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Flow Cytometric Detection of Anti-AB Antibody Titers in Blood Group O Recipients of Blood Group A2 Donor Kidneys
Flow Cytometric Detection of Anti-AB Antibody Titers in Blood Group O Recipients of Blood Group A2 Donor Kidneys P. Ata, F. Cetinkaya, T. Ozgezer, L. Ozel, A. Tulunay, E. Eksioglu, and M.I. Titiz ABSTRACT Aim. ABO-incompatible kidney transplantation has been accepted for end-stage renal failure patients who have no ready opportunity for a deceased or living donor. Antibody titration for ABO-incompatible renal transplantation is not only difficult but also lacks conformity among laboratories. Herein we analyzed 20 living related renal transplant couples to detect recipient anti-A2 antibody using flow cytometric analysis. Materials and methods. Patients were admitted to our center for renal transplantation between January 1999 and December 2010. All but four of them had undergone a previous renal transplantation from an ABO-compatible donor but experienced graft failure. All donor blood groups were subtyped by our blood bank using a lectin-based dilution assay. To detect recipient anti-A2 antibody titers we used a tube hemagglutination method. A/B antibody titer analysis by flow cytometry incubated serially diluted serum samples with donor erythrocytes. Each analysis was repeated three times over a 2-week period using an older and the last sera simultaneously. Results. The 13 male and 7 female patients showed our overall mean age of 32 ⫾ 12 years. All patients had panel-reactive antibody levels below 15%. The level of flow cytometric antibody titers did not vary upon repeated analysis (P ⫽ .01). When compared with the tube method there was a discrepancy of the level at which the antibody titer became negative. Discussion. Flow cytometric antibody titration is a practical and rapid technique to determine the amount of anti-A2 antibody in renal recipients. HE ABO SYSTEM IS IMPORTANT in solid organ transplantation because these antigens are expressed on almost all cells. According to the law formulated by Landsteiner (1945), humans have antibodies against those ABO(H) antigens that are absent in an individual’s own tissues. These antibodies usually induce hyperacute rejection of grafts expressing foreign A/B antigens.1 When crossing the ABO barrier there are two important factors: one is the donor A–B antigen expression level and the other, the amount of recipient anti-A/B antibody. Herein we sought to compare anti-AB titrations methods against possible donors in 20 patients, searching for a technique that yielded an unique cutoff level for negativity.
T
MATERIALS AND METHODS Waiting list patients registered at our center between January 1999 and December 2010 were admitted for anti-AB antibody analysis together with their ABO-incompatible possible donor. All subjects
except four had undergone a previous renal transplantation from an ABO-compatible donor but had experienced graft failure. All possible donor blood groups were subtyped by our blood bank using lectin-based dilution assays. To detect recipient anti-A2 antibody titer, we used a tube hemagglutination method. Each analysis was repeated three times for all patients within a 2-week period using an older and the last sera for the same analysis. We
From the Department of Molecular Genetics (P.A.) Haydarpasa Numune Research and Training Hospital, Genetic Diseases Diagnosis Center; Department of Immunology (P.A., A.T., E.E.), Marmara University; Blood Bank (F.C.), Haydarpasa Numune Research and Training Hospital; and Department of Transplantation (T.O., L.O., M.I.T.), Haydarpasa Numune Research and Training Hospital, Turkey. Address reprint requests to Pinar Ata, MD, PhD, Genetic Diseases Diagnosis Center, Haydarpasa Numune Research, and Training Hospital, Uskudar, Istanbul, Turkey. E-mail: pinaren@ gmail.com
0041-1345/12/$–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.05.051
© 2012 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1706
Transplantation Proceedings, 44, 1706 –1709 (2012)
FLOW CYTOMETRIC DETECTION OF ANTI-AB ANTIBODIES
1707
Fig 1. Flow cytometric analysis of the serial diluted patient serum with their possible donors’ erythrocytes. collected 38 serum samples from 20 patients. Analysis of A/B antibody titers with flow cytometry consisted of serial dilutions of recipient sera from 1/2 to 1/256 for testing against donor erythrocytes (80,000/mm3). Recipient erythrocytes at the same concentration were used as a negative control and against blood group B erythrocytes as a positive control. Glycophorin A FITC ab was used as an isotype control. Sera and erythrocytes incubated at 37°C for 30 minutes were stained with anti-human immunoglobulin G (IgG) FITC. Statistical analysis of IgG titers was performed using Student t test with P ⬍ .05 considered to be statistically significant2 (Fig 1). Receiver operating characteristic curve analysis was performed to estimate positive and negative predictive values for both methods at the 1/8 and lower titrations.
