False-Positive Flow CytometryCrossmatch Results With Pronase-Treated T Cells in Human Immunodeficiency Virus-Infected Patients

Abstract

A positive flow cytometrycrossmatch (FCXM) is often a contraindication for transplantation. Nonspecific binding of IgG antibodies to Fc receptors on B cells decreases sensitivity and specificity of the B-cell crossmatch. To prevent this, pronase treatment (PT) of lymphocytes is widely used. Effects of PT on T-cell FCXM are not well documented. We have observed a high positive T-cell FCXM rate in the absence of donor-specific antibodies (DSA) in HIV+ patients. This study was designed to determine the probable cause of these aberrant results. Twenty-six HIV+ and 30 HIVpatients were included. Three hundred forty-eight serum samples from HIV+ patients were crossmatched with PT cells, and 81 serum samples were crossmatched without PT (NT) cells from 196 deceased donors. Sixty serum samples from HIV-patients were crossmatched with PT and NT cells from 48 deceased donors. Selected serum samples were pretreated with reducing agents and Adsorb-Out beads. Ninety-seven percent of HIV+ patients exhibited positive T-cell FCXM with PT cells in the absence of DSA; 17% had a weak positive result with NT cells. Only 9% of HIV+ patients had a weak positive B-cell FCXM with PT cells in the absence of DSA; 16% had the same result with NT cells. Positive FCXM results were observed with PT but not with NT CD4+ and CD8+ T-cells. Pretreatment of serum samples with reducing agents or Adsorb-Out beads showed no effect. Nonspecific IgG binding does not cause falsepositive T-cell FCXMs in HIV+ patients since it is not observed on B cells and reducing agents and Adsorb-Out beads did not have any effect. These results are most likely due to autoreactive antibodies recognizing cryptic epitopes exposed by PT on T cells. Thus, a positive T-cell FCXM with PT cells should not be considered a contraindication for transplantation in HIV+ patients.