Molecular testing for Rhesus D antigen (RhD) in patients with ambiguous results in serological testing for RhD: implications for transfusion strategy

Background

According to the Guideline "Hemotherapy", patients should be tested for Rhesus D antigen (RhD) by two different monoclonal IgM antibodies which cannot detect the D category VI. In case of an ambiguous reaction, a patient is preliminary declared as RhD-negative and transfusion of RhD-positive red blood cells (RBC) should be avoided before clarification. This approach is time-consuming and presumably results in urgent transfusion of RhD-negative RBC´s to RhD-weak positive patients.

Methods

Patient samples were tested for RhD by manual tube testing (IgM Anti-D Mono-Type, Grifols, Barcelona, Spain) and automated by two monoclonal IgM antibodies with a solid-phase or (since May 2022) a column agglutination assay (Erytype S ABO+D, BioRad, Dreieich, Germany or DG Gel ABO/Rh, Grifols). Samples showing a discrepant result between tube and automated testing or weak reaction with at least one monoclonal antibody were considered ambiguous and forwarded to molecular RhD-testing by PCR (RBC-Fluogene Dweak/variant and RBC-Fluogene CDE, Inno-Train, Kronberg, Germany). Patients were transfused with RhD-negative RBC until the PCR result was available. RhD type of transfused RBC was determined by database analysis.

Results

Between September 2019 and September 2022, 119 patient samples showed ambiguous results. Molecular RhD-testing revealed that 71 (59.7 %) were weak D type 1, 16 (13.4 %) had type 2 and 14 (11.8 %) type 3. Two (1.7 %) patients were type 4.0 and seven (5.9 %) were normal D. Four patients (3.4 %) were dd. One (0.8 %) patient each was type 4.2, type 15, type 45.1, partial D DVII and DAR-E. In 33/119 (27.7 %) patients, 118 RBC`s were transfused, 21 of those patients received 89 RhD-negative RBC´s as the result of molecular RhD-typing was pending. 9/21 had weak D type 1, two type 2, two type 3, one type 4.0 and another two had a normal D. Two patients were dd and one each weak D type 15, type 4.2 and partial D DAR-E.

Conclusion

The majority of patients (110, 92.4 %) had a normal RhD or weak D type 1 – 4.0 and thus could be safely transfused with RhD-positive RBC´s. Only a minority of patients should precautionary be declared as RhD-negative, as transfusions of RhD-positive RBC´s might induce anti-D formation. Urgent transfusions with RhD-positive RBC`s in patients with ambiguous results in the serological RhD-testing is reasonable, safe in the majority of patients and avoids wastage of RhD-negative RBC´s.

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