHD BeadChip (Immucor). Complex samples that cannot be resolved are referred to CBS. These services are available to all hospitals in the region served by the Ottawa Hospital.

For patients in whom genotyping is being considered, a full phenotype should be performed first whenever possible. Because genotyping is complimentary to serologic phenotyping, all genotyping results should have the predicted phenotype confirmed serologically.

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