Naturally occuring non-radioactive Calcium isotope ratios in the human body as a biomarker for bone metastasis of prostate cancer -a pilot study-
B. Brandt (Kiel, DE)
Aims: Prostate cancer (PCa) is the second most frequent malignancy in men worldwide. Although 5-year survival in patients with organ-confined PCa is nearly 100%, metastasis to the bones still remains incurable. Therefore, there is an urgent need for markers able to predict bone metastasis (BM), in order to personalize patients’ (pts) treatment. The ratios of Calcium isotopes like 44Ca and 42Ca ( δ44/42Ca ), can be used to identify unbalanced bone mineral disorders, e.g. osteoporosis. This is based on the kinetic isotope fractionation factor between blood and bones being constant in humans. In this study we tested how sensitive δ44/42Ca changes in serum reflect PCa bone metastasis. Materials and Methods: Sera from 20 pts were measured by plasma-mass spectroscopy for δ44/42Ca-values. For QC all data were compared and calibrated to international standards. The long-term standard reproducibility was in the order of 0.06 ‰. Nine pts (6 with PCa, 3 without) had no signs of bone metastasis. Eleven pts had moderate to multiple BM. BM were evaluated by PSMA-PET/CT or bone scintigraphy. BM pts received various androgen deprivation therapies, and, 4 received also taxane therapy. For osteoprotection Denosumab , Zoledronic acid and Vit D3 was given in 2, 1 and 1 pt., resp.. Results: Six of 9 pts w/o BM had normal age adjusted δ44/42Ca values, 2 showed Ca resorption and 1 Ca absorption ratios. Ca resorption occurred in 3 BM pts with moderate numbers of BM and low PSA levels (< 0.05 – 8 ng/ml). Eight pts with multiple BM and high PSA levels (31 – 1443 ng/ml) had Ca absorption. In this high δ44/42Ca subgroup 3 pts had Denosumab/ Zoledronic acid as bone protection. Unexpectedly, Vitamin D3 supplementation made no δ44/42Ca increase in 1 BM pt. Discussion (Conclusion): We applied for the first time Ca isotope ratios as a biomarker for PCa bone metastasis. Overall, 2/3 of non-metastatic pts showed balanced δ44/42Ca, whereas BM pts were found in both groups representing Ca resorption and absorption as well. From published reports it might be hypothesized that Ca resorption show earlier phases of metastases which is in agreement with the lower number of BM and PSA values of the pts. In contrary, pts with multiple metastases and high PSA levels where found in the Ca absorption group indicating the final osteoblastic/sclerotic phase of PCa bone metastases. This hypothesis might be substantiated by additional δ44/42Ca measurements from pts only displaying BM in PET-PSMA scan. The δ44/42Ca values in the absorptive range for pts with osteoprotective treatment by Denosumab and Zoledronic acid might also support this assumption. Nevertheless, an extended study is needed to control preanalytical influencing factors which may cause Ca disequilibrium. Those are benign gastrointestinal, kidney, endocrine and bone diseases and Ca supplementation. Possible influencing factors might explain why some patients in the study are misclassified by their δ44/42Ca measurements.
Influence of coagulation proteases and signalling on cognitive function
S. Zimmermann (Leipzig, DE)
Aims: Since effects of thrombin on the blood brain barrier (BBB) have been proposed, we want to define the relevance of the thrombomodulin (TM) - activated protein C (aPC) system for BBB integrity and neuronal cell function. In Germany, one million patients are treated with lifelong anticoagulant therapy (anticoagulants). We speculate that anticoagulants, via influencing coagulation factors, may interfere with neuronal function, impair cognition or convey other CNS-related side-effects. Clinical reports suggested that anticoagulation affects cognition. We want to define (i) the impact of thrombin on neuronal function and cognitive performance in mice and (ii) whether the changed behavior of the animals is a consequence of the increased coagulation activation.
