HLA and MICA allosensitization patterns among patients supported by ventricular assist devices

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HLA and MICA allosensitization patterns among patients supported by ventricular assist devices Medhat Askar, MD, PhD,a Eileen Hsich, MD,b Patrick Reville, MPH,a Amy S. Nowacki, PhD,c William Baldwin, MD, PhD,d Suzanne Bakdash, MD, MPH,e Jenna Daghstani, BSc,f Aiwen Zhang, PhD,a Lynne Klingman, BS,a Nicholas Smedira, MD,g Nader Moazami, MD,g David O. Taylor, MD,b Randall C. Starling, MD, MPH,b and Gonzalo Gonzalez-Stawinski, MDh From the aAllogen Laboratories Transplant Center; Departments of bCardiovascular Medicine; cQuantitative Health Sciences; dImmunology and eTransfusion Medicine, Cleveland Clinic, Cleveland, Ohio; fCollege of Engineering, University of Toledo, Toledo, Ohio; gDepartment of Thoracic and Cardiovascular Surgery, Cleveland Clinic, Cleveland, Ohio; and hDepartment of Thoracic and Cardiovascular Surgery, Baylor University Medical Center, Dallas, Texas.

KEYWORDS: heart transplantation; VAD; allosensitization; HLA antibodies; MICA antibodies; antibody detection

BACKGROUND: Ventricular assist devices (VADs) are increasingly being used as a bridge to transplantation and have been implicated as a risk factor for allosensitization to human leukocyte antigens (HLA). We investigate the association between VAD and allosensitization to human leukocyte antigens (HLA) and major-histocompatibility-complex (MHC) class I-related Chain A (MICA) antigens. METHODS: We considered all patients who received a VAD at our institution between 2000 and 2009; 89 of them had pre-VAD and post-VAD (r6 months after implant) HLA antibody screening. A control group of non-VAD heart transplant candidates was constructed with at least 2 pre-transplant panel-reactive antibody (PRA) tests within 8 months. Two controls were randomly selected/VAD patient matched for year (n ¼ 178). Patients and controls with available sera from these time-points were tested by Luminex/flow PRA single-antigen beads and by MICA antibody Luminex single-antigen beads. Medical records were reviewed for comparison of pre-transplant immunologic risk factors and post-transplant outcomes between the 2 groups. RESULTS: Compared with controls, VAD patients had greater Class I differences between peak and initial PRA (18% vs 0%, p o 0.0001) and higher peak PRA (24% vs 6%, p o 0.0001). The differences between the 2 groups in Class II were less pronounced than in Class I. Of patients who had single-antigen testing, VAD implantation was significantly associated with development of new HLA antibody specificities (Class I and/or Class II) post-VAD with an increase in calculated PRA (cPRA) post-VAD compared with controls (16% vs 0%, p o 0.0001). This risk was still present after adjusting for age, gender, pre-VAD PRA, transfusion and duration of follow-up in a multivariate analysis (p o 0.0001 and 0.02, respectively). There were no differences in development of MICA antibodies between the 2 groups (14% in both). There was no significant difference in the incidence of pre-transplant positive T-cell crossmatch, pre-transplant donorspecific HLA antibodies, rejection episodes or graft survival between the 2 groups. CONCLUSION: Our results suggest that VAD is associated with significant HLA allosensitization independent of common risk factors. J Heart Lung Transplant 2013;32:1241–1248 r 2013 International Society for Heart and Lung Transplantation. All rights reserved.

Reprint requests: Medhat Askar, MD, PhD, Department of Surgery, Cleveland Clinic Lerner College of Medicine, CWRU, 9500 Euclid Avenue, C100 MZA, Cleveland, OH 44195. Telephone: 216-444-5918. Fax: 216-444-8261. E-mail address: [email protected]

Ventricular assist devices (VADs) are increasingly being used as a bridge to heart transplantation in patients with advanced heart failure who otherwise may not survive long enough to undergo transplantation. VAD implantation has

1053-2498/$ - see front matter r 2013 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2013.08.014

