OBJECTIVES:A T-cell-positive and B-cell-negative flow cytometry crossmatch result remains a conundrum since HLA class I antigens are expressed on both T and B cells. We investigated the frequency, donor HLA specificity of the antibodies, and mechanisms for these crossmatch results.MATERIALS AND METHODS:We analyzed 3073 clinical flow cytometry crossmatch tests performed in an American Society of Histocompatibility and Immunogeneticsaccredited histocompatibility laboratory. The sera associated with the T-cell positive and B-cell negative flow cytometry crossmatches were also tested for donor HLA immunoglobulin G antibodies using LABScreen single antigen assays.RESULTS:Among the 3073 test results, 1963 were T-cell negative and B-cell negative, 811 were T-cell negative and B-cell positive, 274 were T-cell positive and B-cell positive, and 25 were T-cell positive and B-cell negative. The LABScreen single antigen assay detected HLA class I immunoglobulin G donor-specific antibodies in 23 of 25 sera associated with a T-cell positive and B-cell negative flow cytometry crossmatch result, and donorspecific antibodies directed at not only HLA-Cw but also at HLA-A or HLA-B were observed. In addition, we identified that the B-cell channel shift threshold used to classify a B-cell flow cytometry crossmatch was a potential contributor to a T-cell-positive and B-cellnegative flow cytometry crossmatch result.CONCLUSIONS:Our analysis of 3073 flow cytometry crossmatches, in addition to demonstrating that HLA antibodies directed at the HLA-A, -B, or -Cw locus were associated with a T-cell-positive and B-cell-negative result, identified mechanisms for the surprising T-cell-positive and B-cell-negative flow cytometry crossmatch result.

Desensitization using plasma exchange can remove harmful antibodies prior to transplantation and mitigate risks for hyperacute and severe early acute antibody-mediated rejection. Traditionally, the use of plasma exchange requires a living donor so that the timing of treatments relative to transplant can be planned. Non-HLA antibody is increasingly recognized as capable of causing antibody-mediated renal allograft rejection and has been associated with decreased graft longevity. Our patient had high-strength non-HLA antibody deemed prohibitive to transplantation without desensitization, but no living donors. As the patient was eligible to receive an A2 ABO blood group organ and was willing to accept a hepatitis C positive donor kidney, this afforded a high probability of receiving an offer within a short enough time frame to attempt empiric desensitization in anticipation of a deceased donor transplant. Fifteen plasma exchange treatments were performed before the patient received an organ offer, and the patient was successfully transplanted. Hepatitis C infection was treated posttransplant. No episodes of rejection were observed. At one-year posttransplant, the patient maintains good graft function. In this case, willingness to consider nontraditional donor organs enabled us to mimic living donor desensitization using a deceased donor.

Interfering factors in sera may confound crossmatch (XM) results. Heat inactivation (HI), DTT and dialysis treatment of sera have been utilized to minimize these confounders. We investigated the impact of HI on donor T cell complement dependent cytotoxicity (CDC) XM (T Cell AHG XM) and donor B cell CDC XM (B Cell NIH XM). We queried our electronic database for all donor CDC T cell and B cell XM performed during 2011–2017 in which potential kidney transplant recipient’s serum was tested in parallel with or without HI and using potential donor’s T cells and B cells as target cells. A total of 6295 living donor XMs and 15880 deceased donor XMs performed in our laboratory were reviewed. Table 1 shows the impact of HI on the 22175 consecutive XM results. 394 of the 6295 living donor T Cell AHG XMs were positive using neat sera and 303 were positive following HI at 63C for 6 min, a 23% conversion from positive to negative results. 698 of the 15880 deceased donor T Cell AHG XMs were positive using neat sera and 540 XMs were positive following HI, a 23% reduction. 1372 of the 6295 living donor B Cell NIH XMs were positive using neat sera and 539 were positive following HI, a 61% reduction. 3015 of the 15880 deceased donor B Cell NIH XMs were positive using neat sera and 842 XMs were positive following HI, a 72% reduction from a positive result to negative result. A substantial percentage of XMs are converted from a positive CDC result to negative result and such conversion is more frequent with B cell XM compared to T cell XM. Transplant centers seldom transplant across a positive T cell CDC XM even when HI converts a positive result to a negative result. On the other hand, it is not uncommon to proceed with a kidney transplant when HI converts a positive B cell CDC to a negative CDC provided there are no other immune contraindications. There is an urgent need to establish the clinical significance of XMs converted from a positive to a negative result by heat inactivation.

