Molecular immunohematology at Canadian Blood Services: Red cell antigen genotyping

Author: Mindy Goldman, MD, FRCPC
Online publication date: 2016 revisions by: Kirsten Hannaford, MLT; Judith Hannon, MD, FRCPC and Philip Berardi, MD, PhD, FRCPC.

Introduction

Molecular immunohematology refers to the detection of the molecular genetic basis of an antigen, rather than the antigen alone.  Use of molecular testing in clinical laboratories requires knowledge of the molecular basis of blood group antigens and the availability of suitable genotyping methods that can be used in our testing environment.  Molecular testing has essentially replaced serologic typing for the extremely polymorphic HLA system antigens.  High volume methods are used to perform mass genotyping for unrelated donor registries, such as the Canadian Blood Services’ OneMatch registry.  Molecular typing may also be used for platelet and neutrophil antigen systems, as the genetic basis of these systems is further characterized.  This may be particularly useful since commercial antisera are not readily available for typing for many of these antigen systems.

The increased use of red blood cell (RBC) genotyping has become an integral part of many transfusion medicine services.1-3 Many of the serologically determined RBC antigens are related to single nucleotide polymorphisms (SNPs) which result in a single amino acid change in RBC antigens.  However, depending on the blood system, the relationship between the RBC antigen gene and the corresponding antigen may be fairly complicated.  For example, in some antigen systems, such as ABO, multiple alleles may encode the same phenotype.  Molecular events other than SNPs in the antigen coding region of the gene may also give rise to blood group antigens, or affect antigen expression.1,3  These include nucleotide changes in the binding site for transcription factors necessary for the expression of the gene, nucleotide changes in splice sites, gene deletions or insertions, and gene recombination events.  Therefore, results obtained by targeted genotyping may differ from those obtained by phenotyping.  Manufacturers have modified their test kits to be able to detect some of the more frequent genetic variations, as new information becomes available.  For example, the Duffy (Fy) genotyping testing platforms detect several different SNPs that influence Fya and Fyantigen expression: a SNP in exon 2 of the Duffy gene that directly encodes for the Fya or Fyb antigens, a SNP in the promoter region of the Duffy gene that prevents expression of the FYB gene in red cells but not in other tissues (GATA-1 mutation), and SNPs in exon 2 that affect the translation and stability of the Fyb antigen, Fyx.4

Red cell antigen genotyping has many applications in blood donor testing, as well as in various diagnostic settings.  In Canada, some high-throughput RBC genotyping platforms and companion test kits are Health Canada approved and licensed for RBC antigen assessment.  Genotyping results can therefore be used as a reliable adjunct to serologic testing and will become increasingly used in red cell antigen assignment.  There remains the occasional discordant result between genotyping and phenotyping that may require further investigation.  These should be discussed with the transfusion medicine physician in your hospital or with the personnel at the National Immunohematology Reference Laboratory (NIRL). This article focuses on the use of molecular genotyping at the Canadian Blood Services NIRL (determination non-RHD) and at the Canadian Blood Services’ Diagnostic Services Laboratory in Edmonton (determination RHD).  Testing is being performed using the Progenika ID CORE XT™ assay (distributed by Grifols) for non-RHD genotyping requests and BioArray Solutions RHD BeadChip™ technology (distributed by Immucor) for RHD genotyping.  Both of these are Health Canada licensed. Several excellent reviews cover the use of genotyping for other indications such as fetal blood group typing and donor typing.1-3

RBC blood group genotyping testing platforms

For non-RHD genotyping the Progenika ID CORE XT™ assay uses the Luminex testing platform.5 The Luminex system is a modified flow cytometer that uses oligonucleotide probes attached to microspheres. It is also used for HLA and human platelet antigen genotyping. The system is highly automated, and includes integrated analysis software.6  Samples are analyzed in small batches following an automated DNA extraction process. The laboratory should be made aware of urgent samples so these can be prioritized. Table 1 summarizes the blood system antigens that are detected using the ID CORE XT™.  The genotyping report lists the respective alleles assayed (see Illustrative Cases section).

