A modeling approach for mean fluorescence intensity value harmonization and cutoff prediction for luminex single antigen bead assays of two different vendors

Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non-linear modeling approach to accurately convert MFI values between two vendors and to establish user-independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA-treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non-linear hyperbola model on raw MFI corrected by locus-specific highest self MFI subtraction yielded the highest correlation (class I r2: 0.946, class II r2: 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r2: 0.952, class II r2: 0.911). Furthermore, in an independent cohort of post-transplant serum samples (n = 11) using the vendor-specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead-specific reactivity assignments by the two vendors. We recommend using the non-linear hyperbola modeling approach with self HLA correction and locus-specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended.</p

A modeling approach for mean fluorescence intensity value harmonization and cutoff prediction for luminex single antigen bead assays of two different vendors

Luminex single antigen bead (SAB) kits from One Lambda (OL) and Lifecodes (LC) are widely used for HLA antibody detection but have substantial differences in design and assay protocol resulting in different mean fluorescence intensity (MFI) values. Here, we present a non-linear modeling approach to accurately convert MFI values between two vendors and to establish user-independent MFI cutoffs when analyzing big datasets. HLA antibody data from a total of 47 EDTA-treated sera tested using both OL and LC SAB kits were analyzed. MFI comparisons were made for the common 84 HLA class I and 63 class II beads. In the exploration set (n = 24), a non-linear hyperbola model on raw MFI corrected by locus-specific highest self MFI subtraction yielded the highest correlation (class I r2: 0.946, class II r2: 0.898). Performance of the model was verified in an independent validation set (n = 12) (class I r2: 0.952, class II r2: 0.911). Furthermore, in an independent cohort of post-transplant serum samples (n = 11) using the vendor-specific MFI cutoffs dictated by the current model, we found 94% accuracy in bead-specific reactivity assignments by the two vendors. We recommend using the non-linear hyperbola modeling approach with self HLA correction and locus-specific analyzes to harmonize MFI values between two vendors in particular research datasets. As there are considerable variations between the two assays, using MFI conversion for individual patient samples is not recommended.</p

Anti-HLA Class II Antibodies Are the Most Resistant to Desensitization in Crossmatch-positive Living-donor Kidney Transplantations:A Patient Series

Background. In HLA-incompatible kidney transplantation, the efficacy of desensitization in terms of anti-HLA antibody kinetics is not well characterized. We present an overview of the course of anti-HLA antibodies throughout plasma exchange (PE) desensitization in a series of crossmatch-positive patients. Methods. All consecutive candidates in the Dutch HLA-incompatible kidney transplantation program between November 2012 and January 2022 were included. The eligibility criteria were a positive crossmatch with a living kidney donor and no options for compatible transplantation. Desensitization consisted of 5-10 PE with low-dose IVIg. Results. A total of 16 patient-donor pairs were included. Patients had median virtual panel-reactive antibody of 99.58%. Cumulative donor-specific anti-HLA antibody (cumDSA) mean fluorescence intensity (MFI) was 31 399 median, and immunodominant DSA (iDSA) MFI was 18 677 for class I and 21 893 for class II. Median anti-HLA antibody MFI response to desensitization was worse in class II as compared with class I (P &lt; 0.001), particularly for HLA-DQ. Class I cumDSA MFI decreased 68% after 4 PE versus 53% in class II. The decrease between the fifth and the 10th PE sessions was modest with 21% in class I versus 9% in class II. Antibody-mediated rejection occurred in 85% of patients, with the iDSA directed to the same mismatched HLA as before desensitization, except for 3 patients, of whom 2 had vigorous rebound of antibodies to repeated mismatches (RMMs). Rebound was highest (86%) in RMM-DSA with prior grafts removed (transplantectomy n = 7), lower (39%) in non-RMM-DSA (n = 30), and lowest (11%) for RMM-DSA with in situ grafts (n = 5; P = 0.018 for RMM-DSA transplantectomy versus RMM-DSA graft in situ). With a median follow-up of 59 mo, 1 patient had died resulting in a death-censored graft survival of 73%. Conclusions. Patients with class II DSA, and particularly those directed against HLA-DQ locus, were difficult to desensitize.</p

Memory B-cell derived donor-specific antibodies do not predict outcome in sensitized kidney transplant recipients: a retrospective single-center study

Background: Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). Methods: We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. Results: At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan–Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). Conclusion: This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs

The genetic architecture of membranous nephropathy and its potential to improve non-invasive diagnosis

