Improved specificity and sensitivity when using pronase-digested lymphocytes to perform flow-cytometric crossmatch prior to renal transplantation

Abstract

Several laboratories have resorted to flow-cytometric crossmatch (FCXM) in an effort to prevent hyperacute and accelerated renal allograft rejections. The currently employed FCXM has problems with both false-positive and -negative reactions, largely as a result of irrelevant IgG binding to Fc IgG receptors. In 1980, we circumvented this problem by digesting Fc IgG receptors with pronase, and demonstrated that, with immunofluorescence microscopy (IF), detection of IgG anti-HLA antibodies was highly sensitive and specific. In 1995, we introduced the pronase technique to FCXM and showed that this enzyme did not decrease HLA expression. We present herein a prospective study at our institution to determine whether FCXM using pronase-digested (PD) lymphocytes is as sensitive and more specific than FCXM with undigested (UD) lymphocytes when compared with the highly sensitive and specific I F assay. In analyzing the 186 donor-specific prerenal-transplant crossmatches, we found that PD FCXM was as sensitive and specific as I F and was able to detect weak IgG anti-HLA antibodies that bound to B cells. Fourteen of these patients would have been denied transplants if one were to have relied on UD FCXM. The data clearly indicate that PD FCXM can reliably be used to detect weak IgG anti-HLA antibodies before renal transplantation.

Systematic study of cell isolation from bovine nucleus pulposus: Improving cell yield and experiment reliability

“…In cell characterization by surface markers, the use of pronase may not be preferred as it may damage the surface proteins due to its relatively low substrate specificity. A number of studies have been performed to investigate the effect of pronase in different applications and reported that proteases affected the cell properties of Lymnaea neurons, extracellular matrix of chondrocytes, lipopolysaccharide receptor of Kupffer cells, and Fc IgG receptors, CD16 and CD32 of lymphocytes (details given in the supplementary file). Thus we also tested the effect of pronase on surface antigens using human mesenchymal stem cells (MSC) from bone marrow.…”

Biomarkers in transplantation: Prospective, blinded measurement of predictive value for the flow cytometry crossmatch after negative antiglobulin crossmatch in kidney transplantation

“…11,15,16 During this time, evidence has accumulated to indicate that this assay is more sensitive than conventional CDC techniques, may detect antibodies which are undetected by other routine crossmatch methods, and may provide important predictive information regarding patient risk and 30 However, this information is complicated by clinical, methodological, and analytical differences between reports. Variable inter-laboratory consensus and false-negative rates point to concerns of assay precision and specificity 30,31 that may reflect differences in methodology or equipment, 30,[32][33][34] in operator technique, or in criteria for defining a positive result in different centers. 33,35,36 In the absence of clear evidence founded on prospective-blinded studies, the objective prognostic performance of FCXM remains unclear.…”

Liver transplantation with a strongly positive crossmatch: Case study and literature review

“…Flow cytometry crossmatching was performed on the day of transplantation with a current serum sample. Briefly, donor cells treated with pronase (1 mg/dL) were incubated with a patient's serum, and HLA antibodies were detected with fluorescein isothiocyanate–conjugated goat [F(ab)′ 2 ] anti‐human immunoglobulin G (IgG; Fc‐specific) and acquired with FACSCalibur. Anti‐CD3 peridinin chlorophyll protein and anti‐CD19 R‐phycoerythrin (BD Biosciences) were used to gate T and B cell populations, respectively.…”