Table 1. Red cell antigen typing using the Progenika ID CORE XT™ (Grifols)

Blood Group

Variants and ISBT number

RhCE

C(RH2), E(RH3), c(RH4), e(RH5),
CW(RH8), V(RH10), hrS(RH19), VS(RH20), hrB(RH31)

Kell

K(KEL1), k(KEL2), Kpa(KEL3), Kpb(KEL4), Jsa(KEL6), Jsb(KEL7)

Kidd

Jka(Jk1), Jkb(Jk2),
JKB_null(IVS5-1a), JKB_null(871C)

Duffy

Fya(FY1), Fyb(FY2),
FYB_GATA, FYB[265T]_FYX

MNS

M(MNS1), N(MNS2), S(MNS3), s(MNS4), U(MNS5), Mia(MNS7)

Diego

Dia(DI1), Dib(DI2)

Dombrock

Doa(DO1), Dob(DO2),
Hy(DO4), Joa(DO5)

Colton

Coa(CO1), Cob(CO2)

Cartwright

Yta(YT1), Ytb(YT2)

Lutheran

Lua(LU1), Lub(LU2)

For RHD genotyping of selected patient samples, testing may be performed by the Diagnostic Services Laboratory in Edmonton using Immucor’s BioArray BeadChip™ Assay which interrogates for many clinically significant RHD gene variants responsible for normal and altered/absent expression of the RhD human red cell antigen. This kit is a qualitative test which uses the proprietary elongation-mediated multiplexed analysis of polymorphisms (eMAP®) technology for allele status determination. The BioArray Solutions Array Imaging System (AIS) is used to image and capture the fluorescent signal from individual beads within the entire BeadChip™ array. There are currently 68 different genotypic variants identified using this approach, including many weak and partial RhD phenotypes. Table 2 summarizes the blood system antigens that are detected with this platform and figure 1 identifies the BeadChip names and RHD variants. This assay is also Health Canada approved.

Table 2. RHD Assay Variant Coverage using the BioArray BeadChip™ (Immucor)

RHD Variant

Variants Interrogated by the Chip

Weak D Types

1, 1.1, 2, 3, 4.0, 4.1, 4.2/DAR, 4.3, 5, 11, 14, 15, 17, 25, 29, 34, 40, 47, 51

D negative and mosaics/hybrid

RHDΨ; W16X; D-CE(3-7)-D; D-CE(4-7)-D; (C)dceS;
DCE(3-9)-D; CE(1-3)-D(4-10); rG; RHD(Y269X)

Del Variants

1227 G>A; IVS3+1G>A; M295I

Partial D Types

DAR/weak D 4.2; DIIIa (DIII type 5), DIII type 4,6; DIIIc;
DIVa; DIVa -2; DIV type 3, 4, 5; DIVb; DV type 1,2,3 (DBS-0),4,5
(DHK),6,7,8,9; DVI type 1,2,3,4; DNB; DHMi; DUC-2; DAU 1,2,3,4,5;
DBT 1,2; DCS 1, 2; DOL; DOL-3; DFR; DFR -2; DTO; DBS-0,1,2

Additional genotyping services available to Canadian Blood Services include Progenika BLOODchip™ technology which is capable of detecting 58 ABO variants, and over 110 allelic variants coding for RhD+, D-, weak D, partial D and Del phenotypes. Turnaround time is estimated at 7-14 working days, and the laboratory should be made aware of any urgent requests. 

Figure 1. RHD variants

Indications for genotyping

Table 3 summarizes the indications for genotyping in patients requiring transfusion or in the prenatal setting that may be sent to Canadian Blood Services NIRL or Diagnostic Services Edmonton. Genotyping requests should include the summary of serologic studies that have already been performed. If you are uncertain if a case corresponds to these indications, please contact NIRL or the Diagnostic Services Edmonton prior to sending samples. 

Table 3. Indications for non-RHD and RHD genotyping

Non-RHD Genotyping (Canadian Blood Services NIRL in Ottawa)

- Serological antigen typing/phenotyping cannot be determined due to chronic transfusion requirement (e.g. Thalassemia major, Blackfan Diamond anemia).

- Serologically complex patients - with multiple or unidentified antibodies who require ongoing transfusion support.

- Patients with autoimmune hemolytic anemia and/or with a positive DAT (in spite of chemical treatment), and circulating autoantibody.

- Patients with a suspected alloantibody against an antigen for which no commercial antisera is available (e.g. Possible anti-Doa).

- Select patients with variable or null reactivity using serological methods when a variant allele is suspected (e.g. Sickle cell anemia).

RHD Genotyping (Canadian Blood Services Diagnostic Services Laboratory  in Edmonton)

- Prenatal patients with discrepant, weak or inconclusive serological RhD testing results where RHD genotyping may modify their blood product or Rh Immune Globulin (RhIG) requirements. For example: prenatal patients with weak or discrepant RhD serology may be Weak D type 1, 2 or 3 and would not require RhIG.

