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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 19  |  Issue : 4  |  Page : 571-573
Retrospective Analysis of T and B Cells Flow-Cross Matches in Renal Transplant Recipients


Global Hospitals, Lakdi-ka-Pool, Hyderabad (A.P), India

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   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 2019 Sep 20];19:571-3. Available from: http://www.sjkdt.org/text.asp?2008/19/4/571/41316

   Introduction Top


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. [1],[2] 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 Top


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 Top


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 Top


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. [1],[2],[3],[4] Further, Mahoney et al., reported that a positive B-cell FCXM has an adverse clinical impact only for high-risk second allo­graft recipients. [5] 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. [6],[7] 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. [8] 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. [9]

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 Top

1.Bittencourt MC, Rebibou JM, Saint-Hiller Y, et al. Impaired renal graft survival after a positive B-cell flow cytometry crossmatch. Nephrol Dial Transplant 1998;13(8):2059-64.  Back to cited text no. 1    
2.Utzig MJ, Blumke M, Wolff-Vorbeck G, Lang H, Kirste G. Flow cytometry cross match: a method for predicting graft reject­tion. Transplantation 1997;63(4):551-4.  Back to cited text no. 2    
3.Buabat B, Chiewsilp P, Patanapanyasat K, et al. Cross matching technique facilitating kidney transplantation. J Med Assoc Thai 1997;80(Suppl 1);S35-61.  Back to cited text no. 3    
4.Kimball P, Rhodes C, King A, et al. Flow cross matching identifies patients at risk for postoperative elaboration of cytotoxic anti­bodies. Transplantation 1998;65(3):444-6.  Back to cited text no. 4    
5.Lefor WM, Ackerman JR, Alveranga DY, et al. Flow cytometry cross matching and primary cadaver kidney graft outcome: relevance of T and B cell targets, historic sera and auto­logous controls. Clin Transplant 1996;10(6 Pt2):601-6.  Back to cited text no. 5    
6.Mahoney RJ, Norman DJ, Colombe BW, et al. Identification of high and low risk second kid­ney grafts. Transplantation 1996;61(9):1349-55.  Back to cited text no. 6    
7.Kerman HR, Susskind B, Buyse I, et al. Flow cytometry-detected IgG is not a contra­indication to renal transplantation: IgM may be beneficial outcome. Transplantation 1999; 68(12):1855-8.  Back to cited text no. 7    
8.Cook DJ, Fettugh HI, Gjertson DW, Cecka JM. Flow cytometry cross matching (FCXM) in the UNOS kidney transplant registry. Clin Transpl 1998;413-9.  Back to cited text no. 8    
9.Kotb M, Russell WC, Hataway DK, Gaber LW, Gaber AO. The use of positive B cell flow cytometry cross match in predicting rejection among renal transplant recipients. Clin Transpl 1999;13(1Pt2):83-9.  Back to cited text no. 9    

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Correspondence Address:
C Lakshmi Kiran
Transplant Immunology Laboratory, Global Hospitals, Lakdi-ka-Pool, Hyderabad–500 004, (A.P)
India
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PMID: 18580015

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    Abstract
    Introduction
    Material and Methods
    Results
    Discussion
    References
    Article Tables
 

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