Flow cytometric crossmatch results using lymphocytes isolated from donor peripheral blood and spleen tissue on five consecutive days

Abstract

A negative flow crossmatch (FXM) is considered as a “go ahead” to transplant solid organs at many centers. FXM is a biologic assay where donor lymphocytes are reacted with recipient sera to identify donor specific antibodies (DSA). Lymphocytes can be derived from different sources like peripheral blood (PB), lymph node or spleen. Mix of cellular components from all three sources are different and also may have different HLA expression depending on the age of the sample. We present our data to understand this variability. Lymphocytes were isolated from PB and spleen of the same donor on five consecutive days using a different sample aliquot. Lymphocytes from PB were isolated by Ficoll–Hypaque technique. Lymphocytes from spleen tissue were isolated using a cell separation kit (Stemcell Technologies). Recovered cells were treated with 1mg/2mL pronase and re-suspended for testing in PBS containing 2% fetal calf serum. FXM was performed in a tray after mixing 3×105 cells with 20 μl of serum. After washing, cells were labeled with FITC goat anti-human IgG, anti-CD3 PerCP and anti-CD19 PE then acquired on a FACS Calibur to detect DSA. A comparison of the FXM mean channel shift (MCS) results for PB and spleen with a cutoff of 52 for T-cells and 106 for B-cells were made for 5 consecutive days. The negative control mean fluorescence intensity (MFI) increased with specimen age for PB and spleen (Figure A), which caused a decrease in MCS in the positive control over time for both lymphocyte sources (Figure B). Patient samples with FXM results close to the T-cell and B-cell cutoffs were discrepant based on lymphocyte source and age of specimen (Table 1). Older samples have a higher negative control which results in a decrease in the MCS and may cause borderline positive results to be reported as negative. The isolation of lymphocytes from PB by day 5 proved to be difficult as the prep was contaminated with whole blood components. Our data showed that the interpretation is dependent on the donor sample source and quality.Download : Download full-size image