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57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr. med.

Vorsitz

Prof.

PD Dr. rer. nat.

Prof. Dr.

Prof. Dr. med. Dipl.-Kfm.

Phenotypical characterization of PROC mutations causing protein C deficiency by assessment of activated protein C formation in an endothelial ex vivo model

Hemostaseology

Phänotypische Charakterisierung von Protein-C-Mangel verursachenden PROC-Mutationen mittels Untersuchung der Bildung von aktiviertem Protein C in einem endothelialen Ex-vivo-Modell

A broad variety of PROC gene mutations is known to cause hereditary protein C deficiency (PCD), a classical type of thrombophilia. In routine diagnostics, PCD is tested for by measuring functional protein C (PC) levels in plasma. These methods, however, do not allow for an assessment of how PROC mutations affect activated PC (APC) formation, which takes place on the endothelial surface. This study aimed to assess endothelium-dependent APC formation ex vivo in patients with PROC mutations.Next Generation Sequencing was applied to identify PROC mutations in patients with PCD (n=25). Healthy blood donors (n=12) served as controls. Apart from reduced PC levels in the patient cohort, all study participants were required to have normal levels of other coagulation factors and inhibitors. For the ex vivo model, human umbilical vein endothelial cells (HUVECs) were cultured in 48-well plates and overlaid with defibrinated, citrated plasma obtained from patients and healthy controls. APC formation in the supernatant was induced by tissue factor, CaCl2, and phospholipids and monitored over 120 minutes via oligonucleotide-based enzyme capture assay. A dilution series of purified PC in PC-deficient plasma was assessed as a reference.Endothelial APC formation was significantly reduced in PCD patients than in healthy controls (Fig. 1). PC plasma levels correlated strongly with the area under the curve of APC formation (AUC APC) in the ex vivo model (r=0.824). When comparing the AUC APC in study participants against the reference curve obtained with purified PC, it did not exceed a range of +/-25% in healthy controls. In the PCD cohort, the AUC APC lay below this range in 7/15 (47%) patients with missense mutations, 4/7 (57%) with nonsense mutations, and in all patients with deletions (n=2) or splice-site mutations (n=1) (Fig. 2). No patient showed a relative increase of the AUC APC when compared against the reference curve.In the majority of patients with PCD, APC formation was disproportionately impaired in the ex vivo model. These data indicate that the assessment of residual PC levels might underestimate the prothrombotic potential in hereditary PCD. Moreover, different mutations might affect APC formation at different extents despite similar residual PC levels in routine tests. Further studies are warranted to understand the interplay between PROC variants and APC formation in PCD.None

Abstract lecture (Abstract winner)

Dr. med.

Transfusion demand related to diagnosis-related groups and gender – an analysis from the Blut-MV study

Hemotherapy and Patient Blood Management

Einfluss von Diagnosebezogenen Fallgruppen und Geschlechtsunterschieden auf den Transfusionsbedarf – eine Analyse der Blut-MV Studie

Patient demographics have a major influence on transfusion demands in an aging population. The Blut-MV study is a prospective longitudinal study conducted to monitor patient and donor demographics and to forecast blood donations and transfusion demands in the federal state of Mecklenburg-Western Pomerania (MWP). Here, we aimed to investigate the impact of diagnosis-related groups and gender on transfusion demands.Patient demographics (date of birth, gender, patient classification [medical, critically-ill, surgical and pediatric]) and diagnosis-related groups (DRG) were analyzed for each transfusion recipient receiving red blood cell concentrates (RBC) in 2020.In 2020, a total of 70,555 red blood cell concentrates (RBCs) were transfused in MWP, corresponding to a decrease of -14.6% compared to 2015. For 55,213 transfused RBCs (78.3%) DRGs of RBC recipients were available. Overall, transfusion demands in medical patients increased by 2.6% since 2015. Four DRG major diagnostic categories are responsible for almost 2/3 (58.7%) of the transfusion demand: diseases of the digestive system (15.7%, G), musculoskeletal diseases (15.0%, I), special cases (14.3 %; A, e.g. ventilation) and diseases of the circulatory system (13.6%, F) (Fig. 1). In female patients, most RBCs were transfused to patients with musculoskeletal diseases (22.5%,I), whereas in males special cases showed the highest demand (17.3%,A).Patient demographics, gender, and diagnosis-related groups are items that allow to identify new developments in blood transfusion demands over time. Monitoring of these items will be crucial to design management strategies addressing transfusion requirements in an aging population.Dr. Schönborn was supported within the Gerhard-Domagk-Research-Program by the University Medicine Greifswald.Andreas Greinacher reports grants and non-financial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc., Rovi, Sagent, Biomarin/Prosensa, personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, Werfen (Instrumentation Laboratory), and non-financial support from Boehringer Ingelheim, Portola, Ergomed, GTH e.V., Universitätsmedizin Greifswald is one of the owners of a patent for a solid phase assay to detect HIT and VITT antibodies.All other authors declare no conflict of interest.

