- Email: [email protected]
Falsely Incompatible B-cell Flow Cytometry Crossmatch After Pronase Treatment: A Case Report
Falsely Incompatible B-cell Flow Cytometry Crossmatch After Pronase Treatment: A Case Report J.D. Harta, C.T. Lutzb,*, C.D. Jenningsa, J.R. Maya, K. Nelsona, S. Jacobsa, and C.W. Hoopesc a Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, Kentucky; bDepartment of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky; and cDivision of Cardiothoracic Surgery, University of Kentucky, Lexington, Kentucky
ABSTRACT This report presents a falsely incompatible B cell crossmatch by flow cytometry in a lung transplant recipient. The patient was a 35-year-old Caucasian male with end-stage lung disease secondary to cystic fibrosis whose pretransplantation serologic workup did not disclose the presence of anti-HLA class II antibodies by single antigen bead testing. Unexpectedly, crossmatch of recipient sera with pronase-treated donor lymphocytes resulted in antibody binding to B cells only. The positive reactivity was reproducible in pronasetreated autologous B cells. Recipient sera did not react with nontreated donor or autologous lymphocytes. Herein, we describe our approach to this unexpected crossmatch result and consider the implications of false-positive crossmatch results on transplantation.
S
INCE its introduction several decades ago [1], the flow cytometric crossmatch (FCXM) has been a very sensitive assay [2] to evaluate donor-specific antibodies to HLA class I and class II antigens. In the assay, donor lymphocytes are incubated with patient sera, and bound immunoglobulin G (IgG) antibodies are detected using a fluorescent antibody to human IgG. T cells and B cells can be distinguished by staining with lineage-specific monoclonal antibodies (mAbs), such as anti-CD3 to detect T cells and anti-CD19 to detect B cells. This allows for a T-cell crossmatch and a B-cell crossmatch to be performed simultaneously without the need to physically separate T and B lymphocytes. The FCXM method has been modified by treatment of donor lymphocytes with the proteolytic enzyme, pronase [3]. This enzyme has a preference for cell surface Fc receptors on B cells. The use of pronase decreases high background fluorescence sometimes caused by immune complexes in patient serum that bind to Fc receptors on the B cell surface and result in a falsely incompatible B-cell FCXM (B-FCXM). In higher concentrations pronase also removes CD20, which may cause falsely incompatible B-FCXM in patients who have undergone therapy with the humanized IgG anti-CD20 antibody, rituximab [4]. Hetrick et al investigated the impact of pronase on flow cytometric crossmatch tests [4] and found that pronase treatment (1 mg/mL) usually increased antibody binding to T cells, whereas it decreased antibody binding to B cells. This finding elucidated the potential for a falsely ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 47, 831e833 (2015)
incompatible T-cell FCXM (T-FCXM). Subsequently, Park et al reported a high rate of incompatible T-FCXM with pronase treatment (1 mg/mL), including in two HLAA, B, and DR-identical donor-recipient pairs. Because the sera tested did not cause incompatible T-FCXM with nontreated cells, the results suggested that pronase treatment caused false-positive T-cell reactivity, which was observed in 13% of cases [5]. A recent abstract reported that sera from 97% of HIV-infected patients caused incompatible FCXM results with pronase-treated T cells [6]. Incompatible T-FCXM results were observed with either allogeneic or autologous cells. With most sera the FCXM was compatible, using either allogeneic or autologous T cells that had not been pronase-treated. The apparent autoantibody could be absorbed with pronasetreated autologous T cells, suggesting that the incompatible results were due to autoantibodies that recognized cryptic T-cell epitopes exposed by pronase. To our knowledge, falsely incompatible B-FCXM has not been reported specifically with pronase-treated cells. Here we describe a patient serum without detectable anti-HLA class II antibodies, which produced incompatible B-FCXM using pronase-treated donor lymphocytes.