- Patients likely to require chronic transfusion, or with complex transfusion needs, where RHD genotyping may modify their blood product requirements.

- Patients who likely require transfusion with an anti-D who appear serologically D positive.

Genotyping may be useful in a variety of situations where a phenotype cannot be determined by serological means.1-3  Particularly in the pediatric population, patients may not have had a phenotype done prior to becoming chronically transfusion dependent with syndromes such as thalassemia major or Blackfan Diamond anemia. Genotyping allows these patients to receive partly phenotyped matched units to avoid alloimmunization. Genotyping may also assist in the resolution of complex serological findings in patients who have not had a phenotype performed pre-transfusion, and have now developed new alloantibodies.  Similarly, patients with autoimmune hemolytic anemia, circulating autoantibody, and a positive DAT in spite of chemical treatment may be easier to support with ongoing transfusions if a phenotype can be predicted from genotyping. Finally, for some antigen systems such as Dombrock where there are no commercial antisera available for typing, genotyping can be used to confirm phenotype. 

Patients with sickle cell anemia may present with a variety of transfusion challenges. Subgroups of sickle cell anemia patients will require chronic transfusion therapy, and have a high incidence of alloimmunization. Since variant RHD and RHCE alleles are more common in sickle cell anemia patients and certain ethnic groups, transfusion support may be optimized following identification of these variants using genotyping.  Furthermore, approximately 67% of African Americans have the Fy (a- b-) phenotype due to a mutation in the GATA-1 binding site, which prevents expression of the Fyb antigen in red cells but not in other tissues.  Since Fyb glycoprotein is still present on other cells through the body, these individuals do not produce anti-Fyb. Dropping the requirement for Fy (b-) units may facilitate provision of red cells that are phenotype matched for the other antigen systems.7

Table 4. Situations where genotyping is usually not indicated

- Patients who will not require transfusion (with the exception of sickle cell anemia patients suspected of having variant alleles).

- Patients with weak or discrepant RhD serology who require immediate transfusion or will only have one transfusion episode (for example, pre-operative sample). In these cases, a decision to use D positive or D negative units should be made based on the clinical context.

Indications for RHD determination

RHD genotyping may be useful in the prenatal setting, for patients who have weak, variable or discrepant test results on routine prenatal D typing.  In Caucasians, approximately 80% of weak D individuals are types 1, 2, or 3.8-11 These individuals are highly unlikely to become alloimmunized against RhD.  Recently the College of American Pathologists and an AABB Work Group on RHD Genotyping was convened to develop recommendations for the management of individuals with a serological weak D typing.12 This group recommended that RHD genotyping be phased in for patients with a serologic weak D phenotype, and that patients with weak D type 1, 2 or 3 should be considered Rh positive for the purposes of RhIG prophylaxis and transfusion.

RHD genotyping may also be indicated in patients with discordant D results, for example, a patient who appears to be D positive but has an anti-D.  Possibilities may include a partial D individual with an alloantibody, or a D positive individual with an autoantibody.13  Hospitals that cannot resolve serological D typing results may send a sample to the Diagnostic Services Laboratory in Edmonton for RHD genotyping.

Genotype/phenotype discordant results

There may be instances when the predicted phenotype, as determined using the genotyping data, will not correspond with phenotyping results using serological methods. The scenarios in which this may occur include, but are not limited to, situations where serological reagents are not able to detect variants with partial antigen expression or variants that are not interrogated by the genotyping technology employed. These cases may require additional work-up possibly including targeted gene sequencing and will be addressed on a case-by-case basis.

Illustrative Cases

Case #1: John Doe is admitted to hospital with a diagnosis of gastrointestinal bleed and a hemoglobin level of 80 g/l.  He was transfused four units of RBCs three weeks previously.  John Doe’s antibody screen and DAT are positive and an investigation determines the presence of anti-C, anti-K plus an unidentified antibody.  A sample is referred to NIRL for genotyping.  The ID CORE XT™ assay is performed. Results are shown in Figure 2.

Figure 2. Red Blood Cells Typing report for case#1

Case #2: Jane Doe is pregnant and in her first trimester. A routine group and screen performed on Jane’s sample identified a weak reaction with the hospital’s routine anti-D. The hospital repeats testing with the routine anti-D and extends the room temperature testing to 5 minutes (as allowed by the package insert) and the initial results are confirmed. Additional anti-D from two other manufacturers are tested and variable reactivity is observed with the patient’s red cells while the controls react as expected. The hospital’s policy is to treat weak D types 1, 2 or 3 as RhD+, and patients with these weak D types do not receive RhIg. A sample is sent to the Diagnostic Services Laboratory in Edmonton for RHD genotype testing. The BioArray BeadChip assay is performed. Results are shown in Figure 3.

