Detection of donor-specific HLA antibodies: A retrospective observation in 350 renal transplant cases

BackgroundThe main objective of this study was to determine the results of the cell-based assay (CDC-XM and FC-XM), and correlate with the results of solid phase assay (L-SAB).MethodsIn this retrospective study, 350 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, FC-XM and L-SAB screening with their corresponding donor.ResultsT-cell-FC-XM showed a sensitivity of 71.43% and a specificity of 91.50% for detecting class I L-SAB (+), while B-cell-FCXM showed a sensitivity of 94.94% and a specificity of 61.99% for detecting class II L-SAB (+). On the other hand, T-CDC-XM showed a sensitivity of 32.14% and a specificity of 98.64% for detecting class I L-SAB (+), while B-CDC-XM showed a sensitivity of 44.30% and a specificity of 94.83% for detecting class II L-SAB (+). In this study, the results indicated that DSA class I MFI value of 2845 and above significantly (p ≤0.001) correlated with T-cell-FC-XM positivity, while MFI value of 4585 and above (p ≤0.001) showed strong predictive accuracy of a positive T-cell-CDC-XM. However, DSA class II MFI cut-off of 1988 and above significantly (p ≤0.001) correlated with B-cell-FC-XM positivity, while MFI value of 5986 and above (p ≤0.001) showed strong predictive accuracy of a positive B-cell-CDC-XM.ConclusionsOur study showed that CDC-XM has poor sensitivity, while FC-XM has poor specificity to detect DSA. L-SAB has good correlation with T-cell-FC-XM (p < 0.0001) but not with B-cell-FC-XM (P = 0.31). DSA strength >2845 and > 1988 significantly correlated with T-cell-FC-XM positivity and B-cell-FC-XM positivity, respectively. While, a MFI value of >4585 and > 5986 significantly correlated with T-cell-CDC-XM positivity and B-cell-CDC-XM positivity, respectively. These MFI cut-off values could serve as a surrogate marker for CDC-XM and FC-XM tests and may help in resolving the limitations of cell-based techniques. In conclusion, we found that L-SAB is more sensitive and specific than CDC-XM and FC-XM and therefore may be used as a test of choice.     

Correlation of Luminex-Based Single Antigen Based Results With Complement-Dependent Cytotoxicity Crossmatch and Flow Cytometry Crossmatch Results: A Single-Center Experience From Istanbul

BackgroundThis study aimed to retrospectively investigate the correlation of mean Class I donor-specific antibody (DSA) intensity values detected in Luminex-based techniques with the results of complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FC-XM) results.MethodsA total of 335 patients with kidney failure and their living donors whose CDC-XM, FC-XM, and single antigen based (SAB) tests were studied between 2018 and 2020 for transplant preparation from living donor candidates were included in the study. Patients were divided into 4 groups according to their mean fluorescence intensity (MFI) values of SAB assay.ResultsAnti-HLA antibodies (class I and/or class II) were detected using SAB in 91.6% patients included in the study (MFI >1000). Class I DSA was positive in 34.8% of patients with anti-HLA antibodies. When CDC-XM and FC-XM results were evaluated in the 4 groups separated according to MFI values, 3 patients with DSA MFI <1000 had negative CDC-XM and T-B-FC-XM results. Of 32 patients with DSA-MFI between 1000 and 3000, 93.75% (n = 30) had T-B-FC-XM or CDC-XM-negative results, and 6.25% (n = 2) had B-FC-XM-positive results. The CDC-XM, T, and B-FC-XM were negative in all 17 patients with DSA-MFI between 3000 and 5000. Our results showed that MFI >5834 DSA values were significantly correlated with positive T-FC-XM (P < .001), and MFI >6016 values were significantly correlated with positive CDC-XM (P = .002). In addition, MFI values >5000 were associated with both CDC-XM and FC-XM in our study.ConclusionsThe MFI values >5000 correlated with both CDC-XM and FC-XM.     