Aim Emerging data suggest that DSA development is a frequent complication post BKV replication, and most DSA are directed against donor HLA-DQ. In the current investigation, we quantified DQ epitope mismatches between the recipient and donor in individuals with BKVN diagnosis and investigated the association between DQ epitope load and development of DSA. Methods In this pilot study, using the most probable 4-digit HLA typings, we investigated epitope mismatches in individuals who developed DSA post BKVN diagnosis compared to those who did not and correlated them to DSA development and graft dysfunction defined as an increase in serum creatinine by u003e 0.5 mg/dL at 12 months post-BKVN diagnosis. We had SSOP based intermediate HLA typing available for recipients and donors in 13 recipients who developed BKVN confirmed by renal allograft biopsy. Of the 13 recipients, 2 were excluded: 1 for early graft loss and 1 who had zero DQ mismatches. None of the 11 recipients with BKVN diagnosis had a positive XM or DSA at time of transplantation. Results Time from kidney transplantation to BKVN diagnosis was 8.5 ± 5.1 months. Serum creatinine at time of diagnosis was 1.78 ± 0.56. Of the 11 with BKVN, 36% developed de novo DSA. All DSAs were directed against DQ mismatches. Time from BKVN diagnosis to DSA development was 5.5 ± 2.3 months. Table below lists the epitope mismatch number at each locus. Of the 4 patients with de novo DSA, 75% experienced graft dysfunction during the 12 months post BKVN and of the 7 patients without de novo DSA, 29% experienced graft dysfunction by 12 months. Our data demonstrated that the most frequent DQ epitope mismatches in those with de novo DSA were 52PL3, 45EV, 52PQ2, and 52PR. Conclusions In our pilot study, the epitope load was not different between BKVN patients who developed or did not develop de novo DSA. The most frequent target of de novo antibodies were DQ locus mismatched epitopes. Knowledge of epitope targets of de novo DSA in patients with BKVN may facilitate targeted therapies and improve kidney allograft outcomes. Download high-res image (148KB) Download full-size image

Luminex Single Antigen bead assay (LSAB) derived mean fluorescence intensity (MFI) values of anti-HLA antibodies directed at the potential donor’s HLA are the primary parameter used in the virtual crossmatch to predict physical crossmatch (XM) outcome. We aimed to develop statistically validated LSAB MFI cutpoints for predicting CDC XM and FCXM results. We leveraged the ASHI proficiency testing (PT) 80% consensus results of 7156 T FCXM, 6758 B FCXM, 2917 T CDC XM and 2233 B CDC XM as the reference results to investigate whether LSAB MFI of IgG anti-HLA antibodies predict validated physical XM results. LSAB MFI was determined in our laboratory using One Lambda Single Antigen HLA Class I and Class II beads. The 80% consensus results are from 8 consecutive challenges during 2013 to 2016, and 107 laboratories across USA tested sera and HLA typed cells distributed by ASHI, and reported the results to ASHI for assessment of the laboratory’s proficiency. Data analysis included: summing of MFI of IgG antibodies directed at HLA-A, B and C for investigating their association with T FCXM and T CDC XM results; summing of MFI of antibodies directed at HLA-A, B, C, DR, DQ and DP for investigating their association with B FCXM and B CDC XM results; assigning alternate ASHI challenges to the Discovery set and the Validation set; investigating the association between LSAB MFI and physical XM results by logistic regression analysis corrected for overdispersion; and identification of LSAB MFI cutpoint for maximizing the sum of sensitivity and specificity. LSAB MFI cutpoints derived from the Discovery set to predict FCXM and CDC XM results were investigated in an independent validation set. Table 1 demonstrates that LSAB MFI cutpoints from the Discovery set predicts XM outcomes in the Validation set (ROC AUC range from 0.974 to 0.999). We have developed and validated LSAB MFI cutpoints for use in the virtual crossmatch to accurately predict physical T FCXM, B FCXM, T CDC XM and B CDC XM results.