RESULTS
There were 13 male and 7 female patients of overall mean age of 32 ⫾ 12 years. All patients displayed panel-reactive antibody levels below 15%. All donors were of the A2 blood type. The level of flow cytometric antibody titers did not vary on the first versus the repeated analysis (P ⫽ .01). When compared with the tube method, there was a discrepancy of the level at which the antibody titer became negative (Table 1). If we consider the cutoff level of positivity as 1/8 dilution versus lower titrations and compare the first analysis results of the flow cytometric and tube methods separately, the true positive rate was higher for flow cytometric analysis (0.83) compared with the tube method (0.7). For the same cutoff level (1/8 and lower titrations) the positive predictive value (PPV) of the flow cytometric analysis was 0.83 and the negative predictive value (NPV) was 0.75. But for the tube method PPV was 0.7 and NPV was 0.62 (Fig 2). Our data supported the conclusion that there were consistent results for the flow cytometric analysis versus the tube method and that it could be used for anti-AB antibody detection.
DISCUSSION
ABO blood group compatibility has for decades been considered a prerequisite for successful renal transplantation. Early rejection of ABO-incompatible renal transplantations is described as hyperacute, delayed hyperacute, acute humoral (vascular) and acute cellular. It can be triggered by binding of anti-A/B antibodies to renal vascular endothelial cells thereby activating complement, platelet aggregation and inflammation, leading to intravascular thrombosis and occlusion of blood flow.1,3 Determining the anti-AB antibody level determines the dose of immunosuppression and the number of plasmapheresis or intravenous immunoglobulin therapies. Circulating anti-A/B antibodies in the recipient’s serum must be reduced prior to transplantation to overcome anti-A/B Ab-mediated rejection and early graft loss in ABO-incompatible kidney transplantation.4,5 A technique to determine ABO antibodies in the clinical setting must be simple to perform, in routine use, and cheap, fast, reliable, and reproducible. A number of methods have been employed to study and determine ABO antibodies, including routine blood banking techniques (titration), flow cytometry and enzyme-linked immunosor-
Table 1. Number of Variable Analysis at the Repeated Testing
Methods
Tube hemagglutination method Flow cytometric analysis* *P ⫽ .01.
First Analysis of Serum Samples Repeated 3 Times (n ⫽ 20)
Analysis of New Serum Samples Taken After 3 mo (n ⫽ 20)
14
11
1
2
1708
ATA, CETINKAYA, OZGEZER ET AL
Fig 2. Receiver operating characteristic analysis of the flowcytometric and tube test results. True positive rates were higher for flow cytometric analysis compared to gel method when three repeated sample values were analyzed.
bent assay. There is, however, no absolute correlation between ABO antigen expression on erythrocytes and in kidneys. Titration fulfills the requirements of being simple, in routine use, cheap, and fast, but not all criteria for ABO antibody determinations. Titration is associated with several drawbacks. Comparisons among centers are usually not possible, as shown recently in a Japanese study, where there was great variability among titers performed on a single serum sample at different institutions.6 Possible explanations for the results are various technologies (tube method, gel column, solid phase), titration end point, Coombs reagent serum or plasma use, incubation time, panel or patient target cells freezing thawing of samples, and, finally, subjective readings. It is clear that the expression of incompatible antigens on the graft is important as is the amount of anti-A/B antibody in the recipient. However, there is no absolute correlation between these parameters and outcomes. Present-day determinations of ABO antibodies make it difficult compare results among centers. Better methods are needed to quantify antibodies. However, titration is a simple, fast, and easy way to measure ABO antibodies in the clinical care of patients.7 Kumlien et al detected reduced intercenter variation in A/B antibody titrations from a median of three (range 0 – 6) titer steps using various hemagglutination techniques to a median of one (range 0 – 4) titer step using a gel hemagglutination technique on the one sample of red blood cells (RBCs).8 Considering the inherent variability in hemagglu-
tination techniques, it is probably necessary to use exactly the same method to achieve similar results in different centers. Because it is impossible to use the same test RBCs worldwide, kidney donor RBCs have been suggested for the test for A/B antibody titration in ABO-incompatible kidney transplant patients. A more reproducible method for A/B antibody quantification than hemagglutination is needed to develop a protocol for ABO-incompatible kidney transplantation.8 In conclusion, to determine the amount of anti-A2 antibody in renal recipients, flow cytometric antibody titrations are a practical, rapid technique that can be used prior to ABO incompatible renal transplantation. They are safe for detection of invariate cut-off levels of antibody negativity.
