57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr. med.

Prof.

Prof. Dr.

PD Dr. rer. nat.

Sequential vaccination against Streptococcus pneumoniae appears as immunologically safe in clinically stable kidney transplant recipient

Organ Transplatation

Die sequenzielle Impfung gegen Streptococcus pneumoniae erscheint bei klinisch stabilen Nierentransplantat-Empfängern als immmunologisch sicher

Infections and rejection of the allograft are two major problems in organ transplant recipients. Vaccination against Streptococcus pneumoniae is recommended in this risk group, to reduce morbidity and mortality from invasive pneumococcal disease. Currently, sequential vaccination against pneumococci is recommended, consisting of vaccination with a 13-valent conjugate vaccine, followed by a 23-valent polysaccharide vaccine. Data on alloantibodies after sequential pneumococcal vaccination are still lacking.In the current study, we sequentially determined HLA class I and II and MICA antibodies in 41 clinically stable kidney transplant recipients prior to and after first and second vaccination against pneumococci, comprising a 12-months period. Antibodies were measured by Luminex mixed bead assay. We summed up the score values of each bead (1=negative, 4=undetermined and 8=positive) for HLA class I, HLA class II or MICA, respectively; yielding antibody score values of 12–96, 5–40 or 2–16, and we added MFI values for the respective beads. In parallel, pneumococcal IgG antibodies were determined by ELISA.During the 12-months period the sequential analysis yielded no significant change in HLA class I and II and MICA antibodies, irrespective of how the data were quantified (Figure 1). Figure 1: Antibodies prior to and after sequential pneumococcal vaccination in 41 kidney transplant recipients.One patient developed de novo donor-specific antibodies (DSA) 1.5 months after first vaccination (MFI 1600), but no allograft rejection was detected in this patient. A biopsy proven rejection was detected 7 months after the first vaccination in another patient (histopathological: borderline rejection with Banff category 3), who did not develop de novo DSA. Both showed a minor increase in HLA class I and II antibodies at month 1. As expected, pneumococcal antibodies showed a significant increase already at month 1 after vaccination (p = 0.0006) and the increase was even stronger at month 6, 7 and 12 (p &lt; 0.0005).In conclusion, HLA class I and II and MICA antibodies remained overall constant after sequential pneumococcal vaccination. Considering the general risk of alloimmune processes in solid organ transplant recipients, we would not attribute the de novo DSA or borderline rejection that occurred in 2 of 41 patients to the administered vaccines. Therefore, we consider the sequential administration of both vaccines in our cohort to be immunologically safe.None.

Poster

Validation data for rapid high-resolution HLA nanopore sequencing using NGS-Turbo®

Validierungsdaten für die schnelle hochauflösende HLA-Nanoporen-Sequenzierung mit NGS-Turbo®

HLA epitopes on donor HLA molecules play a crucial role in solid organ transplantation, and high-resolution typing of all 11 HLA loci is essential for enhanced allocation. However, for deceased organ donors, there has not yet been sufficient time to perform high-resolution sequencing, hence only intermediate resolution typing from SSP-PCR techniques has been available.In this study, we present validation data on the NGS-Turbo Kit (GenDX) for high-resolution HLA typing with Oxford Nanopore Technologies devices in less than 4 hours.The method was tested on 41 samples previously typed by Illumina sequencing.The high-resolution 3-field HLA typings obtained with Nanopore sequencing were 100% concordant compared to short-read sequencing using the NGSgo MX11-3 kit (GenDX), and in certain cases, the resolution was even higher. Notably, the re-run rate was a minimal 2.4%, comparable to alternative methods.In conclusion, the adoption of high-resolution HLA typing for deceased donors holds promise for enhancing organ allocation strategies, potentially yielding significant clinical benefits for transplant recipients.The authors declare that they have no relevant or material financial interests related to the research described in this paper.

Physical/de facto cross-matching in order to complement its primary virtual variant prior to kidney allografting: Substantial benefit or only fishing for diagnostic artifacts?

Die physische Verträglichkeitsprobe im Nachgang ihrer virtuellen Variante als diagnostische Vorbereitung von Nierentransplantationen: Nutzen oder Artefaktfischerei?

