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1 Correlation of Circulating Complement-Fixing Donor-Specific Antibodies Identified by the C1q Assay and Presence of C4d in Endomyocardial Biopsy Specimens Renee Frank, MD, 1 Priti Lal, MD, 1 Jane Kearns, CHS, 1 Maria R. Molina, MSN, CRNP, 2 Joyce W. Wald, DO, 2 Lee R. Goldberg, MD, MPH, 2 and Malek Kamoun, MD, PhD 1 From the 1 Department of Pathology and Laboratory Medicine, and 2 Heart Failure and Cardiac Transplant Program, Division of Cardiovascular Medicine, University of Pennsylvania, Philadelphia, PA Key Words: Endomyocardial biopsy; C4d; C1q assay; Donor-specific antibody Am J Clin Pathol January 2016;145:62-68 DOI: /AJCP/AQV016 ABSTRACT Objectives: Donor-specific antibodies (DSAs) are associated with increased cardiac graft loss. We applied a C1q solidphase assay in parallel with the standard immunoglobulin G (IgG) single antigen bead (SAB) assay to examine the correlation of circulating complement-fixing donor-specific antibodies and the presence of C4d in endomyocardial biopsy (EMB) specimens. Methods: We retrospectively studied the relationship of C1qþ DSAs and C4d immunofluorescence (IF) in 49 EMB specimens from 44 heart transplant recipients who had concurrent EMB, C4d IF, and DSA measurements. We applied a C1q SAB in parallel with the standard IgG SAB assay to examine the DSA profiles in heart transplant patients posttransplant. Results: A better concordance is observed between C1qþ DSAs with C4d IFþ compared with IgG DSAs with C4d IF þ (40% vs 24%, P ¼.02). However, the correlation of C1q DSAs with C4d IF is not statistically significant (P ¼.24). Importantly, C1qþ DSAs were observed in 16 of 17 cases with C4d IFþ; 24 cases had circulating C1qþ DSAs without detectable C4d staining, suggesting that that the presence of C1qþ DSAs may precede the detection of C4d deposition in EMB specimens and/or the development of antibody-mediated rejection. Conclusions: In this cohort of 44 patients, no significant correlation was observed between circulating C1q DSAs and C4d IF in EMB specimens. Additional studies are needed to further evaluate the association of C1q DSAs with EMB specimens and C4d staining. Donor-specific antibodies (DSAs) are associated with increased cardiac allograft injury and loss, including vascular injury and graft dysfunction. Detection of antibody-mediated rejection (AMR) relies in part on graft dysfunction, endomyocardial biopsy (EMB) histopathologic criteria, and circulating DSAs. 1,2 DSAs can be against anti class I human leukocyte antigen (HLA), anti class II HLA, or non anti-hla antibodies. 3 Since Patel and Terasaki 4 demonstrated a significant correlation between a positive crossmatch and hyperacute and accelerated acute grafts dysfunction in kidney transplants using a complement-dependent cytotoxicity (CDC) assay in 1969, there has been no highly sensitive andspecificassaypredictive of clinical outcomes in transplantation patients. The currently available assays for detection of DSAs uses either CDC, or flow cytometry or Luminex (Luminex Corporation, Austin TX) technology using beads coated with single HLA antigens. While these assays are highly sensitive, they fail to differentiate antibodies that fix complement and cause graft injury or dysfunction from antibodies that do not cause graft injury or dysfunction. In the end, it is the pathogenicity, or ability of the antibody to fix complement, that is most pertinent to the acute and chronic effects of DSAs and ultimately clinical outcome. 5-8 Detection of antibodies capable of binding complement, specifically antibodies binding the first component of the classic pathway, C1q, gives the earliest indication of the potential for complement-mediated injury. 9,10 A C1q single antigen bead (SAB) assay to detect complement-fixing HLA antibodies has recently been developed. 9,11 This assay is highly sensitive and specific for distinguishing subsets of immunoglobulin G (IgG) antibodies that can fix complement and assess risk of early clinical AMR in cardiac 62 Am J Clin Pathol 2016;145:62-68 American Society for Clinical Pathology, All rights reserved. For permissions, please journals.permissions@oup.com