Material and Methods: We performed snRNA sequencing of wildtype (WT) versus TMPro/Pro (mutation leads to endothelial dysfunction due to reduced function of the TM-protein C pathway murine brains. We use mice with genetically altered activity of the TM-PC system to study its impact on cognitive function in mice, investigating locomotor activity, fear-related exploratory behavior, sensorimotor gating and learning patterns. Evans Blue (EB) extravasation test will show us, how (i) genotypes are affected regarding BBB integrity and (ii) whether anticoagulation affects integrity. Using CRISPR/Cas9 mediated cell-specific deletion of EPCR or PARs (PAR1-4) we want to identify the receptor through which aPC and thrombin modulate the function of brain cells, given the available data on IIa`s and aPC`s effect on endothelial barrier.
Results: We observed reduced EB extravasation in APChigh (aPC overexpression) mice in a chronic kidney disease mouse model as compared to WT or TMPro/Pro mice. Single nuclei RNA-sequencing of wildtype and TMPro/Pro murine brains revealed striking differences in various cell type clusters, comprising neurons, microglia cells, glia cells, pericytes and endothelial cells. Functional annotation revealed downregulation of genes related to “learning” in the TMPro/Pro mice. Indeed, we saw impairment in behavioral testing of TMPro/Pro mice.
Discussion: Our data establish that coagulation proteases differentially regulate the BBB in vivo. Altered coagulation activation is associated with structural defects in the CNS and with altered cognition. Recent data suggest that different anticoagulants differentially regulated coagulation protease dependent signaling, which may affect disease outcome. A prime example is activated protein C (APC), a blood protease with anticoagulant activity and cell-signaling activities. Of note, receptors for coagulation proteases are widely expressed at the blood brain barrier (BBB) and by various cells in the central nervous system (CNS). APC variants have shown benefits in preclinical models of ischemic stroke, brain trauma, multiple sclerosis and amyotrophic lateral sclerosis. Our data may show, for the first time, how coagulation may affect cognition.
Using quantitative morphometry data, the impaired formation of lamellipodia in Glanzmann thrombasthenia patients could be evaluated
K. von Bargen (Bad Oeynhausen, DE)
After vascular injury, platelet adhesion to the extracellular matrix leads to platelet activation, which in turn induces platelet shape change. Thereby the reorganization of the cytoskeleton is mediated by a variety of signaling pathways. Among other things, the fibrinogen receptor (GPIIb/IIIa complex) is also involved. This complex plays a crucial role in primary hemostasis by mediating platelet aggregation. Therefore, the study aimed to investigate the shape change (spreading) in dependence of the GPIIb/IIIa complex. For this purpose, platelets from Glanzmann thrombasthenia patients (GT-patients) were studied. This is a platelet dysfunction with a quantitative or qualitative defect of the GPIIb/IIIa complex.
In the present study, platelet shape change was investigated in a total of five healthy donors and six GT patients. For this purpose, platelets were allowed to spread on fibrinogen under different conditions (without activator, with ADP, or with TRAP), the actin cytoskeleton was stained with phalloidin, and then 40 immunofluorescence images were acquired per condition and time point. The immunofluorescence images were then evaluated using an algorithm (automated quantitative morphometry analysis) by determining various parameters such as area, fractal dimension, number of pseudopodia, etc. In addition, the morphometry of the platelets was examined by electron microscopy.
Analysis of the immunofluorescence images shows that the GT platelets have a spreading defect, which is particularly characterized by the absence of lamellipodia formation. Whereas the healthy platelets have mostly a fully spread shape after 45 min, the GT platelets persist in the early phase of spreading, which is characterized by a large number of long pseudopodia. Overall, the results of the algorithm show that the individual parameters (such as number of pseudopodia, FD, circularity, and area) describing the morphometry differ significantly between the studied collectives (healthy and GT). Here, the differences are particularly distinct at the late spreading time points. Thus, the GT platelets are much smaller due to the large number of pseudopodia, and the FD is increased. Electron microscopy also shows the altered morphometry of the GT platelets.
Spreading analysis show that platelet shape change is impaired in GT platelets. Both immunofluorescence microscopy and electron microscopy could show an absence of lamellipodia formation. Quantitative morphometry analysis was used to better describe cytoskeletal reorganization and to show the differences between the two collectives. Overall, quantitative morphometry analysis is a useful tool to better describe the different stages of platelet shape change in patients with thrombocytopathy.
