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| Year : 2009 | Volume
: 20
| Issue : 4 | Page : 662-665 |
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| Pronase-free B-cell flow-cytometry crossmatch |
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AH Hajeer1, S Saleh2, P Sutton2, A Shubaili2, H Anazi2
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 Publication | 8-Jul-2009 |
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 Abstract | | |
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 | |  |
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 | | |
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 | | |
[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 | | |
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 | | |
| 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.  [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. |
| 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. |
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| 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. |
| 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. |
| 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. |
| 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. |
| 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. |
| 13. | Narahashi Y. Pronase. Methods in Enzymology 1970;19:651-64. |
| 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. |
| 15. | Hajeer AH. Panel Reactive Antibody test (PRA) in renal transplantation. Saudi J Kidney Dis Transpl 2006;17(1):1-4. |
| 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. |
| 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. |
Correspondence Address:
A H Hajeer
Dept. of Pathology and Laboratory Medicine, King Abdulaziz Medical City, P.O. Box 22490, Riyadh11426
Saudi Arabia
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 19587513 
[Table 1] |
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| This article has been cited by | | 1 |
Generation of donor-specific anti-human leukocyte antigen antibodies after the transplantation of a fully matched kidney allograft and its impact on the selection of a subsequent renal regraft: A case report |
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| Schlaf, G. and Radam, C. and Wahle, A. and Altermann, W.W. | | Transplantation Proceedings. 2012; 44(5): 1442-1445 | | [Pubmed] | | | 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 |
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| Schlaf, G. and Mauz-Körholz, C. and Ott, U. and Leike, S. and Altermann, W. | | Histology and Histopathology. 2012; 27(1): 31-38 | | [Pubmed] | | | 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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| Schlaf, G. and Pollok-Kopp, B. and Manzke, T. and Schurat, O. and Altermann, W. | | NDT Plus. 2010; 3(6): 527-538 | | [Pubmed] | |
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