2 allografts. 12,13 A strong correlation between preformed complement-fixing DSAs detected by a C1q SAB, positive CDC crossmatch, and early clinical AMR has also been shown. 12,13 Interestingly, in the kidney transplantation literature, complement-fixing DSAs detected by a C1q SAB fixation and C4d deposition in kidney transplant biopsy specimens do not appear to be directly correlated. 14 In the cardiac transplant literature, studies have evaluated early clinical AMR with C4d staining of EMB specimens and complement-fixing DSAs detected by a C1q SAB assay; however, to our knowledge, there are no systematic studies evaluating DSAs detected by a C1q SAB with C4d staining and other parameters, including demographics and comorbidity factors. 15,16 We applied a C1q SAB in parallel with the standard IgG SAB to examine the correlation of circulating complement-fixing donor-specific antibodies and presence of C4d in EMB specimens. Materials and Methods Patient Population In this retrospective analysis, we studied the relationship of complement-fixing DSAs and C4d immunofluorescence (IF) in 40 EMB specimens from 38 cases of previously selected heart transplant recipients. 17 Heart transplant patients ( ) with concurrent endomyocardial biopsy, immunofluorescence for C4d, and DSA measurements were initially selected. Thirty-eight patients had stored serum samples available for this analysis. Clinical data gathered included patient demographics, presence and HLA class specificity of DSAs, ejection fraction (EF), nonimmunologic comorbidities, and cause of death. Nonimmunologic comorbidities gathered from the electronic medical record included presence or absence of hypertension, diabetes mellitus, cytomegalovirus (CMV) viremia, obesity, and hyperlipidemia CMV viremia was defined as symptomatic patients with a quantitative CMV viral load of more than 5,000 copies/ml who were treated with intravenous (IV) ganciclovir. Allograft dysfunction was defined as a precipitous decline in EF by at least 10% or an EF 35% or less, detected by transthoracic echocardiogram or transesophageal echocardiogram. To be included in the cohort, the measurement of EF was done within 61 month of the EMB. Cardiovascular mortality was defined by death due to acute AMR, acute myocardial infarction, and progressive allograft dysfunction and heart failure. For the purposes of this study, patients who died of noncardiac causes were censored from the graft failure analysis. Official cause of death was obtained from our electronic medical record systems, which were recorded directly from the patient death certificate and autopsy report when applicable. The maintenance immunosuppression protocols, including a calcineurin inhibitor, antimetabolite, or a cell cycle inhibitor, have been described in our previous study. 17 At our institution, all patients are on maintenance immunosuppression protocols, including a calcineurin inhibitor, antimetabolite, or a cell cycle inhibitor. The target tacrolimus level is 10 to 15 lg/l at 0 to 6 months, 10 to 12 lg/l at 6 to 12 months, 8 to 10 lg/l at 12 to 24 months, and 5 to 8 lg/l at more than 24 months. The target cyclosporine level is 200 to 300 ng/ml at 1 to 6 months, 150 to 200 ng/ ml at 6 to 12 months, 120 to 175 ng/ml at 12 to 24 months, and 80 to 100 ng/ml at more than 24 months. All patients who exhibit acute cellular rejection (ACR) grade 2R are treated with IV solumedrol, 1 g, for 3 days if the patient is status posttransplant less than 6 months, or prednisone, 100 mg, for 5 days if the patient is status posttransplant more than 6 months. If the patient is exhibiting ACR grade 2R or 3R with hemodynamic compromise and a decrease in EF of more than 10%, then the patient will also be treated with received rabbit anti thymocyte globulin (thymoglobulin). There was no difference in treatment since all treatments were similar for each patient, with the exception of plasmapheresis and photopheresis. Access to patient medical record information and review of archived myocardial biopsy tissue was granted approval by the Hospital of the University of Pennsylvania Institutional Review Board. Endomyocardial IF IF for C4d is performed on one to three pieces of fresh tissue submitted by the clinical team in addition to tissue for routine H&E evaluation. C4d IF staining is not performed in our institution within the first 2 weeks of transplantation. In general, fresh-frozen tissue was analyzed by indirect IF microscopy technique by using antibodies directed against C4d primary affinity-purified monoclonal antibody (dilution, 1:50; 30-minute incubation at room temperature; Sigma Aldrich, St Louis, MO). The fresh-frozen tissues were cut at 3 to 4 lm thickness and mounted on single-ring positively charged slides (Fisher, Pittsburgh, PA). Staining was performed according to standard procedures. Indirect IF microscopy