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The Journal of Heart and Lung Transplantation, Vol 32, No 12, December 2013

been shown to increase survival time and end-stage organ function with no significant difference in post-transplant survival of heart transplant recipients.1 What remains unclear is whether VAD implantation affects a patient’s candidacy for heart transplantation due to the development of allosensitization to human leukocyte antigens (HLA). HLA allosensitization is defined as the development of HLA antibodies after exposure to a sensitizing event such as pregnancy, blood/product transfusion and organ transplantation. The implantation of VADs has been implicated in HLA allosensitization.2–4 Allosensitization often limits the donor pool available for a given patient to avoid donors with HLA antigens corresponding to the patient’s antibodies and potentially increases the waiting time to transplantation. Allosensitization has also been implicated in allograft rejection and worse survival post-transplant.5–7 Majorhistocompatibility-complex (MHC) class I-related Chain A (MICA) antigens are a set of polymorphic antigens expressed on the endothelium and have been shown to be recognized by antibodies produced by transplant recipients.8 MICA antibodies have been reported in association with increased incidence of transplant coronary artery disease.9 In this study we investigated the changes in pre-transplant HLA and MICA allosensitization in association with VAD implantation and compared with the findings with data from patients who did not receive VAD. We also compared the post-transplant allograft outcomes between these 2 groups.

Methods Patients We performed a retrospective analysis on all consecutive patients who underwent left ventricular device implantation at our institute between January 2000 and December 2009. We included all VAD patients who had HLA antibody screening prior to VAD implantation and within 6 months after VAD implantation. The control group consisted of heart failure patients without a VAD who were on the transplant waiting list at the Cleveland Clinic during the same time period and had at least 2 pre-transplant panelreactive antibody (PRA) tests within 8 months. Two controls were randomly selected for each VAD patient and matched for year of initial HLA antibody testing to account for era effect on advances in laboratory technology, medical practice and surgical practice. The duration of follow-up for VAD patients is defined as the time interval between VAD implantation and the peak PRA testing date post-VAD. For controls, follow-up duration is defined as the time interval between the first and last PRA testing. We excluded patients with a history of prior transplantation and technically unsatisfactory HLA antibody tests (e.g., negative control bead reacting positively to the patient serum). The final cohort consisted of 89 VAD patients and 178 matched controls. Patients’ demographics, including gender, race and primary diagnosis, along with outcome measures, such as overall survival and rejection episodes, were obtained from electronic medical records. Red blood cell and platelet transfusion history for VAD and control patients was obtained from the blood bank. This study was approved by the institutional review board at the Cleveland Clinic and, because all data were collected and recorded as part of routine clinical care, the requirement for informed consent was waived.

Ventricular assist devices There were 60 patients who underwent left ventricular VAD implantation with pulsatile (P) devices and 29 patients with continuous-flow (CF) devices. Among the patients with P devices, there were 43 HeartMate IP-ELEC; 5 each of Thoratec, HeartMate XVE and Novacor PC; and 3 Abiomed AB devices. One of these patients had 2 devices placed during the study period, an Abiomed AB followed by a HeartMate IP-ELEC. Among those with CF devices, 24 were HeartMate II, 3 were Micromed DeBakey and 2 were Jarvik 2000. The CF devices were valveless and the only P devices with bio-prosthetic valves included in the study were HeartMate IP-ELEC, HeartMate XVE and Novacor PC.

HLA antibody testing and calculated PRA calculation HLA antibody testing was performed for all study subjects using FlowPRA screening beads (One Lambda, Canoga Park, CA). In addition, for a subset of the subjects, stored sera were available for HLA antibody single-antigen bead testing (and, consequently, percent calculated PRA calculation). HLA antibody (Class I and II) specificities were identified using FlowPRA (OneLambda) or LABScreen single-antigen beads (OneLambda) and detected on a flow cytometer (BD FACSCalibur, San Jose, CA) or Luminex (IS 200; Luminex, Austin, TX) instruments, respectively. For patients who had single-antigen testing performed (58 VAD patients and 63 control patients), a calculated combined Class I and Class II panelreactive antibody percentage (cPRA) was determined using an inhouse cPRA calculator. The algorithm for cPRA calculations is based on HLA antigen frequency in a historical cohort of 46,000 deceased donors tested in our laboratory and closely resembles the cPRA calculator developed by the Organ Procurement and Transplantation Network (OPTN). Comparison between VAD cases and controls included peak PRA, increase in PRA and increase in cPRA values. The pPRA was defined as the highest PRA value after VAD implantation or the highest of the 3 measured PRA values in the control group. The increase in PRA was calculated based on the difference between the peak and the initial PRA measured.

