Figure 815

Techniques of crossmatch testing. Early crossmatch testing provided a means to prevent most but not all hyperacute rejections. These early tests were performed with a technique of rather low sensitivity. Subsequently, more sensitive techniques were employed in an attempt to not only prevent all hyperacute rejections but also improve graft survival rates. Techniques that have been used include variations of the lymphocytotoxicity test that incorporate wash steps, change in incubation times or temperatures, or both, or add an antiglobulin reagent. Flow cytometry and an array of other methods such as antibody-

dependent cellular cytotoxicity also have been tried. Two of the most sensitive techniques are the antiglobulin-augmented lym-phocytotoxicity (AHG) and flow cytomet-ric crossmatching. A, The use of flow cytometry to define the lymphocyte population by light scatter parameters, followed by a specific marker for T lymphocytes, ie, CD3 (B) allows this technique to be highly specific for human leukocyte antigen (HLA) class I-positive cells. The donor lymphocytes have been preincubated with recipient serum, washed, and subsequently stained with AHG-Fluorescsin isothio-cyanate (FITC), a fluorochrome-labeled antihuman globulin. C, Results of flow cytometric cross-matching are evaluated as shifts in the fluorescence from negative sera and are interpreted as positive or negative based on independently defined cutoffs above the negative. D, Multiple studies in renal transplantation have shown correlations between positive AHG or flow cyto-metric cross-matches and decreased graft survival at 1 year or more. The largest differences are seen when patients are grouped as primary grafts versus repeat grafts. In some instances the effect of using a more sensitive cross-match technique only can be seen in patients having repeat grafts or those with a higher immunologic risk. CD3 PE—monoclonal antibody to CD3 fluorescent labelled with phycoery-thrin; FC—constant fragment of IgG molecule; FITC—fluorescent labelled with fluorescein isothiocynate; FSC—forward scatter; R1—region 1; R2—region 2; SSC—side scatter. (Panel D adapted from Cook [3]; with permission.)

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