The use of pronase-digested human leukocytes to improve specificity of the flow cytometric crossmatch

Peter I. Lobo, Clinton E. Spencer, William C. Stevenson, Christopher McCullough, Timothy L. Pruett

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Two-color fluorescence cytometry (FCXM) has recently been introduced to improve the detection of anti-HLA antibodies that react to donor cells, especially in recipients receiving kidney allografts. Although this assay system is highly sensitive, it lacks specificity. Between 70% and 90% of potential kidney recipients with a positive FCXM would have been denied transplant if such an assay had been used alone to detect antidonor antibodies. Lack of specificity is principally due to normal or irrelevant IgG in aggregates or immune complexes binding to Fcλ R receptors on lymphocytes including B cells and a significant subset of T cells. To circumvent this problem, we digested Fcλ R receptors on lymphocytes with pronase. We present data demonstrating that pronase digestion of lymphocytes does not alter HLA antigenicity. In addition, pronased lymphocytes allow one to use either single- or two-color FCXM. With single-color FCXM, one can quantitate antibody reactivity to lymphocytes via a cursor (on the fluorescence histogram) that separates lymphocytes that do not bind to antibodies. We present data demonstrating that this modification renders FCXM highly sensitive and specific. In addition, one can discriminate between IgG and IgM antibodies that react to lymphocytes.

Original languageEnglish (US)
Pages (from-to)472-480
Number of pages9
JournalTransplant International
Volume8
Issue number6
DOIs
StatePublished - Nov 1 1995

Keywords

  • Crossmatch pronase
  • Flow cytometry, pronase
  • Pronase, crossmatch

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