The correlation of results of panel reactive antibody,identification, and single antigen beads in detection of anti-HLA antibodies: Istanbul Faculty of Medicine,tissue typing laboratory experience

BackgroundWe have performed a retrospective analysis of anti-HLA class I MHC and class II MHC antibodies measured using a single antigen bead (SAB) assay and a panel reactive antibody (PRA) assay.Material and methodsA group of 256 patients with end-stage renal disease (ESRD) was tested for anti-HLA antibodies in the tissue typing laboratory between 2017 and 2020. In the cohort, the serum samples of patients waiting for transplantation were tested. Both the PRA and SAB tests of these patients were analyzed using the Luminex (Immucor) method. The threshold of positivity was accepted as median fluorescence intensities (MFI) ≥1000 for PRA screening and MFI ≥750 for SAB screening.ResultsOverall, antibodies to HLA antigens were detected in 202 (78.9%) out of 256 patients in the PRA study. Antibodies against both class I/II antigens were detected only in 15.6% of these patients, whereas antibodies against only against class I HLA in 31.3% and only against class II HLA in 32.0%. By comparison, the SAB study found that 66.8% of patients were positive for HLA antigens. Furthermore, donor-specific antibodies (DSA) were detected in 52.0% of PRA-positive patients and 52.6% of SAB-positive patients. It was shown that 168 patients (83.2%) out of 202 PRA-positive patients were found to be SAB-positive. In addition, 51 patients negative in the SAB assay (94.4%) were also negative in the PRA assay. Statistical analysis established a significant correlation between the PRA and SAB positivity (p > 0.001).It was also shown that MFI ≥3000 PRA positivity for class I HLA antigens (p = 0.049) and MFI ≥5000 PRA positivity for class II antigens (p < 0.001) correlated with the SAB positivity in patients.ConclusionOur results showed the importance of both PRA and SAB assays to define the status of sensitization in patients.     

Influence of pretransplant class I and II non-donor-specific anti-HLA immunization on immunologic outcome and graft survival in kidney transplant recipients

BackgroundAnti-HLA immunization determined by Panel Reactive Antibody (PRA) is known to have a negative impact on patient and graft survival. The predictive value of peak PRA (pPRA) on immunologic outcome, however, and the individual effects of anti-HLA class I and II antibodies remain uncertain.MethodsThe influence of HLA immunization on immunologic outcome parameters and graft survival was investigated in 1150 adult patients without pretransplant donor-specific antibodies (DSA) and in a subgroup of elderly kidney recipients aged ≥ 65 (n = 264). Anti-HLA immunization was defined as a pPRA > 0%. We investigated the influence of class I and II pPRA by dividing all kidney recipients into four pPRA groups (0%, 1–20%, 21–80%, >80%).ResultsPatients with non-donor-specific pretransplant anti-HLA immunization were at a higher risk for developing de novo DSA (49.9% vs. 18.7% p < 0.001), antibody mediated rejections (ABMR) (15.7% vs. 5.1%; p < 0.001), had a poorer death censored graft survival (69.2% vs. 86.2%; p < 0.001) and a higher decline of the calculated GFR. In elderly patients anti-HLA immunization only had a significant influence on the development of DSA (40.5% vs. 27.4%; p = 0.004). A multivariate model adjusted for all relevant factors revealed only class I but not class II pretransplant HLA immunization as a significant independent risk factor for de novo DSA, ABMR and death censored graft loss (HR 2.76, p < 0.001, HR 4.16, p < 0.001 and HR 2.07, p < 0.001, respectively).ConclusionMainly non-donor-specific pretransplant HLA class I immunization is an independent risk factor for the development of de novo DSA, ABMR and graft loss.     

Effects of different sensitization events on HLA alloimmunization in solid organ transplantation patients

Objective

HLA alloimmunization is caused by various sensitization events, such as transfusion, pregnancy, or organ transplantation. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using an identical test method. We evaluated how different sensitization events affect the panel-reactive antibody (PRA) status in solid organ transplantation candidates.

Methods

PRA identification tests were performed on 674 patients (354 males and 320 females) using Luminex assay kits (LIFECODES, Gen-Probe, Stamford, CT, United States). PRA-positive rates (HLA-A, B, or DR antibodies of median fluorescence intensity [MFI] values of ≥1000) and antibody strengths in PRA-positive cases were analyzed according to the different sensitization events and gender.

Results

PRA (class I and/or II)-positive rates were significantly higher in patients with transfusion (33.0%; P = .001), pregnancy (71.4%; P < .001), or transplantation events (76.9%; P < .001) than in controls without any identifiable sensitization events (5.6%). Transplantation had the strongest immunization effect, especially for class II HLA antigens. Female compared with male patients (60.3% vs 34.2%; P < .001) and retransplantation compared with first transplantation candidates of kidney transplantation (80.2% vs 41.1%; P < .001) showed a significantly higher PRA-positive rate. Retransplantation candidates (MFI 14,164) showed significantly stronger antibody strength than first transplantation candidates (MFI 5456) and those with single sensitization events of transfusion (MFI 4185) or pregnancy (MFI 5548; P < .001 for each).