Preformed Donor-specific anti-HLA antibodies (DSA) represent the most prominent cause for humoral hyper-acute and acute allograft rejections and, therefore, have strictly to be avoided prior to kidney allografting. Since April 2023 the so-called virtual crossmatch (vXM) as a procedure aligning the recipients' antibody specificities with the HLA-antigens of their donors has principally replaced the physical/de facto crossmatch regarding the detection/identification of Non Acceptable HLA Antigens. In spite of this pivotal meaning of vXM, the Lymphocytotoxicity- (LCT/CDC-) based cross-matching has not been disestablished but is still required by the guidelines of the German Federal Medical Association as a second catch-all in order to detect potential DSA. Due to well documented drawbacks of LCT cross-matching in terms of artifacts caused by recipients' autoimmune diseases and phamacological treatment this approach must critically be challenged.We here present our data of ten positive LCT-based crossmatch outcomes appearing in our laboratory's emergency duties between April 2023 and 2024, all of which were observable in spite of prior negative virtual cross-matching. For the virtual approach donors' typing results were available for the 11 gene loci HLA-A*, B*, C*, DRB1*, DRB3*,4*,5*, DQB1*, DQA1*, DPB1* and DPA1* at the intermediate level of resolution defined by Eurotransplant. Antibody specifications of the respective recipients had been performed at the corresponding allelic level of resolution.In all ten cases the source of LCT-based positive crossmatch outcomes was doubtlessly definable. Nine out of ten could be ascribed to artificial origins such as underlying autoimmune diseases (n=6). Furthermore, immunosuppressive treatment by rituximab (n=2) or intravenous immunoglobulins (IVIG) (n=1) also showed its falsifying effect. Only one case exhibited DSA which had not been included by prior vXM since a transfusion as immunizing event had not been adequately considered by a follow up antibody specification. However, representing a serious failure of the respective transplant center, this case should be beyond occurrence.We conclude that the highly specific vXM taking into account all immunizing events represents an adequate tool in order to exclude DSA whereas subsequent LCT-based physical cross-matching rather raises artificial outcomes which with the utmost probability prevent recipients from receiving adequate allografts.No disclosure statement!!

Defining of epitope matches and mismatches with epiTOol

Bestimmung von Epitopübereinstimmungen und -mismatche mit epiTOol

Following immune challenge, specific HLA antibodies might be produced causing humoral problems such as rejection and early graft loss in organ transplants or no take in the case of platelet substitution. We already demonstrated the benefit of high-resolution HLA typing in kidney transplantation for a determination of these antibodies (Lehmann et al. doi: 10.3389/fimmu.2023.1094862). Here we present the potential use of &quot;epiTOol&quot;, an interactive analytics tool for all-in-one processing of HLA typing and antibody data.We analyzed a cohort of 108 living transplants regarding their antibody verified epitopes matches (EM) as well as mismatches (EMM). The recipients were 59% related and out of them 43% 1st degree related to the donor. All recipient and donors were HLA typed for HLA-A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 at high resolution. HLA antibody data was taken from existing routine data determined as part of quarterly screening or post-monitoring.For HLA class I, we found unique epitopes for HLA-A (n=33), -B (n=28) and -C (n=20), with 144KR being the most frequently mismatched epitope (n=29) in our cohort. For HLA class II, the highest number of different epitopes is observed for HLA-DR (n=37), followed by -DQ (n=28) and -DP (n=13). The number of antibody-verified total EMM ranged from 0 (6 pairs) to 50 (1 pair). The largest number of EMM was observed for the DR locus (n=522), followed by A (n=440), DQ (n=418), B (n=323), C (n=217) and DP (n=213). The number of EM is not proportional to the number of EMM. The DR locus has the most matched epitopes n=1736, followed by DQ (n=1307), A (n=1183), B (n=888), C (n=798) and DP (n= 663). Finally, via epiTOol we mapped the donor specific epitopes. Interestingly, 33% (n=36) of the recipients have not formed anti-HLA antibodies following kidney transplantation although up to 40 epitope mismatches were present. In contrast, recipients with only nine EMM had produced antibodies towards the donor.Defining epitopes is helpful for understanding the humoral response but it is not a matter of numbers but a matter of quality and could be a useful tool for donor selection in the future.none

Taboo concept 2.0: Homozygosity of any HLA locus is associated with dedicated HLA antibody patterns

Tabu-Konzept 2.0: Homozygotie eines beliebigen HLA-Locus ist mit bestimmten HLA-Antikörpermustern assoziiert

