Molecular testing of serologic RHD discrepant females within childbearing age-A facility's experience

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Conference Proceeding

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Vox Sanguinis


Background: The RH system is very complex, and follows the ABO system in immunogenicity. The RHD protein is very multifaceted, and depending on a person's genetics, the way in which the RHD antigen is expressed on the red blood cell (RBC) membrane can vary. From this variation, patients can be classified as having a weak expression of the D antigen (formally Du) through serologic testing. Weak reactions with Anti-D can also be caused by a variant (part of the antigen is missing) RhD. Serologic testing, mostly automated, are available to help detect potential RHD discrepancies in patients. Automated testing using two different clones of monoclonal Anti-D reagent provide the ability to detect discrepancies in RHD patient samples. However, only molecular testing can differentiate the RHD gene as being fully expressed or having a variant expression. Serologically detected RHD discrepancies pose the most concern for women of child bearing age (<50 years old). Depending on the RHD variant identified, these women have the potential to develop Anti-D if exposed to the RHD antigen during pregnancy. This facility developed a pilot program to identify the genetic RHD status of qualifying RHD discrepant patients. Determining the RHD status of women <50 years old will aid in accurately conserving Rh-negative RBCS and allow for the proper administration of RhIg prophylaxis in this patient population. Aims: Identify women of child bearing age at risk of developing Anti-D due to the present of a variant RHD gene. Methods: Beginning January 2015, patient samples showing inconclusive results with Anti-D through automated testing are subject to manual serologic investigation via tube testing. Patients who qualified for manual analysis had their RBCS tested with the Anti-D monoclonal reagent validated for tube testing: A) Women <50 years old with manual reactions of ≥2+ had samples sent for molecular RHD determination. Until results are known, these patients are classified as Rh-negative and will receive Rh-negative RBCS and will be candidates for RhIg prophylaxis evaluation; B) Women >50 years old and all men with manual reactions ≥2+ will be classified as Rh-positive and receive Rh-positive RBCs; C) All patients with manual reactions <2+ will be classified as Rh-negative and receive Rh-negative RBCs. Molecular testing results were interpreted for patient support as per molecular laboratory recommendations. Results: As of February 2018, there have been 97 female patients of child bearing age evaluated for RHD testing discrepancy. Seven patients were classified as Rh-negative by manual serological testing. Molecular testing was performed on 90 patients: of which, 8 patients were identified as Rh-positive with no variant, and variant RHD was identified in 82 patients. Variant RHD was identified in 91% of qualified patient samples. Summary/Conclusions: Serologic RHD discrepancies via automated methods is a good predictor for the presence of variant RHD, recommending follow-up and confirmation with molecular testing.



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