Martina Schnaidt

HLA Antibody Specification Using Single-Antigen Beads—A Technical Solution for the Prozone Effect

Background. Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect. Methods. Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone. Results. The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q. Conclusion. Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.

Expression of blood group genes by mesenchymal stem cells

Incompatible blood group antigens are highly immunogenic and can cause graft rejections. Focusing on distinct carbohydrate‐ and protein‐based membrane structures, defined by blood group antigens, we investigated human bone marrow‐derived mesenchymal stem cells (MSCs) cultured in human serum. The presence of H (CD173), ABO, RhD, RhCE, RhAG, Kell, urea transporter type B (SLC14A1, previously known as JK), and Duffy antigen receptor of chemokines (DARC) was evaluated at the levels of genome, transcriptome and antigen. Fucosyltransferase‐1 (FUT1), RHCE, KEL, SLC14A1 (JK) and DARC mRNA were transcribed in MSCs. FUT1 mRNA transcription was lost during differentiation. The mRNA transcription of SLC14A1 (JK) decreased during chondrogenic differentiation, while that of DARC increased during adipogenic differentiation. All MSCs synthesized SLC14A1 (JK) but no DARC protein. However, none of the protein antigens tested occurred on the surface, indicating a lack of associated protein function in the membrane. As A and B antigens are neither expressed nor adsorbed, concerns of ABO compatibility with human serum supplements during culture are alleviated. The H antigen expression by GD2dim+ MSCs identified two distinct MSC subpopulations and enabled their isolation. We hypothesize that GD2dim+ H+ MSCs retain a better ‘stemness’. Because immunogenic blood group antigens are lacking, they cannot affect MSC engraftment in vivo, which is promising for clinical applications.

Successful transplantation of highly selected CD34+ peripheral blood stem cells in a HLA-sensitized patient treated with immunoadsorption onto protein A.

BACKGROUND Haploidentical bone marrow transplantation with preexisting anti-HLA antibodies is associated with a high risk of graft failure. METHODS A 27-year-old female patient with chronic myeloid leukemia and evidence of several osseous chloromas had no suitable matched bone marrow donor, and fluorescence cytometric cross-match (FCXM) revealed antibodies against donor-specific HLA-molecules. Immunoadsorption onto staphylococcal protein A was applied to remove these antibodies, and peripheral stem cell transplantation was performed from her haploidentical sister after a negative FCXM was documented after immunoadsorption and conditioning treatment. RESULTS FCXM for donor lymphocytes and stem cells remained negative throughout the posttransplant period, and engraftment of donor cells was documented on day +69. CONCLUSION Immunoadsorption onto protein A should be considered in stem cell transplantation even from an haploidentical donor where anti-HLA antibodies and a positive FCXM are documented.

High‐dose immunoglobulin in the antenatal treatment of neonatal alloimmune thrombocytopenia: case report and review

GIFT can be easily and economically introduced into hospitals, and has therefore become widely available in Units unable to offer IVF or cryopreservation facilities (Interim Licensing Authority 1991). However, the number of oocytes transferred back to the patient should be limited to a maximum of three (Interim Licensing Authority 1991) to reduce the risk of multiple pregnancy after GIFT. The use of the IVC (intravaginal culture) technique to transfer supernumerary oocytes and sperm to a local IVF unit with the facilities for cryopreservation provides a simple and cost-effective method for maximizing the chance of achieving a pregnancy per surgical procedure. This has important implications in the structuring ofinfertility services, both within the National Health Service and the private sector. The present report describes the first viable pregnancy achieved following the intra-vaginal transport of embryos from a GIFT unit to an IVF unit for cryopreservation. The modified IVC protocol used in the present study in which the transported supernumerary oocytes were examined for evidence of fertilization 20-24 h after insemination, enables screening for polyspermy. In addition, cryopreservation at the 1 cell pronucleate stage offers improved embryo survival rates compared with cryopreservation at early cleavage stages (Troup et al. 1990). This differs from the original method of IVC (Ranoux ct al. 1988) which involved the culture of embryos for 2 days before they were checked. Whilst it is unlikely that every GIFT unit will be able to provide a cryopreservation service, the present report of a viable pregnancy shows it is possible for IVF centres to provide embryo storage facilities for the GIFT units within their region, with the IVC technique providing a convenient method for transport and culture of oocytes between the two units.

Alpha-synuclein levels in blood plasma decline with healthy aging.