Anti-MICA antibody testing Patients who had stored sera from the study specific time-points were tested for circulating anti-MICA antibodies. Serum circulating immunoglobulin G (IgG) MICA antibodies were identified using LSA-MIC Luminex beads (Hologic Gen-Probe, Inc., San Diego, CA) according to the manufacturer’s instructions. The GenProbe kit detects 28 MICA antigenic specificities, which cover MICA antigens in approximately 95% of the population.

Pre-transplant crossmatch status and presence/ development of DSA All patients who receive a heart transplant at our institute (including VAD patients and controls) are initially evaluated for organ offers using a virtual crossmatch method by comparing the mismatched donor HLA antigens to the HLA antibodies identified in the recipient’s most current serum to determine the presence of pretransplant DSA (pDSA), as described elsewhere.10 In addition, they are tested for T- and B-cell flow crossmatch that is performed prospectively (with local donors) or concurrently/retrospectively (with imported donors). Post-transplant HLA antibody testing is performed

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on an as-needed basis to determine the development of de novo DSA (dDSA). We reviewed electronic medical records of all VAD patients and controls for flow crossmatch, pDSA and dDSA results.

Statistical analysis Continuous variables are expressed as the median (Q1 to Q3) and categorical variables are presented as percent. Associations among VAD status and patient demographics, laboratory measures and primary diagnosis were determined using 2-tailed Mann–Whitney U-tests and 2-tailed Fisher’s exact tests. Analyses to investigate the association among VAD status (and type) and allosensitization were performed 2 ways due to concerns of possible differences among patients treated over the study period: (1) linear regression assuming independence; and (2) generalized estimating equations with a firstorder autoregressive correlation structure. Results of the 2 analyses are similar and the latter are presented as unadjusted data and data adjusted for pre-VAD PRA, gender, age, number of red blood cell and platelet units transfused and duration of follow-up. Kaplan–Meier survival curves were constructed and differences between groups were assessed by log-rank test. Cox proportional hazards regression was utilized to investigate the adjusted association among VAD status and survival. All survival information is current as of August 2012. All analyses were done using SAS, version 9.3 (SAS Institute, Cary, NC), and all tests were considered significant at the p o 0.05 significance level.

Results Baseline characteristics The baseline characteristics of patients with VAD and without VAD (control) are shown in Table 1. Age, race and Table 1

HLA allosensitization results by VAD implantation status and type Unadjusted HLA allosensitization data are shown in Table 2 comparing VAD to control (Table 2A) and different device types (Table 2B). The median difference (peak  baseline) in Class I PRA was 18% (Q1 to Q3 ¼ 3% to 52%) in the VAD patients compared with a median of 0% (Q1 to Q3 ¼ 0% to 1%) in the control group (p o 0.0001). The median difference in Class II PRA was 2% (Q1 to Q3 ¼ 0% to 13%) in the VAD patients compared with a median of 0% (Q1 to Q3 ¼ 0% to 0%) in the control group (p o 0.0001). The difference between the 2 groups in Class II was less pronounced than in Class I. The median difference in cPRA between the initial evaluation and peak due to development of new HLA antibody specificities was also higher in the VADs than in the controls (16% vs 0%, p o 0.0001). No VAD patient for which we had single-antigen results showed any reactivity to self-HLA (data not shown). The median peak Class I PRA values were higher in the VAD patients than in controls (24% vs 6%, p o 0.0001). The median peak Class II

Patients’ Demographics, Laboratory Measures and Primary Diagnosis for VAD and Control Patients

a

Age, years Maleb Raceb White Black Asian Hispanic Other Follow-up duration, daysa Initial PRA Class Ia Initial PRA Class IIa Initial cPRAa RBC units transfuseda Platelet units transfuseda Total productsa Primary diagnosisb Coronary artery disease Congenital heart diagnosis Dilated cardiomyopathy Hypertrophic cardiomyopathy Restrictive cardiomyopathy Valvular heart diagnosis Other a Data b

initial Class I and II PRA were similar between the 2 groups. Compared with the control group, the VAD cohort had more male patients (82% vs 70%, p ¼ 0.04), and more red blood cell (median of 13 vs 1 U, p o 0.0001) and platelet transfusions (median of 3 vs 0 U, p o 0.0001). Follow-up duration was longer in the controls by design to capture any transient fluctuations in PRA in non-VAD potential heart recipients (53 vs 28 days, p ¼ 0.001).

expressed as median (interquartile range). Data expressed as number (%).

