Blood donation sustains an estimated 118 million annual donations worldwide, and while advances in infectious disease screening have greatly reduced recipient risk, the long-term health of repeat donors has received comparatively less attention. This cumulative narrative review synthesises current evidence on the principal acquired complications of regular blood donation across all modalities — whole blood, plasmapheresis, and plateletpheresis — addressing iron deficiency, plasma protein depletion, apheresis-related lymphopenia, clonal haematopoiesis of indeterminate potential (CHIP), and micronutrient deficiency. Iron deficiency affects 15–36% of frequent whole blood donors and is systematically underdetected by haemoglobin-based screening alone; ferritin-guided interval adjustment and oral supplementation are effective countermeasures. Regular plasmapheresis depletes immunoglobulins (IgG below normal in 5–15% of high-frequency donors), albumin, and coagulation factors, compounded by cumulative citrate-mediated hypocalcaemia. Plateletpheresis causes measurable subset-selective T-cell and NK-cell depletion, with CD4⁺ counts below clinically significant thresholds in a minority of the most frequent donors and attenuated vaccine responses documented in affected individuals. Emerging molecular data indicate that repeated haematopoietic stress selectively enriches low-risk, EPO-responsive DNMT3A clonal variants without increasing malignant risk. Regular blood donation if properly applied is a safe process. Across all domains, current eligibility criteria based on haemoglobin alone turn out to be inadequate; a shift to multiparameter, individualised and targeted donor monitoring — integrating ferritin, serum proteins, differential leucocyte counts, and micronutrient profiling — is both evidence-based and ethically imperative to sustain the long-term health of voluntary donor populations.

ABSTRACT:Managing patients with suspected heparin-induced thrombocytopenia (HIT) poses significant clinical challenges. Limited evidence exists on how management decisions impact clinical outcomes, leading to treatment recommendations based on low-certainty evidence. This study aimed to evaluate the treatment strategies and clinical outcomes of patients with suspected HIT in a contemporary multicenter cohort. We conducted a prospective, multicenter cohort study including consecutive patients with suspected HIT from 11 centers. Patients were stratified into 3 groups: (1) HIT confirmed, (2) HIT-negative but heparin/platelet factor 4 (PF4) antibody-positive, and (3) HIT-negative without antibodies. Clinical and laboratory data were systematically collected. HIT was diagnosed using the washed-platelet heparin-induced platelet activation test as the reference standard. Among 1393 patients (46% female, median age 67 years), HIT was confirmed in 119 (8.5%). Most patients were in intensive care (37%), or had undergone cardiac surgery (32%). Argatroban was the predominant treatment (70%), and platelet recovery occurred in 77% of patients with HIT. Among patients with HIT, subsequent venous thromboembolism occurred in 23%, arterial thromboembolism in 9%, major bleeding in 12.6%, and mortality in 18%, with no significant differences between anticoagulants. Treatment with argatroban, bivalirudin, or direct oral anticoagulants (DOACs) significantly reduced arterial thromboembolism risk. Outcomes did not differ between patients who were HIT-negative with or without heparin/PF4 antibodies. HIT, as well as the mere suspicion of HIT, remains a serious condition with a high risk of adverse outcomes, including death. Our findings provide further evidence supporting the effectiveness of DOACs, argatroban, and bivalirudin in reducing arterial thromboembolism risk.

Background: Following the current guidelines, immunoassays for the diagnosis of heparin-induced thrombocytopenia (HIT) are interpreted dichotomously, with test results categorized as either positive or negative. However, the extent to which test results hold diagnostic significance across the entire dynamic range remains unclear. Objectives: We utilized data from the prospective towards precise and rapid diagnosis of heparin-induced thrombocytopenia study, comprising 1393 consecutive patients with suspected HIT, to assess the diagnostic significance of 2 heparin/platelet factor 4 immunoassay test results across their respective dynamic ranges: HemoSil Acustar HIT IgG (chemiluminescence immunoassay [CLIA]) and Lifecodes PF4 immunoglobulin G (enzyme-linked immunosorbent assay [ELISA]). Methods: HIT diagnosis was determined by a washed platelet heparin-induced platelet activation assay. For each measurement point in the dataset, we computed likelihood ratios (LRs), sensitivities, and specificities. To provide posttest probabilities for indi- vidual test results, we calculated interval-specific LRs and integrated them into a web- based calculator. Results: The prevalence of HIT was 8.5% (n = 119). An LR of >= 10 was first achieved at 0.3% of the dynamic range (0.4 U/mL; CLIA) and then at 16% (0.64 optical density; ELISA). An LR of >= 100 was present at 9.4% (12 U/mL; CLIA) and 75.0% (3.0 optical density; ELISA). The slope of the linear regression line (LR ti dynamic range) was 9.5 (CLIA) and 0.9 (ELISA). Conclusion: Despite both immunoassays showing an association between results and diagnostic significance, the strength of the association varies by assay. CLIA has a larger increase per measurement unit. Posttest probabilities for individual patients can be estimated using a web-based calculator: https://pcd-research.shinyapps.io/Bayesian Calculator/.