Abstract lecture

Outpatient Red Cell Transfusion in Patients with Blood disorders or Cancer: The difference between one or two units of red blood cell concentrates

Ambulante Erythrozytentransfusion bei hämato-onkologischen Patienten: Der Unterschied zwischen einer oder zwei Einheiten von Erythrozytenkonzentraten

There are significantly fewer studies on red blood cell (RBC) transfusion in the outpatient setting than in the inpatient setting. In this retrospective study, we investigate factors influencing transfusion strategy and transfusion triggers in outpatients. The here presented data, focused on the comparison of a strategy of transfusion of one (n=919 visits) versus two RBC (n=2752 visits) in outpatients with cancer or blood disorders (n=571).Data on outpatient RBC transfusions performed in the years 2021 and 2022 were retrospectively collected in a single centre. Multiple linear regression or multiple logistic regression was used to determine factors that contribute to decision making for RBC transfusion. For the logistic regression data is given as Odds Ratio (OR) and 95% confidence intervals. Data are given as median and interquartile ranges if not stated otherwise. N=778 patients with n=7296 visits are included in the whole cohort of the study. One unit was transfused in n=919 visits and 2 units in n=2752 visits.The strongest factor to avoid RBC transfusion was a higher Hb with an OR of 0.13 (0.11-0.14). The OR was highest for fatigue (6.0, 4.9-7.3) and dyspnoea (2.1, 1.7-2.8). Increased OR was observed for concomitant heart disease (1.6, 1.3-2.0) and male sex (1.3, 1.1-1.6). Age had a minor effect (1.04, 1.03-1.04). Two instead of 1 RBC units were more likely to be given in females and patients with dyspnoea, fatigue or cytopenia. Selecting episodes with transfusion intervals ≤ 28 days showed distances of 9 days after 1 RBC and 14 days after 2 RBC units. A linear modelling showed shorter intervals in males, in cases with lower post-Hb levels, lower reticulocyte counts and presence of dyspnoea, concomittant transfusion of Plts and administration of 1 instead of 2 RBC units. In this model the transfusion of 2 instead of 1 RBC units prolonged the interval by 2.1 days.This is the largest published cohort in outpatient transfusions. Different factors contribute to the transfusion of RBC with Hb-level and the occurrence of symptoms among them. Transfusion intervals were shortened by accompanying Plt transfusions, male sex and lower Hb. The potential of reducing RBC units by a strategy of administering 1 unit per visit can be estimated at 21-42% based on our retrospective data. However, this potential needs to be determined in a prospective clinical trial.All authors are employed by a blood transfusion service.

Univ.-Prof. Dr. med.

Clinical relevance of platelet function testing in cardiac surgery

Klinische Relevanz der Thrombozytenfunktionsdiagnostik in der Kardiochirurgie

Bleeding therapy with platelet (PLT) concentrates is routinely guided by the PLT count (PLT-C). However, doubts are increasing as to the suitability of the PLT-C for reflection of the haemostatic PLT capacity. We used cardiac surgery as a model to compare PLT-C with function (PLT-F).Impedance aggregometry (Multiplate®, TRAPtest) was used to assess PLT function in 3.212 cardiac surgery patients. Thrombin receptor activating peptide (TRAP) together with PLT count was assessed sequentially at incision (start, "S") and before administration of protamine ("bP"), as well as during haemostasis after protamine application ("aP"). Subgroup analyses comprised both surgery type as well as comparison of bypass surgery with or without heart lung machine (HLM). Red blood cell concentrate (RBC) transfusion needs during the intra-operative and 24hrs postoperative period were put into relation with aggregation findings.The relation from "S" to "bP" shows the PLT count to decrease in 83.8% of patients whereas aggregation capacity (TRAP) increases in 55.6% of patients. This mobilisation of PLT function is strongly and rapidly reversible in 68.0% of patients with aggregation values dropping below the values at "S". Significant differences in aggregation dynamics are found with respect to the type of surgery. Unexpectedly, HLM induced decrease of platelet function is moderate and observed only after departure from the extracorporeal circulation (p&lt;0.001), but not at "bP" (p&gt;0.05). RBC transfusion volumes are correlated inversely with aggregation capacities and dynamics independently from PLT counts. Notably this is true even for the PLT-F at "S".PLT haemostasis as judged by aggregometry deviates from PLT count. Aggregometry allows for inter-individual as well as surgery type-specific distinction of PLT function. HLM induced loss of platelet function is delayed until departure from the HLM. According to an inverse correlation of aggregometry with RBC transfusion, PLT function predicts bleeding intensity. PLT transfusion tailored by aggregometric testing may allow for optimisation of intra-operative patient blood management.There is no conflict of interest.