VAD patients (n ¼ 89)

Controls (n ¼ 178)

54 (46–62) 73 (82%)

56 (46–63) 125 (70%)

78 10 1 0 0 28 4 1 0 13 3 15

(88%) (11%) (1%) (0%) (0%) (14–67) (2–13) (1–4) (0–22) (7–23) (2–6) (8–29)

151 24 0 1 2 53 4 1 11 1 0 2

(85%) (13%) (0%) (1%) (1%) (23–116) (2–20) (1–4) (0–40) (0–3) (0–0) (0–4)

24 0 55 6 0 4 0

(27%) (0%) (62%) (7%) (0%) (4%) (0%)

33 10 102 10 12 4 6

(19%) (6%) (58%) (6%) (7%) (2%) (3%)

p-value 0.95 0.04 0.73

0.001 0.76 0.55 0.01 o0.0001 o0.0001 o0.0001 0.003

1244 Table 2A

The Journal of Heart and Lung Transplantation, Vol 32, No 12, December 2013 Allosensitization by VAD Implantation Status

Peak PRA Class I Difference in PRA Class I Peak PRA Class II Difference in PRA Class II Difference in cPRAa

VAD (n ¼ 89)

Control (n ¼ 178)

p-value

24 18 4 2 16

6 0 2 0 0

o0.0001 o0.0001 0.04 o0.0001 o0.0001

(7–72) (3–52) (1–16) (0–13) (0–41)

(2–26) (0–1) (1–5) (0–0) (0–0)

Results are expressed as percent [median (interquartile range)]. Difference is defined in the VAD group as (peak value post-VAD) – (value pre-VAD), and in the control group as the difference between the highest PRA – the lowest PRA during the follow-up period regardless of the sample sequence. a VAD patients, n ¼ 58; controls, n ¼ 63.

PRA values were low in both the VAD patients and controls (4% vs 2%, p ¼ 0.04). Multivariate models were constructed to assess the association among VAD status and differences in Class I and II PRA and cPRA after adjusting for common risk factors of allosensitization and potentially confounding factors, including age, gender, initial PRA, red blood cell and platelet units transfused, and duration of device implantation or followup (in the control group). The significance of the observed differences between VAD patients and controls in Class I and II PRA and cPRA remained unchanged in the adjusted models (p o 0.0001, 0.02 and 0.0002, respectively; Table 3). When devices were stratified by bio-prosthetic valve containment, compared with valveless devices, valvecontaining devices were associated with a larger difference in both Class I and Class II PRA (26% vs 7%, p ¼ 0.04, and 5% vs 1%, p ¼ 0.001, respectively; Table 2B) and peak Class I and Class II PRA value (31% vs 19%, p ¼ 0.04, and 9% vs 2%, p ¼ 0.003, respectively). Similar associations with Class II were observed when patients were stratified by device type. P devices, compared with CF devices, were associated with a greater difference in Class II PRA and peak Class II PRA value (5% vs 1%, p ¼ 0.002, and 8% vs 2%, p ¼ 0.003, respectively; Table 2B). Similar trends that did not reach statistical significance were also observed in Class I PRA differences and peak PRA values in association with P devices compared with CF devices.

MICA allosensitization by VAD placement To investigate whether VAD placement was associated with an increase in non-HLA antibodies, we compared MICA antibody development among the VAD and control patients. Forty VAD patients and 14 controls had sera available for Table 2B

MICA testing. In the VAD group, only 1 patient had MICA antibodies pre-VAD and 6 patients (14%) who had no MICA antibodies pre-VAD developed MICA antibodies post-VAD. In the control group, 5 patients had MICA antibodies on initial testing and, at the end of follow-up, 2 patients (14%) who previously had MICA antibodies developed new MICA antibody specificities. No distinguishable patterns of MICA antibody specificities were observed in either group.

Pre-transplant crossmatch status and presence/ development of DSA by VAD implantation status There was no evidence of significant differences among the proportions of VAD patients and controls who proceeded to transplant with positive T- and B-cell flow crossmatch (11% vs 7%), with positive B-cell flow crossmatch only (43% vs 34%), and with negative T- and B-cell flow crossmatch (46% vs 59%). Compared with controls, the VAD patients had relatively lower pre-transplant pDSA (10% vs 19%, p ¼ 0.15) and dDSA (3% vs 16%, p ¼ 0.03), as shown in Table 4. It is noteworthy that it is not uncommon in our program to transplant in the face of a weak DSA and/or positive B-cell flow crossmatch with negative T-cell flow crossmatch. These transplants are performed in patients with an urgent need for a transplant and in which they typically receive induction immunosuppression of anti-thymocyte globulin. However, all transplants in this study except 2 had negative complement-dependent cytotoxicity (CDC) crossmatches.

