Assessing reagents and techniques for identifying RhCE variants in routine serological testing

Identifying RhCE variants is essential to prevent alloimmunization and manage complex cases. Unfortunately, these variants are often only detected after antibody formation, as they may go unnoticed in serological tests. This study aimed to assess monoclonal antisera using various methodologies to define the reactivity patterns of some variants by variable expression of RhCE antigens.

Samples were chosen based on atypical reactivity on routine RhCE typing of donors, screening of Afro-descendant donors using tube testing and patient samples with antibodies against their own antigens. All 53 samples were tested using tube, gel and microplate tests with five antisera. Antigen expression was assessed by flow cytometry, and RhCE variants were molecularly classified.

Tube test screening of African descent donors proved more effective in identifying a broad range of weak or partial antigens, particularly when using anti-e composed of MS-21, MS-16, MS63 clones and anti-c from the MS8011531019 clone. Automated instrument phenotyping successfully identified samples with RHCE*ceJAL allele, while most other variants were detected as positive (4+), similar to gel test, which intensified most reactions. When comparing methods and antisera for detecting variant e antigens, tube test identified a higher percentage of weak samples (63%-77%) compared with microplate (35%) and gel tests (14%).

The results highlight the critical role of tube test in serological routines and the need to select clones capable of identifying RhCE variants. Detecting reduced RhCE antigen expression during routine serological testing can guide further molecular investigations and help prevent Rh alloimmunization.