LSAB mean fluorescence intensity (MFI) is often used to predict physical FCXM results, and LSAB MFI cutpoints are used to list unacceptable antigens. We therefore determined the accuracy of LSAB assay MFI cutpoint used to score the LSAB assay as positive or negative in foretelling FCXM results. One hundred and forty-six pre-transplant sera from 146 patients with end stage renal disease were tested against potential kidney donor’s T cells and B cells using a 3-color flow cytometry clinical protocol; the same 146 sera were tested using LSAB assay and the cumulative MFI directed at donor HLA class I and/or II antigens were determined. A T cell FCXM with a median channel shift (MCS) ≥ 40 with the patients serum vs. negative control serum was classified as a positive test; a B cell FCXM with MCS ≥ 50 with the patients serum vs. negative control serum was classified as a positive test. In the LSAB assay, MFI > 2000 directed at donor HLA antigens was scored as positive DSA. These cutpoints are our laboratory’s cutpoints for both ASHI proficiency testing and for clinical use. Statistical analysis demonstrated a significant association between LSAB results and FXCM results (Table 1). LSAB assay results predicted T Cell FCXM results with a sensitivity of 70% and a specificity of 100% (Fischers Exact Test, P < 0.0001, Positive Predictive Value (PPV = 100%, Negative Predictive Value (NPV) = 91%). LSAB assay results also predicted B Cell FCXM results with a sensitivity of 86% and a specificity of 72% (Fischers Exact Test, P < 0.0001, PPV = 62%, NPV = 91%) (Table 1). In a subset of 31 patients, both donor FCXM and auto FCXM results were available and 17 of 31 donor positive FCXM were also auto B cell FCXM positive (17/17) and auto T cell FCXM positive (1/17). LSAB assay results are significantly associated with FCXM results. Auto B cell FCXM and high resolution HLA typing should further refine the relationship between these two high sensitive assays.Download : Download high-res image (138KB)Download : Download full-size image

Luminex Single Antigen bead assays (LSAB) are robust for detecting and identifying IgG anti-HLA antibodies, and the LSAB mean fluorescence intensity (MFI) is currently used to list unacceptable antigens and to predict physical crossmatch (XM) results. Concerns exist regarding the use of MFI as the criterion because MFI thresholds for accurately predicting physical XM results are far from established. We aimed to address this unmet need. We leveraged the XM results from ASHI Proficiency Testing (PTs) to investigate whether LSAB MFI cutpoints are predictive of physical XM results. ASHI graded (⩾80% consensus) XM results from 8 consecutive ASHI PTs in which up to 30 labs tested 40 sera and 16 cells distributed by ASHI were used to examine whether LSAB MFI data we generated in the same PTs predict physical XM results. Cumulative MFIs of DSA directed at HLA-A, B, and C were tested for their ability to predict T cell Flow XM and T cell AHG XM results; cumulative MFIs of DSA directed at HLA-A, B, C, DRB1, DRB3/4/5, DQB and DP were tested for their ability to predict B cell Flow XM and B cell CDC XM results. Our data analysis identified that cumulative MFI of DSA less than 6000 directed at HLA-class I antigens predicted T cell Flow XM results with a 100% negative predictive value (NPV) and MFI > 7000 predicted T cell Flow XM results with a 100% positive predictive value (PPV) (Chi-Square for trend, X2 = 70, df = 1, P < 0.0001) (Table 1). Cumulative MFI of DSA less than 5000 directed at HLA-class I and II antigens predicted B cell Flow XM result with a 100% NPV and MFI > 8000 predicted B Flow XM result with a 100% PPV (X2; =; 67, df = 1, P < 0.0001) (Table 1). Cumulative MFIs predicted T cell AHG XM results and B cell CDC XM results with a slightly lesser accuracy (Table 1).Download : Download high-res image (343KB)Download : Download full-size image Our data demonstrating the feasibility of developing LSAB MFI cutpoints for the accurate prediction of T cell Flow XM, B cell Flow XM, T cell AHG XM, and B cell CDC XM results advance a strategy for the interpretation of virtual crossmatches and the prediction of physical XM results.

