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Saudi Journal of Kidney Diseases and Transplantation
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SPECIAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 4  |  Page : 662-665
Pronase-free B-cell flow-cytometry crossmatch

1 College of Medicine, King Saud bin Abdulaziz University for Health Sciences and Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh, Saudi Arabia
2 Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh, Saudi Arabia

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Date of Web Publication8-Jul-2009


Detection of anti-class II antibodies by panel response assay (PRA) and flow cross­match techniques carries an important value in terms of graft function. Even low levels of pre­formed alloantibodies to HLA class II antigens represent a risk of rejection. We present here a method for blocking non-specific flow crossmatch reactions using pooled, heat-inactivated rabbit serum. This method shows very low background and minimal non-specific reactions. In addition, it avoids the use pronase enzyme that can non-specifically digest different cell surface proteins.

How to cite this article:
Hajeer A H, Saleh S, Sutton P, Shubaili A, Anazi H. Pronase-free B-cell flow-cytometry crossmatch. Saudi J Kidney Dis Transpl 2009;20:662-5

How to cite this URL:
Hajeer A H, Saleh S, Sutton P, Shubaili A, Anazi H. Pronase-free B-cell flow-cytometry crossmatch. Saudi J Kidney Dis Transpl [serial online] 2009 [cited 2022 Jul 6];20:662-5. Available from: https://www.sjkdt.org/text.asp?2009/20/4/662/53297

   Introduction Top

HLA class II molecules are not normally ex­pressed on tubular tissues. However, HLA class II expression is induced in renal tubular epi­thelial cells in different conditions including renal allograft rejection. [1],[2],[3],[4]

The presence of pre-formed cytotoxic antibo­dies to donor HLA antigens was first recog­nized in the 1960s as the strongest predictors of graft rejection. [5] Since then, more sensitive cross­matching techniques have been developed, in­cluding flow-cytometry crossmatch testing.

Flow cytometry crossmatch (FCXM) testing was developed in 1983, it is a very sensitive technique for the detection of IgG antibodies to both T and B lymphocytes. [6] Class I specific anti­bodies are detected by both T and B lympho­cytes, however, class II specific antibodies are usually detected by B-cell crossmatches. Posi­tive B cell crossmatch does not mean class II specific antibodies only, but it could be due to class I specific antibodies; B cells do express more class I than T cells or even non-specific (non-HLA) antibodies. [7]

Positive results of B-cell flow crossmatch have been found to be associated with hyper-acute, acute vascular and chronic rejection. [8],[9],[10],[11] These are usually due to anti-HLA class II anti-bodies.

B-cell staining for flow-cytometry analysis can be hampered by non-specific reactions due to the presence of the Fcy receptors on B cells. Pronase treatment was introduced to circumvent this technical issue. [12]

This is not without disadvantages, as pronase enzyme activity is not specific to the Fcy re­ceptors. It is a non-specific protease with pro­teolytic activity that extends to both denatured and native proteins resulting in their break down into individual amino acids. [13]

We have used pooled rabbit serum to block the Fcγ receptors on B cells, therefore, reduced the non specific reactions to the minimum. We present data showing that this modification renders FCXM highly sensitive and specific.

   Methods Top

Donor lymphocytes are incubated with 5% pooled, heat-inactivated rabbit serum (pooled from 5 unimmunized rabbits) for 30 min at 37°C. The cells are then washed one time with PBS/ 0.1%BSA (washing solution). Patients and con­trols sera are added and incubated at 37°C for 30 min. Cells are then washed twice with washing solution. Then, Anti-IgG-FITC together with PE­conjugated anti-CD20 antibodies are added and incubated for 30 min at 37°C. Cells are finally washed three times and resuspended in flow medium. Finally, cells are analyzed for mean fluorescence intensity using BD FACSCanto II machine using 256-channel. Cutoff values for B-cell crossmatch were determined as shift of MFI channel of > 30.

   Results Top

[Table 1] shows mean fluorescent intensity (MFI) and MFI shift of negative and positive serum using rabbit serum as a blocking agent of non­specific flow crossmatch reactions. Results of negative control cells were relatively low with MFI channels in the range of 100. Results of samples with autoantibody against B cells and true positive B-cell flow crossmatch results are also shown.

   Discussion Top

We present here a method for blocking non­specific flow crossmatch reactions using pooled, heat-inactivated rabbit serum. This method shows very low background and minimal non-specific reactions.

Detection of anti-class II antibodies by PRA and flow crossmatch techniques carries an im­portant value in terms of graft function. Even low levels of pre-formed alloantibodies to HLA class II antigens represent a risk of rejection. [14]

It is worth noting that interpreting crossmatch results can not be accurate without the results of a sensitive PRA technique. It is recommended that the PRA method should be of the same sen­sitivity as the crossmatching technique. [15]

Mahoney et al [16] analyzed retrospectively 9031 cadaveric kidney graft recipients who had B­cell crossmatching results. All had negative T­cell crossmatch result. They found that both primary and regraft recipients who were posi­tive B-cell crossmatch were at risk of rejection incidents and early graft loss.

