Günther F. Körmöczi

Anti‐D immunization by DEL red blood cells

BACKGROUND: No data are available on the immunogenicity of extremely weak D variants called DEL. Evaluation of alloanti‐D formation in a D– female patient after transfusion of apparently D– blood from an Austrian donor led to discovery of a so far unknown DEL type.

Presence of RHD in serologically D–, C/E+ individuals: a European multicenter study

BACKGROUND: RHD blood group alleles with reduced or absent antigen expression are a clinically significant and heterogeneous group.

A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization

BACKGROUND: The D antigen of the polymorphic Rh blood group system is of particular clinical importance regarding transfusion‐ and pregnancy‐induced alloimmunization. Different RhD variants with specific clinical implications have been characterized. The least expressed D variants collectively called DEL are serologically detectable only by adsorption‐elution techniques, with so far only poorly defined antigenic properties.

Flow cytometry based detection of HLA alloantibody mediated classical complement activation

Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment-IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 degrees C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 degrees C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.

Pivotal Role of Complement-Fixing HLA Alloantibodies in Presensitized Kidney Allograft Recipients

Recipient presensitization represents a major risk factor for kidney allograft loss. Complement fixation may be a critical attribute of deleterious alloantibodies. We investigated clinical impact of complement‐fixing HLA presensitization employing [C4d]FlowPRA, a novel assay permitting selective detection of HLA panel reactive antibody (PRA)‐triggered C4 complement split product deposition. A cohort of 338 kidney transplants was evaluated for presensitization applying [C4d]FlowPRA together with [IgG]FlowPRA and complement‐dependent cytotoxicity (CDC)‐PRA. Analysis of HLA class I alloreactivities revealed a high incidence of C4d‐positive graft dysfunction in [IgG]FlowPRA(+)/[C4d]FlowPRA(+) and [IgG]FlowPRA(+)/[C4d]FlowPRA(−) recipients (23% and 22% vs. 3% in [IgG]FlowPRA(−) patients). Only patients with complement‐fixing HLA class I immunization had inferior graft survival [75% (3 years) vs. 91% and 89%, respectively (p = 0.036)]. Despite frequent finding of capillary C4d deposition (28%), complement‐fixing HLA class II immunization was not associated with inferior survival rates. This may have been due to reduction of clinical effects by intense immunosuppression in presensitized patients. Evaluating CDC, 29% of CDC‐PRA(+)/[C4d]FlowPRA(+) recipients had C4d‐positive graft dysfunction. For these patients 3‐year graft survival was worst, followed by CDC‐PRA(+)/[C4d]FlowPRA(−) and CDC‐PRA(−) patients (76% vs. 81% vs. 90%, p = 0.014). Results highlight a strong impact of complement‐fixing HLA presensitization. Discerning complement‐activating abilities of HLA alloantibodies, [C4d]FlowPRA may help identify recipients at particularly high risk for graft rejection and loss.

Effect of the proteasome inhibitor bortezomib on humoral immunity in two presensitized renal transplant candidates.

Background. Recipient presensitization represents a major hurdle to successful renal transplantation. Previous case series have suggested that the proteasome inhibitor bortezomib directly affects the alloantibody-secreting plasma cells in rejecting allograft recipients. However, the ability of this agent to desensitize nonimmunosuppressed transplant candidates before transplantation is currently unknown. Methods. In this analysis, two sensitized hemodialysis patients were selected to receive two subsequent bortezomib cycles. Bortezomib was given at 1.3 mg/m2 on days 1, 4, 8, and 11. Dexamethasone was added to the second cycle to enhance treatment efficiency. Serial immune monitoring included cytotoxic panel reactive antibody testing, Luminex single antigen testing for anti-human leukocyte antigen (HLA) IgG with or without C4d-fixing capability, and ABO antibody detection. Results. During a half-year follow-up period, cytotoxic panel reactive antibody decreased from 87% to 80% (patient 1) and 37% to 13% (patient 2). Patient 1 showed a 40% reduction in binding intensities of identified Luminex HLA single antigen reactivities and, in parallel, slight reductions in ABO blood group antibody and total immunoglobulin levels. In patient 2, bortezomib did not affect circulating antibody levels in a meaningful way. Both patients showed a more than 50% reduction in the levels of anti-HLA antibody-triggered C4d deposition to Luminex beads. Conclusion. Our initial experience suggests that, without additional immunosuppressive measures, bortezomib has modest effects on circulating antibodies against HLA or blood group antigens. The reduced levels of antibody-triggered complement fixation, however, imply potential clinical relevance of proteasome inhibition for recipient desensitization.