Incidence of rejection by VAD implantation status We analyzed rejection episodes for patients with available data (VAD, n ¼ 78; control, n ¼ 152; Table 5). There was a

Allosensitization by VAD Type

Peak PRA Class I Difference in PRA Class I Peak PRA Class II Difference in PRA Class II Difference in cPRAa

Pulsatile (n ¼ 60)

Continuous flow (n ¼ 29)

p-value

Valveb (n ¼ 52)

No valveb (n ¼ 36)

p-value

27 22 8 5 5

22 7 2 1 21

0.44 0.22 0.003 0.002 0.60

31 26 9 5 11

19 7 2 1 19

0.04 0.04 0.003 0.001 0.79

(6–77) (4–64) (2–19) (1–16) (0–41)

(7–61) (0–43) (1–7) (0–4) (0–48)

(6–88) (4–64) (2–20) (1–17) (0–41)

(7–55) (0–32) (1–6) (0–5) (0–44)

Data expressed as percent [median (interquartile range)]. Difference defined in the VAD group as (peak value post-VAD) – (value pre-VAD), and in the control group as the difference between the highest PRA – the lowest PRA during the follow-up period regardless of the sample sequence. a Pulsatile, n ¼ 36; continuous flow, n ¼ 22; valve, n ¼ 31; no valve, n ¼ 26. b Excludes 1 patient who had two devices (1 valve, 1 no valve) placed during the study period.

Askar et al. Table 3

HLA and MICA Patterns

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Multivariate Comparison of Allosensitization by VAD Implantation Status Model 1

Model 2

Model 3

Difference in Class I PRA (%)

Difference in Class II PRA (%)

Difference in cPRA

Slope (95% CI)

p-value

Slope (95% CI)

VAD vs control 25.2 (15.2 to 35.1) o0.0001 11.2 (1.7 to 20.8) Age, years –0.02 (–0.16 to 0.11) 0.73 –0.11 (–0.20 to –0.03) Male –4.6 (–11.0 to 1.8) 0.16 –0.6 (–6.0 to 4.8) Initial PRA Class I –0.08 (–0.16 to 0.01) 0.09 –– Initial PRA Class II –– –– 0.05 (–0.03 to 0.12) RBC units transfused 0.19 (–0.18 to 0.56) 0.32 –0.01 (–0.21 to 0.20) Platelet units transfused –0.49 (–1.05 to 0.08) 0.09 –0.17 (–0.46 to 0.13) Follow-up duration (days) 0.03 (–0.01 to 0.06) 0.09 0.02 (–0.01 to 0.05)

non-significantly higher proportion of VAD patients with at least 1 rejection episode compared with controls (33% vs 24%, p ¼ 0.24). There was no evidence of an association between device type (continuous-flow or pulsatile) and rejection episodes (data not shown).

Overall survival by VAD implantation status Kaplan–Meier survival curves were constructed to compare post-transplant survival of individuals bridged to transplant with a VAD (Figure 1). There was no difference in overall survival between those bridged to transplant with a VAD and those who were not (p ¼ 0.62). The multivariate Cox regression analysis adjusted for the same covariates listed previously and indicated that VAD status remained nonsignificantly associated with survival (hazard ratio [HR] ¼ 0.65, confidence interval [CI] 0.34 to 1.20, p ¼ 0.17). Covariates found to be associated with survival were number of red blood cell units transfused (HR ¼ 1.02, CI 1.01 to 1.04, p ¼ 0.01) and age (HR ¼ 1.02, 95% CI 1.01 to 1.05, p ¼ 0.005); initial Class I and II PRA, gender and length of follow-up were non-significant.