Conclusion

Solid organ transplantation appears to have the strongest HLA alloimmunization effect followed by pregnancy and transfusion, especially for class II HLA antigens.     

Pre-transplant donor specific anti-HLA antibody is associated with antibody-mediated rejection,progressive graft dysfunction and patient death

BackgroundThe long term effect of donor specific antibodies (DSA) detected by Luminex Single Antigen Bead (SAB) assay in the absence of a positive complement-dependant cytotoxicity (CDC) crossmatch is unclear.DSA at the time of transplant were determined retrospectively in 258 renal transplant recipients from 2003 to 2007 and their relationship with rejection and graft function prospectively evaluated.After a median of 5.6 years follow-up 9% of patients had antibody mediated rejection (AMR) (DSA 11/37 (30%), DSA-Neg 13/221 (6%), HR 6.6, p < 0.001). Patients with anti-HLA class II (HR 6.1) or both class I + II (HR 10.1) DSA had the greatest risk for AMR. The Mean Fluorescent Intensity (MFI) of the DSA was significantly higher in patients with AMR than those with no rejection (p = 0.006). Moreover, the strength of the antibody was shown to be important, with the risk of AMR significantly greater in those with DSA > 8000 MFI than those with DSA < 8000 MFI (HR 23, p < 0.001).eGFR progressively declined in patients with DSA but was stable in those without DSA (35.7 ± 20.4 mls/min vs 48.5 ± 22.7) and composite patient and graft survival was significantly worse in those with class II (HR 2.9) or both class I + II (HR 3.7) but not class I DSA. Class II DSA alone, or in combination with class I DSA had the strongest association with graft loss and patient death.Patients with DSA not only have increased rates of acute AMR, but also chronic graft dysfunction, graft loss and death. Antibody burden quantified by SAB assay may identify patients at highest immunological risk and therefore influence patient management and improve long-term patient outcome.     

Novel Flow Cytometry-Based Method for Detection of Anti-HLA Complement Activating Donor-Specific Antibodies in Renal Transplant Recipients and Its Comparison With Other Conventional Detection Methods

BackgroundPresence of preformed donor specific antibodies (DSAs) detected by complement-dependent cytotoxicity (CDC-XM) is a strong contraindication for transplant. However, it has limitations including its sensitivity and its inability to distinguish between HLA-specific and other non-HLA-specific antibodies. In this study, we standardized CDC-XM by flow cytometry and determined its relevance by comparing its results with other methods of DSA detection, such as routine CDC-XM, antibody binding assay by flow cytometry (FC-XM), and Luminex-based crossmatch assays, such as Luminex crossmatch (LXM) and virtual crossmatch (VXM).Materials and MethodsA total of 79 serum samples were tested for DSAs by the flow cytometric complement-dependent cytotoxicity crossmatch assay (FC-CDC-XM) and then the results of FC-CDC-XM were compared with other detection methods such as CDC-XM, FC-XM, LXM, and VXM.ResultsWe found that the FC-CDC-XM assay is more sensitive than routine CDC-XM. Out of total 79 sera, 24 sera were detected positive (T cells positive: 1 case and B cells positive: 23) by FC-CDC-XM as compared with 3 sera using CDC-XM; these 3 sera also showed positivity by FC-CDC-XM. After FC-XM assay, 23 samples were positive by FC-XM and out of these 23 samples, 13 were also positive by FC-CDC-XM. On comparing the FC-CDC-XM results with VXM and LXM, 10 sera of 24 FC-CDC-XM positive had HLA class II antibodies detected on a Luminex platform.ConclusionsThe FC-CDC-XM is a more sensitive and specific method for detection of HLA-specific complement-fixing antibodies than CDC-XM and FC-XM. FC-CDC-XM should be used in tissue-typing laboratories after intra- and inter- laboratory validation.     