*Address correspondence to Charles T. Lutz, MD, PhD, 800 Rose Street, MS 117, Lexington, Kentucky 40536-0298. E-mail: [email protected] 0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2014.12.022
831
832
CASE REPORT A 35-year-old Caucasian male with end-stage lung disease secondary to cystic fibrosis was considered for lung transplantation. The patient clinically deteriorated before transplantation, requiring frequent hospital admissions due to multiple infections for which he had received various antibiotics, including the chronic use of azithromycin. Prior antibody screening and single-antigen bead testing (LABScreen Single Antigen Class I and Class II kits; One Lambda, Canoga Park, CA) on the Luminex 200 instrument (Luminex, Austin, TX) disclosed anti-A80 antibody and the absence of anti-HLA class II antibodies. After potentially suitable organs were found, a crossmatch was performed. Mononuclear cells were isolated using routine laboratory methods with the aid of Histopaque lymphocyte separation media (Sigma Diagnostics, St. Louis, MO, United States) and treated with pronase (0.5 mg/mL for 10 min at 37 C, Sigma). Treated mononuclear cells were incubated with 30 mL patient sera or control sera at room temperature for 30 min. After washing, bound human IgG was detected with fluorescein isothiocyanatelabeled F(ab’)2 goat antihuman IgG (Jackson Immuno Research Laboratories, West Grove, Pa, United States), and lymphocyte subsets were identified with antiCD3 mAb labeled with peridinin-chlorophyll proteins (BD Biosciences, San Jose, Calif, United States) and anti-CD19 mAb labeled with phycoerythrin (BD Biosciences). Samples were analyzed on a BD FACSCanto II flow cytometer (BD Biosciences). An initial FCXM showed unexpected B-FCXM incompatibility with both current serum and a historic serum that had been stored frozen for 10 months. The concurrent T-FCXM was compatible (Table 1). Testing the same sera using Luminex single-antigen beads did not reveal any significant antiHLA class II antibodies. The crossmatch was repeated using both pronase-treated and nontreated cells from both the prospective organ donor and from the patient. With both sources of lymphocytes, pronase treatment caused incompatible B-FCXM, whereas nontreated cells were B-FCXM compatible. In all cases, the T-FCXM was compatible (Table 1). This suggested an autoantibody reacting with a cryptic antigen that was expressed on only pronase-treated B cells. To test the hypothesis that the incompatible FCXM was the result of autoantibodies recognizing cryptic B-cell epitopes exposed by pronase treatment, we conducted an auto-absorption experiment. In the experiment, 0.4 mL of the patient’s more highly reactive historic serum sample was incubated at 37 C for 2 hours with 5 106 fresh patient cells that either had been pronase-treated or nontreated. Crossmatches were then performed using both preabsorbed and nonabsorbed sera and autologous or allogeneic cells that were either pronase-treated or nontreated. The sera produced incompatible B-FCXM with both autologous and allogeneic pronase-treated cells. Antibody reactivity was not removed by incubation with either pronase-treated or nontreated autologous cells (data not shown).
DISCUSSION
The effect of pronase on HLA expression and FCXM reactivity has been fairly well documented, including the effect of varying pronase concentrations. Whereas previously described pronase treatment protocols used concentrations of 1 mg/mL [3e5] or 2 mg/mL [4] pronase at 37 C for 30 min, our procedure was performed with a pronase concentration of only 0.5 mg/mL at 37 C for only 10 min. Our use of a low-pronase concentration for only 10 min likely accounts for the fact that we have not observed falsely incompatible T-FCXM reported by Park et al [5]. Therefore,
HART, LUTZ, JENNINGS ET AL Table 1. The Effect of Pronase Treatment on Flow Cytometry Crossmatch With Allogeneic and Autologous Cells Cell Source
Cell
Treatment
Serum
MCS*
Allogeneic
T T B B T T B B T T B B T T B B
Pronase Pronase Pronase Pronase None None None None Pronase Pronase Pronase Pronase None None None None
Current Historic Current Historic Current Historic Current Historic Current Historic Current Historic Current Historic Current Historic
6 16 ¼ 10 7 16 ¼ 9 58 27 ¼ 31 195 27 ¼ 168 9 12 ¼ 3 11 12 ¼ 1 39 46 ¼ 7 45 46 ¼ 1 9 13 ¼ 4 9 13 ¼ 4 66 29 ¼ 37 138 29 ¼ 109 11 13 ¼ 2 11 13 ¼ 2 37 38 ¼ 1 51 38 ¼ 13
Autologous
*MCS refers to the mean channel shift (value with test serum value with negative control serum). Incompatible levels are denoted by bold print.
