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

European Renal Best Practice Guideline on kidney donor and recipient evaluation and perioperative care

The European Best Practice Guideline group (EBPG) issued guidelines on the evaluation and selection of kidney donor and kidney transplant candidates, as well as post-transplant recipient care, in the year 2000 and 2002. The new European Renal Best Practice board decided in 2009 that these guidelines needed updating. In order to avoid duplication of efforts with kidney disease improving global outcomes, which published in 2009 clinical practice guidelines on the post-transplant care of kidney transplant recipients, we did not address these issues in the present guidelines. The guideline was developed following a rigorous methodological approach: (i) identification of clinical questions, (ii) prioritization of questions, (iii) systematic literature review and critical appraisal of available evidence and (iv) formulation of recommendations and grading according to Grades of Recommendation Assessment, Development, and Evaluation (GRADE). The strength of each recommendation is rated 1 or 2, with 1 being a ‘We recommend' statement, and 2 being a ‘We suggest' statement. In addition, each statement is assigned an overall grade for the quality of evidence: A (high), B (moderate), C (low) or D (very low). The guideline makes recommendations for the evaluation of the kidney transplant candidate as well as the potential deceased and living donor, the immunological work-up of kidney donors and recipients and perioperative recipient care. All together, the work group issued 112 statements. There were 51 (45%) recommendations graded ‘1', 18 (16%) were graded ‘2' and 43 (38%) statements were not graded. There were 0 (0%) recommendations graded ‘1A', 15 (13%) were ‘1B', 19 (17%) ‘1C' and 17 (15%) ‘1D'. None (0%) were graded ‘2A', 1 (0.9%) was ‘2B', 8 (7%) were ‘2C' and 9 (8%) ‘2D'. Limitations of the evidence, especially the lack of definitive clinical outcome trials, are discussed and suggestions are provided for future research. We present here the complete recommendations about the evaluation of the kidney transplant candidate as well as the potential deceased and living donor, the immunological work-up of kidney donors and recipients and the perioperative recipient care. We hope that this document will help caregivers to improve the quality of care they deliver to patients. The full version with methods, rationale and references is published in Nephrol Dial Transplant (2013) 28: i1-i71; doi: 10.1093/ndt/gft218 and can be downloaded freely from http://www.oxfordjournals.org/our_journals/ndt/era_edta.htm

Identification of a unique intervillous cellular signature in chronic histiocytic intervillositis

Introduction: Chronic histiocytic intervillositis (CHI) is a rare histopathological lesion in the placenta characterized by an infiltrate of CD68+ cells in the intervillous space. CHI is associated with adverse pregnancy outcomes such as miscarriage, fetal growth restriction, and (late) intrauterine fetal death. The adverse pregnancy outcomes and a variable recurrence rate of 25-100% underline its clinical relevance. The pathophysiologic mechanism of CHI is unclear, but it appears to be immunologically driven. The aim of this study was to obtain a better understanding of the phenotype of the cellular infiltrate in CHI.Method: We used imaging mass cytometry to achieve in-depth visualization of the intervillous maternal immune cells and investigated their spatial orientation in situ in relation to the fetal syncytiotrophoblast.Results: We found three phenotypically distinct CD68+HLA-DR+CD38+ cell clusters that were unique for CHI. Additionally, syncytiotrophoblast cells in the vicinity of these CD68+HLA-DR+CD38+ cells showed decreased expression of the immunosuppressive enzyme CD39.Discussion: The current results provide novel insight into the phenotype of CD68+ cells in CHI. The identification of unique CD68+ cell clusters will allow more detailed analysis of their function and could result in novel therapeutic targets for CHI.Research into fetal development and medicin

Restricted specificity of peripheral alloreactive memory B cells in HLA-sensitized patients awaiting a kidney transplant

International audienceThe contribution of memory B cells in alloreactive humoralresponses remains poorly understood. Here we testedthe presence of circulating alloreactive memory B cells in69 patients with end-stage renal disease under renalreplacement therapy, using an in vitro memory Bcell–stimulation assay combined with identification of IgGhuman leukocyte antigen (HLA) antibodies in culturesupernatant. HLA antibody–producing memory B cells wereevidenced only in patients carrying serum HLA antibodiesfollowing multiple classical HLA-immunizing events. Inpatients with a previous renal allograft, alloreactive memoryB cells could be detected ranging from 6 to 32 years (mean13.2 years) after transplantation. HLA antibodies produced bymemory B cells were also detected in the corresponding seraand showed a restricted reactivity, targeting only a fewepitopes shared by several HLA antigens. In contrast, serumHLA antibodies, not associated with the detection of specificmemory B cells, showed a broader pattern of specificities.Thus, expansion and survival of alloreactive memory B cells isalloantigen driven, and their frequency is related to the‘strength’ of HLA immunization

Novel insights into non-HLA alloimmunity in kidney transplantation

International audienceRecognition of non‐self structures on donor cells represents the main immunological barrier in solid organ transplantation. The human leukocyte antigens (HLA) are considered the most important non‐self (allo)antigens in transplantation. Long‐term graft attrition is mainly caused by the formation of alloreactive antibodies that are directed against non‐self structures (i.e., epitopes) on cell surface proteins. Recently published data provided evidence for a similar importance of non‐HLA mismatches between donors and recipients in acute rejection as well as long‐term kidney allograft survival. These data suggest a broader concept of immunological non‐self that goes beyond HLA incompatibility and expands the current concept of polymorphic non‐self epitopes on cell surface molecules from HLA to non‐HLA targets. Amino acid substitutions caused by single nucleotide variants in protein‐coding genes or complete loss of gene expression represent the basis for polymorphic residues in both HLA and non‐HLA molecules. To better understand these novel insights in non‐HLA alloimmunity, we will first review basic principles of the alloimmune response with a focus on the HLA epitope concept in donor‐specific antibody formation before discussing key publications on non‐HLA antibodies

Broad-Spectrum Sunscreens Offer Protection Against Urocanic Acid Photoisomerization by Artificial Ultraviolet Radiation in Human Skin1

SummaryCis-urocanic acid (UCA) has been indicated as an important mediator of ultraviolet (UV)-induced immunosuppression. In this study we describe a rapid, noninvasive method for the determination of the protective capacity of various sunscreens against the UV-induced isomerization of trans-UCA into its cis form. For this purpose we applied sunscreens prior to in vivo exposure of human volunteers with single or repeated broadband UVB irradiations of 100 mJ per cm2. We found significant but different levels of protection against UCA photoisomerization by all sunscreens that correlated with the sun protection factor. A comparison of various sunscreens with a sun protection factor of 10, showed that the best protection was offered by the sunscreens (containing organic UV filters or TiO2) with broad absorption spectra. The ability to inhibit cis-UCA formation was not influenced by the penetration characteristics of sunscreens, as determined by application of the sunscreen on quartz glass that was placed on the skin, preventing penetration of sunscreen in the skin. In addition ex vivo UV exposure of human skin was employed to permit other tests of immunomodulation, in this case the mixed epidermal cell lymphocyte reaction. The advantage of this ex vivo method is that there is no need to take biopsies from volunteers. Ex vivo irradiation of human skin with a single dose of 200 mJ per cm2 resulted in similar protection by the sunscreens against cis-UCA formation as in the in vivo system. Furthermore, the mixed epidermal cell lymphocyte reaction data correlated with the cis-UCA findings. We conclude that UCA isomerization is an excellent method to determine sunscreen efficacy and that broad-spectrum sunscreens offer good immunoprotection

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