Differential role for competitive reverse transcriptase-polymerase chain reaction and intracellular cytokine staining as diagnostic tools for the assessment of intragraft cytokine profiles in rejecting and nonrejecting heartallografts

The early and reliable diagnosis of allograft rejection is a difficult task and the assessment of cytokine expression in the grafts can he a helpful p arameter. We have compared competitive reverse transcriptase-polymerase cha in reaction (RT-PCR) with intracellular cytokine staining by flow cytometry as tools to measure cytokine expression in rejecting and nonrejecting muri ne cardiac allografts. Both techniques gave comparable results for cytokine expression in rejecting allografts and syngeneic controls. Grafts from mic e pretreated with anti-CD4 antibody and donor-specific blood transfusion sh owed a marked reduction in cytokine expression, as assessed by competitive RT-PCR, even though a cellular infiltrate was present in the graft. In cont rast, the cytokine production measured by intracellular cytokine staining o f the isolated graft-infiltrating cells was high and exceeded even that of the rejecting allografts. We conclude that intracellular cytokine staining of graft-infiltrating leukocytes by flow cytometry does not necessarily ref lect accurately the cytokine milieu in the graft. This technique might ther efore have a limited clinical application in contrast to competitive RT-PCR for the differentiation between graft acceptance and graft rejection.