Early and late post transplant complications have been associated with antibodies (Abs) directed against donor HLA; however, the impact of pre-transplant Abs directed against donor HLA-C remains unresolved. In view of diminished display of HLA-C antigens on the cell surface compared to HLA-A or-B antigens, we hypothesized that the impact of pre-transplant Abs to donor HLA-C will be lesser compared to Abs directed at donor HLA-A and B. In the current investigation we studied the impact of circulating pre-transplant IgG antibodies to donor HLA-C locus antigens alone as compared to Abs to HLA- or B locus antigens. We conducted a retrospective review of records of 1252 kidney allograft recipients transplanted at our center between January 2010 and January 2016 to identify patients with circulating pre-transplant IgG Abs directed at kidney donor HLA-A, B or C locus antigens. Abs were detected and reported using the Luminex Single Antigen Bead assay for HLA Class I. Pre and post-transplant data were collected and graft outcomes of 16 kidney graft recipients with Abs to HLA-C locus antigens alone were compared to the outcomes of 56 recipients with Abs to HLA-A or B locus antigens. The mean pre-transplant MFI value of DSA in those with Abs to HLA- A or B antigens was 3880 ± 3023 and 4331 ± 5540 in those with Abs to HLA- C antigens (P = 0.4). The one-year acute rejection rate was 6% in those with DSA to HLA-C antigens and 20% in those with DSA to HLA-A or B antigens (Fig. 1A). Incidence of AMR was increased in those with DSA-MFI value greater than 6000 (Fig. 1B). Kidney allograft survival rate was 100% in those with DSA to HLA-C antigens and 95% in those with DSA to HLA-A or B locus antigens (P > 0.05). Our observations support the concept that circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus alone do not negatively impact kidney allograft outcomes and suggests that listing unacceptable HLA C locus antigens based on MFI of Single Antigen Bead Assay requires additional scrutiny and due caution.Download : Download high-res image (125KB)Download : Download full-size image

The relationship between circulating pre-transplant immunoglobulin G (IgG) antibodies to donor human leukocyte antigen (HLA) -C locus determined antigens alone and acute rejection, kidney allograft function, and graft survival is not fully defined. Also, the impact of circulating pre-transplant IgG antibodies to donor HLA-C locus antigens alone on these outcomes has not been compared with the impact of circulating pre-transplant IgG antibodies to donor HLA-A or -B locus antigens. We conducted a retrospective review of records of 1252 kidney allograft recipients transplanted at our center between January 2010 and January 2016 to identify patients with circulating pre-transplant IgG antibodies directed at kidney donor HLA-A, -B, or -C locus determined antigens. Antibodies were detected and reported using the LABScreen Single Antigen Bead assay with microbeads coated with single HLA class I antigens. Pre-transplant and post-transplant data were collected and the graft outcomes of 16 kidney graft recipients with antibodies to HLA-C locus antigens were compared to the outcomes in 56 recipients with antibodies to HLA-A or -B locus determined antigens. The one-year acute rejection rate was 6% in those with donor-specific antibodies (DSA) to HLA-C locus antigens and 20% in those with DSA to HLA-A or -B locus antigens. The graft survival rate was 100% in those with DSA to HLA-C locus antigens and 95% in those with DSA to HLA-A or -B locus antigens. None of the numerical differences were statistically significant (p>0.05). The presence of circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone may not be associated with an increased risk of acute rejection or a decreased graft survival rate. Our observations support the concept that circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone do not negatively impact kidney allograft outcomes and that the mean fluorescence intensities of the antibodies directed at HLA-C locus alone should not be used to list unacceptable HLA-C locus antigens for kidney allocation. A study with a larger cohort is needed to investigate our hypothesis.

