The American Red Cross AABB-accredited CLIA licensed National Molecular Laboratory offers state-of-the-art testing services and expertise to help you care for patients in challenging situations. We support complex antibody identifications, help guide selection of compatible donors and determine risk of alloimmunization to blood group and platelet antigens.
The laboratory also screens whole blood and platelet donors to identify donors of antigen negative blood products. The laboratory has been using red blood cell genotyping of donors since 2008 and in the last 5 years, has screened more than 150,000 blood donors. In 2016, genotyping using matrix-assisted laser desorption ionizing time of flight (MALDI-TOF) was implemented, allowing more donors to be screened for more antigens.
For patients, molecular testing is a useful adjunct to complex serologic cases. It can resolve typing discrepancies and predict a red cell phenotype when serologic typing is unreliable.
National Molecular Laboratory
700 Spring Garden Street
Philadelphia, PA 19123
Phone: (215) 451-4917
Fax: (215) 451-2506
M-F 7:00 am – 6:30 pm
Director: Dr Martin CH Chou, PhD
CLIA Director: Shraddha Patel Babariya, MD
CLIA License: 39D0194473
Available Molecular Testing
Molecular genotyping is useful to predict the presence of red cell and platelet antigens. HEA (human erythrocyte antigen) and HPA (human platelet antigen) testing allows simultaneous testing for multiple antigens in one assay. Higher resolution testing, including genomic and cDNA sequence analysis is used to characterize variant antigens and resolve typing discrepancies. This testing can identify novel variants in many blood group and platelet antigen systems.
Human Erythrocyte Antigen (HEA) Genotyping Panel
Test Code SREF-IVD
Recommended CPT Code 0001U, as of Feb. 1, 2017
An FDA-cleared multi-analyte test performed on DNA from whole blood provides a predicted phenotype for 36 antigens. This testing is useful when antigen status cannot be determined serologically, either because of recent transfusion, positive DAT or when reagents are not available. The HEA genotyping panel is useful in patients being treated with the anti-CD38 drug daratumamab. The extended antigen phenotype prediction is also useful in patients on chronic transfusion regimens. In alloimmunized patients, it can be used to assess the additional alloimmunization risk.
RHD Genotyping for D variants
Test Code SREF-RHD
Recommended CPT Code 81403)
RHD Genotyping is used to identify RHD variant alleles and predict the D antigen phenotype. There are many RHD variant alleles and variants can have altered antigen expression and/or altered epitopes. RHD variants are found in all ethnic groups but are most common in individuals of African descent. Weak or altered antigen expression can lead to a serologic weak D phenotype. This is especially important in pregnant women and women of child-bearing age, as the decision regarding RhIg prophylaxis is informed by RHD variant type. Specifically, individuals who are weak D types 1, 2 and 3 are not at risk of D alloimmunization. Weak or altered RhD antigen expression can also result in a typing discrepancy, either between test methods, reagents, laboratories or between a current and historic type. RHD genotyping is performed using a commercially-available “Research Use Only” genotyping panel that can detect many weak, Del and partial alleles including weak D types 1, 2 and 3 plus one or more lab-developed tests to rule out weak or partial alleles not interrogated on the commercial panel.
RHCE Genotyping for C, c, E and e variants including hrB and hrS status
RHCE Genotyping is used to identify RHCE variant alleles and can predict C, c, E, e, V, VS, hrB and hrS antigen phenotypes. There are many RHCE variant alleles and variants can have weakened or altered antigen expression, be associated with loss of a high prevalence antigen or gain of a low frequency antigen. RHCE variants are found in all ethnic groups but are most common in individuals of African descent. Weak antigen expression can lead to typing discrepancies, for example with e and C antigens. Patients who express altered antigens can be at risk of alloimmunization (eg. e+ with anti-e or C+ with anti-C). RHCE genotyping is performed using a commercially-available “Research Use Only” genotyping panel that can detect many common altered alleles plus one or more lab-developed tests to rule out variant alleles not interrogated on the commercial panel.
Test Codes SREF-RHD and SREF450; may also include SREF750, SREF-SEQ, SREF500, SREF550
Recommended CPT Code 81403
RHD variant and RHCE variant alleles are often co-inherited and are not uncommon in individuals of African descent. Patients who are found to have D variants may be at risk of expressing altered RHCE antigens and visa versa. Patients of African descent who are likely to be multiply or chronically transfused may benefit from RH characterization. RH characterization includes use of RHD and RHCE commercially-available “Research Use Only” genotyping panels and may also include performance of one or more lab-developed tests to rule out variant alleles not interrogated on these commercial panels.