was performed on a Nikon OPTISHOT with a Nikon Coolscan V ED camera (Nikon, Melville, NY) using Image Pro Plus (Media Cybernetics, Rockville, MD). Interstitial capillary C4d staining was semiquantitatively graded on a scale of 0 to 3þ for intensity, and a percentage of interstitial capillary staining over the entire parenchyma was assigned. Any EMB specimen exhibiting at least 2þ C4d staining over 50% of the interstitial capillaries was considered significant or positive for C4d deposition, an example of which is shown as Image 1. In this study, AMR was defined as clinical evidence of graft dysfunction, along with positive DSA and IF C4d (as defined above). C4d evaluation is performed at the request of the clinical team in (1) patients with clinical evidence of graft dysfunction (new American Society for Clinical Pathology Am J Clin Pathol 2016;145:

3 Frank et al /DSAS BYC1Q ASSAY WITH C4D INEMB SPECIMENS heart failure symptoms, new S3 murmur, increased jugular venous distention, and a decrease in EF of >10%), (2) patients who exhibit high percent reactive antibody, and (3) all patients with ACR of International Society for Heart and Lung Transplantation grade 3R. Most currently, AMR has been proposed to be a diagnosis rendered by pathologists, without the requirements of clinical dysfunction or positive DSAs, which was required in the past. 3,23,24 It must be emphasized that the pathologic AMR (pamr) grading system represents an initial guideline with the intention of accumulating data, validation, and patient treatment regimens. Furthermore, the recognition and diagnosis of AMR by histopathology, even among experienced transplant pathologists, is challenging. A number of studies have highlighted the problems of key morphologic criteria for the purposes of AMR screening. 25,26 The threshold for therapeutic intervention in patients with AMR remains unknown, particularly in the setting of asymptomatic AMR. For these reasons, we have not used the pamr grade to evaluate the utility of the C1q assay. HLA Typing and Evaluation of Anti-HLA Antibodies HLA typing was performed on all donors and recipients using DNA-based methods. Initial HLA typing included HLA-A, B, C, DRB1, DRB3/4/5, and DQB1. For recipients with antibodies against HLA-DQA, DP, or allele specific antibodies, additional typing of these loci for donors and recipients was also performed on a subset of these patients. Identification of anti-hla antibodies was performed using Image 1 Endomyocardial biopsy specimen exhibiting at least 2þ C4d staining over 50% of the interstitial capillaries, considered positive for C4d deposition. A, Unremarkable myocardium (H&E, 5). B, Myocardium with diffuse endothelial cell proliferation (arrows) and interstitial myxoid change (arrowheads) (H&E, 5). C, C4d immunofluorescence in interstitial capillaries. 64 Am J Clin Pathol 2016;145:62-68 American Society for Clinical Pathology 64

4 solid-phase class I and class II assays, which included flow cytometry bead and/or Luminex bead assays. At least two antibody solid-phase testing methods were used, one of which was a mixed-bead class I and class II assay or a class I and class II phenotype assay. HLA specificities were confirmed by single-antigen bead assays (One Lambda, Canoga Park, CA). Evaluation of complement-binding anti-hla antibodies was detected by using C1q single antigen bead assays (One Lambda). For sensitized patients, antibody testing included HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, and DPB1 specificities. A retrospective or prospective flow cytometric crossmatch was performed on all patients. The strength of antibody determined using solid-phase assays was correlated with the crossmatch test results. Donor-specific anti-hla antibodies were considered positive if the median fluorescence intensity assessed by Luminex single antigen bead assays was greater than 1,000 for IgG and higher than 400 for the C1q assays. Statistical Analysis Statistical analysis was performed using Fisher s exact t test and a paired samples t test. Statistical significance was defined as P <.05 (MedCalc, version (MedCalc Software, Ostend, Belgium)). Results Patient Characteristics Upon review of our pathology archives between 2005 and 2011, a total of 109 patients had concurrent EMB, DSA evaluation, and C4d IF. 17 Of these 109 patients, 44 had stored serum samples for C1q SAB analysis. The current cohort comprises 49 allografts from 44 patients, including 11 women and 33 men. Patient demographics and characteristics