BACKGROUND:Characteristics of pretransplant antibodies directed at donor human leukocyte antigen (HLA) donor-specific antibodies (DSA) associated with adverse outcomes in kidney transplant recipients are being elucidated but uncertainties exist.METHODS:We prospectively screened pretransplant sera from 543 kidney recipients using single antigen bead assays and identified 154 patients with and 389 without DSA. We investigated the association of DSA features to acute rejection and graft failure.RESULTS:One-year acute rejection incidence was higher in DSA-positive group (P < 0.001), primarily due to antibody-mediated rejection (AMR, 13% vs. 1.8%, P < 0.001) and not T cell-mediated rejection (ACR, 5% vs.6%, P = 0.65). The sum of mean fluorescence intensity of DSA (DSA MFI-Sum) of 6,000 or higher (OR, 18; 95% CI, 7.0-47; P < 0.001) and the presence of DSA against both HLA class I and II (OR, 39; 95% CI, 14-106; P < 0.0001) predicted 1-year AMR, independent of other covariates. Calculated panel reactive antibody and a positive flow cytometry cross-match result were associated with AMR by bivariate analysis but neither was an independent predictor in a multivariable regression analysis that included DSA-MFI-Sum or HLA DSA class. In multivariable Cox proportional hazards models, the covariate-adjusted hazard ratio for graft failure was 2.03 (95%CI, 1.05-3.92; P = 0.04) for DSA MFI-Sum of 6,000 or higher and 2.23 (95% CI, 1.04-4.80; P = 0.04) for class I and II DSA. Prediction of graft failure was not independent of AMR.CONCLUSION:Our study suggests that DSA MFI-Sum and HLA class of DSA are characteristics predictive of AMR and graft failure. The elevated risk of graft failure in those with the identified features of DSA is attributable to increased risk of AMR.

pact of hepatitis C infection on outcomes after heart transplantation. Transplantation 2009; 88: 1137. 3. Lee I, Localio R, Brensinger CM, et al. Decreased post-transplant survival among heart transplant recipients with pre-transplant hepatitis C virus positivity. J Heart Lung Transplant 2011; 30: 1266. 4. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: 975. 5. Fagiuoli S, Pevere S, Minniti F, et al. Natural leukocyte interferon alfa for the treatment of chronic viral hepatitis in heart transplant recipients. Transplantation 2003; 75: 982. 6. Kim EY, Ko HH, Yoshida EM. A concise review of hepatitis C in heart and lung transplantation. Can J Gastroenterol 2011; 25: 445. 7. Slaughter MS, Pagani FD, Rogers JG, et al. Clinical management of continuousflow left ventricular assist devices in advanced heart failure. J Heart Lung Transplant 2010; 20: S1.

The predominant anti-HLA IgG subclass during antibody mediated rejection episodes is IgG1. Class I DSA IgG subclasses respond to single cycle of bortezomib-based therapy more frequently as compared to Class II DSA IgG subclasses. High titer Class II DSA IgG subclasses are less likely to respond to single cycle bortezomib-based therapy. Future studies are needed to determine if the response to bortezomib-based therapy is dependent on the type of DSA and/or the strength of the DSA at the time of antibody mediated rejection episode.

Limited data exist on the effect of intravenous immunoglobulin (IVIg) on anti‐HLA antibodies as determined by solid‐phase assays. We reviewed our experience treating sensitized wait‐listed kidney transplant recipients with IVIg as a method for desensitization and report our results utilizing Luminex single antigen (LSA) bead assay to quantify antibody reactivity (MFI). Fifteen patients with a cPRA > 40% received 2 g/kg IVIg per month for four months or until transplanted. LSA testing was performed before and after IVIg. Median MFI for anti‐class I antibodies fell in 11 (73%) and increased in 4 (27%) patients after IVIg. Similar significant changes in MFI for anti‐class II antibodies were observed in 10 patients (66%). Administration of IVIg was associated with a modest decrease in reactivity to both class I and II HLA antigens (median MFI change 493 and 1110, respectively; p < 0.0001) but did not significantly alter mean cPRA (85% before IVIg vs. 80% after IVIg; p = 0.1). Our data suggest a smaller effect of IVIg on HLA antibody reactivity than previously described, leading us to question how best to measure the efficacy of a desensitization protocol in current practice.