Test Code SREF-RHZ
Recommended CPT Code 81403
It is not uncommon to be hemizygous for RHD alleles. The RHD zygosity status of the partner of an RhD negative woman with anti-D can be used to assess risk of alloimmunization. RHD zygosity includes one or more lab-developed tests.
ABO Common Allele Determination
Test Code SREF200
Recommended CPT Code 81403)
Determination of ABO common alleles (A1, A2, B, O1, O2) can be helpful in investigating discrepancies involving ABO blood type. This testing cannot rule out the presence of rare subgroups. ABO common alleles are tested using a lab-developed test.
Test Code SREF550
Recommended CPT Code 81403)
When ABO common allele determination does not resolve an ABO discrepancy or unexpected serologic result, ABO variant testing may be warranted. ABO variant alleles (eg., B(A), Ax, Aw) can be identified by gene sequencing. This testing is currently performed by a subcontracted laboratory using patient DNA prepared at the Red Cross.
Non Rh, Non ABO Variants
Test Codes include SREF-SEQ, SREF750, SREF200
Recommended CPT Code 81403
Typing discrepancies or suspected alloantibodies to non-Rh blood group systems may benefit from molecular testing. Blood group systems include MNS, LU, KEL, FY, JK, DO, KN, and XK. The KLF1 gene can be interrogated to rule out a possible In(Lu) phenotype. For example, a patient who is Jk(a+) with allo-Jka may express a Jka variant that is associated with alloimmunization. In addition, variant testing can be useful in resolving discrepancies between serologic testing and the phenotype predicted by the HEA genotyping panel (described above).
This testing is performed using lab-developed tests using gel-based genotyping including sequence-specific primer PCR (SSP-PCR) and sequence-based typing (SBT).
Human Platelet Antigen 1a/1b (HPA-1a/1b) Genotyping
Test Code SR81400A
Recommended CPT Code 81400
A lab-developed test performed on DNA from whole blood provides a predicted phenotype for HPA-1a/1b. This testing is useful when antigen status cannot be determined serologically. The antigen phenotype prediction is useful in workups of suspected Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT) and in platelet refractory patients in whom HLA antibodies have been ruled out.
HPA-1a/1b assay tests for ITGB3 c.176T/C (p.33L/P) in GPIIIa (with gene name, nucleotide and amino acid information in HGNC nomenclature)
Also known as (aka) PlA1/A2
Human Platelet Antigen (HPA) Genotyping Panel
Test Code SREF-HPA
Recommended CPT Code 81400
A single multi-analyte test performed on DNA from whole blood provides a predicted phenotype for 22 antigens (HPA-1a/1b, HPA-2a/2b, HPA-3a/3b, HPA-4a/4b, HPA-5a/5b, HPA-6a/6b, HPA-7a/7b, HPA-8a/8b, HPA-9a/9b, HPA-11a/11b, HPA-15a/15b). This testing is useful when antigen status cannot be determined serologically. The extended antigen phenotype prediction is useful in workups of suspected Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT). In platelet refractory patients in whom HLA antibodies have been ruled out, it can be used to assess alloimmunization and select platelet products.