are shown in Table 1. Four patients received a second cardiac transplant that was included in the study group. Of our patient cohort, four were treated with photopheresis and five were treated with a combination of plasmapheresis (courses of four to six exchanges), IV immunoglobulin, rituximab, and cytoxan. Histopathology We analyzed a total of 49 EMB and concurrent IF studies from 49 cardiac grafts in 44 patients who had pre- and posttransplant DSA measurements. Mild and moderate acute cellular rejection was seen in 32 and 12 grafts, respectively. There were no EMB specimens with severe acute cellular rejection in this cohort. As shown in Table 1, nonimmunologic comorbidities of the C1q DSA and non-c1q DSA patient groups are similar. The distribution of total IgG DSAs and Table 1 Clinical Information for Patients With C1q Donor-Specific Antibodies and Those Without C1q Donor-Specific Antibodies With Nonimmunologic Comorbidities of Patients With and Without DSAs a C1q DSAs Characteristic Positive (n ¼ 40) Negative (n ¼ 9) Sex Male 31 6 Female 9 3 Age at time of biopsy, mean 48 (21-78) 43 (22-63) (range), y Pretransplant DSAs 5 3 Concurrent episode of ACR 36 8 Etiology of heart failure Ischemic CM 10 3 Dilated CM 12 1 Hypertrophic CM 2 0 Congenital CM 1 0 Restrictive CM 1 0 Nonischemic CM NOS 14 5 Nonimmunologic comorbidities CMV viremia 7 1 Hyperlipidemia 40 8 Obesity (BMI >30 kg/m 2 ) 6 1 Diabetes mellitus 24 6 Hypertension 37 9 ACR, acute cellular rejection; BMI, body mass index; CM, cardiomyopathy; CMV, cytomegalovirus; DSAs, donor-specific antibodies; NOS, not otherwise specified. a Values are presented as numbers unless otherwise indicated. C1qþ DSAs is shown in Figure 1 ; 84% (36/43) of patients who had IgG DSAs had C1qþ complement-fixing DSAs in this selected cohort of patients. Importantly, C1qþ DSAs were observed in 16 of 17 cases with C4d IFþ; 24 cases had circulating C1qþ DSAs without detectable C4d staining Table 2. However, the correlation of C1q DSAs with C4d IF is not statistically significant (P ¼.24). EMB Characteristics and Graft Dysfunction EMB IF results were compared with corresponding graft EFs. Of the 49 biopsies, 28 (70%) EMBs were performed during a clinically significant decline in EF as defined above. Twenty-three (82%) of the 28 EMB specimens evaluated during an episode of graft dysfunction correlated with positive C1q DSAs. As shown in Table 3,in patients with positive C1q DSAs (n ¼ 23), positive C4d staining on the EMB specimen was observed in 13 (56%). In addition, in patients who were tested negative for C1q DSAs, positive C4d staining on the EMB specimen was observed in only one (20%). Graft Failure In this study group of a total of 44 patients with 49 grafts evaluated, 18 patients died or underwent a heart retransplant. Two patients were not included in the graft failure analysis American Society for Clinical Pathology Am J Clin Pathol 2016;145:

5 Frank et al /DSAS BYC1Q ASSAY WITH C4D INEMB SPECIMENS since one patient was lost to follow-up and one did not meet cardiac mortality criteria (died secondary to acute myeloid leukemia). After these two exclusions, 47 allografts were evaluated for graft failure. As shown in Table 4, overall, 38% (18/47) cardiac allografts failed. Graft failure occurred in 14 (37%) of 38 patients who tested positive for C1q DSAs and in four (44%) of nine patients who tested negative for C1q DSAs; three of these four patients developed IgG DSAs. The mean time to graft failure was 56 months and 59 months for grafts with and without C1qþ DSAs, respectively. Of the 28 allografts that survived, 24 (86%) tested positive for C1q DSAs. Similarly, 83% (15/18) of allografts that failed tested positive for C1q DSAs. No. of Cardiac Allografts C1q+ C1q No DSA (n = 6) DSA to HLA Class I (n = 5) DSA to HLA Class II (n = 19) Table 2 C4d Immunofluorescence With and Without C1q Donor- Specific Antibodies a C1q DSAs, No. (%) Characteristic Positive (n ¼ 40) Negative (n ¼ 9) Total No. C4d positive 16 (40) 1 (11) 16 C4d negative 24 (60) 8 (89) 32 Total 40 (100) 9 (100) 49 DSAs, donor-specific antibodies. a The correlation of C1q DSAs with C4d immunofluorescence is not statistically significant (P ¼.24). Table 3 Endomyocardial Biopsy During an Episode of Allograft Dysfunction With Corresponding C4d Immunofluorescence Staining (P ¼.619) C1q DSAs No C4d, No. (%) C4d >50%, No. (%) Positive (n ¼ 23) 13 (56) 10 (44) Negative (n ¼ 5) 4 (80) 1 (20) DSA to Both HLA Class I and II (n = 19) Figure 1 Donor-specific antibodies by human