Stable measurement of chemistry, immunochemistry, and hematology analytes in a heparin-based anticoagulant with iloprost additive: A promising candidate for the polyvalent blood collection tube.

INTRODUCTION: A polyvalent blood collection tube could potentially reduce the number and volume of blood samples drawn from patients and reduce the risk of tube mix-ups in a point-of-care setting in the emergency department and the intensive care unit. METHODS: Four different concentrations of our experimental heparin anticoagulant with iloprost additive (HEP-ILOP 50 nM, 150 nM, 1000 nM, and 10 µM, respectively) were tested for significant differences and bias performance specifications against EDTA for 29 hematology analytes, and the highest concentration (HEP-ILOP 10 µM) against lithium heparin for 14 chemistry and immunochemistry analytes. Samples were drawn from 79 consenting subjects from the Oncology Department (n = 38) and the Intensive and Intermediary Care Unit (n = 41). RESULTS: For hematology analytes, the HEP-ILOP formulation generally provided stable measurement within optimal requirements within 5 h after sampling (mean 104 ± 56 min), with very little difference between the four HEP-ILOP concentrations. Because of differences in platelet and red blood cell swelling between EDTA and HEP-ILOP, all size-dependent analytes required proportional factorization to produce similar results. Platelet count by impedance similarly required factorization, whereas the fluorescent method provided results identical with EDTA. Chemistry and immunochemistry analytes were within optimal requirements except for potassium, lactate dehydrogenase, and glucose, indicating a cytoprotective effect of iloprost reducing cell metabolism and rupture, thereby producing results closer to in vivo conditions. CONCLUSIONS: Our novel dry-sprayed anticoagulant formulation, HEP-ILOP, is a promising candidate for a polyvalent blood collection tube, enabling the analysis of hematology, chemistry, and immunochemistry analytes in the same tube.

Evaluation of a novel heparin-iloprost-based antithrombotic formulation blood collection tube for clinical usage.

BACKGROUND: The objective of our study was to evaluate a single blood collection tube with a novel antithrombotic formulation to measure both hematological, biochemical, and d-dimer analytes. METHODS: Paired samples of gold standard blood tubes (EDTA, lithium heparin, sodium citrate) and a new antithrombotic formulation blood tube were collected from 187 patients. The new antithrombotic tube is a lithium heparin tube preloaded with a liquid form of prostacyclin analog. The novel tube was tested on seventeen hematological parameters and smears against EDTA, on fourteen biochemical parameters against lithium heparin and on d-dimer against sodium citrate. RESULTS: All correlation coefficients were close to 0.99. The Bland-Altman analyses presented a satisfactory correspondence for all analytes. All the hematological examinations demonstrated comparable results between EDTA and the novel formulation, except for platelet counts analyzed by impedance method, but not by fluorescence. We detected lower mean platelet volume with/without outliers (5.06%)/(5.13%) in the novel formulation and increased mean corpuscular hemoglobin concentration (2.55%). All the biochemistry analytes demonstrated comparable results between lithium heparin and the novel tube. d-dimer showed comparable results between citrated blood and the novel formulation after dilution correction. CONCLUSIONS: We describe a novel antithrombotic formulation tube with the potential to be introduced into clinical laboratories for simultaneous analysis of thirty-two blood analytes.

Platelets drive fibronectin fibrillogenesis using integrin αIIbß3.

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbß3 rather than α5ß1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

Clot-entrapped blood cells in synergy with human mesenchymal stem cells create a pro-angiogenic healing response.

Blood clots stop bleeding and provide cell-instructive microenvironments. Still, in vitro models used to study implant performance typically neglect any possible interactions of recruited cells with surface-adhering blood clots. Here we study the interaction and synergies of bone marrow derived human mesenchymal stem cells (hMSCs) with surface-induced blood clots in an in vitro model by fluorescence microscopy, scanning and correlative light and electron microscopy, ELISA assays and zymography. The clinically used alkali-treated rough titanium (Ti) surfaces investigated here are known to enhance blood clotting compared to native Ti and to improve the healing response, but the underlying mechanisms remain elusive. Here we show that the presence of blood clots synergistically increased hMSC proliferation, extracellular matrix (ECM) remodelling and the release of matrix fragments and angiogenic VEGF, but did not increase the osteogenic differentiation of hMSCs. While many biomaterials are nowadays engineered to release pro-angiogenic factors, we show here that clot-entrapped blood cells on conventional materials in synergy with hMSCs are potent producers of pro-angiogenic factors. Our data might thus not only explain why alkali-treatment is beneficial for Ti implant integration, but they suggest that the physiological importance of blood clots to create pro-angiogenic environments on implants has been greatly underestimated. The importance of blood clots might have been missed because the pro-angiogenic functions get activated only upon stimulation by synergistic interactions with the invading cells.

Synergistic interactions of blood-borne immune cells, fibroblasts and extracellular matrix drive repair in an in vitro peri-implant wound healing model.

Low correlations of cell culture data with clinical outcomes pose major medical challenges with costly consequences. While the majority of biomaterials are tested using in vitro cell monocultures, the importance of synergistic interactions between different cell types on paracrine signalling has recently been highlighted. In this proof-of-concept study, we asked whether the first contact of surfaces with whole human blood could steer the tissue healing response. This hypothesis was tested using alkali-treatment of rough titanium (Ti) surfaces since they have clinically been shown to improve early implant integration and stability, yet blood-free in vitro cell cultures poorly correlated with in vivo tissue healing. We show that alkali-treatment, compared to native Ti surfaces, increased blood clot thickness, including platelet adhesion. Strikingly, blood clots with entrapped blood cells in synergistic interactions with fibroblasts, but not fibroblasts alone, upregulated the secretion of major factors associated with fast healing. This includes matrix metalloproteinases (MMPs) to break down extracellular matrix and the growth factor VEGF, known for its angiogenic potential. Consequently, in vitro test platforms, which consider whole blood-implant interactions, might be superior in predicting wound healing in response to biomaterial properties.

Functional modification of fibronectin by N-terminal FXIIIa-mediated transamidation.

A straightforward strategy is presented for the site-specific incorporation of fluorophores or reactive probes into the extracellular matrix (ECM) protein fibronectin (Fn) by using the enzyme-catalyzed transamidation by activated factor XIII. Characterization by SDS-PAGE, western blotting, absorption measurements, mass spectrometry, and stepwise photobleaching for labeling quantification at the single-molecule level showed that the labeling was efficient and restricted to the N-terminal tails. The introduction of labels did not interfere with Fn fibrillogenesis, as verified by the incorporation of fluorescently labeled Fn into ECM and manually pulled Fn fibers. Site-specific incorporation of an azide was used to create a template for bioorthogonal click chemistry reactions in a second bioconjugation step, thus offering versatile modification and application possibilities in the context of matrix biology and tissue engineering.