we were surprised to discover a false-positive B-FCXM under these conditions. To our knowledge this case represents the first description of a falsely incompatible FCXM found exclusively with pronase-treated B lymphocytes. We were able to recapitulate the original B-FCXM results with lymphocytes from both the patient and from a second allogeneic donor (data not shown). However, pre-absorption of patient serum with fresh pronase-treated mononuclear cells at 37 C did not remove the autoantibody (data not shown). This result therefore did not support our hypothesis. We speculate that failure to absorb the autoantibody was due to the relatively small number of B cells in the mononuclear cell mixture. Alternatively, the antibody could have been of very low affinity and failed to be absorbed at 37 C, but was able to cause a false-positive B-FCXM performed at room temperature. The patient’s clinical history of chronic azithromycin therapy has been noted. The immunomodulatory effect of the macrolide class of antibiotics has been described with some suggestion that the macrolide drugs may shift cytokine production from Th1 to Th2 [7]. This would suggest that macrolides, such as azithromycin, may induce antibody and autoantibody formation. Further investigation is needed to ascertain whether macrolide antibiotics induce autoantibody formation. The impact of a falsely incompatible FCXM cannot be overstated because a life-saving organ may be unnecessarily denied to a critically ill patient. After interpretation of the results of repeat FCXM with and without pronase treatment and single HLA antigen bead testing, the transplantation team was confident to proceed. Absent careful investigation, the patient’s outcome may have been severely compromised by delayed transplantation. Our findings suggest that unexpected incompatible B-FCXM be investigated for autoantibodies, either by performing additional crossmatch with and without pronase treatment or by testing autologous cells. These tests will help to rule out clinically insignificant non-HLA autoantibodies.
B CELL FALSE-POSITIVE CROSSMATCH
REFERENCES [1] Garovoy MR, Rheinschmidt MA, Bigos M, et al. Flow cytometry analysis: a high technology crossmatch technique facilitating transplantation. Transplant Proc 1983;15:1939e44. [2] Tait BD, Susal C, Gebel HM, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplantation 2013;95:19e47. [3] Vaidya S, Cooper TY, Avandsalehi J, et al. Improved flow cytometric detection of HLA alloantibodies using pronase. Transplantation 2001;71:422e8.
833 [4] Hetrick SJ, Schillinger KP, Zachary AA, Jackson AM. Impact of pronase on flow cytometric crossmatch outcome. Hum Immunol 2011;72:330e6. [5] Park H, Lim YM, Han BY, et al. Frequent false-positive reactions in pronase-treated T-cell flow cytometric cross-match tests. Transplant Proc 2012;44:87e90. [6] Brooks K, Magas D, Sieg K, et al. Positive flow cytometry crossmatch results with pronase-treated T-cells induced by non-HLA autoantibodies in HIV-infected patients. Hum Immunol 2014;75(abstr):486. [7] Kanoh S, Rubin BK. Mechanisms of action and clinical application of the macrolides as immunomodulatory medications. Clinical Micro Rev 2010;23:590e615.
ABSTRACT This report presents a falsely incompatible B cell crossmatch by flow cytometry in a lung transplant recipient. The patient was a 35-year-old Caucasian male with end-stage lung disease secondary to cystic fibrosis whose pretransplantation serologic workup did not disclose the presence of anti-HLA class II antibodies by single antigen bead testing. Unexpectedly, crossmatch of recipient sera with pronase-treated donor lymphocytes resulted in antibody binding to B cells only. The positive reactivity was reproducible in pronasetreated autologous B cells. Recipient sera did not react with nontreated donor or autologous lymphocytes. Herein, we describe our approach to this unexpected crossmatch result and consider the implications of false-positive crossmatch results on transplantation.
