BACKGROUND: Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the "standard of practice" in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. METHODS: Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. RESULTS: After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78+/-10 to 57+/-4 (P<0.05) and 107+/-11 to 49+/-3 (P<0.00001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-positive reaction observed. In this study, pronase treatment improved the specificity of B cell FCXM for detecting class II antibodies from 75% to 100% (P=0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evaluated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identi. fied in each case. CONCLUSION: Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.
BACKGROUND: Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the "standard of practice" in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. METHODS: Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. RESULTS: After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78+/-10 to 57+/-4 (P<0.05) and 107+/-11 to 49+/-3 (P<0.00001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-positive reaction observed. In this study, pronase treatment improved the specificity of B cell FCXM for detecting class II antibodies from 75% to 100% (P=0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evaluated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identi. fied in each case. CONCLUSION: Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.
Authors: E K Page; A J Page; J Kwun; A C Gibby; F Leopardi; J B Jenkins; E A Strobert; M Song; R A Hennigar; N Iwakoshi; S J Knechtle Journal: Am J Transplant Date: 2012-07-09 Impact factor: 8.086
Authors: Lionel Rostaing; Pablo Massari; Valter Duro Garcia; Eduardo Mancilla-Urrea; Georgy Nainan; Maria del Carmen Rial; Steven Steinberg; Flavio Vincenti; Rebecca Shi; Greg Di Russo; Dolca Thomas; Josep Grinyó Journal: Clin J Am Soc Nephrol Date: 2010-11-04 Impact factor: 8.237
Authors: Eun Young Song; Yu-Joo Lee; Jungwon Hyun; Yon Su Kim; Curie Ahn; Jongwon Ha; Sang Joon Kim; Myoung Hee Park Journal: Ann Lab Med Date: 2012-02-23 Impact factor: 3.464