Procoagulant platelets – Unrecognized potential for bleeding prevention in hemophilia A?

Prokoagulante Thrombozyten – Ein bisher unbeachtetes Potential zur Reduktion der Blutungsneigung in Patienten mit Hämophilie A?

In patients with hemophilia A (PWH), bleeding severity generally correlates with the residual FVIII levels. However, different bleeding phenotypes can be observed in patients with equal FVIII levels, suggestion that other parameters may contribute to the variable bleeding phenotype. In this study, we investigate the role of procoagulant platelets (PLTs) in patients with hemophilia.The potential to form procoagulant PLTs was investigated in PWH and healthy controls (HC) by testing CD62p and Annexin-V (PS) double staining and analysis by flow cytometry (FC) in presence of buffer and after stimulation with TRAP/CVX. We also measured thrombin generation (TG) in a calibrated automated thrombinoscope in platelet rich plasma (PRP) and platelet poor plasma (PPP) samples from PWH and controls.The procoagulant potential of platelets after stimulation with TRAP/CVX (Increase in procoagulant platelets compared to the buffer reaction) in PWH (mean±SEM: 55.32±7.29) are significantly higher compared to healthy controls (HC) (31.97±4.04, p=0.0076). In TG we demonstrate the influence of this procoagulant potential on thrombin generation by first showing the dependence with the inhibition of lactadherin. In low FVIII environment (&lt;=10%) the lag time (Buffer vs. Lactadherin mean±SEM: 5.8±0.15 vs. 7.29±0.3, p=0.0037) and time to peak (ttpeak) (23.96±1.4 vs. 38.9±3.9, p=0.0148) are significantly prolonged and the thrombin peak is significantly lower (p=0.0095) compared to normal factor levels. In HC lag time and ttpeak are were not affected by PS inhibition (data p=0.3, and p=0.07, respectively) indicating that the PS dependency is stronger in low FVIII environments. Activation of PRP samples with TRAP-6+CVX heightened the TG of HC, as well as in PWH.A high proportion of procoagulant platelet formation improves TG in hemophilia patients. This indicates a crucial role of procoagulant PLTs in allowing hemophilia patients to compensate their FVIII deficiency.Hemophilia research is supported by Octapharma and Bayer

Anti-platelet factor 4-induced procoagulant platelets are key mediators of thrombus formation in vaccine-induced thrombotic thrombocytopenia

Anti-Plättchen Faktor 4-induzierte prokoagulante Plättchen sind essentielle Vermittler der Thrombusformation in der Vakzin-induzierten immunthrombotischen Thrombozytopenie

Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare but life-threatening prothrombotic syndrome observed in individuals following vaccination with vector-based SARS-CoV-2 vaccines. VITT is caused by antibodies (Abs) that recognize specific heparin-independent epitopes on platelet factor 4 (PF4) which results in the initiation of a multicellular driven prothrombotic response and onset of thrombosis at unusual sites. Although PF4 has been identified as the primary target of VITT Abs, the mechanisms of VITT Ab-mediated prothrombotic conditions are not fully clarified yet. This study investigates the contribution of anti-PF4-induced procoagulant PLTs in VITT Ab-mediated thrombus formation.For the investigation of VITT patient Ab-induced PLT alterations, a flow cytometry (FC)-based protocol was employed. To study the impact of VITT Ab-mediated PLT alterations on thrombus formation, a microfluidic-based ex vivo model of Ab-mediated thrombosis was developed.VITT patient IgG induced significant formation of procoagulant PLTs in a PF4-dependent manner as procoagulant PLT formation was only detected in the presence of exogenous PF4 and not in vehicle (p=0.0184). Of note, PLTs that were incubated with VITT IgG in the presence of exogenous PF4 and developed a procoagulant PLT phenotype mediated dramatic thrombus formation compared to control (p=0.0022). Most importantly, the relevance of Ab-induced procoagulant PLTs in thrombus was further confirmed as depletion of PLTs in whole blood prior to VITT IgG incubation and specific PLT PS inhibition via dynamin inhibitor MitMab both resulted in a nearly complete abolishment of Ab-induced procoagulant PLT-driven thrombus formation.Taken together, our data indicate that PF4 is the key mediator of VITT Ab-induced procoagulant PLT-driven thrombus formation ex vivo. The finding that PLT depletion and specific PLT PS inhibition results in significant reduction of VITT Ab-induced thrombus directs towards an essential contribution of Ab-induced procoagulant PLTs in VITT.J.Z., K.A and T.B. submitted a patent (PCT/E P2022/06214) for the detection of procoagulant platelets as a diagnostic tool for HIT and vaccine-induced thrombotic thrombocytopenia. All other authors have no conflicts of interest to disclose.

Raum 26

Patient Blood Management and Hemostaseology

Abstractsitzungen

Patient Blood Management and Hemostaseology

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