REFERENCES 1. Rydberg L: ABO-incompatibility in solid organ transplantation. 11:325, 2001 2. Tanabe K: Interinstitutional variation in the measurement of anti-A/B antibodies: Japanese ABO-incompatible transplantation committee survey. Transplantation 84(12 suppl):S13, 2007 3. Alkhunaizi AM, de Mattos AM, Barry JM, et al: Renal transplantation acrossthe ABO barrier using A2 kidneys. Transplantation 67:1319, 1999 4. Gloor JM, Lager DJ, Moore SB, et al: ABO-incompatible kidney transplantation using both A2 and non- A2 living donors. Transplantation 75:971, 2003
FLOW CYTOMETRIC DETECTION OF ANTI-AB ANTIBODIES 5. Yung GP, Valli PV, Starke A, et al: Flow cytometric measurement of ABO antibodies in ABO-incompatible living donor kidney transplantation. Transplantation 84:S20, 2007 6. Kobayashi T, Saito K: A series of surveys on assay for anti-A/B antibody by Japanese ABO-incompatible transplantation committee. Xenotransplantation 13:136, 2006
1709 7. Rydberg L, Skogsberg U, Molne J: ABO antigen expression in graft tissue: is titration against donor erythrocytes relevant? Transplantation 84:S10, 2007 8. Kumlien G, Wilpert J, Säfwenberg J, et al: Comparing the tube and gel techniques for ABO antibody titration, as performed in three European centers. Transplantation 84:S17, 2007
T
MATERIALS AND METHODS Waiting list patients registered at our center between January 1999 and December 2010 were admitted for anti-AB antibody analysis together with their ABO-incompatible possible donor. All subjects
except four had undergone a previous renal transplantation from an ABO-compatible donor but had experienced graft failure. All possible donor blood groups were subtyped by our blood bank using lectin-based dilution assays. To detect recipient anti-A2 antibody titer, we used a tube hemagglutination method. Each analysis was repeated three times for all patients within a 2-week period using an older and the last sera for the same analysis. We
From the Department of Molecular Genetics (P.A.) Haydarpasa Numune Research and Training Hospital, Genetic Diseases Diagnosis Center; Department of Immunology (P.A., A.T., E.E.), Marmara University; Blood Bank (F.C.), Haydarpasa Numune Research and Training Hospital; and Department of Transplantation (T.O., L.O., M.I.T.), Haydarpasa Numune Research and Training Hospital, Turkey. Address reprint requests to Pinar Ata, MD, PhD, Genetic Diseases Diagnosis Center, Haydarpasa Numune Research, and Training Hospital, Uskudar, Istanbul, Turkey. E-mail: pinaren@ gmail.com
0041-1345/12/$–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.05.051
© 2012 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1706
Transplantation Proceedings, 44, 1706 –1709 (2012)
FLOW CYTOMETRIC DETECTION OF ANTI-AB ANTIBODIES
1707
Fig 1. Flow cytometric analysis of the serial diluted patient serum with their possible donors’ erythrocytes. collected 38 serum samples from 20 patients. Analysis of A/B antibody titers with flow cytometry consisted of serial dilutions of recipient sera from 1/2 to 1/256 for testing against donor erythrocytes (80,000/mm3). Recipient erythrocytes at the same concentration were used as a negative control and against blood group B erythrocytes as a positive control. Glycophorin A FITC ab was used as an isotype control. Sera and erythrocytes incubated at 37°C for 30 minutes were stained with anti-human immunoglobulin G (IgG) FITC. Statistical analysis of IgG titers was performed using Student t test with P ⬍ .05 considered to be statistically significant2 (Fig 1). Receiver operating characteristic curve analysis was performed to estimate positive and negative predictive values for both methods at the 1/8 and lower titrations.
RESULTS
There were 13 male and 7 female patients of overall mean age of 32 ⫾ 12 years. All patients displayed panel-reactive antibody levels below 15%. All donors were of the A2 blood type. The level of flow cytometric antibody titers did not vary on the first versus the repeated analysis (P ⫽ .01). When compared with the tube method, there was a discrepancy of the level at which the antibody titer became negative (Table 1). If we consider the cutoff level of positivity as 1/8 dilution versus lower titrations and compare the first analysis results of the flow cytometric and tube methods separately, the true positive rate was higher for flow cytometric analysis (0.83) compared with the tube method (0.7). For the same cutoff level (1/8 and lower titrations) the positive predictive value (PPV) of the flow cytometric analysis was 0.83 and the negative predictive value (NPV) was 0.75. But for the tube method PPV was 0.7 and NPV was 0.62 (Fig 2). Our data supported the conclusion that there were consistent results for the flow cytometric analysis versus the tube method and that it could be used for anti-AB antibody detection.
