Monocyte monolayer assay using plateletpheresis by-products and application to anti-Di^a antibody

The monocyte monolayer assay (MMA) is a key method for predicting the clinical significance of red blood cell (RBC) antibodies. However, its broader implementation is hindered by the challenge of securing a sustainable peripheral blood mononuclear cell (PBMC) source. We assessed plateletpheresis by-products as an alternative PBMC source for MMA and applied the established MMA to assess the clinical significance of anti-Di^a antibodies.

Plateletpheresis by-products were collected from two systems (Trima and Amicus) and categorized into four types: leukoreduction system chamber (LRSC) and thrombocyte apheresis cassette (TAC) from Trima, and air trap (AT) and return line filter (RLF) from Amicus. MMA was performed under varying conditions based on by-product type, PBMC isolation date (D0 or D1), and RBC source (autologous or allogeneic). Anti-Di^a antibodies were evaluated using sera from confirmed cases.

MMA results from 14 plateletpheresis sessions (112 positive and 112 negative control wells using two 8-chamber slides per session) consistently demonstrated expected positivity and negativity across different experimental conditions. The proportion of reactive monocytes was significantly higher when PBMCs from LRSC (22.3 ± 5.7 %) were used, compared to those from TAC (19.0 ± 5.8 %), AT (18.3 ± 4.4 %), and RLF (16.6 ± 2.9 %). No significant differences were observed based on RBC source or PBMC isolation dates. Among 12 cases with anti-Di^a antibodies, 75 % (9/12) showed positive MMA results, suggesting their clinical relevance.

Plateletpheresis by-products are a reliable and sustainable PBMC source for MMA, enhancing its practicality and providing a foundation for PBMC-based immunological studies.