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Published on: January 7, 2019
Flow cytometry PRA using lymphocyte pools from random donors
Dong Il Won1, Hee Du Jung, Ok-Ju Jung
1Department of Clinical Pathology, Kyungpook National University School of Medicine, Daegu, Republic of Korea. wondi@knu.ac.kr
Insights
A new flow cytometry panel reactive antibody (FC PRA) assay using randomly selected donors (RD FC PRA) accurately predicts positive HLA crossmatches. This method is cost-effective and simplifies panel preparation for HLA antibody detection.
Area of Science:
- Immunology
- Transplantation Immunology
- Clinical Chemistry
Background:
- Flow cytometry (FC) panel reactive antibody (PRA) assays traditionally use lymphocytes from selected donors.
- Predicting positive HLA crossmatches is crucial for successful organ transplantation.
Purpose of the Study:
- To develop and evaluate a novel FC PRA assay utilizing mixed lymphocyte pools from a large number of randomly selected donors (RD FC PRA).
- To assess the RD FC PRA assay's accuracy in predicting positive HLA crossmatches compared to existing methods.
Main Methods:
- Prepared lymphocyte pools from 120 randomly selected donors.
- Calculated %PRA based on anti-IgG FITC histogram of T cells.
- Compared RD FC PRA with bead FC PRA, antiglobulin-augmented CDC (AHG-CDC) PRA, and calculated %PRA.
Main Results:
- RD FC PRA demonstrated 100% positivity rate in 29 FC crossmatch-positive sera, similar to bead FC PRA.
- RD FC PRA detected low-level HLA antibodies in sensitized patients with negative FC crossmatches (21% positivity rate).
- RD FC PRA showed comparable performance to bead FC PRA in identifying HLA antibodies.
Conclusions:
- The RD FC PRA assay simplifies panel preparation and reduces costs.
- This assay naturally reflects the probability of a positive crossmatch within the relevant donor population.
- RD FC PRA offers a valuable alternative approach for determining % PRA and improving crossmatch prediction.
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.