Hematopoietic stem cell transplantation (HCT) represents a curative treatment option for certain malignant and nonmalignant hematological diseases. Conditioning regimens before HCT, the development of graft-versus-host disease (GVHD) in the allogeneic setting, and delayed immune reconstitution contribute to early and late complications by inducing tissue damage or humoral alterations. Hemostasis and/or the complement system are biological regulatory defense systems involving humoral and cellular reactions and are variably involved in these complications after allogeneic HCT. The hemostasis and complement systems have multiple interactions, which have been described both under physiological and pathological conditions. They share common tissue targets, such as the endothelium, which suggests interactions in the pathogenesis of several serious complications in the early or late phase after HCT. Complications in which both systems interfere with each other and thus contribute to disease pathogenesis include transplant-associated thrombotic microangiopathy (HSCT-TMA), sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD), and GVHD. Here, we review the current knowledge on changes in hemostasis and complement after allogeneic HCT and how these changes may define clinical impact.

Patients with active cancer have an increased risk of arterial occlusion, venous thromboembolism (VTE) and bleeding events. Traditionally, these patients were treated with low-molecular-weight heparins to reduce the risk of VTE, while vitamin K antagonists (VKAs) were used to prevent ischemic stroke in patients with atrial fibrillation (AF). A recent addition to this anticoagulant landscape is the new class of direct oral anticoagulants (DOACs), which not only effectively prevent and treat VTE but also reduce the risk of arterial thromboembolism and stroke in patients with AF. DOACs have several advantages over heparins or VKAs. This includes rapid onset of action, no requirement for routine laboratory monitoring and fewer drug-drug interactions. In this review, we focus on the use of anticoagulants in cancer patients, providing an overview of the available anticoagulants, and discuss coagulation assays, tools for assessing thrombosis/bleeding risk and perioperative considerations. Cancer patients represent an extremely heterogenous and challenging population to treat with anticoagulants. The risk/benefit evaluation has to be individualized for each patient, requiring a thorough understanding of the coagulation system, the available compounds and monitoring tests, as well as the potential influence of the underlying cancer and anti-neoplastic treatments. PEER REVIEWED ARTICLE Peer reviewers: Dr Lukas Graf, Haemostasis and Haemophilia Centre St. Gallen, St. Gallen, Switzerland One anonymous peer reviewer Received on May 30, 2024; accepted after peer review on October 14, 2024; published online on October 30, 2024.

Background: Von Willebrand factor (vWF) is an important part of blood coagulation since it binds platelets to each other and to endothelial cells. In traumatic and surgical haemorrhage, both blood cells and plasmatic factors are consumed, leading to consumption coagulopathy and fluid resuscitation. This often results in large amounts of crystalloids and blood products being infused. Additional administration of vWF complex and platelets might mitigate this problem. We hypothesize that administration of vWF concentrate additionally to platelet concentrates reduces blood loss and the amount of blood products (platelets, red blood cells [RBC], fresh frozen plasma [FFP]) administered. Methods: We conducted a monocentric 6-year retrospective data analysis of cardiac surgery patients. Included were all patients receiving platelet concentrates within 48 h postoperatively. Patients who additionally received vWF concentrates were allocated to the intervention group and all others to the control group. Groups were compared in mixed regression models correcting for known confounders, based on nearest neighbour propensity score matching. Primary endpoints were loss of blood (day one and two) and amount of needed blood products on day one and two (platelets, RBC, FFP). Secondary endpoints were intensive care unit (ICU) and in-hospital length of stay, ICU and in-hospital mortality, and absolute difference of platelet counts before and after treatment. Results: Of 497 patients analysed, 168 (34%) received vWF concentrates. 121 patients in both groups were considered for nearest neighbour matching. Patients receiving additional vWF were more likely to receive more blood products (RBC, FFP, platelets) in the first 24 h after surgery and had around 200 mL more blood loss at the same time. Conclusion: In this retrospective analysis, no benefit in additional administration of vWF to platelet concentrates on perioperative blood loss, transfusion requirement (platelets, RBC, FFP), length of stay, and mortality could be found. These findings should be verified in a prospective randomized controlled clinical trial (www.clinicaltrials.gov identifier NCT04555785).

Hematopoietic stem cell transplantation (HSCT) and chimeric antigen receptor-T (CAR-T) immunotherapy are widely used for the management of hematological malignancies. HSCT can be complicated by endothelial injury syndromes, such as HSCT-thrombotic microangiopathy (HSCT-TMA) and sinusoidal obstructive syndrome/ veno-occlusive disease (SOS/VOD), which are life-threatening. Moreover, venous thromboembolic events (VTEs) are common in HSCT recipients due to endothelial injury, use of central venous catheters, prolonged hospitalization, and the development of a procoagulant state. VTEs have also been reported post-CAR-T infusion. The management of thrombotic events in these patients is challenging, due to the high risk of bleeding that is present. CAR-T immunotherapy might be followed by toxicities, such as cytokine release syndrome (CRS) and immune effector cell-associated neuro-toxicity syndrome (ICANS). Endothelial dysfunction is implicated in the pathogenesis of these syndromes. Early recognition and management of the above complications are crucial for better outcomes for our patients.

