| Abstract|| |
Complement-mediated cytotoxic antibodies in conventional cross match, often result in misappropriation of true positives and borderline positives which are detrimental to allograft survival. Flow cross matches (FCXM) are sensitive to capture even non complement fixing cytotoxic antibodies. This retrospective study evaluates the utility of FCXM in effectively predicting acute allograft rejection. A total of 17 cases were processed for FCXM (T and B cell) of whom seven had no rejection episodes, while the remaining 11 had acute rejection despite negative cross match and panel reacting antibodies being negative (less than 20%). The sensitivity and specificity of the FCXM outcome demonstrated that positive B-cell FCXM has potential to be a good tool in pre-transplant screening. The current analysis proposes the possible utility of B-cell positive FCXM as a more sensitive parameter in predicting acute allograft rejection prior to transplantation.
Keywords: FCXM, Acute allograft rejection, T-cell flow cross match, B-cell flow cross match
|How to cite this article:|
Lakshmi Kiran C, Kamaraju SR, Kancherla R. Retrospective Analysis of T and B Cells Flow-Cross Matches in Renal Transplant Recipients. Saudi J Kidney Dis Transpl 2008;19:571-3
|How to cite this URL:|
Lakshmi Kiran C, Kamaraju SR, Kancherla R. Retrospective Analysis of T and B Cells Flow-Cross Matches in Renal Transplant Recipients. Saudi J Kidney Dis Transpl [serial online] 2008 [cited 2020 Jan 29];19:571-3. Available from: http://www.sjkdt.org/text.asp?2008/19/4/571/41316
| Introduction|| |
Flow cytometry cross match (FCXM) is currently accepted as the most sensitive cross match assay to detect circulating antibodies against prospective donors. Conventional complement-mediated cytotoxicity (CDC) does not distinguish between IgG and IgM type of antibodies as well as non complement fixing antibodies. Identification of the pre-sensitized status of the recipient is mandatory for good graft survival. Flow cross match is more sensitive than CDC and thus is ideal as the primary cross match technique. , Nevertheless, the utility of cross match as one of the pre-transplant diagnostic tools needs further documentation for its applicability, simplicity, reproducibility, particularly in living related donors.
The current retrospective analysis was performed utilizing T and B cell flow cytometry cross match (3 color) in 17 patients. The role of B-cell cross match in predicting graft rejection was also addressed.
| Material and Methods|| |
CDC XM/PRA screening
The CDC cross match was performed using the ASHI technique with whole blood PBL and separated T and B cells using Dyna beads. Lymphocytes from cadaver tissue were isolated from six cases. The remaining 11 were living related donors. A positive cross match was defined as any degree of cytotoxicity above the negative controls that are not reduced by DTT. PRA screening was performed using trays made in-house as well as commercially available ones (one Lambda, USA). Patients negative by CDC cross match, AHG and PRA, were further processed by FCXM.
Flow cytometry cross match (3 color)
Whole blood PBL (50 µl) and lymphocytes from lymph nodes (50 µl), whose concentration was adjusted to 10 x 10 6 cells/ml, were distributed in each of six tubes constituting two negative controls (NHS-HLAFlow), one positive control, two test sera (most recent and near past) and one tube with PBS-azide. The cells were incubated with the respective sera for 20 minutes at 4° C. The cells were then washed thrice with PBS-azide in serofuge at 1000g. Rabbit antihuman IgG FITC (1:40) 50 µl, 10 µl of CD3 PC5 (1:4) and 10 µl of CD19 ECD were added to the cell pellet and further incubated for 20 minutes at 4° C. Three washes with PBS-azide were performed to the cells, which were then fixed with 500 µl of para formaldehyde. Acquisition and analysis was performed using Coulter EPICS XL with System II software. Collection was done using LIN FSC, LIN SSC, LOG FL1, LOG FL2, LOG FL3 and LOG FL4 instrument settings. Median values were converted into MESF value for the NHS (number of MESF to span 99% confidence interval was used to determine the cut off between a negative and positive result).
| Results|| |
The CDC cross match was negative, including PRA less than 20%, prior to renal transplantation in all the 17 subjects under study. Chi-square analysis of the T and B cell flow cross match showed significant difference between the rejected grafts and grafts that were not rejected (p< 0.05) [Table 1],[Table 2].
| Discussion|| |
Cytotoxic IgG against Class I antigens can contribute to renal dysfunction or failure after transplantation. However, the clinical relevance of IgG measured by FCXM is controversial, particularly the positive B-cell cross match. The results of the present study demonstrate 40% sensitivity for positive Tcell cross match only, while it was 90% for positive B-cell cross match. The chi-square value of 9.75 with positive B-cell cross match significantly correlates with graft predictability. These observations are in confirmation with the studies conducted by Kimball et al., Buabat et al., Utzig et al. and Lefor et al. ,,, Further, Mahoney et al., reported that a positive B-cell FCXM has an adverse clinical impact only for high-risk second allograft recipients.  Another study by Kerman et al., suggested that, after a negative donorspecific IgG-AHG cross match, an IgGpositive FCXM is not a contra-indication for transplantation. , The presence of IgM may be beneficial in reducing the occurrence of rejection episodes and improving graft survival. The UNOS Scientific Transplant Registry reported 8.8% B-cell FCXM positivity of the total 13% FCXM positive results.  The FCXM outcome not only could be utilized in predicting graft survival, but also as a post-transplant graft monitoring tool, especially in recipients who have other risk factors for rejection. 
T and B cell FCXM's are currently performed using 1024-channel or 256-channel flow cytometer and the shift in median channel fluorescence (SMCF) over the negative control. The current study makes an attempt at more objective assessment of the channel shifts on Coulter Epics XL by further modulating the data with the aid of Windows based software Quickcal over the two negative controls. The relative fluorescence intensity is converted into molecules of equivalent soluble flurochrome (MESF) values before they are incorporated into Microsoft software (Access) for appropriate interpretation of the fluorescence intensities.
The data demonstrates the value of a positive B-cell FCXM for predicting post transplant rejections particularly when evaluated in the context of prior sensitization and DR mismatching.
| References|| |
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C Lakshmi Kiran
Transplant Immunology Laboratory, Global Hospitals, Lakdi-ka-Pool, Hyderabad500 004, (A.P)
[Table 1], [Table 2]