Discussion In this study we have investigated HLA allosensitization in association with VAD implantation using solid-phase–based sensitive and specific antibody detection methods. We also compared the observed changes in HLA allosensitization Table 4

0.02 0.01 0.83 –– 0.27 0.94 0.26 0.15

16.8 0.20 –5.1 –0.06 –0.19 –0.23 0.73 –0.07

p–value

(8.1 to 21.5) 0.0002 (0.09 to 0.30) 0.00 (–13.8 to 3.6) 0.25 (–0.15 to 0.02) 0.15 (–0.38 to –0.01) 0.04 (–0.56 to 0.11) 0.18 (0.38 to 1.09) o0.0001 (–0.11 to –0.03) 0.001

level in the VAD group with a comparison group who were evaluated in the same study period but had not received VAD or any other sort of mechanical support devices, such as a total artificial heart (TAH) and extracorporeal mechanical oxygenation (ECMO). Our data indicate that VAD implantation was associated with Class I and, to a lesser extent, Class II allosensitization, independent of known allosensitization risk factors and potential confounders. VADs have been implicated in HLA allosensitization in a number of studies. However, most of these studies were underpowered and relied on cell-based, less-sensitive/ specific antibody detection methods.2,4,11 They also did not account for confounding factors such as blood product transfusion. In our study, VAD patients and controls were comparable with regard to most common risk factors, except for blood product transfusions, which were significantly more prevalent in the VAD group. Notably, all of the blood products transfused to VAD patients and controls during the study period were leukocyte-reduced. There were more men in the VAD group compared with the controls, which is consistent with other reports of women receiving fewer mechanical support devices, possibly due to the increased adverse effects in women.12–14 Despite this finding, our multivariate analysis has shown for the first time that the observed association between VAD and allosensitization remained significant even after adjusting for red blood cell and platelet transfusions, as well as other potential confounders, such as gender, age and duration of VAD implantation (or follow-up in controls).

Pre-transplant Crossmatch Status and Presence/Development of DSA by VAD Implantation Status

Pre-transplant crossmatch status T- and B-cell XM-positive B-cell XM-positive only T-cell XM-positive only T- and B-cell XM-negative Pre-transplant donor-specific HLA antibodiesa De novo donor-specific HLA antibodiesb patients, n ¼ 82; controls, n ¼ 63. VAD patients, n ¼ 58; controls, n ¼ 63.

a VAD b

p-value Slope (95% CI)

VAD (n ¼ 89)

Control (n ¼ 172)

10 38 0 41 8 2

13 58 0 101 12 10

p-value 0.13

(11%) (43%) (46%) (10%) (3%)

(7%) (34%) (59%) (19%) (16%)

0.15 0.03

1246 Table 5

The Journal of Heart and Lung Transplantation, Vol 32, No 12, December 2013 Rejection Episodes by VAD Implantation Status

Rejection episodes (yes/no) [n (%)] Number of rejection episodes [median (IQR)]

VAD (n ¼ 78)

Control (n ¼ 152)

p-value

26 (33%) 0 (0–1)

36 (24%) 0 (0–0)

0.24 0.46

In our study, P devices and bio-prosthetic valve– containing devices were associated with higher HLA allosensitization compared with CF and non–valve-containing devices, respectively. However, given the fact that not all CF devices contained bio-prosthetic valves, we were not able to determine whether the observed increased allosensitization was attributed to the P design or the containment of bio-prosthetic materials. CF VADs have been suggested to have a less allosensitizing effect compared with P devices.2,4,15 P devices cause more shear stress in circulation than CF devices, which have been shown to cause endothelial and platelet activation.16 Both endothelial activation and platelet activation have long been linked to HLA antibody formation.17 It has also been suggested that the immunologic sensitizing potential with VADs is related to the device’s surface texture and biomaterials.11,18,19 Surface contact may cause B-cell hyper-reactivity and antibody production.19 Studies have shown that the surface of VADs becomes populated by T cells, which can be activated to induce B-cell help.18 We have also investigated the changes in breadth of alloantibody production post-VAD using the cPRA, which provides an estimate of the percentage of deceased organ donors that will be crossmatch-incompatible for a candidate.20 Our results indicate a significant increase in the incidence of development of new HLA antibody specificities in association with VAD implantation; however, there was no evidence that the VAD-associated cPRA increase was influenced by device type. In the setting of short cold ischemic time during heart transplantation, virtual crossmatch often substitutes for a prospective actual crossmatch. The virtual crossmatch allows for comparison of recipient HLA antibodies to HLA antigens of a potential donor to predict with high degree of sensitivity and specificity the result of a crossmatch without performing one prospectively.21 It is reasonable to assume that increased cPRA would increase the proportion of donors predicted to be virtual crossmatch-positive. Consequently, donors who have the corresponding HLA antigens in their phenotype would be excluded, leading to a limited donor pool and potentially increasing the transplant wait time. A recent International Society for Heart and Lung Transplantation (ISHLT) registry analysis indicated that approximately 18% of recipients for whom PRA data were reported were transplanted with PRA 410%.22 However, due to the retrospective design of the present study and the multifactorial nature of accepting a potential donor, the direct impact of increased cPRA in VAD patients on waiting time to transplant could not be investigated. MICA antibodies have been implicated in both renal and heart allograft failure.23,24 We had a relatively small number

