A micro colorimetric assay using cryopreserved monocytes to evaluate antibody-mediated red cell-monocyte interaction

A number of in vitro assays based on the interaction of red cells with monocytes have been used to determine the clinical significance of red cell antibodies. When used in our laboratory, one of these assays (the monocyte-macrophage phagocytosis assay-MMPA), was very time consuming and showed great variability.

We set up a monocyte phagocytosis colorimetric assay (MPCA), using standard microtiter plate wells coated at 37 degrees C for 1 hour with monocytes from healthy donors. After washing the wells to remove non-adherent monocytes, test red cells are added to the wells. Sensitized red cells bind to the monocytes, which are lysed after incubation to measure red cell phagocytosis. This is done by hemoglobin detection in the lysate through reaction with o-phenylenediamine and absorbance evaluation with a colorimeter. The results are expressed as the phagocytosis index (PI), which is calculated with the following formula: PI = [1-(A450 unsensitized red cells/A450 sensitized red cells)] x 100. In this study we determined: the source of MPCA variability; the precision of MPCA results; the correlation between MMPA and MPCA results; the MPCA reference values and the MPCA and MMPA execution times.

MPCA variability depended largely on the monocyte source. The smallest variation coefficient of the results of replicate assays (19-21%) was found using pooled, cryopreserved monocytes. When performed with a pool of cryopreserved monocytes from 10 subjects, the SD of PI values obtained in replicate assays showed little variation (11-13) over the range of anti-D concentrations tested (from 18.75 to 300 ng/mL). A linear correlation coefficient r of 0.96 was obtained when MPCA and MMPA were performed in parallel, and the 95th centile of PI reference values determined with red cells of 40 non-transfused surgical patients free of irregular red cell antibodies was 7. MPCA execution time was 56% of that needed to perform MMPA.

These studies show that MPCA is an easy and reproducible assay which allows objective and automated evaluation of red cell phagocytosis.