Impact of pretransplant donor‐specific antibodies on kidney allograft recipients with negative flow cytometry cross‐matches

The Luminex test can detect low levels of donor‐specific antibody (DSA) that cannot be detected by flow‐cytometric cross‐matching (FCXM) in kidney transplantation (KT). This study evaluated the impact of DSA on clinical outcomes in KT recipients negative on FCXM. Of 575 consecutive patients who underwent living donor KT between January 2013 and July 2016, 494 (85.9%) were DSA‐negative and 81 (14.1%) were DSA‐positive. Although rates of acute cellular rejection (ACR) at 1 year were similar in the 2 groups (P = .54), the incidence of antibody‐mediated rejection (ABMR) was significantly higher in the DSA‐positive group (P < .01). There was no statistically significant association between rejection‐free graft survival (RFGS) rates and pretransplant class I DSA. However, evaluation of pretransplant class II DSA showed that RFGS rates were significantly lower in patients with mean fluorescence intensity (MFI) >3000 than in patients with DSA‐negative (P < .01). On multivariate analyses, class II DSA MFI ≥5000 was a significant risk factor for acute rejection (hazard ratio, 7.48; P < .01). These findings suggested that pretransplant DSA alone did not affect graft survival in KT recipients without desensitization. However, class II DSA MFI >5000 was an independent predictor of acute rejection in DSA‐positive patients.     

Improved detection of donor-specific HLA-class II antibody in kidney transplant recipients by modified immunocomplex capture fluorescence analysis

Immunocomplex capture fluorescence analysis (ICFA) which basic principle is same as Luminex crossmatch (LXM), could detect donor-specific HLA antibody (DSA). The advantages of ICFA are (i) detection of DSA and (ii) no requirement of viable cells over the flow cytometry crossmatch (FCXM). However, FCXM has been widely used because of its higher sensitivity than ICFA, in particular HLA-class II antibody detection. In this study the accuracy of DSA detection against HLA-class II was investigated by modifying the original method of ICFA. Increment of the sensitivity was found when purified peripheral blood mononuclear cells (PBMCs) were used instead of whole blood. An ICFA-PBMC in addition to FCXM-T/B was conducted for 118 patients before kidney transplantation and 13 patients with de novo DSA against HLA-class II after transplantation. Significantly positive correlation was observed between the values of ICFA-PBMC and DSA mean fluorescence intensity (MFI) targeting class II (p < 0.0001). When the cutoff level of 1.4 was determined by receiver operating characteristic curve analysis, the average DSA MFI was found to be significantly higher in the ICFA-PBMC (class II) positive group comparing to that in the negative group (12,217 vs 3885, p = 0.0027). ICFA-PBMC and optimized cutoff level could provide valid information in cases of suspected DSA.     

Human leukocyte antigens antibodies after lung transplantation: Primary results of the HALT study

Donor‐specific antibodies (DSA) to mismatched human leukocyte antigens (HLA) are associated with worse outcomes after lung transplantation. To determine the incidence and characteristics of DSA early after lung transplantation, we conducted a prospective multicenter observational study that used standardized treatment and testing protocols. Among 119 transplant recipients, 43 (36%) developed DSA: 6 (14%) developed DSA only to class I HLA, 23 (53%) developed DSA only to class II HLA, and 14 (33%) developed DSA to both class I and class II HLA. The median DSA mean fluorescence intensity (MFI) was 3197. We identified a significant association between the Lung Allocation Score and the development of DSA (HR = 1.02, 95% CI: 1.001‐1.03, P = .047) and a significant association between DSA with an MFI ≥ 3000 and acute cellular rejection (ACR) grade ≥ A2 (HR = 2.11, 95% CI: 1.04‐4.27, P = .039). However, we did not detect an association between DSA and survival. We conclude that DSA occur frequently early after lung transplantation, and most target class II HLA. DSA with an MFI ≥ 3000 have a significant association with ACR. Extended follow‐up is necessary to determine the impact of DSA on other important outcomes.     

