EFFECT OF INTRAVENOUS IMMUNOGLOBULIN THERAPY ON FLOWPRA ASSAYS.
P Warner and E Klohe, HLA Laboratory, Inland Northwest Blood Center, Spokane, WA.
FlowPRA assays have been demonstrated to be a highly sensitive and specific means to determine the presence of anti-HLA antibodies in the serum of transplant candidates. However, we and others have noted that high or low serum protein levels may cause histogram peaks to shift substantially to the right or left, respectively, compared to normal serum. Our thoracic transplant candidate population includes many patients who have been sensitized by blood transfusions during mechanical assist device implantation. These patients often receive repeated high dose intravenous immunoglobulin (IVIG) to reduce their PRA level and increase their opportunity to be transplanted. We evaluated the effect of IVIG on the FlowPRA I/II assay to determine its utility in PRA monitoring. IVIG was diluted with normal serum to obtain a range of IVIG concentrations of 10-50%, which mimics concentrations of those achieved with in vivo IVIG therapy. Histogram peaks exhibited concentration dependent right-hand (positive) shifts of 20-100% from the normal serum control with both FlowPRA I and II beads. However, normal histogram peak architecture is maintained in that the entire population of beads in each sample is shifted uniformly. This suggests non-specific immunoglobulin binding.
Mixtures of positive PRA sera and IVIG were tested by FlowPRA I/II to determine whether HLA antibody detection would be masked. The multiple peaks of high PRA sera, representing multiple antibody specificities and titers, assumed a bell-shaped histogram and shifted even further to the right with the addition of IVIG. Typical histograms of low PRA sera have a major negative peak (overlaying the normal control) and one or more minor peaks exhibiting a right-hand shift. The addition of IVIG shifts all events to the right and reduces the number of minor peaks in the tail.
In conclusion, IVIG appears to non-specifically bind to FlowPRA I and II beads, causing histogram peaks to uniformly shift to the right. Specific HLA antibody peaks are partially or totally obscured. Therefore, neither channel shift or peak architecture may be used to elucidate HLA antibodies by FlowPRA methodology in the presence of IVIG. FlowPRA methodology is not recommended to monitor PRA following IVIG therapy.