Validation of flow cytometry to quantify the potency of anti-D immunoglobulin preparations.

Abstract

Flow cytometry has been recommended as an alternative to that of autoanalyser methodology for estimation of anti-D potency. This investigation was performed to validate the flow cytometry method based on a quality assurance system. A flow cytometry method based on indirect labelling of Rh(D)-positive red blood cells was validated using the parameters precision and accuracy and was compared to the autoanalyser data of manufacturers of anti-D immunoglobulin preparations. The experiment first investigated the possible differences between assays from single donors compared with a pooled assay. Red blood cells of four individual donors and their pooled red blood cells were interchangeable. There was no significant difference between donors, on one hand, and between the use of a single donation and the pooled red blood cells, on the other hand (P = 0.695). The two-sided 95% confidence intervals (CIs) of the difference between single donors and the pool ranged from -4.6% to 4.7%. The intermediate variability was determined by standard deviation (SD) = 48.4 IU/ml [coefficient of variation (CV) = 3.8%]; the repeatability was SD = 34.3 IU/ml (CV = 2.7%). Using a spiking experiment, the second part of the experiment investigated recovery of a known anti-D potency. The recovery of samples spiked with defined amounts of reference preparation was 97.7-101%, with a mean bias of -1.3 (95% CI: -4.1 to 1.6). The results of the flow cytometry assay, as compared to those of the autoanalyser performed by the manufacturers of anti-D immunoglobulin preparations for those manufacturers who have their method validated in-house, ranged from 87 to 129%, indicating good correlation. Flow cytometry is a suitable quality control method for polyclonal anti-D immunoglobulins, which can be standardized in a quality control laboratory using a quality assurance system.