DISCUSSION
ABO blood group compatibility has for decades been considered a prerequisite for successful renal transplantation. Early rejection of ABO-incompatible renal transplantations is described as hyperacute, delayed hyperacute, acute humoral (vascular) and acute cellular. It can be triggered by binding of anti-A/B antibodies to renal vascular endothelial cells thereby activating complement, platelet aggregation and inflammation, leading to intravascular thrombosis and occlusion of blood flow.1,3 Determining the anti-AB antibody level determines the dose of immunosuppression and the number of plasmapheresis or intravenous immunoglobulin therapies. Circulating anti-A/B antibodies in the recipient’s serum must be reduced prior to transplantation to overcome anti-A/B Ab-mediated rejection and early graft loss in ABO-incompatible kidney transplantation.4,5 A technique to determine ABO antibodies in the clinical setting must be simple to perform, in routine use, and cheap, fast, reliable, and reproducible. A number of methods have been employed to study and determine ABO antibodies, including routine blood banking techniques (titration), flow cytometry and enzyme-linked immunosor-
Table 1. Number of Variable Analysis at the Repeated Testing
Methods
Tube hemagglutination method Flow cytometric analysis* *P ⫽ .01.
First Analysis of Serum Samples Repeated 3 Times (n ⫽ 20)
Analysis of New Serum Samples Taken After 3 mo (n ⫽ 20)
14
11
1
2
1708
ATA, CETINKAYA, OZGEZER ET AL
Fig 2. Receiver operating characteristic analysis of the flowcytometric and tube test results. True positive rates were higher for flow cytometric analysis compared to gel method when three repeated sample values were analyzed.
bent assay. There is, however, no absolute correlation between ABO antigen expression on erythrocytes and in kidneys. Titration fulfills the requirements of being simple, in routine use, cheap, and fast, but not all criteria for ABO antibody determinations. Titration is associated with several drawbacks. Comparisons among centers are usually not possible, as shown recently in a Japanese study, where there was great variability among titers performed on a single serum sample at different institutions.6 Possible explanations for the results are various technologies (tube method, gel column, solid phase), titration end point, Coombs reagent serum or plasma use, incubation time, panel or patient target cells freezing thawing of samples, and, finally, subjective readings. It is clear that the expression of incompatible antigens on the graft is important as is the amount of anti-A/B antibody in the recipient. However, there is no absolute correlation between these parameters and outcomes. Present-day determinations of ABO antibodies make it difficult compare results among centers. Better methods are needed to quantify antibodies. However, titration is a simple, fast, and easy way to measure ABO antibodies in the clinical care of patients.7 Kumlien et al detected reduced intercenter variation in A/B antibody titrations from a median of three (range 0 – 6) titer steps using various hemagglutination techniques to a median of one (range 0 – 4) titer step using a gel hemagglutination technique on the one sample of red blood cells (RBCs).8 Considering the inherent variability in hemagglu-
tination techniques, it is probably necessary to use exactly the same method to achieve similar results in different centers. Because it is impossible to use the same test RBCs worldwide, kidney donor RBCs have been suggested for the test for A/B antibody titration in ABO-incompatible kidney transplant patients. A more reproducible method for A/B antibody quantification than hemagglutination is needed to develop a protocol for ABO-incompatible kidney transplantation.8 In conclusion, to determine the amount of anti-A2 antibody in renal recipients, flow cytometric antibody titrations are a practical, rapid technique that can be used prior to ABO incompatible renal transplantation. They are safe for detection of invariate cut-off levels of antibody negativity.
REFERENCES 1. Rydberg L: ABO-incompatibility in solid organ transplantation. 11:325, 2001 2. Tanabe K: Interinstitutional variation in the measurement of anti-A/B antibodies: Japanese ABO-incompatible transplantation committee survey. Transplantation 84(12 suppl):S13, 2007 3. Alkhunaizi AM, de Mattos AM, Barry JM, et al: Renal transplantation acrossthe ABO barrier using A2 kidneys. Transplantation 67:1319, 1999 4. Gloor JM, Lager DJ, Moore SB, et al: ABO-incompatible kidney transplantation using both A2 and non- A2 living donors. Transplantation 75:971, 2003
FLOW CYTOMETRIC DETECTION OF ANTI-AB ANTIBODIES 5. Yung GP, Valli PV, Starke A, et al: Flow cytometric measurement of ABO antibodies in ABO-incompatible living donor kidney transplantation. Transplantation 84:S20, 2007 6. Kobayashi T, Saito K: A series of surveys on assay for anti-A/B antibody by Japanese ABO-incompatible transplantation committee. Xenotransplantation 13:136, 2006
1709 7. Rydberg L, Skogsberg U, Molne J: ABO antigen expression in graft tissue: is titration against donor erythrocytes relevant? Transplantation 84:S10, 2007 8. Kumlien G, Wilpert J, Säfwenberg J, et al: Comparing the tube and gel techniques for ABO antibody titration, as performed in three European centers. Transplantation 84:S17, 2007