ImportanceHeparin-induced thrombocytopenia (HIT) is a life-threatening condition that requires urgent diagnostic clarification. However, knowledge of the diagnostic utility of the recommended diagnostic tests is limited in clinical practice.ObjectiveTo evaluate the current diagnostic practice for managing the suspicion of HIT.Design, Setting, and ParticipantsThis prospective diagnostic study was conducted from January 2018 to May 2021 among consecutive patients with suspected HIT from 11 study centers in Switzerland, Germany, and the United States. Detailed clinical data and laboratory information were recorded. Platelet factor 4/heparin antibodies were quantified using an automated chemiluminescent immunoassay (CLIA). A washed-platelet heparin-induced platelet activation (HIPA) test was used as a reference standard to define HIT.ExposuresSuspicion of HIT.Main Outcomes and MeasuresThe primary outcome was the diagnostic accuracy of the 4Ts score, the CLIA, and the recommended algorithm serially combining both tests.ResultsOf 1448 patients included between 2018 and 2021, 1318 were available for the current analysis (median [IQR] age, 67 [57-75] years; 849 [64.6%] male). HIPA was positive in 111 patients (prevalence, 8.4%). The most frequent setting was intensive care unit (487 [37.0%]) or cardiovascular surgery (434 [33.0%]). The 4Ts score was low risk in 625 patients (46.8%). By 2 × 2 table, the numbers of patients with false-negative results were 10 (9.0%; 4Ts score), 5 (4.5%; CLIA), and 15 (13.5%; recommended diagnostic algorithm). The numbers of patients with false-positive results were 592 (49.0%; 4Ts score), 73 (6.0%; CLIA), and 50 (4.1%; recommended diagnostic algorithm), respectively.Conclusions and RelevanceIn this diagnostic study of patients suspected of having HIT, when the recommended diagnostic algorithm was used in clinical practice, antibody testing was required in half the patients. A substantial number of patients were, however, still misclassified, which could lead to delayed diagnosis or overtreatment. Development of improved diagnostic algorithms for HIT diagnosis should be pursued.

New analytical techniques can assess hundreds of proteins simultaneously with high sensitivity, facilitating the observation of their complex interplay and role in disease mechanisms. We hypothesized that proteomic pro filing targeting proteins involved in thrombus formation, in flammation, and the immune response would identify potentially new biomarkers for heparin-induced thrombocytopenia (HIT). Four existing panels of the Olink proximity extension assay covering 356 proteins involved in thrombus formation, in flammation, and immune response were applied to randomly selected patients with suspected HIT (con firmed HIT, n = 32; HIT ruled out, n = 38; and positive heparin/platelet factor 4 [H/PF4] antibodies, n = 28). The relative difference in protein concentration was analyzed using a linear regression model adjusted for sex and age. To con firm the test results, soluble P-selectin was determined using enzyme-linked immunosorbent assay (ELISA) in above mentioned patients and an additional second data set (n = 49). HIT was de fined as a positive heparin-induced platelet activation assay (washed platelet assay). Among 98 patients of the primary data set, the median 4Ts score was 5 in patients with HIT, 4 in patients with positive H/PF4 antibodies, and 3 in patients without HIT. The median optical density of a polyspeci fic H/PF4 ELISA were 3.0, 0.9, and 0.3. Soluble P-selectin remained statistically signi ficant after multiple test adjustments. The area under the receiver operating characteristic curve was 0.81 for Olink and 0.8 for ELISA. Future studies shall assess the diagnostic and prognostic value of soluble P-selectin in the management of HIT.

Die hämostatischen Funktionen werden auf natürliche Art im Verlauf der Schwangerschaft verstärkt. Diese Veränderung erhöht spürbar neben dem erwünschten blutstillenden Effekt auch die Inzidenz von unerwünschten Thrombosen. Ursächlich spielen hier Venenstauung, Gefäßwandschäden und Hyperkoagulabilität interaktiv eine kausale Rolle. Thrombosen treten in allen 3 Trimestern auf, etwa 5-mal häufiger als außerhalb der Schwangerschaft, die Inzidenz ist im Wochenbett am höchsten. Zusätzliche transiente oder persistierende Risikofaktoren können das Thromboserisiko weiter erhöhen. Gegen die Thrombosen kann der Therapeut prophylaktisch oder therapeutisch wirken. Schwerpunkt dieser Intervention stellt die parenterale Antikoagulation mit Heparinen dar. Im folgenden Text werden Pathogenese, Diagnose und Behandlung der Thrombosen in Schwangerschaft und Wochenbett veranschaulicht. Vorhandene nationale und internationale Leitlinien werden dargestellt und vergleichsweise als Praxistipps zusammengefasst.