Predictors of hypercoagulability in prediabetes
S. Hörber (Tübingen, DE)
Background: Obesity and insulin resistance predispose for arterial and venous thrombosis that can be explained by a hypercoagulable state. However, data about underlying associations of the hypercoagulability with metabolic alterations is limited. Therefore, the aim of the present study was to identify metabolic predictors of hypercoagulability in prediabetes.
Methods: Endogenous thrombin potential (ETP) was determined in 141 subjects with impaired glucose tolerance and/or impaired fasting glucose using a commercially available thrombin generation assay. All subjects were metabolically characterized including an oral glucose tolerance test. Furthermore determination of body fat distribution and liver fat content was performed using magnetic resonance imaging and spectroscopy, respectively.
Results: ETP was significantly associated with fasting plasma glucose, insulin sensitivity and body fat distribution. In particular, increased amounts of total adipose tissue, visceral adipose tissue and subcutaneous adipose tissue were significantly associated with an increase in ETP. Increased liver fat content was also related to higher ETP. Subjects with fatty liver had higher levels of ETP compared to subjects without fatty liver. Adjusting for insulin sensitivity, fasting plasma glucose and body fat compartments ETP remained significantly and independently elevated in subjects with fatty liver compared to controls.
Conclusion: ETP is closely linked to metabolic alterations in prediabetes. Body fat distribution, particular increased liver fat content is significantly and independently associated with hypercoagulability in prediabetes and may therefore contribute to the increased risk for arterial and venous thrombosis. Further analysis will focus on the underlying mechanisms including molecular associations of hypercoagulability and liver fat content in prediabetes.
Tumour derived prothrombin interacts with tumour PAR1 receptors
Y. Mouhish (Mainz , DE)
Thrombin is a liver-derived serine protease involved in hemostasis, acting through catalytic activation of soluble substrates (fibrinogen) and circulating cells (platelets). In addition, thrombin has a host of actions on cells with functions in development, angiogenesis, wound healing, inflammation, atherosclerosis, brain disorders, and tumour biology through activation of membrane-bound G-protein coupled protease-activated receptors (PARs). Previously, we uncovered extrahepatic prothrombin expression in emerging fibrosarcoma tumours, which drives tumour proliferation and invasiveness (Nourse et al.,bioxiv 2021). Here we investigated the interaction between endogenous tumour-derived (pro)thrombin and PAR1 on the surface of these cells by using bioluminescence resonance energy transfer (BRET).
To establish the BRET reporter assay system, we produced a prothrombin-luciferase (emission max 535 nm) and a PAR1-turbo fluorescent protein (emission at 635 nm) fusion construct, which were then used in transient transfection experiments of HEK cells. A bioluminogenic substrate (coelenterazine) was added to the co-transfected cells to elicit the energy transfer between the prothrombin-luciferase and the PAR1 turbo FP, resulting in a specific red fluorescent signal upon the interaction of (pro)thrombin with PAR1.
Subsequently, we transfected endogenously prothrombin-luciferase expressing fibrosarcoma cells (obtained from a newly generated transgenic reporter mouse model after chemical tumor induction with methylcholanthrene (Nourse et al.,bioxiv 2021)) with the PAR1 turbo FP construct to study the interaction between endogenous (pro)thrombin and PAR1 using the established BRET assay system.
First, we confirmed the production of a functional prothrombin-luciferase fusion protein and the presence of PAR1-turbo FP on the membrane of the cells. After establishing optimal BRET assay conditions, we were able to observe red-shifted signal in transfected cells, which could be modulated with ecarin (increase in red signal) and hirudin (reduced red signal), respectively. These findings confirm a successful function of the established BRET assay principle. Furthermore, we could show an emission stroke shift in endogenously prothrombin expressing cancer cells, suggesting that prothrombin binds to PAR1 receptors located on the membrane of these cells.
We successfully established a BRET reporter assay system to monitor (pro)thrombin-PAR1 interaction. We demonstrate that tumor-derived-prothrombin binds to PAR1 receptors expressed on the membrane of the tumor cells. Regarding the wide-spread clinical use of thrombin-targeting by direct oral anticoagulants, determination of the role and underlying mechanisms of thrombin in tumour growth may reveal previously unidentified benefits of selective therapeutic targeting of the hemostatic system in cancer
Automated 24/7 screening and quantification of DOACs in plasma in a single run on CLAM2030 – LCMS8050
A. Abratis (Göttingen, DE)
I. Markovic (Göttingen, DE)
In recent years, therapeutic drug monitoring (TDM) as part of DOAC therapy has gained in importance, since the outcome can be improved through individual dose adjustment, especially when treating critically ill emergency patients. At present, the effects of various DOACs are usually assessed indirectly and insufficiently (e.g. by determining the thromboplastin time or the activated partial thromboplastin time). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is an appropriate system for simultaneous measurement of multiple drugs. Until today, however, the use of LC-MS/MS Systems in emergency labs were not established.