In Vitro Detection of C4d‐Fixing HLA Alloantibodies: Associations With Capillary C4d Deposition in Kidney Allografts

Capillary C4d deposition is a valuable marker of antibody‐mediated rejection (AMR). In this analysis, flow cytometric detection of alloantibody‐triggered C4d deposition to HLA antigen‐coated microparticles ([C4d]FlowPRA) was evaluated for its value as a marker for C4d deposition in renal allografts. For comparative analysis, 105 first renal biopsies performed for graft dysfunction and an equal number of concurrent sera were subjected to immunohistochemistry and [C4d] plus standard [IgG]FlowPRA, respectively. C4d deposition/fixation was detected in 17 biopsies and, applying [C4d]FlowPRA HLA class I and II screening, also in a small number of corresponding sera (N = 20). IgG reactivity detected by standard [IgG]FlowPRA was more frequent (49% of sera). Comparing [C4d]FlowPRA screening with capillary C4d staining, we found a high level of specificity (0.92 [95% confidence interval: 0.86–0.98]), which far exceeded that calculated for [IgG]FlowPRA (0.60 [0.50–0.70]). [IgG]FlowPRA screening, however, turned out to be superior in terms of sensitivity (0.94 [0.83–1.05] vs. 0.76 [0.56–0.97] calculated for C4d‐fixing panel reactivity). Remarkably, posttransplant single antigen testing for identification of complement‐fixing donor‐specific alloreactivities failed to improve the predictive value of FlowPRA‐based serology. In conclusion, our results suggest that detection of complement‐fixing HLA panel reactivity could provide a specific tool for monitoring of C4d‐positive AMR.

Clinical relevance of preformed C4d-fixing and non-C4d-fixing HLA single antigen reactivity in renal allograft recipients

Donor‐specific alloantibodies (DSA), especially those fixing complement, may pose a particular immunologic risk to transplant recipients. To assess the clinical impact of C4d‐ or non‐C4d‐fixing (IgG) HLA sensitization, pretransplant sera obtained from 338 kidney allograft recipients prescreened by FlowPRA were retrospectively evaluated by Luminex single antigen (SA) testing using a novel fluorescent‐labeled anti‐C4d reagent for detection of antibody‐triggered C4d deposition in addition to IgG binding. Recipients with [IgG]DSA (n = 39) showed a substantially higher rate of C4d positive rejection (33%) than 16 patients with [IgG] non‐DSA (0%) or 283 antibody‐negative patients (4%, multivariate analysis excluding retransplantation because of high co‐linearity: P < 0.0001), and adversely affected 5‐year death‐censored graft survival (74% vs. 81% and 90%, respectively, multivariate model: P < 0.05). [C4d] DSA (n = 21) and [C4d] non‐DSA (n = 25) increased rates of C4d positive rejections to a similar extent (24% and 28% vs. 4% in recipients without C4d‐fixing reactivity; multivariate analysis: P ≤ 0.002) with a trend towards adverse 5‐year graft survival (76% and 76% vs. 90%; P ≤ 0.2). In conclusion, Luminex‐based characterization of HLA sensitization may be a useful strategy for risk stratification. Possibly as a result of intensified immunosuppression in presensitized recipients, identification of C4d‐fixing DSA was not associated with a further increase of rejection and graft loss rates.

Influence of clinical factors on the haemolysis marker haptoglobin

Background  Plasma haptoglobin determination is clinically used as parameter for haemolysis. To date, however, the influence of the mode of haemolysis (extravascular vs. intravascular) and of nonhaemolytic conditions on haptoglobin concentration and its reliability as a haemolysis marker remain poorly defined.

Stability of coagulation factors in thawed, solvent/detergent-treated plasma during storage at 4 °C for 6 days

Background and Objectives  Transfusion of fresh‐frozen plasma is still a pillar in emergency medicine for using to prevent dilutional coagulopathy or disseminated intravascular coagulation after severe blood loss, but thawing procedures can delay its availability. On the other hand, the wastage of plasma, once thawed and not transfused within a defined time‐period, represents an inefficient handling of economic resources and is contradictory to blood donor intentions. In this study we investigated the stability of coagulation factor activities and plasma protein levels during 6 days of storage of thawed solvent/detergent (S/D)‐treated plasma at +4 °C. Our results may form the basis for reconsideration of expiry times of thawed S/D‐treated plasma.