S
INCE its introduction several decades ago [1], the flow cytometric crossmatch (FCXM) has been a very sensitive assay [2] to evaluate donor-specific antibodies to HLA class I and class II antigens. In the assay, donor lymphocytes are incubated with patient sera, and bound immunoglobulin G (IgG) antibodies are detected using a fluorescent antibody to human IgG. T cells and B cells can be distinguished by staining with lineage-specific monoclonal antibodies (mAbs), such as anti-CD3 to detect T cells and anti-CD19 to detect B cells. This allows for a T-cell crossmatch and a B-cell crossmatch to be performed simultaneously without the need to physically separate T and B lymphocytes. The FCXM method has been modified by treatment of donor lymphocytes with the proteolytic enzyme, pronase [3]. This enzyme has a preference for cell surface Fc receptors on B cells. The use of pronase decreases high background fluorescence sometimes caused by immune complexes in patient serum that bind to Fc receptors on the B cell surface and result in a falsely incompatible B-cell FCXM (B-FCXM). In higher concentrations pronase also removes CD20, which may cause falsely incompatible B-FCXM in patients who have undergone therapy with the humanized IgG anti-CD20 antibody, rituximab [4]. Hetrick et al investigated the impact of pronase on flow cytometric crossmatch tests [4] and found that pronase treatment (1 mg/mL) usually increased antibody binding to T cells, whereas it decreased antibody binding to B cells. This finding elucidated the potential for a falsely ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 47, 831e833 (2015)
incompatible T-cell FCXM (T-FCXM). Subsequently, Park et al reported a high rate of incompatible T-FCXM with pronase treatment (1 mg/mL), including in two HLAA, B, and DR-identical donor-recipient pairs. Because the sera tested did not cause incompatible T-FCXM with nontreated cells, the results suggested that pronase treatment caused false-positive T-cell reactivity, which was observed in 13% of cases [5]. A recent abstract reported that sera from 97% of HIV-infected patients caused incompatible FCXM results with pronase-treated T cells [6]. Incompatible T-FCXM results were observed with either allogeneic or autologous cells. With most sera the FCXM was compatible, using either allogeneic or autologous T cells that had not been pronase-treated. The apparent autoantibody could be absorbed with pronasetreated autologous T cells, suggesting that the incompatible results were due to autoantibodies that recognized cryptic T-cell epitopes exposed by pronase. To our knowledge, falsely incompatible B-FCXM has not been reported specifically with pronase-treated cells. Here we describe a patient serum without detectable anti-HLA class II antibodies, which produced incompatible B-FCXM using pronase-treated donor lymphocytes.
*Address correspondence to Charles T. Lutz, MD, PhD, 800 Rose Street, MS 117, Lexington, Kentucky 40536-0298. E-mail: [email protected] 0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2014.12.022
831
832
CASE REPORT A 35-year-old Caucasian male with end-stage lung disease secondary to cystic fibrosis was considered for lung transplantation. The patient clinically deteriorated before transplantation, requiring frequent hospital admissions due to multiple infections for which he had received various antibiotics, including the chronic use of azithromycin. Prior antibody screening and single-antigen bead testing (LABScreen Single Antigen Class I and Class II kits; One Lambda, Canoga Park, CA) on the Luminex 200 instrument (Luminex, Austin, TX) disclosed anti-A80 antibody and the absence of anti-HLA class II antibodies. After potentially suitable organs were found, a crossmatch was performed. Mononuclear cells were isolated using routine laboratory methods with the aid of Histopaque lymphocyte separation media (Sigma Diagnostics, St. Louis, MO, United States) and treated with pronase (0.5 mg/mL for 10 min at 37 C, Sigma). Treated mononuclear cells were incubated with 30 mL patient sera or control sera at room temperature for 30 min. After washing, bound human IgG was detected with fluorescein isothiocyanatelabeled F(ab’)2 goat antihuman IgG (Jackson Immuno Research Laboratories, West Grove, Pa, United States), and lymphocyte subsets were identified with antiCD3 mAb labeled with peridinin-chlorophyll proteins (BD Biosciences, San Jose, Calif, United States) and anti-CD19 mAb labeled with phycoerythrin (BD Biosciences). Samples were analyzed on a BD FACSCanto II flow cytometer (BD Biosciences). An initial FCXM showed unexpected B-FCXM incompatibility with both current serum and a historic serum that had been stored frozen for 10 months. The concurrent T-FCXM was compatible (Table 1). Testing the same sera using Luminex single-antigen beads did not reveal any significant antiHLA class II antibodies. The crossmatch was repeated using both pronase-treated and nontreated cells from both the prospective organ donor and from the patient. With both sources of lymphocytes, pronase treatment caused incompatible B-FCXM, whereas nontreated cells were B-FCXM compatible. In all cases, the T-FCXM was compatible (Table 1). This suggested an autoantibody reacting with a cryptic antigen that was expressed on only pronase-treated B cells. To test the hypothesis that the incompatible FCXM was the result of autoantibodies recognizing cryptic B-cell epitopes exposed by pronase treatment, we conducted an auto-absorption experiment. In the experiment, 0.4 mL of the patient’s more highly reactive historic serum sample was incubated at 37 C for 2 hours with 5 106 fresh patient cells that either had been pronase-treated or nontreated. Crossmatches were then performed using both preabsorbed and nonabsorbed sera and autologous or allogeneic cells that were either pronase-treated or nontreated. The sera produced incompatible B-FCXM with both autologous and allogeneic pronase-treated cells. Antibody reactivity was not removed by incubation with either pronase-treated or nontreated autologous cells (data not shown).