Therefore, this study aims to develop and to validate a new LCMS-based method to screen and quantify different DOACs simultaneously in a single run. Similar methods have so far been restricted to research purposes, usually presupposing trained staff and long running times. Connecting the LCMS to an automated sample preparation module, we assessed its suitability for DOAC screening on a routine 24/7 basis. We compared the method for three of the available DOACs using the LCMS-8050 system coupled to CLAM-2030 (both Shimadzu) versus commercially available chromogenic tests.
The plasma samples from 56 anesthesiologically and nephrologically supervised patients were collected. DOAC plasma concentrations of Apixaban, Dabigatran and Rivaroxaban were measured on a LCMS system through an automated sample preparation module. Sample protein precipitation and chromatographic separation with a sharp linear gradient on a fused core column at 45°C were performed automatically by CLAM-2030. The target compounds were identified by parent ions and optimized MRM transitions. Quantification was performed by using deuterated internal standards. Quality controls were checked twice a day. In parallel, the quantitative determination of DOACs were assessed using conventional automated chromogenic tests (DTI-Assay (Dabigadran), Anti-Xa-Assay (Apixaban, Rivaroxaban), HemosIL (Werfen Company) on a IL Coagulation System (ACL TOP 750). Patient plasma samples were placed on the devices according to their arrival in the lab.
The Screening results have shown a good correspondence with the patients’ data. Passing–Bablok regression analysis revealed good comparability between the methods (Apixaban r=0.984, y=1.019*x–1.354; Rivaroxaban r=0.986, y=1.063*x–1.663; Dabigadran r=0.988, y=0.856*x–0.362). The precision of calibrations (range about 10 to 500ng/ml) and controls was within the manufacturer limit. The automated system was straightforward and proved easy to handle after short training periods. Running time including sample preparation was approx. six minutes.
The presented method is convincing in its easy handling and is conceivable for (24/7) routine measurements. In contrast to previously used methods, particularly the contemporaneous assignment and quantification of different DOACs is innovative.
Review: The Peripheral Blood Smear
E. Parulan Holzhueter (Achterwehr, DE)
Review: The Peripheral Blood
Case Report: 74 yr. old female on a yearly medical check. Date : 25th of October 2021
Differentialblutbild autom.% Neutrophile Granulozyten: 52%, Eosiphile Granulozyten:3.5%,
Basophile Granulozyten: 1.2%, Monozyten: 12.3%, Lymphozyten: 31%
Light microscopic examination revealed presence of plasma cells and some mitotic cellular changes.
Immunfixation revealed: Monoclonal Gammopathy Type IgM Lambda
Relevance of this presentation:
1. The examination of the cells with the naked eye through the microscope remains a standard procedure to detect early behavioural cellular changes in the human cells.
2. To develop the skill to recognize the presence of normal and abnormal cells as early as possible.
3. to emphasize the importance of laboratory diagnosis in health care and clinical decision making.
Procoagulant platelets as a diagnostic tool for heparin-induced thrombocytopenia using platelet-rich plasma by flow cytometer
L. Pelzl (Tuebingen, DE)
Heparin-induced thrombocytopenia (HIT) is caused by anti- PF4/heparin IgG antibodies, which activate the platelets and lead to thrombocytopenia and thrombosis. The diagnosis of HIT is usually confirmed by using functional assays such as the Heparin-Induced Platelet Activation assay (HIPA assay). However, functional assays are time consuming and routinely available only in specialized laboratories.