Paroxysmal Nocturnal Hemoglobinuria (PNH) constitutes a rare bone marrow failure syndrome characterized by hemolytic anemia, thrombotic events (TEs), and bone marrow aplasia of variable degrees. Thrombosis is one of the major clinical manifestations of the disease, affecting up to 40% of individuals with PNH. Venous thrombosis is more prevalent, affecting mainly unusual sites, such as intrabdominal and hepatic veins. TEs might be the first clinical manifestation of PNH. Complement activation, endothelial dysfunction, hemolysis, impaired bioavailability of nitric oxide, and activation of platelets and neutrophils are implicated in the pathogenesis of TEs in PNH patients. Moreover, a vicious cycle involving the coagulation cascade, complement system, and inflammation cytokines, such as interleukin-6, is established. Complement inhibitors, such as eculizumab and ravulizumab (C5 inhibitors), have revolutionized the care of patients with PNH. C5 inhibitors should be initiated in patients with PNH and thrombosis, while they constitute a great prophylactic measure for TEs in those individuals. Anticoagulants, such as warfarin and low-molecular-weight heparin, and, in selected cases, direct oral anticoagulants (DOACs) should be used in combination with C5 inhibitors in patients who develop TEs. Novel complement inhibitors are considered an alternative treatment option, especially for those who develop extravascular or breakthrough hemolysis when terminal inhibitors are administered.

Background Diagnosing heparin-induced thrombocytopenia (HIT) at the bedside remains challenging, exposing a significant number of patients at risk of delayed diagnosis or overtreatment. We hypothesized that machine-learning algorithms could be utilized to develop a more accurate and user-friendly diagnostic tool that integrates diverse clinical and laboratory information and accounts for complex interactions. Methods We conducted a prospective cohort study including 1393 patients with suspected HIT between 2018 and 2021 from 10 study centers. Detailed clinical information and laboratory data were collected, and various immunoassays were conducted. The washed platelet heparin-induced platelet activation assay (HIPA) served as the reference standard. Findings HIPA diagnosed HIT in 119 patients (prevalence 8.5%). The feature selection process in the training dataset (75% of patients) yielded the following predictor variables: (1) immunoassay test result, (2) platelet nadir, (3) unfractionated heparin use, (4) CRP, (5) timing of thrombocytopenia, and (6) other causes of thrombocytopenia. The best performing models were a support vector machine in case of the chemiluminescent immunoassay (CLIA) and the ELISA, as well as a gradient boosting machine in particle-gel immunoassay (PaGIA). In the validation dataset (25% of patients), the AUROC of all models was 0.99 (95% CI: 0.97, 1.00). Compared to the currently recommended diagnostic algorithm (4Ts score, immunoassay), the numbers of false-negative patients were reduced from 12 to 6 (-50.0%; ELISA), 9 to 3 (-66.7%, PaGIA) and 14 to 5 (-64.3%; CLIA). The numbers of false-positive individuals were reduced from 87 to 61 (-29.8%; ELISA), 200 to 63 (-68.5%; PaGIA) and increased from 50 to 63 (+29.0%) for the CLIA. Interpretation Our user-friendly machine-learning algorithm for the diagnosis of HIT (https://toradi-hit.org) was substantially more accurate than the currently recommended diagnostic algorithm. It has the potential to reduce delayed diagnosis and overtreatment in clinical practice. Future studies shall validate this model in wider settings. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd.

BACKGROUND: Evidence-based hemostatic treatment for intracerebral hemorrhage (ICH) associated with non–vitamin K antagonist oral anticoagulants (NOACs) is lacking. Tranexamic acid (TXA) is an antifibrinolytic drug potentially limiting hematoma expansion. We aimed to assess the efficacy and safety of TXA in NOAC-ICH. METHODS: We performed a double-blind, randomized, placebo-controlled trial at 6 Swiss stroke centers. Patients with NOAC-ICH within 12 hours of symptom onset and 48 hours of last NOAC intake were randomized (1:1) to receive either intravenous TXA (1 g over 10 minutes followed by 1 g over 8 hours) or matching placebo in addition to standard medical care via a centralized Web-based procedure with minimization on key prognostic factors. All participants and investigators were masked to treatment allocation. Primary outcome was hematoma expansion, defined as ≥33% relative or ≥6 mL absolute volume increase at 24 hours and analyzed using logistic regression adjusted for baseline hematoma volume on an intention-to-treat basis. RESULTS: Between December 12, 2016, and September 30, 2021, we randomized 63 patients (median age, 82 years [interquartile range, 76–86]; 40% women; median hematoma volume, 11.5 [4.8–27.4] mL) of the 109 intended sample size before premature trial discontinuation due to exhausted funding. The primary outcome did not differ between TXA (n=32) and placebo (n=31) arms (12 [38%] versus 14 [45%]; adjusted odds ratio, 0.63 [95% CI, 0.22–1.82]; P =0.40). There was a signal for interaction with onset-to-treatment time ( P interaction =0.024), favoring TXA when administered within 6 hours of symptom onset. Between the TXA and placebo arms, the proportion of participants who died (15 [47%] versus 13 [42%]; adjusted odds ratio, 1.07 [0.37–3.04]; P =0.91) or had major thromboembolic complications within 90 days (4 [13%] versus 2 [6%]; odds ratio, 1.86 [0.37–9.50]; P =0.45) did not differ. All thromboembolic events occurred at least 2 weeks after study treatment, exclusively in participants not restarted on oral anticoagulation. CONCLUSIONS: In a smaller-than-intended NOAC-ICH patient sample, we found no evidence that TXA prevents hematoma expansion, but there were no major safety concerns. Larger trials on hemostatic treatments targeting an early treatment window are needed for NOAC-ICH. REGISTRATION: URL: https://clinicaltrials.gov ; Unique identifier: NCT02866838.

