Improved flow cytometric detection of HLA alloantibodies using pronase: potential implications in renal transplantation.
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.


CONCLUSIONS
Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.
Vaidya
S
S
Department of Pathology, University of Texas Medical Branch, Galveston 77555-0178, USA.
Cooper
T Y
TY
Avandsalehi
J
J
Barnes
T
T
Brooks
K
K
Hymel
P
P
Noor
M
M
Sellers
R
R
Thomas
A
A
Stewart
D
D
Daller
J
J
Fish
J C
JC
Gugliuzza
K K
KK
Bray
R A
RA
D005188:False Negative Reactions; D005434:Flow Cytometry / Q000379:methods*; D006084:Graft Rejection / Q000175:diagnosis; D006680:HLA Antigens / Q000276:immunology*; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D007518:Isoantibodies / Q000032:analysis*; D016030:Kidney Transplantation / Q000276:immunology; D008214:Lymphocytes / Q000187:drug effects; D011402:Pronase / Q000627:therapeutic use*; D012680:Sensitivity and Specificity
D006680:HLA Antigens; D007518:Isoantibodies; D011402:Pronase