leukocyte antigen (HLA) class as related to C1q positivity. Discussion In this report, we present a single-institution study of a subset of 44 patients who underwent cardiac transplants and had comprehensive clinical, pathologic, and immunologic data to study the relationship of these parameters with cardiac allograft transplantation outcomes. Our data suggest no significant correlation between circulating complement-fixing DSAs detected by C1q SAB and C4d IF in EMB specimens. A better concordance is observed between C1qþ DSAs with C4d IF þ compared with total IgG DSAs with C4d IF þ (40% vs 24%, P ¼.02). Patients with concurrent data for graft dysfunction, EMB, IF for C4d, and C1q DSA were analyzed. Nonimmunologic comorbidities were also evaluated to overcome possible confounding factors, and were similar in patients who had circulating C1qþ DSAs and those who remained negative for C1q DSAs (Table 1). However, the correlation of C1q DSAs with C4d IF was not statistically significant (P ¼.24). Importantly, C1qþ DSAswereobservedin16of17caseswithC4dIFþ; 24 cases had circulating C1qþ DSAs without detectable C4d staining (Table 2), suggesting that the presence of C1qþ DSAs may precede the detection of C4d deposition in EMB specimens and/or the development of AMR. In this cohort of 38 patients, no significant correlation was observed between circulating C1q DSAs and C4d IF in EMB specimens. In patients with graft dysfunction, 82% had circulating C1q DSAs. There was no significant correlation observed between circulating C1q DSAs and graft failure; this lack of correlation may in part be due to a patient selection bias since our cohort was focused on patients who had a graft dysfunction, EMB, and IF C4d staining. The clinical utility of novel assays detecting complement binding anti-hla antibodies was recently reported in heart transplantation. 12,15 However, the role of complement-fixing Table 4 Cardiac Allograft Failure With Corresponding C1q DSA Status and C4d Immunofluorescence Staining (P ¼.716) C1q DSAs Graft Failure, No. (%) Time to Graft Failure, Mean (Range), mo Negative C4d, No./ Total No. (%) Positive C4d, No./ Total No. (%) IgG DSAs, No./ Total No. (%) Positive (n ¼ 38) a 14 (37) 56 (9-162) 9/14 (64) 5/14 (36) 14/14 (100) Negative (n ¼ 9) 4 (44) 59 (17-139) 4/4 (100) 0 3/4 (75) DSAs, donor-specific antibodies; IgG, immunoglobulin G. a Two patients censored from graft failure analysis: one patient died of acute myeloid leukemia, and the second patient was lost to follow-up. 66 Am J Clin Pathol 2016;145:62-68 American Society for Clinical Pathology 66

6 vs non complement-fixing DSAs and its correlation with C4d IF staining had not yet been elucidated. All DSAs are not equal in terms of detriment to allograft function. 27 The ability to activate the complement cascade appears to be a critical factor in differentiating clinically relevant and nonrelevant DSAs. 12 A positive C1q DSA with a negative C4d staining in EMB specimens could be due to a number of factors, including low sensitivity of IF staining, low sensitivity of the threshold used for a positive read on IF, or lack of C3d testing in our cohort. It should also be noted that a patient could have positive C4d staining without circulating DSAs secondary to non-hla antiallograft antibodies or due to complement fixation secondary to infections or other types of injuries. 28 In this cohort of 44 patients, no significant correlation was observed between circulating C1q DSAs and C4d IF in EMB specimens. Prospective studies are needed to further evaluate the association of C1q DSAs with EMB and IF C4d staining, as well as with long-term outcomes in heart transplant recipients. Because our study is limited to a small cohort, additional studies of the impact of treatment strategies of AMR with circulating C1qþ DSAs are necessary to transform the diagnostic data into an actionable treatment plan that improves long-term heart transplant outcomes. Corresponding author: Renee Frank, MD, University of Pennsylvania, 3400 Spruce St, 6th Floor, Founders Building, Philadelphia, PA 19104; renee.frank@uphs.upenn.edu. We thank Insuk Choe, JD, for help in compiling and in the critical evaluation of HLA data. We appreciate the assistance of Lynita Thomas and Christopher Mignogna with the IF processing protocols. These data were presented in part at the American Society for Histocompatibility and Immunogenetics, 39th Annual Meeting, Chicago, IL, November 17-21, References 1. 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