Figure 3. RHD genotype testing report for case# 2.

Sample requisition forms (Non-RHD genotyping)

Below is an example of the Requisition for Blood Group Genotyping (Patient) form. To access the most up-to-date form please visit - https://www.blood.ca/en/hospitals/ottawa-reference-laboratory.

Sample requisition forms (RHD Genotyping)

Below is an example of the Requisition for RHD Genotyping form. To access the most up-to-date form please visit - https://www.blood.ca/sites/default/files/Request_for_RHD_Genotyping.pdf

Specimen shipping requirements

Sample Requirements

Notification

Shipping Address

RHD: EDTA (purple top) whole blood specimen (minimum 2 mL).

Samples must be received within 14 days of sample collection.

Samples must be labeled with DOB/name/ID #.

Notify Diagnostic Services Laboratory prior to shipment by faxing copy of completed requisition to 780-431-8779 or email genotyping.edm@blood.ca or by phone at 780-431-8765.

Send completed Request for RHD Genotyping requisition form for each specimen.

Diagnostic Services Laboratory

Canadian Blood Services

8249-114 Street

Edmonton, Alberta

T6G 2R8

Red Cell antigen (Non-RHD): EDTA (purple top) whole blood specimen (2 mL to 7 mL).

Samples must be received within 14 days of sample collection.

Samples must be labeled with DOB/name/ID #.

Notify NIRL prior to shipment at

613-739-2460 or email to NIRL@blood.ca.

Send completed Request for Blood Group Genotyping form for each specimen.

National Immunohematology Reference Laboratory

1800 Alta Vista Drive

Ottawa, Ontario

K1G 4J5

Sample receipt at laboratory is Monday to Friday 08:00 to 16:00.

References

  1. Hillyer CD, Shaz BH, Winkler AM, Reid M.  Integrating Molecular Technologies for Red Blood Cell Typing and Compatibility Testing Into Blood Centers and Transfusion Services.  Transfus Med Rev 2008; 22(2):117-132.
  2. van der Schoot CE, de Haas M, Engelfriet CP, et al. International Forum: Genotyping for red blood cell polymorphisms. Vox Sang 2009; 96:167-179.
  3. Westhoff CM.  The potential of blood group genotyping for transfusion medicine practice.  Immunohematology 2008; 24:190-195.
  4. Castilho L.  The value of DNA analysis for antigens in the Duffy blood group system.  Transfusion 2007; 47(Supplement 1) 28S-31S.
  5. Goldman M, Nogues N and Castilho LM. An overview of the Progenika ID CORE XT: an automated genotyping platform based on a fluidic microarray system. Immunohematology 2015; 31:62-68.
  6. Avent ND, Martinez A, Flegel WA, et al.  The BloodGenproject: toward mass-scale comprehensive genotyping of blood donors in the European Union and beyond.  Transfusion 2007; 47:40S-46S.
  7. Wilkinson K, Harris S, Gaur P, et al.  Molecular blood typing augments serologic testing and allows for enhanced matching of red blood cells for transfusion in patient with sickle cell disease.  Transfusion 2012; 52:381-388.
  8. Daniels G.  Variants of RhD- current testing and clinical consequences.  British J of Haem 2013; 161,461-470.
  9. Flegel WA, Denomme GA, Yazer MH.  On the Complexity of D Antigen Typing: A Handy Decision Tree in the Age of Molecular Blood Group Diagnostics.  J ObstetGynaecol Can 2007; 29(9):746-752.
  10. Nance ST, Lomas-Francis C.  Where are we in efforts to unravel the complexity of Rh to guide transfusion decisions?  Transfusion 2013; 53:2840-2843.
  11. Berardi P, Hannon J, Clarke G et al.  Resolution of maternal D typing using serology and genotyping.  Transfusion 2013; 53(25), 107A.
  12. Sandler SG, Flegel WA, Westhoff CM, et al; College of American Pathologists Transfusion Medicine Resource Committee Work Group. It's time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion 2015; 55: 680-689.
  13. Pham B-N, Roussel M, Payrard T, et al. Anti-D investigations in individuals expressing weak D Type I or weak D Type 2: allo- or autoantibodies?  Transfusion 2011; 51:2679-2685.