The HPA-Panel tests for the following (with gene name, nucleotide and amino acid information in HGNC nomenclature):
- HPA-2a/2b in GPIba
- GP1BA c.482C/T (T145M)
- aka Kob/a
- HPA-3a/3b in GPIIb
- ITGA2B c.2621T/G (I843S)
- aka Baka/b
- HPA-4a/4b in GPIIIa
- ITGB3 c.506G/A (R143Q)
- aka Pena/b, Yukb/a
- HPA-5a/5b in GPIa
- ITGA2 c.1600G/A (E505K)
- aka Brb/a
- HPA-6a/6b in GPIIIa
- ITGB3 c.1544G/A (R489Q)
- HPA-7a/7b in GPIIIa
- ITGB3 c.1297C/G (P407A)
- HPA-8a/8b in GPIIIa
- ITGB3 c.1984C/T (R636C)
- HPA-9a/9b in GPIIb
- ITGA2B c.2602G/A (V837M)
- HPA-11a/11b in GPIIIa
- ITGB3 c.1976G/A (R633H)
- HPA-15a/15b in CD109
- CD109 c.2108C/A (S682Y)
- aka Govb/a
Indications for Patient Care
- Determining extended red cell phenotype in a patient who
- Was recently transfused
- Has a positive direct antiglobulin test
- Has autoimmune hemolytic anemia
- Is taking (or is going to begin taking) anti-CD38 (daratumamab) or anti-CD47 therapeutic agent
- has sickle cell disease or thalassemia
- needs long-term transfusion support
- has had a bone marrow transplant and for whom engraftment is not complete and/or has produced a red cell alloantibody
- Investigation of antigen typing discrepancy
- Resolution of weak D typing to determine risk of alloimmunization
- Rule in/out weak D types 1, 2, and 3
- Identify partial D status
- Determine candidacy for Rh immunoprophylaxis
- Determination of paternal RHD gene zygosity
- Identification of Rh variants (C, c, E, e, hrB, hrS)
- RHCE variant allele assignments for purposes of Rh genotype matching
- Evaluation of risk of hemolytic disease of the fetus or newborn (HDFN)
- Evaluation of risk of neonatal alloimmune thrombocytopenia (NAIT)
- Human Platelet Antigen (HPA) prediction for platelet refractoriness and post-transfusion purpura (PTP)
- Sequence-specific primer (SSP)-Polymerase chain reaction (PCR)
- PCR-restriction fragment length polymorphism (RFLP)
- Array-based genotyping for red cell and platelet antigens
- Sequence-based testing (SBT) using genomic DNA and cDNA
- Plasmid cloning of PCR products followed by Sanger Sequencing
How to Order Testing
- For customers using Connect, please select Molecular Testing and complete the electronic form. Print a copy of the form and include with the sample.
- To submit a sample using a paper request, please complete the Molecular Testing Request Form.
- Orders placed without an ordering physician or test type selected will not be accepted.
- All samples and the Request for Molecular Testing form must be clearly labeled with the full name of the individual and a unique identification number. The information on the tube must match the information on the request form. Sample labels should also include date and time of collection.
- Samples without sufficient information for unique identification will be rejected.
- Samples less than 10 days old are preferred. DNA yield of older specimens may be low or QNS.
- Whole Blood: 5-10 ml EDTA (lavender top) or ACD type A (yellow top) whole blood tube. If submitting pre- and post-transplant samples, clearly label them as such and submit separate request forms. Lithium heparin sample tubes are NOT acceptable for testing
- Amniocytes: 1-5 ml amniotic fluid or 1-5x106 cultured amniocytes. A maternal blood sample is required when submitting fetal sample. Submit amniocytes and maternal samples with separate request forms
- Buccal swabs may be acceptable depending on the testing requested; contact the laboratory for more information. Buccal swabs should be air dried completely before putting in bag or envelope.
- Ship according to Department of Transportation regulations for biological specimens.
- Whole blood sample can be shipped at room temperature or refrigerated using ice packs or wet ice sealed in plastic bags
- Amniocytes must be shipped refrigerated using ice packs or wet ice sealed in plastic bags, according to DOT regulations for biological specimens.
- Air-dried buccal swabs can be shipped at room temperature
- Wrap all samples in absorbent materials to safeguard from freezing or breakage.
- Ship all samples “Next Day” delivery or via American Red Cross courier, if applicable.
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Kacker S, Vassallo R, Keller MA, Westhoff CM, Frick KD, Sandler SG, Tobian AA. Financial implications of RHD genotyping of pregnant women with a serologic weak D phenotype. Transfusion. 2015 Sep;55(9):2095-103. doi: 10.1111/trf.13074. Epub 2015 Mar 21. PubMed PMID: 25808011; PubMed Central PMCID: PMC4739823.
Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller MA, Johnson ST, Katz L, Queenan JT, Vassallo RR, Simon CD; 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. College of American Pathologists Transfusion Medicine Resource Committee Work Group. Transfusion.
2015 Mar;55(3):680-9. doi: 10.1111/trf.12941. Epub 2014 Dec 1. PubMed PMID:25438646; PubMed Central PMCID: PMC4357540.
Joint Statement on RHD Genotyping