There is unequivocal evidence that alpha-synuclein plays a pivotal pathophysiological role in neurodegenerative diseases, and in particular in synucleinopathies. These disorders present with a variable extent of cognitive impairment and alpha-synuclein is being explored as a biomarker in CSF, blood serum and plasma. Considering key events of aging that include proteostasis, alpha-synuclein may not only be useful as a marker for differential diagnosis but also for aging per se. To explore this hypothesis, we developed a highly specific ELISA to measure alpha-synuclein. In healthy males plasma alpha-synuclein levels correlated strongly with age, revealing much lower concentrations in older (avg. 58.1 years) compared to younger (avg. 27.6 years) individuals. This difference between the age groups was enhanced after acidification of the plasmas (p<0.0001), possibly reflecting a decrease of alpha-synuclein-antibody complexes or chaperone activity in older individuals. Our results support the concept that alpha-synuclein homeostasis may be impaired early on, possibly due to disturbance of the proteostasis network, a key component of healthy aging. Thus, alpha-synuclein may be a novel biomarker of aging, a factor that should be considered when analyzing its presence in biological specimens.

Reactivation of antibodies of donor and recipient origin to platelet antigens early after allogeneic bone marrow transplantation: a case report

Summary. Reactivation of platelet‐reactive antibodies of donor and recipient origin is described in a patient following allogeneic BMT (donor: anti‐HPA‐5b; recipient: anti‐HLA, anti‐HPA‐1b). The antibodies were detected around day 15 after BMT, peaked around day 25, and then decreased. These antibodies are interpreted as an antigen‐independent reactivation of secondary B‐cell responses, activated in the context of recognition of host antigens by the graft.

Difficulties in the antenatal assessment of neonatal alloimmune thrombocytopenia.

In this paper we present a patient with an initially questionable history of neonatal alloimmune thrombocytopenia (NAT) due to materno‐fetal HPA‐la (PLAl) incompatibility. No circulating antibodies were detectable in untreated maternal serum, but an adsorption/elution technique enabled the demonstration of the platelet‐specific anti‐HPA‐la (anti‐PLAl) in maternal serum. Cordocentesis at 35 weeks of gestation revealed a fetal platelet count of 18 × 109/1. Intrauterine platelet transfusion with HPA‐Ia (PLAl)‐negative donor platelets was performed prior to cesarean section.

Reactivation of Recipient Antibody to Blood Cell Antigens Soon After Allogeneic Bone Marrow Transplantation

Reactivation of recipient antibody to HLA and red blood cell antigens is described in 8 patients after allogeneic bone marrow transplantation. These IgG antibodies can be detected between day 10 and day 40 after transplantation and, in 1 patient, can be shown to be antigen‐independent. We hypothesize that, induced by graft recognition of recipient antigens, antigen‐independent activation of sensitized recipient B cells takes place leading to transient antibody production.

Serological Screening, Using Three Different Test Systems of Platelet‐Transfused Patients with Hematologic‐Oncologic Disorders

Sera of hematologic‐oncologic patients were tested regularly after platelet transfusions in three test systems: lymphoeytotoxicity test, platelet adhesion immunofluorescence test, and — only selected sera — in the monoclonal antibody‐specific immobilization of platelet antigen test. Of 388 patients 53 (14%) had HLA antibodies 5 of these in combination with platelet‐specific alloantibodies. Lymphocyte‐restricted (non‐HLA) reactions were observed in 20 patients, the majority of which was attributed to lymphocyte‐specific auto‐ or alloantibodies. Sera of 27 patients showed platelet‐specific reactions, usually cold‐reacting autoantibodies which have no effect in vivo.

Post‐transfusion purpura following liver transplantation

The human platelet antigen-1 (HPA-1) system is localized on platelet glycoprotein (GP)IIIa or the β 3 integrin. The HPA-1a and -1b alleles differ by a single nucleotide polymorphism (SNP), represented by the leucine33proline replacement in the mature protein. β 3 is also expressed on endothelial cells in the context of an alternative α chain, α V [1]. Approximately one in 350 pregnant women form HPA-1a antibodies [2]. In some HPA-1a alloimmunized women, blood transfusion may precipitate a post-transfusion purpura (PTP). The mechanism for the destruction of the autologous platelets is unknown, but two have been proposed: either autologous platelets may scavenge HPA-1/anti-HPA-1 immune complexes, or short-lived autoantibodies are generated during the HPA-1a recall immune response [3]. This report describes the clinical and serological data in a case of PTP following liver transplantation.