Staining B cells for flow cytometry can suffer from false positive reactions due to the presence of Fcy receptors on the cell surface. Several techniques were attempted to overcome this problem, including pronase treatment [12] and pro­nase plus milk proteins. [17]

Up to our knowledge this is the first study to report the use of rabbit serum, in the absence of pronase, to reduce non-specific B-cell flow-cy­tometry crossmatch reactions. We have used this technique for the last seven years with suc­cessful transplantation program and external QC reports through College of American Pathologists (CAP). We strongly recommend this modifi­cation.

   References Top

1.Forsum U, Claesson K, Jonsson R, et al. Diffe­rential tissue distribution of HLA DR, DP and DQ antigens. Adv Exp Med Biol 1987;216A: 233-9.  Back to cited text no. 1  [PUBMED]  
2.Muller CA, Markovic-Lipkovski J, Risler T, Bohle A, Muller GA. Expression of HLA DQ, DR, and DP antigens in normal kidney and glo­merulonephritis. Kidney Int 1989;35(1):116-24.  Back to cited text no. 2    
3.Von Willebrand E, Salmela K, Isoniemi H, Taskinen E, Krogerus L, Hayry P. Expression of activation markers, HLA class II and IL-2R in acute vascular rejection of human renal allo­grafts. Transpl Int 1992;5 Suppl 1:S690-1.  Back to cited text no. 3    
4.Wadgymar A, Ritchie S, Cattran DC, Fenton S, Halloran PF. Patterns of HLA antigen expr­ession in human kidney disease. Transplant Proc 1987;19(4):3410-4.  Back to cited text no. 4    
5.Patel R, Terasaki PI. Significance of the posi­tive cross match test in kidney transplantation. N Engl J Med 1969;280(14):735-9.  Back to cited text no. 5    
6.Garovoy MR, Bigos M, Perkins H, Colombe BN, Salvatierra O. Flow cytometry analysis: A high technology cross match technique facili­tating transplantation. Transplant Proc 1983;15: 1939-44.  Back to cited text no. 6    
7.Pellegrino MA, Belvedere M, Pellegrino AG, Ferrone S. B peripheral lymphocytes express more HLA antigens than T peripheral lymphocytes. Transplantation 1978;25(2):93-5.  Back to cited text no. 7    
8.Ghasemian SR, Light JA, Currier CB, et al. The significance of the IgG anti-B-cell cross match on renal transplant outcome. Clin Transplant 1997;11(5 Pt 2):485-7.  Back to cited text no. 8    
9.Scornik JC, LeFor WM, Cicciarelli JC, et al. Hyperacute and acute kidney graft rejection due to antibodies against B cells. Transplantation 1992;54(1):61-4.  Back to cited text no. 9    
10.Kotb M, Russell WC, Hathaway DK, Gaber LW, Gaber AO. The use of positive B cell flow cytometry cross match in predicting rejection among renal transplant recipients. Clin Trans­plant 1999;13(1 Pt 2):83-9.  Back to cited text no. 10    
11.Bittencourt MC, Rebibou JM, Saint-Hillier Y, et al. Impaired renal graft survival after a positive B-cell flow cytometry cross match. Nephrol Dial Transplant 1998;13(8):2059-64.  Back to cited text no. 11    
12.Lobo PI, Spencer CE, Stevenson WC, McCu­llough C, Pruett TL. The use of pronase­digested human leukocytes to improve speci­ficity of the flow cytometric cross match. Transpl Int 1995;8(6):472-80.  Back to cited text no. 12    
13.Narahashi Y. Pronase. Methods in Enzymology 1970;19:651-64.  Back to cited text no. 13    
14.Lobashevsky AL, Senkbeil RW, Shoaf J, et al. Specificity of preformed alloantibodies causing B cell positive flow cross match in renal trans­plantation. Clin Transplant 2000;14(6):533-42.  Back to cited text no. 14    
15.Hajeer AH. Panel Reactive Antibody test (PRA) in renal transplantation. Saudi J Kidney Dis Transpl 2006;17(1):1-4.  Back to cited text no. 15    
16.Mahoney RJ, Taranto S, Edwards E. B-Cell cross matching and kidney allograft outcome in 9031 United States transplant recipients. Hum Immunol 2002;63(4):324-35.  Back to cited text no. 16    
17.Youngs D, Nelson K, Warner P. Got Milk. A simple method for reducing the undesirable effects of pronase treatment of lymphocytes. ASHI Quarterly 2008;32:42-4.  Back to cited text no. 17    

Correspondence Address:
A H Hajeer
Dept. of Pathology and Laboratory Medicine, King Abdulaziz Medical City, P.O. Box 22490, Riyadh11426
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

PMID: 19587513

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  [Table 1]

This article has been cited by
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2 General insufficiency of the classical CDC-based crossmatch to detect donor-specific anti-HLA antibodies leading to invalid results under recipientsæ medical treatment or underlying diseases
Schlaf, G. and Mauz-Körholz, C. and Ott, U. and Leike, S. and Altermann, W.
Histology and Histopathology. 2012; 27(1): 31-38
3 Novel solid phase-based ELISA assays contribute to an improved detection of anti-HLA antibodies and to an increased reliability of pre-and post-transplant crossmatching
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