The aim of the current study was to establish a flow cytometer-based assay to determine procoagulant platelets in platelet-rich plasma (PRP) for the diagnosis of HIT. Sera samples from patients with HIT (HIT group) were incubated with PRP from healthy donors for different durations with (30, 60, 90 minutes). Procoagulant platelets were determined by analyzing double expression of P-selectin (CD62p) and phosphatidylserine (PS) externalization by flow cytometry. CD32a-mediated cross-link and platelet stimulation with ionomycin were used as positive controls.
Sera from HIT-diagnosed patients but not from the control-group induced a significant increase in the procoagulant platelet subpopulation in the presence of 0.2 U/mL heparin (% double positive CD62/PS: 1.2±1.1 vs 18.5±8.1, p=0.0021). The optimal incubation time was 60 minutes. A donor dependency of the flow cytometric method was not observed (control vs HIPA+: 0.7±0.59 vs19.0±3.2, p=0.0129). In addition, the use of washed platelets and PRP with HIT-sera led to the same results in this flow cytometric method (34.2±6.3 vs 30.1±2.2, ns).
Our data reflects towards suitability of aPRP-based protocol that can be used to detect the ability of HIT antibodies to induce procoagulant platelets by flow cytometry. In our ongoing studies, we are currently investigating the potential clinical implementation of this protocol in the diagnostic work up for HIT.
Entfällt: Calcium, calcium-sensing receptor and its role in leukaemia progression
Introduction: 99% of the body’s calcium is stored in bone, and calcium is released during bone remodeling. The calcium sensing receptor (CaSR) plays a role in the localization of normal haematopoietic stem cells in the BM microenvironment (BMM). However, the role of this receptor and its associated pathways for leukaemia development, therapy success and whether modulation of this receptor may be beneficial therapeutically, is not known.
Methods: Hypothesizing that the CaSR contributes to development, progression and response to therapy in leukaemia, we employed various in vitro assays, in vivo microscopy, leukaemia induction and in vivo treatment assays to test this question.
Results: The local calcium concentration forms a gradient in the BMM with highest calcium concentrations close to the endosteum. The calcium concentration in the BMM, CaSR expression on leukaemia cells, CaSR sensitivity to extracellular calcium, adhesion and migration in various concentrations of calcium differ between leukaemia types. CaSR acts as tumor suppressor or an oncogene in different leukaemias. In acute myeloid leukaemia (AML), limiting dilution transplantation of CaSR-deficient AML-initiating cells revealed a 7-fold reduction of leukaemic stem cells. Downstream of CaSR, we implicated filamin A and other proteins as important signaling molecules. Treatment of mice with a CaSR agonist or antagonist differentially impacted myeloid leukaemias.
Conclusion: In summary, our results suggest that the CaSR and possibly calcium ions from the BMM strongly and differentially influence leukaemia progression, with filamin A playing an essential role in AML. As an adjunct to existing treatment strategies, targeting of CaSR with specific pharmacologic agents may be beneficial in different leukaemias.
Liquid profiling of circulating tumor DNA in colorectal cancer: steps needed to achieve its full clinical value as standard care
M. Hedtke (Mannheim, DE)
Introduction: The analysis of circulating tumor DNA (ctDNA) is at the threshold of implementation into standard care for colorectal cancer (CRC) patients. However, data about the clinical utility of liquid profiling (LP), its acceptance by clinicians, and its integration into clinical workflows in real-world settings remain limited.
Methods and Results: In total, 243 LP tests for 168 CRC patients were performed as part of standard care for RAS using beads, emulsification, amplification, and magnetics (BEAMing), for BRAF V600 by digital droplet PCR (ddPCR), or for both molecular targets. LP tests requested as part of routine care since 2016 were retrospectively evaluated. Results show restrained request behavior that improved moderately over time, as well as reliable diagnostic performance comparable to translational studies, with an overall agreement of 91.7%. Extremely low ctDNA levels at < 0.1% in over 20% of cases, a high frequency of concomitant driver mutations (in up to 14% of cases), and ctDNA levels reflecting the clinical course of disease were revealed. However, certain limitations hampering successful translation of ctDNA into clinical practice were uncovered, including the lack of clinically relevant ctDNA thresholds, appropriate time points of LP requests, and integrative evaluation of ctDNA, imaging, and clinical findings.
Conclusion: These results highlight the potential clinical value of LP for CRC patient management and demonstrate issues that need to be addressed for successful long-term implementation in clinical workflows.