In a collaborative study of the University Hospital Leipzig and the Charité Berlin on a large cohort of kidney transplant recipients, we aimed to analyse the occurrence of HLA-specific antibody patterns with respect to the HLA repertoire of the patients.Patients typed for the relevant HLA determinants using next generation sequencing. Antibody screening was performed by multiplex-based technology using microspheres coupled with the HLA alleles of HLA class I and II.Patients homozygous for HLA-A*02, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*18, HLA-B*35, HLA-B*44, HLA-C*03, HLA-C*04, HLA-C*07 for class I and for class II: HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*07, HLA-DRB1*15, HLA-DQA1*01, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB1*03(7), HLA-DQB1*06, HLA-DPA1*01, and HLA-DPB1*04 were found to have a significant higher antibody production compared to the heterozygous ones. All HLA determinants are affected. Remarkably, HLA-A*24 homozygous patients can produce antibodies towards all HLA-A determinants, while HLA-B*18 homozygous make antibodies towards all HLA-B and selected HLA-A and -C antigens and are associated with an elevation of HLA-DRB1, partly -DQB1, and -DPB1 alleles. Homozygosity for the HLA class II HLA-DRB1*01, and HLA-DRB1*15 seems to increase the risk for antibody responses against most of the HLA class I antigens (HLA-A, -B and -C) in contrast to HLA-DQB1*03(7) where a lower risk towards few HLA-A, and B alleles is observed.The widely observed differential antibody response is to be accounted to the patient's HLA repertoire. Homozygous patients are at risk to produce HLA-specific antibodies hampering the outcome of transplantation or any substitution. Including this information in the allocation procedure might reduce antibody mediated immune reactivity and prevent graft loss. In addition, all patients in need of HLA based substitution and being homozygous in any of the HLA loci should treated in a way to avoid sensitization.none

PD Dr.

Development of de novo donor-specific anti-HLA antibodies and ABMR in renal transplant patients depends on CYP3A5 genotype

Entwicklung der de novo donorspezifischen anti-HLA Antikörper and der humoralen Abstoßung bei nierentransplantierten Patienten ist abhängig vom CYP3A5-Genotyp

The single-nucleotide polymorphism CYP3A5 rs776746 is related to a reduction in the metabolizing activity of the CYP3A5 enzyme. Persons carrying at least one copy of the wild-type allele, defined as CYP3A5 expressers, exhibit higher clearance and lower trough concentrations of tacrolimus than homozygous nonexpressers, and this difference may affect alloimmunization and allograft function.We retrospectively studied 400 kidney transplant recipients treated with a tacrolimus-based immunosuppression regimen to detect CYP3A5 genotype, de novo formation of human leukocyte antigen (HLA) antibodies and donor-specific antibodies (DSA), and clinical outcome up to 5 years after transplant. CYP3A5 genotype was determined by pyrosequencing.We found that 69 (17%) of the 400 patients were CYP3A5 expressers. During the first 3 years after transplant, CYP3A5 expressers tended to have lower tacrolimus trough levels than non-expressers, even though their tacrolimus dosage was as much as 80% higher. De novo anti-HLA antibodies and DSAs were found more frequently in CYP3A5 expressers than in nonexpressers (de novo anti-HLA antibodies, 24/69 [35%] vs. 83/331 [25%], p = 0.05; de novo DSAs, 13/69 [19%] vs. 33/331 [10%], p = 0.02). De novo anti-HLA antibody–free (p = 0.03) and DSA-free survival rates (p = 0.02) were significantly lower for expressers than for nonexpressers. CYP3A5 genotype had no effect on allograft failure, but CYP3A5 expressers exhibited a significantly higher frequency of antibody-mediated rejection. CYP3A5 expresser status was an independent risk factor for the development of de novo DSAs (relative risk, 2.34, p= 0.01).Early detection of CYP3A5 expressers, enabling genotype-based dose adjustment of tacrolimus immediately after renal transplant, may be a useful strategy for reducing the risk of de novo DSA production and antibody-mediated rejection.No disclosures.