Background: Anecdotal reports suggest that the correlation between heparin/platelet factor 4 (PF4) antibody assays for the diagnosis of heparin-induced thrombocytopenia (HIT) is limited. Objectives: To investigate the correlation between widely used assays and examine possible factors contributing to variability.Methods: This is a large, prospective cohort study with 10 participating tertiary care hospitals including 1393 patients with suspected HIT in clinical practice. HIT was defined by a positive heparin-induced platelet activation (HIPA) assay (washed platelet reference standard test). Three different immunoassays were used to measure heparin/ PF4 antibodies: chemiluminescent immunoassay, enzyme-linked immunosorbent assay, and particle gel immunoassay. Various factors that could influence the assays were examined: sex (male or female), age (<65 years or =65 years), unfractionated heparin exposure, presence of thrombosis, cardiovascular surgery, and intensive care unit. Spearman's correlation coefficients were calculated. Z-scores and diagnostic odds ratios were determined in the aforementioned subgroups of patients.Results: Among 1393 patients, 119 were classified as HIT-positive (prevalence, 8.5%). The median 4Ts score was 5 (IQR, 4-6) in patients with HIT compared with 3 (IQR, 2-4) in patients without HIT. Correlations (rs) between immunoassays were weak (0.53-0.65). Inconsistencies between immunoassays could not be explained by further analyses of z-scored test results and diagnostic odds ratios in subgroups of patients.Conclusion: The correlation between widely used heparin/PF4 antibody assays was weak, and key factors could not explain this variability. Standardization of immuno-assays is requested to improve comparability.

INTRODUCTION:von Willebrand Factor (vWF) is a key protein mediating platelet adhesion on the surface of damaged endothelia. To the best of our knowledge, no trial exists that investigated the effect of platelet transfusion in combination with the administration of balanced vWF in severe blood loss, despite being widely used in clinical practice. The Basel Will-Plate study will investigate the impact of the timely administration of balanced vWF (1:1 vWF and FVIII) in addition to platelet transfusion on the need for blood and coagulation factor transfusion in patients admitted to the intensive care unit (ICU) who suffer from severe bleeding. The study hypothesis is based on the assumption that adding balanced vWF to platelets will reduce the overall need for transfusion of blood products compared to the transfusion of platelets alone.METHODS AND ANALYSIS:The Will-Plate study is an investigator-initiated, single-centre, double-blinded randomised controlled clinical trial in 120 critically ill patients needing platelet transfusion. The primary outcome measure will be the number of fresh frozen plasma (FFP) and red blood cell (RBC) transfusions according to groups. Secondary outcome measures include the number of platelet concentrates transfused within the first 48 h after treatment of study medication, quantity of blood loss in the first 48 h after treatment with the study medication, length of stay in ICU and hospital, number of revision surgeries for haemorrhage control, ICU mortality, hospital mortality, 30-day mortality and 1-year mortality. Patients will be followed after 30 days and 1 year for activities of daily living and mortality assessment. The sample size was calculated to detect a 50% reduction in the number of blood products subsequently transfused within 2 days in patients with Wilate® compared to placebo.ETHICS AND DISSEMINATION:This study has been approved by the Ethics Committee of Northwestern and Central Switzerland and will be conducted in compliance with the protocol, the current version of the Declaration of Helsinki, the ICH-GCP or ISO EN 14155 (as far as applicable) and all national legal and regulatory requirements. The study results will be presented at international conferences and published in a peer-reviewed journal.TRIALS REGISTRATION:ClinicalTrials.gov NCT04555785.PROTOCOL VERSION:Clinical Study Protocol Version 2, 01.11.2020. Registered on Sept. 21, 2020.