Flow cytometry crossmatching to investigate kidney-biopsy-proven,antibody-mediated rejection in patients who develop de novo donor-specific antibodies

IntroductionThe appearance of de novo donor-specific anti-human leukocyte antigen antibodies (dnDSAs) after kidney transplantation is independently associated with poor long-term allograft outcomes. The objective of the present study was to evaluate the predictive value of a flow cytometry crossmatching (FC-XM) assay after the appearance of dnDSAs related to antibody-mediated allograft rejection (ABMR) after kidney transplantation.Materials and methodsA total of 89 recipients with dnDSAs after transplantation were included. The crossmatching results were compared with the dnDSA profile (the mean fluorescence intensity (MFI), the complement-binding activity, and the IgG subclass profile) and the biopsy's morphological features.ResultsOf the 89 patients, 59 (66%) were positive in an FC-XM assay, 17 (19%) had complement-binding DSAs, 55 (62%) were positive for IgG1 and/or IgG3 in a solid phase assay, and 45 (51%) had morphological biopsy features linked to ABMR.ConclusionAn FC-XM assay was unable to discriminate between cases with or without ABMR on biopsy findings; it had a low positive predictive value (<70%) and a low negative positive predictive value (<42.9%), taking into account the sensitivity of our assay (limit of detection: DSAs with an MFI >3000). In this context, the height of the MFI of the dnDSAs might be enough for a high positive predictive value for ABMR and additional testing for complement binding activity can remain optional.     

Association Between HLA Antibodies and Different Sensitization Events in Renal Transplant Candidates

Background

Human leukocyte antigen (HLA) allo-immunization is caused by various events such as blood transfusions, pregnancies, or organ transplantations, which can lead to sensitization. In this retrospective study, we evaluated different sensitization models and their effects on panel-reactive antibody (PRA) profiles of renal transplantation candidates.

Ibrutinib suppresses alloantibody responses in a mouse model of allosensitization

BackgroundIbrutinib is a Bruton's tyrosine Kinase (BTK) antagonist that inhibits B cell receptor (BCR) signaling. Complete BTK deficiency is associated with absence of B-cells. Ibrutinb is currently approved by FDA for treatment of B-cell malignancies, including Waldenström macroglobulinaemia. We recently carried out studies to determine if ibrutinib could modify alloantibody responses.Materials and methodsA mouse model of allogenic sensitization using a C57BL/6 mouse as the recipient of a skin allograft from an HLA-A2 transgenic mouse was utilized to examine the effects of ibrutinib on alloantibody responses and B cell effector functions. Donor-specific antibody (DSA) levels were measured in a flow-cytometric antibody binding assay. Splenic T and B cell subsets and plasma cells were analyzed in flow cytometry.ResultsControl mice developed peak levels of DSA IgM at day 14 PTx while the ibrutinib treated mice had significantly lower levels of DSA IgM (p = 0.0047). Control mice developed HLA.A2-specific IgG antibodies at day 14 (230 ± 60 MFI) and reached peak levels at day 21 (426 ± 61 MFI). In contrast, mice in the treatment group had low levels of HLA.A2-specific IgG at day 14 (109 ± 59 MFI, p = 0.004) and day 21 (241 ± 86 MFI, p = 0.003). FACS analysis found a reduction of B220⁺ or CD19⁺ B cell population (p < 0.05). In addition, ibrutinib attenuated recall DSA IgG responses to re-sensitization (p < 0.05) and reduced CD38⁺ CD138⁺ plasma cells (p < 0.05) in the spleens.ConclusionsIbrutinib is effective in suppressing alloantibody responses through blocking BTK-mediated BCR signaling, leading to reduction of B cells and short-lived plasma cells in the spleens. Use of ibrutinib may provide benefits to HLA-sensitized transplant patients for alloantibody suppression.     

Safety and Efficacy of a Steroid Avoidance Immunosuppression Regimen in Renal Transplant Patients With De Novo or Preformed Donor-Specific Antibodies: A Single-Center Study

Although interest in the role of donor-specific antibodies (DSAs) in kidney transplant rejection, graft survival, and histopathological outcomes is increasing, their impact on steroid avoidance or minimization in renal transplant populations is poorly understood. Primary outcomes of graft survival, rejection, and histopathological findings were assessed in 188 patients who received transplants between 2012 and 2015 at the Scripps Center for Organ Transplantation, which follows a steroid avoidance protocol. Analyses were performed using data from the United Network for Organ Sharing. Cohorts included kidney transplant recipients with de novo DSAs (dnDSAs; n = 27), preformed DSAs (pfDSAs; n = 15), and no DSAs (nDSAs; n = 146). Median time to dnDSA development (classes I and II) was shorter (102 days) than in previous studies. Rejection of any type was associated with DSAs to class I HLA (P < .05) and class II HLA (P < .01) but not with graft loss. Although mean fluorescence intensity (MFI) independently showed no association with rejection, an MFI >5000 showed a trend toward more antibody-mediated rejection (P < .06), though graft loss was not independently associated. Banff chronic allograft nephropathy scores and a modified chronic injury score were increased in the dnDSA cohort at 6 months, but not at 2 years (P < .001 and P < .08, respectively). Our data suggest that dnDSAs and pfDSAs impact short-term rejection rates but do not negatively impact graft survival or histopathological outcomes at 2 years. Periodic protocol post-transplant DSA monitoring may preemptively identify patients who develop dnDSAs who are at a higher risk for rejection.     