Early genotype-based dose adjustment of tacrolimus reduces risk for de novo donor-specific HLA antibodies and rejection in CYP3A5 expressers among renal transplant patients

Frühe Genotypen-basierte Tracrolimus Dosierung reduziert das Risiko von de novo Donor-spezifischen HLA Antikörpern und Abstoßungen bei CYP3A5 Expressoren nach Nierentransplantation

The single nucleotide polymorphism rs776746 in the CYP3A5 gene slows down metabolism of tacrolimus via CYP3A5 enzyme. Our previous retrospective single-center cohort study showed a trend towards low tacrolimus trough levels and an increased risk for development of de novo donor-specific anti-HLA antibodies (DSA) and antibody-mediated rejection in CYP3A5 expressers carrying at least one copy of the wild-type allele at a follow-up of 3 years. We hypothesized that detection of CYP3A5 expresser status immediately post renal transplant, allowing early genotype-based dose adjustment of tacrolimus, might prevent the occurrence of de novo DSA and ABMR and improve renal transplant outcome.A total of 160 renal allograft recipients who underwent renal transplantation between May 2019 and 2022 were genotyped for CYP3A5 rs776746 polymorphism within the first two weeks post-transplant, and consequently genotype-based dose adjustment of tacrolimus was performed. The CYP3A5 genotype was determined by Pyrosequencing. After determination of CYP3A5 expresser status, patients were followed up for 2 years and development of calcineurin inhibitor (CNI) nephrotoxicity, rejections and de novo DSA was retrospectively analyzed.CYP3A5 expresser status was detected in 33 (21%) patients, whereas 127 (79%) patients were non-expressers. Tacrolimus trough levels were similar in CYP3A5 expressers and non-expressers over the follow-up period of 2 years. However, we observed a trend towards slightly increased tacrolimus trough levels in CYP3A5 expressers, who as expected required tacrolimus dosages twice as high as non-expressers. Correspondingly, CYP3A5 expressers had significantly lower concentration-to-dose ratios compared to non-expressers at all indicated time points. CNI nephrotoxicity-free survival rates were comparable between CYP3A5 expressers and non-expressers (p=0.49). Rejection-free survival rates (p=0.89) and de novo anti-HLA antibody-free survival rates (p=0.57) also did not differ between the two groups. The frequency of de novo DSA (2 (6%) vs. 5 (4%), p=0.6) and de novo DSA-free survival (p=0.61) were similar in CYP3A5 expressers and non-expressers.Early detection of CYP3A5 expresser status resulting in genotype-based adaption of tacrolimus dosage was not associated with increased incidence of CNI toxicity in CYP3A5 expressers but protected them from developement of transplant rejection and de novo DSA formation.K.S. has no conflict of interest.

Adverse immunisation by 3 erythrocyte concentrates while waiting for a kidney transplant – a patient case from the HLA lab

Auf der Nierentransplantations-Warteliste durch 3 Erythrozyten-Konzentrate unglücklich immunisiert – Ein Fallbeispiel aus dem HLA-Labor

For patients with end-stage renal disease only a kidney transplant is a chance of prolonging and improving their lives. Especially problematic are highly immunized patients who often spend a long time on dialysis while waiting for a deceased donor's transplant.A patient (f, 53) was listed by Eurotransplant (ET) for 10 years waiting for a kidney transplantation. As requested by ET, the patient was screened quarterly by Luminex HLA antibody screening / identification where strong but constant antibody burden was found. In recent years, the complement‐dependent cytotoxicity assay (CDC) was negative.In autumn 2023 she had an acute life-threatening closure of the dialysis shunt requiring immediate surgery. She received 3 erythrocyte concentrates intra- and post-operatively. Unfortunately, neither the transplant centre nephrologists nor the HLA lab were informed. 10 weeks after surgery she was offered a deceased donor organ by ET. In the run-up to the transplantation she was routinely interviewed and she told the team of the blood transfusions. A crossmatch against the donor with freshly taken serum was highly positive, while the archived quarterly serum taken a few days before the transfusion was negative. Eventually the kidney was transplanted into another patient. Closer analysis of the crossmatch using the Luminex assay (Labscreen SA1, One Lambda) detected some of the antibodies even in 1:1,000 dilution. Furthermore, a new sample 6 months after the transfusion showed that the antibody profile was not yet back to pre-transfusion levels. In the CDC analysis, both with or without DDT, the PRA was 96% respectively 100%, and no specificities could be defined.This patient case shows that as few as 3 leukocyte-depleted erythrocyte concentrates can lead to a massive immunisation. Diligence on the part of the regional DSO-HLA lab avoided an impending acute organ failure after transplantation. This case study also highlights first the importance of open communication between all the teams treating a patient on the kidney waiting list, and second the importance of using freshly taken serum if there is a risk of an uncommunicated immunising event.no conflict of interest

Dr. med.