Zusammenfassung Einleitung Nabelschnurblut (NSB) enthält hämatopoetische Stammzellen mit therapeutischem Potenzial und einzigartigen zellulären Eigenschaften. Aufgrund der begrenzten Anzahl an Stammzellen im NSB (Surrogatmarker total nucleated cells, TNC) eignet sich nur jede fünfte Spende für eine Transplantation. Ziel dieser Studie war es, zu untersuchen, ob prädiktive Faktoren für eine TNC-Zahl über der 99. Perzentile existieren. Material und Methodik Retrospektive Datenanalyse der 100 größten NSB-Spenden (Top100-Kohorte) aus 2299 registrierten Einheiten. Unterschiede zwischen maternalen, fetalen und geburtshilflichen Faktoren wurden analysiert und mit einer standardisierten Kohorte von 731 NSB-Spenden verglichen. Ergebnisse Das mütterliche Alter und der BMI in der Top100-Kohorte waren höher als in der Vergleichskohorte (32 vs. 31 Jahre, p=0,007; 30 kg/m2 vs. 29 kg/m2, p=0,024). Es gab mehr Erstgebärende (76,0 vs. 62,8%, p=0,013) und Gestationsdiabetikerinnen (5,00 vs. 1,65%, p=0,044). Die Schwangerschaftswoche, das Geburtsgewicht, der Anteil vaginal-operativer Geburten und sekundärer Sectiones war in der Top100-Kohorte höher (40+4 vs. 40+1 SSW, p=0,002), (3700 vs. 3450 g, p<0,001), (53,0 vs. 22,7%, p<0,001) (10 vs. 6,2%, p=0,014). Fazit Für eine erfolgreiche Transplantation ist die Höhe der TNC-Zahl entscheidend. Vaginal-operative Entbindungen, sekundäre Sectiones und ein Geburtsgewicht über 3700 g sind günstige Faktoren. Gerade bei Geburten mit einem pathologischen Verlauf sollte nach sicherer Versorgung von Mutter und Kind nicht auf eine Entnahme verzichtet werden.