DISCUSSION
The effect of pronase on HLA expression and FCXM reactivity has been fairly well documented, including the effect of varying pronase concentrations. Whereas previously described pronase treatment protocols used concentrations of 1 mg/mL [3e5] or 2 mg/mL [4] pronase at 37 C for 30 min, our procedure was performed with a pronase concentration of only 0.5 mg/mL at 37 C for only 10 min. Our use of a low-pronase concentration for only 10 min likely accounts for the fact that we have not observed falsely incompatible T-FCXM reported by Park et al [5]. Therefore,
HART, LUTZ, JENNINGS ET AL Table 1. The Effect of Pronase Treatment on Flow Cytometry Crossmatch With Allogeneic and Autologous Cells Cell Source
Cell
Treatment
Serum
MCS*
Allogeneic
T T B B T T B B T T B B T T B B
Pronase Pronase Pronase Pronase None None None None Pronase Pronase Pronase Pronase None None None None
Current Historic Current Historic Current Historic Current Historic Current Historic Current Historic Current Historic Current Historic
6 16 ¼ 10 7 16 ¼ 9 58 27 ¼ 31 195 27 ¼ 168 9 12 ¼ 3 11 12 ¼ 1 39 46 ¼ 7 45 46 ¼ 1 9 13 ¼ 4 9 13 ¼ 4 66 29 ¼ 37 138 29 ¼ 109 11 13 ¼ 2 11 13 ¼ 2 37 38 ¼ 1 51 38 ¼ 13
Autologous
*MCS refers to the mean channel shift (value with test serum value with negative control serum). Incompatible levels are denoted by bold print.
we were surprised to discover a false-positive B-FCXM under these conditions. To our knowledge this case represents the first description of a falsely incompatible FCXM found exclusively with pronase-treated B lymphocytes. We were able to recapitulate the original B-FCXM results with lymphocytes from both the patient and from a second allogeneic donor (data not shown). However, pre-absorption of patient serum with fresh pronase-treated mononuclear cells at 37 C did not remove the autoantibody (data not shown). This result therefore did not support our hypothesis. We speculate that failure to absorb the autoantibody was due to the relatively small number of B cells in the mononuclear cell mixture. Alternatively, the antibody could have been of very low affinity and failed to be absorbed at 37 C, but was able to cause a false-positive B-FCXM performed at room temperature. The patient’s clinical history of chronic azithromycin therapy has been noted. The immunomodulatory effect of the macrolide class of antibiotics has been described with some suggestion that the macrolide drugs may shift cytokine production from Th1 to Th2 [7]. This would suggest that macrolides, such as azithromycin, may induce antibody and autoantibody formation. Further investigation is needed to ascertain whether macrolide antibiotics induce autoantibody formation. The impact of a falsely incompatible FCXM cannot be overstated because a life-saving organ may be unnecessarily denied to a critically ill patient. After interpretation of the results of repeat FCXM with and without pronase treatment and single HLA antigen bead testing, the transplantation team was confident to proceed. Absent careful investigation, the patient’s outcome may have been severely compromised by delayed transplantation. Our findings suggest that unexpected incompatible B-FCXM be investigated for autoantibodies, either by performing additional crossmatch with and without pronase treatment or by testing autologous cells. These tests will help to rule out clinically insignificant non-HLA autoantibodies.
B CELL FALSE-POSITIVE CROSSMATCH
REFERENCES [1] Garovoy MR, Rheinschmidt MA, Bigos M, et al. Flow cytometry analysis: a high technology crossmatch technique facilitating transplantation. Transplant Proc 1983;15:1939e44. [2] Tait BD, Susal C, Gebel HM, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplantation 2013;95:19e47. [3] Vaidya S, Cooper TY, Avandsalehi J, et al. Improved flow cytometric detection of HLA alloantibodies using pronase. Transplantation 2001;71:422e8.
833 [4] Hetrick SJ, Schillinger KP, Zachary AA, Jackson AM. Impact of pronase on flow cytometric crossmatch outcome. Hum Immunol 2011;72:330e6. [5] Park H, Lim YM, Han BY, et al. Frequent false-positive reactions in pronase-treated T-cell flow cytometric cross-match tests. Transplant Proc 2012;44:87e90. [6] Brooks K, Magas D, Sieg K, et al. Positive flow cytometry crossmatch results with pronase-treated T-cells induced by non-HLA autoantibodies in HIV-infected patients. Hum Immunol 2014;75(abstr):486. [7] Kanoh S, Rubin BK. Mechanisms of action and clinical application of the macrolides as immunomodulatory medications. Clinical Micro Rev 2010;23:590e615.