Introduction:

The LMX-SAB has been extensively used for DSA virtual crossmatch (VXM) to predict actual crossmatch (AXM). The extreme pH and harsh chemical reaction conditions used in the preparation of LMX-SAB can cause unpredictable denaturation and configuration changes on HLA-Ag molecules and result in both false positive and false negative VXMs.

A novel cell-based flow cytometry DSA detection method (DSA-FXM) was developed in our lab. It is able to selectively detect HLA-Abs reactive with native HLA molecules presented on the cell surface (Fig. 1) and eliminate false positive DSA called by LMX-SAB.

Methods:

DSA-FXM: 0.2X10⁶ donor PBMCs were incubated with recipient serum at RT for 30', washed, and then the beads conjugated with anti-HLA class-I (CI) and -II (CII) MoAbs were added. The cells were lysed and the complexes of HLA-Ag-Ab were captured by CI or CII beads; and further detected by PE-anti IgG on a flow cytometer.

FXM: Regular IgG FXM procedure

LMX-SAB: One Lambda method

Results:

A total of 116 samples were tested by DSA-FXM, FXM, and LMX-SAB. 140 DSA specificities were identified by DSA-FXM and confirmed by LMX-SAB which covered all class I and II specificities including A, B, C, DR, DQ, and DP (Table 1). 8 DSA+ LMX-SAB samples were found to be false positive (MFI: 5951-10782), proved negative by DSA-FXM and FXM (Table 2).

Conclusions:

The antibodies reacting with non-native HLA molecule can cause false positive DSA results on LMX-SAB leading to an incompatible virtual crossmatch. DSA-FXM was able to eliminate the false positive and detect the true DSA against the native HLA-Ags expressed on the donor cell surface.

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