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by functional insufficiency of coagulation factor VIII (FVIII). Autoantibodies targeting FVIII may neutralize its procoagulant effect, thereby causing severe bleeding. Such inhibitory autoantibodies have been detected in autoimmune diseases, pregnancy, infections, or malignant diseases. Older age and certain drugs are known co‐risk factors.1.Tiede A. Zieger B. Lisman T. Acquired bleeding disorders.Haemophilia. 2021; 27: 5-13Crossref PubMed Scopus (4) Google Scholar To our knowledge, only two reported cases document AHA diagnosed 8 and 20 days after influenza vaccination.2.Moulis G. Pugnet G. Bagheri H. et al.Acquired factor VIII haemophilia following influenza vaccination.Eur J Clin Pharmacol. 2010; 66: 1069-1070Crossref PubMed Scopus (22) Google Scholar, 3.Pirrotta M.T. Bernardeschi P. Fiorentini G. A case of acquired haemophilia following H1N1 vaccination.Haemophilia. 2011; 17: 815PubMed Google Scholar Vaccines have been rarely associated with autoimmune disease occurrence or disease flares. Recently, vaccine‐induced immune thrombocytopenia and thrombosis (VITT) has been characterized as a new entity.4.Pavord S. Scully M. Hunt B.J. et al.Clinical features of vaccine‐induced immune thrombocytopenia and thrombosis.N Engl J Med. 2021; 385: 1680-1689Crossref PubMed Scopus (248) Google Scholar Immunological studies established a pathogenetic role of platelet‐activating autoantibodies targeting platelet factor 4 (PF4) in VITT. VITT‐associated anti‐PF4‐IgG were not cross‐reactive with the SARS‐CoV2 spike antigen, suggesting that the vaccine‐specific antibody response is not directly causing VITT.5.Greinacher A. Selleng K. Mayerle J. et al.Anti‐platelet factor 4 antibodies causing VITT do not cross‐react with SARS‐CoV‐2 spike protein.Blood. 2021; 138: 1269-1277Crossref PubMed Scopus (65) Google Scholar A recent study linked the occurrence of VITT to the interaction of the adenoviral vector with the coxsackie and adenovirus receptor and PF4, thus instigating memory B cell differentiation and the release of anti‐PF4 auto‐antibodies.6.Baker A.T. Boyd R.J. Sarkar D. et al.ChAdOx1 interacts with CAR and PF4 with implications for thrombosis with thrombocytopenia syndrome.Sci Adv. 2021; 7Crossref Scopus (60) Google Scholar Our group recently reported three cases of AHA occurring in temporal association with mRNA COVID‐19 vaccine immunizations.7.Cittone M.G. Battegay R. Condoluci A. et al.The statistical risk of diagnosing coincidental acquired hemophilia A following anti‐SARS‐CoV‐2 vaccination.J Thromb Haemost. 2021; 19: 2360-2362Crossref PubMed Scopus (21) Google Scholar Statistically, we found no strong evidence that the AHA incidence during the COVID vaccination campaign in Switzerland was substantially higher than the background AHA incidence. In our previous report, we did not address the possibility of FVIII cross‐reactivity of the vaccine‐induced anti‐spike IgG (anti‐S‐IgG). Excluding cross‐reactivity of anti‐foreign IgG with a self‐antigen is critical to refute 'molecular mimicry' in the immunopathogenesis of an autoimmune disease. Here, we studied the binding, function, and cross‐reactivity of the vaccine‐induced anti‐S‐IgG in our previously reported three cases of AHA diagnosed in temporal association with COVID vaccination.7.Cittone M.G. Battegay R. Condoluci A. et al.The statistical risk of diagnosing coincidental acquired hemophilia A following anti‐SARS‐CoV‐2 vaccination.J Thromb Haemost. 2021; 19: 2360-2362Crossref PubMed Scopus (21) Google Scholar The main goal was to address whether the vaccine‐induced antibody response against the SARS‐CoV2 spike protein may exhibit FVIII inhibitory functions. The sequence alignment of the FVIII (UniProtKB accession number P00451) and the SARS‐CoV2 spike protein (UniProtKB accession number P0DTC2) revealed minimal sequence similarity. We identified one region (amino acid position 540–570 within the A2 domain of FVIII) with 13/35(37%) amino acid sequence similarity using the NCBI blast sequence alignment tool. In silico antigenic peptide prediction (http://imed.med.ucm.es/Tools/antigenic.pl) revealed 95 and 63 antigenic determinants in the FVIII and spike protein, respectively. Of those, a single overlapping potential epitope was present in both proteins, locating to the region with the sequence similarity (Figure 1A; SDPRCLTRYYS‐S in the FVIII sequence [FVIII 543–554]; underlined amino acids indicate homology to the SARS‐CoV2 spike protein). Since only a few amino acids are shared between the FVIII and spike protein in this region, the likelihood of a cross‐reactive B cell epitope is, however, low. Next, we addressed this experimentally. The presence of vaccine‐specific antibodies is a pre‐requisite for a potential cross‐reactivity to FVIII. Serological analyses proved considerable anti‐Spike IgG (anti‐S‐IgG) levels in the serum of all three vaccinated patients (Figure 1B). Anti‐S‐IgG is the only antigen‐specificity induced by the mRNA COVID vaccines. To explore the FVIII inhibitory potential of the anti‐S‐IgG fraction, we performed a bead‐based antibody pull‐down to deplete and enrich for anti‐S‐IgG (Supplementary Data). The anti‐S‐IgG enrichment and ‐depletion was confirmed in a Luminex assay using spike protein‐coated beads (Figure 1C) and in western blot loaded with recombinant spike protein (Figure 1D). Despite efficient depletion and enrichment, the 'anti‐S‐IgG enriched' fraction contained residual non‐anti‐S‐IgG based on total IgG measurements (mean total‐IgG in the anti‐S‐enriched fraction 0.49g/l. Moreover, we found traces of other serum proteins as assessed by gel electrophoresis (Figure S1). To determine which of the serum fractions contained the FVIII inhibitory factor, we first performed a mixing FVIII assay (Supplementary Methods). The non‐manipulated serum and anti‐S‐IgG‐depleted fractions showed similar FVIII inhibition of 75%. In contrast, only 35% FVIII inhibition was observed using the anti‐S‐IgG‐enriched fraction, indicating that anti‐S‐IgG was not the main mediator of FVIII inhibition in this assay (Figure 1E). The application of an enzyme‐linked immunosorbent assay for total anti‐FVIII IgG yielded detectable levels in serum and anti‐S‐IgG depleted fractions. However, the anti‐S‐IgG enriched fractions either showed no detectable (log10 titer <0.7, n = 1) or about 1.5 log10 (30‐fold, n = 2) lower titers (Figure 1F) compared to the other fractions. Furthermore, the binding of the anti‐S‐IgG enriched fraction to different therapeutic FVIII preparations, as assessed by Luminex‐based analysis, was negative or very low in all cases (Figures S2 and S3). The FVIII inhibitory potential of the binding antibodies was addressed using the Nijmegen‐Bethesda assay (NBA). Using serum rather than plasma in this study, neutralizing anti‐FVIII‐activity could not be detected within the original or derived samples of one of the patients. However, using citrated plasma, an inhibitor level of 1.01 BU/ml was previously described in the same patient.7.Cittone M.G. Battegay R. Condoluci A. et al.The statistical risk of diagnosing coincidental acquired hemophilia A following anti‐SARS‐CoV‐2 vaccination.J Thromb Haemost. 