Membranous Nephropathy (MN) is a rare autoimmune cause of kidney failure. Here we report a genome-wide association study (GWAS) for primary MN in 3,782 cases and 9,038 controls of East Asian and European ancestries. We discover two previously unreported loci, NFKB1 (rs230540, OR = 1.25, P = 3.4 × 10-12) and IRF4 (rs9405192, OR = 1.29, P = 1.4 × 10-14), fine-map the PLA2R1 locus (rs17831251, OR = 2.25, P = 4.7 × 10-103) and report ancestry-specific effects of three classical HLA alleles: DRB1*1501 in East Asians (OR = 3.81, P = 2.0 × 10-49), DQA1*0501 in Europeans (OR = 2.88, P = 5.7 × 10-93), and DRB1*0301 in both ethnicities (OR = 3.50, P = 9.2 × 10-23 and OR = 3.39, P = 5.2 × 10-82, respectively). GWAS loci explain 32% of disease risk in East Asians and 25% in Europeans, and correctly re-classify 20-37% of the cases in validation cohorts that are antibody-negative by the serum anti-PLA2R ELISA diagnostic test. Our findings highlight an unusual genetic architecture of MN, with four loci and their interactions accounting for nearly one-third of the disease risk

Natural and anthropogenic radionuclides in the granitic region of Kapidag peninsula, Western Anatolia, Turkey

Naturally occurring radionuclides of terrestrial origin (also called primordial radionuclides) are present in various degrees in all media in the environment. This study represents the reports on the natural and anthropogenic radionuclides in the Kapidag granitic region. For this purpose, activities of radionuclides in soil, beach sands and rocks of the region have been investigated to assess the radiological hazard of the natural radioactivity. The radium equivalent activities, the absorbed dose rates and the external hazard indexes have been calculated, and also in situ gamma dose rates have been measured in the region. The mean activities of U-238, Th-232 and K-40 with the ranges were determined as 31.115.7 (12.171.9), 42.515.9 (19.794.9), 590.3192.2 (184.7892.5), in the soil, as 16.59.5 (4.940.8), 67.1106.9 (18.5433.0), 569.2212.6 (162.0821.1) in the sand and as 25.412.8 (4.850.7), 37.821.5 (4.596.7), and 592.4285.5 (62.41121.6) Bq kg(1) in the rocks, respectively. It was also observed that the average activities of Cs-137 were ranged 027.8 Bq kg(1) in the soil and 0.63.8 Bq kg(1) in the beach sands. The mean Ra-eq activities of the rocks, sands and soil were found to be 125.159.5, 156.3157.2 and 137.348.8 Bq kg(1), respectively, lower than the recommended maximum value of 370 Bq kg(1) with some exceptions. The maximum contributors to the total absorbed gamma dose rates in air were determined as U-238 (45 ) for the beach sands, U-238 (40 ) for the soil and K-40 (41 ) for rocks. The average outdoor gamma dose rates for the soil due to terrestrial and cosmic radiations were found to be 64.622.7 and 47.19.6 nGy h(1), respectively, with the total of 111.729.5 nGy h(1) outdoor gamma exposure rate and the annual average effective outdoor gamma dose was calculated as 13736.2 Sv for the region. The results of the study were discussed with similar studies in close regions and the worldwide averages

IL-10 and TNF-alpha Gene Polymorphisms in Patients with Celiac Disease

Celiac disease (CD) is an autoimmune disorder characterized by intolerance to ingested gluten. HLA-DQ genes are strongly associated with susceptibility to CD. Non-HLA genes are effective in CD pathogenesis as well as HLA genes. TNF-alpha has a single nucleotidepolymorphism (SNP) at position -308 in promoter region, which has been shown to have association with CD in previous studies. The aim of this study is to investigate the association of TNF-alpha and IL-10 cytokine gene polymorphisms with CD and with DQB1*02 status in patients with celiac disease. Thirty three patients and 93 healthy individuals were included in the present study. GG and AA genotypes in position -308 of TNF-alpha gene had a significantly increased frequency in the patient group and in patients with DQB1*02 when compared to the controls. No significant differences could be established for IL-10 gene polymorphisms within the patients and controls. As a result of all these findings, it might be suggested that there is no significant association of IL-10 gene polymorphism with CD and data on TNF-alpha gene polymorphisms are not sufficient enough to clarify the disease pathogenesis thus indicating roles for other genes within or out of MHC gene region

Donor-specific B Cell Memory in Alloimmunized Kidney Transplant Recipients: First Clinical Application of a Novel Method

Background: HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen re-exposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell-derived HLA antibodies (DSA-M) in renal allograft recipients with pre-transplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (ABMR) using a recently developed method. Methods: Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells (PBMC) were collected at 3 timepoints (pre-transplant, month 6, month 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated PBMC using SAB assays and compared HLA antibody profiles with same day plasma results. Results: Plasma SAB analysis revealed 35 DSA in 20 patients pre-transplant. DSA-M were detected in 9/20 (45%) patients and for 10/35 specificities (29%). While median mean fluorescence intensity (MFI) values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3/6 patients with ABMR and low MFI DSA (<3000) had DSA-M. Overall, pre-transplant DSA/DSA-M pos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical ABMR (p=0.032) and a higher extent (g≥1+ptc≥1) of microvascular inflammation (67% versus 9%, p=0.02). In 17 patients (28 DSA) with post-transplant analyses, persisting DSA post-transplant had more often DSA-M (6/12; 50%) than non-persisting DSA (2/16; 13%). Conclusion: Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pre-transplant risk stratification in patients with DSA