of VAD patients and controls tested for MICA allosensitization and, in this cohort, there was no evidence of increase in MICA allosensitization in the VAD group compared with controls. Further assessment of VAD-associated MICA allosensitization with a larger sample size is needed to validate these results. The higher incidence of Class I and II PRA and cPRA increases after VAD implantation in the absence of evidence of MICA antibody increases may argue against non-specific generalized immune activation. Recently, it has been postulated that VADs are associated with production of clinically irrelevant antibodies that do not bind intact antigens on the cell surface.25 However, even if proven true, the practical utility of this concept will be challenging because VAD-associated HLA allosensitization almost invariably develops in the context of multiple transfusions, a known risk factor for HLA allosensitization. It is almost impossible to determine with any level of confidence which HLA antibodies developed due to transfusion and should be avoided versus those that developed due to VAD and should be considered acceptable. Investigating the clinical relevance of VAD-associated HLA antibodies would require comparing clinical outcomes in an adequately sized cohort of transplant candidates who proceed to transplant in the face of VAD-associated DSA versus those with non–VAD-related DSA, but this was beyond the scope of our study. There were comparable proportions of VAD patients and controls who proceeded to heart transplantation with both positive T- and B-cell flow crossmatch or positive B-cell

Figure 1 There is no evidence of a difference in overall survival between those patients bridged to transplant with VAD and those who were not (p ¼ 0.62).

Askar et al.

HLA and MICA Patterns

flow crossmatch only. Although the proportions of VAD patients who had pDSA and those who developed dDSA were relatively fewer compared with controls, the overall frequency of DSA was not adequate to make meaningful inferences. We also observed that, in both VAD patients and controls, there were proportions of positive B-cell crossmatches in the absence of pDSA, which is not an uncommon finding due to non-HLA and/or non-specific binding to B-cell surface molecules. There was no evidence of a difference in overall allograft survival or incidence of rejection between VAD patients and controls. There was also no difference in rejection episodes by VAD device type. These results are consistent with earlier reports.22,26 There are several limitations to the study. First, it was a retrospective investigation. Second, there is a potential for selection bias in our population as repeat PRA screening in our cohort of patients awaiting heart transplantation may have been performed after blood product transfusions; however, we attempted to mitigate this limitation by including a control group of patients who were tested under the same circumstances during the same time period. In addition, stored sera were not available for the entire cohort to allow HLA single-antigen testing, cPRA calculation and MICA testing. Another significant limitation is the inadequate documentation in our database of how many potential donors were considered for each VAD patient or control included in the study or the cause for decline of any given donor. These data would be necessary to determine whether allosensitization affects transplant waiting time. However, the most recent adult heart transplant report showed a significant barrier to transplant with increasing PRA.22 Another limitation is that the study spanned a 10-year period in which the ISHLT diagnostic criteria for both antibody- and cell-mediated rejection evolved, and this limited our ability to determine the proportion of patients with antibody-mediated rejection vs cell-mediated rejection. Finally, the possibility cannot be ruled out that patients in both the VAD and control groups may have passively acquired HLA antibodies through transfusion of blood products. However, given that patients in the 2 groups received these products under the same conditions and were matched for the year of PRA testing, it is unlikely that passively transfused HLA antibodies would have significantly impacted the observed associations. In conclusion, VAD placement was associated with significant Class I and Class II allosensitization independent of known risk and confounding factors, including blood product transfusion. Among patients receiving a VAD, device type affected allosensitization, with continuous-flow devices and devices without bio-prosthetic valves causing less allosensitization. VAD placement had no effect on posttransplant survival and no significant associations with rejection episodes after transplantation.

Disclosure statement The authors have no conflicts of interest to disclose.

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