2021; 19: 2360-2362Crossref PubMed Scopus (21) Google Scholar For the other two patients, neutralizing anti‐FVIII‐activity was detected in the original and the anti‐S‐IgG depleted serum samples, while no inhibition was observed in the anti‐S‐IgG‐enriched fraction (Figure 1G). In patient AHA01, the inhibitory titer was even higher than in the original publication, which was likely due to a combination of another time‐point of sample acquisition, different sample material (serum vs. plasma), and the use of different FVIII test systems for assay read‐out.7.Cittone M.G. Battegay R. Condoluci A. et al.The statistical risk of diagnosing coincidental acquired hemophilia A following anti‐SARS‐CoV‐2 vaccination.J Thromb Haemost. 2021; 19: 2360-2362Crossref PubMed Scopus (21) Google Scholar In order to ensure assay specificity for anti‐FVIII activity, we applied control measures with respect to the use of serum instead of plasma samples (Supplementary Data). Anti‐phospholipid antibodies may interfere with functional anti‐FVIII assays.8.Scandella D. Gilbert G.E. Shima M. et al.Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid‐binding site.Blood. 1995; 86: 1811-1819Crossref PubMed Google Scholar We therefore screened all samples for anti‐phospholipid antibodies (IgG and IgM) that were found to be negative or only at threshold levels (Table S1). Furthermore, to further increase specificity, a chromogenic FVIII assay was applied to determine (remaining) FVIII‐activities.9.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar In summary, we found that (i) the likelihood of cross‐reactive epitopes between the spike protein and FVIII is low based on in silico protein structures; (ii) the anti‐S‐IgG enriched fraction showed weak FVIII cross‐reactivity in binding assays; (iii) weak cross‐binding of the anti‐S‐IgG enriched fraction did not translate into FVIII inhibition. The FVIII binding in the enriched anti‐S‐IgG fraction may have been due to residuals of anti‐FVIII‐IgG with low cross‐reactivity against the spike protein. The amount of total IgG measured in this fraction indeed indicated a substantial non‐anti‐S‐specific IgG. We conclude that AHA associated with mRNA COVID vaccination was likely not due to vaccine‐induced cross‐reactive, FVIII‐inhibiting anti‐S‐IgG. Alternatively, the broad toll‐like‐receptor stimulation by mRNA vaccines10.Teijaro J.R. Farber D.L. COVID‐19 vaccines: modes of immune activation and future challenges.Nat Rev Immunol. 2021; 21: 195-197Crossref PubMed Scopus (308) Google Scholar may cause polyclonal B cell activation and thereby trigger autoantibody production in pre‐existing self‐reactive B cell clones in persons predisposed to AHA. Indeed, several studies indicated that thymic deletion of T cell clones specific for endogenous ("self") FVIII is incomplete and that these cells may expand in persons with AHA following a corresponding immune response.11.Meunier S. Menier C. Marcon E. Lacroix‐Desmazes S. Maillère B. CD4 T cells specific for factor VIII are present at high frequency in healthy donors and comprise naive and memory cells.Blood Adv. 2017; 1: 1842-1847Crossref PubMed Scopus (17) Google Scholar While we are not aware of comparable data on B cell clones, the detection of natural anti‐FVIII antibodies in healthy subjects strongly argues for the presence of anti‐FVIII specific B cells.12.Algiman M. Dietrich G. Nydegger U.E. Boieldieu D. Sultan Y. Kazatchkine M.D. Natural antibodies to factor VIII (anti‐hemophilic factor) in healthy individuals.Proc Natl Acad Sci USA. 1992; 89: 3795-3799Crossref PubMed Scopus (127) Google Scholar Interestingly, the FVIII sequence (epitope) described above (FVIII 543–554), which shows some similarities to SARS‐CoV2 Spike, has been identified to overlap with a particularly immunogenic FVIII sequence (FVIII 545–559; patent application WO2009071886), and corresponding peptides are therefore part of a proposed strategy to induce immune tolerance in susceptible HA patients.13.Pletinckx K. Nicolson K.S. Streeter H.B. et al.Antigen‐specific immunotherapy with apitopes suppresses generation of FVIII inhibitor antibodies in HLA‐transgenic mice.Blood Adv. 2021; https://doi.org/10.1182/bloodadvances.2021004451Crossref Scopus (2) Google Scholar Thus, due to these sequence similarities, it might be speculated whether the presentation of corresponding SARS‐CoV2 Spike peptides by MHC class II led to activation of FVIII‐specific pre‐existing T cell clones. A detailed analyses of the T cell responses in the patients was, however, limited by sample availability and beyond the scope of this work as extensive T cell assays using peptide arrays would have been needed to experimentally assess this. As another limitation, we studied only three subjects and moreover cannot exclude that ethnical backgrounds or MHC haplotypes may have affected the findings, given that we only studied patients in Switzerland. On a larger scope, the here presented data, combined with our epidemiological analysis,7.Cittone M.G. Battegay R. Condoluci A. et al.The statistical risk of diagnosing coincidental acquired hemophilia A following anti‐SARS‐CoV‐2 vaccination.J Thromb Haemost. 2021; 19: 2360-2362Crossref PubMed Scopus (21) Google Scholar demonstrates that immunological phenotypes occurring related to vaccination may occur unrelated to the vaccine‐antigen. Detailed epidemiological and immunological studies, rather than single clinical case reports, are needed to advance the understanding of adverse events following vaccination. The authors declare no conflict of interest related to this work. JRH, MM, DAT, MR, JO, BP, and JM designed and performed experiments, analyzed, and interpreted the data. M.M., M.G.C, and L.G. contributed samples, helped analyze the data and edited the manuscript. JM, BG, and CTB designed the study, provided funding, analyzed the data, and drafted the manuscript. The work for this study was supported by the Uniscientia Foundation, Switzerland (to CTB).