Flow cytometry antibody screening using pooled red cells.

BACKGROUND: For red cell alloantibody screening, the column agglutination technique (CAT) is used extensively, and flow cytometry (FC) screening has recently been demonstrated to be accurate, rapid, and cost effective. We attempted to determine whether the high sensitivity of FC allows pooling of screening red cells, which is generally not an acceptable technique in CAT. METHODS: For FC screening, a commercial two-cell screening panel was utilized for the preparation of individual cells (CSi), as well as pooled cells diluted 1 in 2 (CSp), and 1 in 3 (CS1/3). Another panel was pooled from 120 randomly selected group O donors (RSp). RESULTS: Comparing the endpoint titrations of serial dilutions, CS1/3 was found to be one dilution, on the average, less sensitive than CSi. In 33 CAT-positive patient samples, the sensitivities of CSi and CSp did not differ significantly without polyethylene glycol (PEG) (30/33, 26/33, respectively, P = 0.125), although they did differ significantly with PEG (32/33, 25/33, respectively, P = 0.016). The percentages of reactive cells among the total cells from RSp were roughly proportional to the relevant antigen frequencies of the local donors. CONCLUSIONS: A trend toward reduced sensitivity was observed using pooled red cells, even via FC. Pooled cells from randomly selected group O donors may be employed as another method by which the characteristics of known antibodies might be assessed.

Flow cytometry PRA using lymphocyte pools from random donors.

BACKGROUND: Pools of lymphocytes from carefully chosen donors have been used for flow cytometry (FC) panel reactive antibody (PRA) assays. We intended to devise an FC PRA assay using mixed lymphocyte pools from a large number of randomly selected donors (RD FC PRA) to accurately predict the likelihood of a positive HLA crossmatch. METHODS: Lymphocyte pools were prepared from randomly selected donors (N = 120). %PRA was calculated based on the anti-IgG FITC histogram of the T cells. The proposed RD FC PRA assay was assessed in comparison with the bead FC PRA, antiglobulin-augmented CDC (AHG-CDC) PRA assay, and the expected %PRA calculated by summing up the antigen frequencies of the known specificities. RESULTS: In 29 FC crossmatch positive sera, the positivity rate for the bead FC, RD FC, and AHG-CDC PRA was 100, 100, and 79%, and the mean %PRA was 77% +/- 0.205). In 19 sensitized patients with a negative FC crossmatch, the positivity rate was 21% using the RD FC PRA and 16% using the bead FC PRA, which suggested that both assays had similar abilities to detect low levels of HLA antibodies. CONCLUSIONS: The RD FC PRA assay allows easy panel preparation, reduces cost, and naturally reflects the probabilities of a positive crossmatch in the population to which the cadaveric donor belongs. Therefore, this new assay is expected to be useful as another approach to determine the % PRA.