PD Dr. med.

Donor-derived cell-free DNA: A promising non-invasive biomarker for the early detection of allograft rejection and damage in lung transplant recipients

Vom Spender stammende zellfreie DNA: ein vielversprechender nicht-invasiver Biomarker für die frühzeitige Erkennung von Abstoßungsreaktionen und Allotransplantatschäden bei Lungentransplantatempfängern

Although more patients are now surviving longer after lung transplantation, chronic allograft dysfunction due to ACR and AMR still remains a significant risk factor. Invasive histopathology of transbronchial biopsies is the method of choice for the detection of acute rejection. The aim of this study is to investigate the potential utility of donor-derived cell-free DNA (ddcfDNA) as a non-invasive biomarker for the detection of allograft function and acute rejection after lung transplantationHome made digital droplet PCR was used to determine the total amount of cell-free DNA as well as the total amount and the proportion of ddcfDNA. In 81 lung recipients from whom blood was taken before and several times after transplantation, median levels of ddcfDNA [%] was related to cellular or humoral rejection, infection or changes in lung function (Wilcoxon and Kruskal-Wallis test).70.4 % of the patients were male and had a mean age of 55.4 years (sd=11.0). The most common underlying disease was ILD (55.6%), followed by COPD (18.5%) and rheumatic diseases (13.6%). The probability of acute rejection was significantly higher in pre-immunised patients (75.0% vs. 30.1%, p=0.02). The patient group with ACR (1.92, IQR:0.70-2.30), AMR (1.27, IQR:0.34-2.29), isolated lymphocytic bronchiolitis (0.54, IQR:0.23-2.18) and infection (0.50, IQR: 0.22-2.35) had significantly increased mean %ddcfDNA values compared to the cohort with healthy allograft function (0.26, IQR:0.09-0.60) (p=0.0003). Patients with FEV1 loss had significantly higher %ddcfDNA values (1.98, IQR:0.50-2.30, p=0.04) compared to patients with stable or improving lung function at 12 months (1.36, IQR:0.27-1.66).These results suggest that ddcfDNA is a promising biomarker for monitoring allograft function to detect allograft damage caused by ACR, AMR or infection at an early stage. Finally, this non-invasive biomarker could help to reduce the number of biopsies in the future.no conflict of interest

Feasibility study for a Flow Cytometry crossmatch procedure: Results of the first workshop

Durchführbarkeitsstudie für ein durchflusszytometrisches Kreuzprobeverfahren

While establishing a flow cytometry crossmatch (FCXM) exchange program for laboratories in solid organ transplantation we conducted a wet bench feasibility study during a national conference in Germany. Aim of this first step was to acquaint participating laboratories in the FCXM procedure. Especially the use of pronase and proper controls was in scope. Currently, the complement dependent cytotoxicity (CDC) assay is the standard method used for crossmatching in solid organ transplantation. CDC is based on laboratory experience and does not comply with the IVDR requirements (in vitro diagnostic medical devices and repealing, Directive 98/79/EC Regulation (EU) 2017/746, consolidated 23/03/2023), which prompted the laboratories to change their policies.17 participants from 9 different institutes were present, and the Halifaster FCXM assay (https://doi.org/10.1016/j.humimm.2017.10.020) was demonstrated in two practical sessions. The use of pronase to eliminate B cell background and to limit the false positive and false negative reactions were demonstrated. In addition, a set of negative and positive control sera was featured that together serve to proper control for washing technique and ensure the validity of the FCXM results.The results indicated that the use of pronase treatment is essential to decrease B cell background and to limit both false positive and false negative B cell reactions in FCXM. Furthermore, a carefully selected and developed set of negative and positive control sera was featured and demonstrated the importance of using proper controls to ensure proper washing technique and the validity of the FCXM results.The Halifaster FCXM method is rapid, cost effective, provides high quality results and is used by many laboratories internationally for pre-transplant crossmatching. Our next step is to conduct a large study with many participating laboratories in central Europe to evaluate the Halifaster FCXM protocol as a replacement method for the CDC crossmatch. We aim to accomplish this by end of August 2024.none

Posterausstellung 2
Organ Transplantation

Termin

Programm

Posterausstellung 2Organ Transplantation

Termin

Programm

Posterausstellung 2
Organ Transplantation