Clinical Significance of C3d Assay in Kidney Transplant Recipients With Donor-Specific Anti–Human Leukocyte Antigen Antibodies

BackgroundAntibody-mediated rejection (AMR) is a major cause of allograft loss in kidney transplant. Although donor-specific anti–human leukocyte antigen antibody (DSA) is a key cause of AMR, not all patients with DSA are diagnosed as having AMR and show poor allograft outcomes. This study aimed to evaluate clinical significance of C3d-binding activity in patients with DSA identified by single-antigen bead (SAB) assay.MethodsA total of 168 recipients screened for DSA from 2015 to 2018 were enrolled. Among them, 52 patients had DSA confirmed by SAB assay. Sera were tested using the C3d assay on Luminex platform. AMR was defined by kidney allograft biopsy results using Banff 2015 criteria.ResultsOf 52 patients, C3d-binding DSAs were detected in 22 patients (42.3%). Indication allograft biopsy was performed in 35 patients, with 31 (88.6%) diagnosed as having AMR. Patients with C3d-binding DSA had more class II SAB-DSA (73.3% vs 100%, P = .015) and showed significantly higher mean (SD) fluorescence intensity of class II SAB-DSA than the C3d-binding DSA(?) group (9606.7 [6096.6] vs 1921.0 [1483.8], P < .001). There was a positive correlation in the highest mean fluorescence intensity between class II SAB-DSA and class II C3d-binding DSA (r = 0.70, P < .001). Patients with C3d-binding DSA showed worse death-censored graft survival than those with non-C3d-binding DSA (P = .023).ConclusionsThis study showed that presence of C3d-binding DSA was significantly associated with allograft loss in SAB-DSA–positive patients. Further trials are warranted.     

Clinical impact of preformed donor‐specific denatured class I HLA antibodies after kidney transplantation

Class I single‐antigen flow beads (SAFB) carry native and denatured human leukocyte antigen (HLA) molecules. Using a cohort of 179 class I HLA‐sensitized kidney recipients, we described incidence and clinical relevance of preformed denatured HLA donor‐specific antibodies (DSA) using two different assays: an acid‐treated SAFB assay (anti‐dHLA DSA) and the iBeads assays (SAFB+/iBeads‐ DSA). Eighty‐five class I DSA were found in 67 patients (median mean fluorescence intensity [MFI] of 1729 [range 520–13 882]). Anti‐dHLA and SAFB+/iBeads‐ DSA represented 11% and 18% of class I DSA and were mainly low MFI DSA (500–1000 MFI). Concordance between these two assays was good (90%). None of the patients with only class I anti‐dHLA DSA or only SAFB+/iBeads‐ DSA developed acute clinical antibody‐mediated rejection in the first‐year post‐transplantation, and their five‐yr death‐censored graft survival was similar to that of patients without DSA. Moreover, all these patients displayed a negative current T‐cell flow cytometry cross‐match. Therefore, both anti‐dHLA DSA and SAFB+/iBeads‐ DSA appear irrelevant, which could explain the good outcome observed in some patients with preformed class I DSA.     

Frequency of Human Leukocyte Antigens and Donor Specific Antibodies in Long-Term Living Donor Kidney Transplantation

