Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab

Rituximab (RIT), a murine/human chimeric monoclonal antibody directed against human CD20 is under investigation for its role in transplantation. RIT causes B-cell crossmatches to appear positive. Pronase, a proteolytic enzyme that targets F c receptors removes CD20 from B cells. After CD20 is remove...

Full description

Saved in:
Bibliographic Details
Published in:Human immunology 2004-08, Vol.65 (8), p.803-809
Main Authors: Bearden, Christopher M., Agarwal, Avinash, Book, Benita K., Sidner, Richard A., Gebel, Howard M., Bray, Robert A., Pescovitz, Mark D.
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal - blood
Antibodies, Monoclonal, Murine-Derived
B cells
Cell Culture Techniques
crossmatch
CV
FCXM
FITC
flow cytometric crossmatches
Flow Cytometry
fluorescein isothiocyanate
Histocompatibility Testing - methods
HLA testing
HLA+
Humans
Isoantibodies - blood
Kidney Transplantation - immunology
Leukocytes, Mononuclear - drug effects
Leukocytes, Mononuclear - immunology
mAb
median channel value
monoclonal antibodies
monoclonal antibody
NHS
normal human serum
PBMC
PBS
percentage coefficient of variation
peripheral blood mononuclear cells
phosphate-buffered saline
phycoerythrin
pooled positive control sera
Pronase - pharmacology
RIT
Rituximab
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Rituximab (RIT), a murine/human chimeric monoclonal antibody directed against human CD20 is under investigation for its role in transplantation. RIT causes B-cell crossmatches to appear positive. Pronase, a proteolytic enzyme that targets F c receptors removes CD20 from B cells. After CD20 is removed, RIT should not bind, making it possible to detect class I or class II antibodies on treated B cells. In this study, we incubated RIT with normal human serum (NHS, negative control) or pooled sera from highly sensitized (>50% panel reactive antibody, HLA+) subjects awaiting renal transplantation (positive control) and then performed B-cell flow cytometric crossmatches using untreated or pronase treated B cells as targets. We observed that untreated B cells incubated with RIT-spiked NHS displayed a significant increase in surface fluorescence compared with NHS without RIT, similar to the fluorescence that occurs with a positive crossmatch. In contrast, when CD20 was cleaved from the B cells with pronase, B cells displayed a negative crossmatch with the RIT-spiked NHS. In addition, there was no change in the crossmatches of pooled high panel reactive antibody (PRA) sera after pronase treatment. RIT could be used without worry about losing the ability to perform transplant immunologic monitoring.
ISSN:0198-8859
1879-1166
DOI:10.1016/j.humimm.2004.06.001