PurposeHLA antibodies have been shown to be associated with late graft loss. In this study, we defined the incidence and profiles of anti-HLA antibodies and their impact on graft outcome in long-term kidney recipients.MethodsThe sera of 118 kidney transplant recipients were screened for anti-HLA antibody presence. The antigen specificity of the detected HLA class I and class II antibodies was identified using a Luminex assay (Luminex Corp, Austin, TX, United States). Presence of donor specific antibodies (DSA) was examined in individuals with anti-HLA antibodies using the Luminex method.ResultsAnti-HLA class I and/or class II antibodies were detected in serum of 16.1% of the kidney transplant patients. The antibodies were directed against HLA class I antigens in 4 patients (21.1%), HLA class II antigens in 9 patients (47.4%), and both class I and class II antigens in 6 patients (31.6%). The overall prevalence of DSA was 10.2%. Anti-HLA antibodies were significantly associated with higher rate of cyclosporine use. Presence of DSA was associated with a lower rate of tacrolimus use, a higher rate of cyclosporine use, and lower donor age. Presence of anti-HLA antibodies was associated with higher acute cellular rejection and higher chronic active humoral rejection rates. Presence of DSA was associated with chronic active humoral rejection.ConclusionThe presence of either HLA antibodies or DSA significantly correlated with lower graft survival, poor transplant function, and proteinuria.     

Deleterious impact of C3d-binding donor-specific anti-HLA antibodies after pediatric liver transplantation

BackgroundThe prevalence and clinical impact of anti-HLA donor-specific antibodies (DSA) after liver transplantation (LT) have not been extensively studied, especially in pediatric population.MethodsThe present cross-sectional study included 100 patients who underwent a first LT in childhood. Anti HLA immunization study was performed at a single time point during routine follow-up using Luminex® single antigen tests with classical anti-IgG conjugate and anti-C3d conjugate.ResultsThe main indication for LT was biliary atresia (52%) and median age at LT was 4.6 years. The median time between LT and DSA assessment was 7.8 years (range 1–21 years). DSA was identified in twenty-four patients (24%) after LT, with a prevalence of 8%, 28%, 33%, 50%, respectively 0–5 years, 5–10 years, 10–15 years and > 15 years after LT. DSA were mainly class II (23/24) with a mean MFI of 9.731 ± 5.489 and 18 (79.3%) were C3d-binding DSA. Multivariate analysis disclosed that time elapsed since LT (p < 0.01) and history of fulminant hepatitis (p = 0.04) were significantly associated with a higher rate of DSA. Liver function tests (at time of DSA assessment) were not different according to the presence or not of DSA (or C3d-binding DSA). Regarding histology, the DSA group had a higher rate of chronic rejection, cirrhosis and centrilobular fibrosis or cirrhosis. In addition, patients with C3d-binding DSA and high MFI (> 10,000) had a significant poorer long-term graft survival (p = 0.03).ConclusionIn our pediatric cohort of LT, prevalence of DSA was high and increased regularly with time. Presence of C3d positive-DSA with high MFI was associated with a higher rate of graft loss.     

Prospective examination of HLA sensitization after VAD implantation in children and adults

BackgroundVentricular assist devices (VADs) have improved survival to heart transplantation (HTx). However, VADs have been associated with development of antibodies against human leukocyte antigen (HLA-Ab) which may limit the donor pool and decrease survival post-HTx. Since HLA-Ab development after VAD insertion is poorly understood, the purpose of this prospective single-center study was to quantify the incidence of and evaluate risk factors for HLA-Ab development across the age spectrum following VAD implantation.MethodsAdult and pediatric patients undergoing VAD placement as bridge to transplant or transplant candidacy between 5/2016 and 7/2020 were enrolled. HLA-Ab were assessed pre-VAD and at 1-, 3-, and 12-months post-implant. Factors associated with HLA-Ab development post-VAD implant were explored using univariate and multivariate logistic regression.Results15/41 (37%) adults and 7/17 (41%) children developed new HLA-Ab post-VAD. The majority of patients (19/22) developed HLA-Ab within two months of implant. New class I HLA-Ab were more common (87% adult, 86% pediatric). Prior pregnancy was strongly associated with HLA-Ab development in adults post-VAD (HR 16.7, 95% CI 1.8–158, p = 0.01). Of the patients who developed new HLA-Ab post-VAD, in 45% (10/22) the HLA-Ab resolved while in 55% (12/22) the HLA-Ab persisted.ConclusionMore than one-third of adult and pediatric VAD patients developed new HLA-Ab early after VAD implant with the majority having class I antibodies. Prior pregnancy was strongly associated with post-VAD HLA-Ab development. Further studies are needed to predict regression or persistence of HLA-Ab developed post-VAD, to understand modulation of individuals' immune responses to sensitizing events, and to determine whether transiently detected HLA-Ab post-VAD recur and have long-term clinical impact post-heart transplantation.