Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies.

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

A prospective, blinded study of a PF4-dependent assay for HIT diagnosis.

Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14-labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.

Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis.

Antibodies specific for platelet factor 4 (PF4)/heparin complexes are the hallmark of heparin-induced thrombocytopenia and thrombosis (HIT), but many antibody-positive patients have normal platelet counts. The basis for this is not fully understood, but it is believed that antibodies testing positive in the serotonin release assay (SRA) are the most likely to cause disease. We addressed this issue by characterizing PF4-dependent binding of HIT antibodies to intact platelets and found that most antibodies testing positive in the SRA, but none of those testing negative, bind to and activate platelets when PF4 is present without any requirement for heparin (P < .0001). Binding of SRA-positive antibodies to platelets was inhibited by chondroitinase ABC digestion (P < .05) and by the addition of chondroitin-4-sulfate (CS) or heparin in excess quantities. The findings suggest that although all HIT antibodies recognize PF4 in a complex with heparin, only a subset of these antibodies recognize more subtle epitopes induced in PF4 when it binds to CS, the major platelet glycosaminoglycan. Antibodies having this property could explain "delayed HIT" seen in some individuals after discontinuation of heparin and the high risk for thrombosis that persists for weeks in patients recovered from HIT.

3D-Printed, Modular, and Parallelized Microfluidic System with Customizable Scaffold Integration to Investigate the Roles of Basement Membrane Topography on Endothelial Cells.

Because dysfunctions of endothelial cells are involved in many pathologies, in vitro endothelial cell models for pathophysiological and pharmaceutical studies have been a valuable research tool. Although numerous microfluidic-based endothelial models have been reported, they had the cells cultured on a flat surface without considering the possible three-dimensional (3D) structure of the native extracellular matrix (ECM). Endothelial cells rest on the basement membrane in vivo, which contains an aligned microfibrous topography. To better understand and model the cells, it is necessary to know if and how the fibrous topography can affect endothelial functions. With conventional fully integrated microfluidic apparatus, it is difficult to include additional topographies in a microchannel. Therefore, we developed a modular microfluidic system by 3D-printing and electrospinning, which enabled easy integration and switching of desired ECM topographies. Also, with standardized designs, the system allowed for high flow rates up to 4000 µL/min, which encompassed the full shear stress range for endothelial studies. We found that the aligned fibrous topography on the ECM altered arginine metabolism in endothelial cells and thus increased nitric oxide production. There has not been an endothelial model like this, and the new knowledge generated thereby lays a groundwork for future endothelial research and modeling.

Microfibrous Extracellular Matrix Changes the Liver Hepatocyte Energy Metabolism via Integrins.

Cell line-based liver models are critical tools for liver-related studies. However, the conventional monolayer culture of hepatocytes, the most widely used in vitro model, does not have the extracellular matrix (ECM), which contributes to the three-dimensional (3D) arrangement of the hepatocytes in the liver. As a result, the metabolic properties of the hepatocytes in the monolayer tissue culture may not accurately reflect those of the hepatocytes in the liver. Here, we developed a modular platform for 3D hepatocyte cultures on fibrous ECMs produced by electrospinning, a technique that can turn a polymer solution to the micro/nanofibers and has been widely used to produce scaffolds for 3D cell cultures. Metabolomics quantitation by liquid chromatography-mass spectrometry (LC-MS) indicated that Huh7 hepatocytes grown in microfibers electrospun from silk fibroin exhibited reduced glycolysis and tricarboxylic acid (TCA) cycle, as compared to the cells cultured as a monolayer. Further mechanistic studies suggested that integrins were correlated to the ECM's effects. This is the first time to report how an ECM scaffold could affect the fundamental metabolism of the hepatocytes via integrins.

3D-Printed Microfluidic Devices for Enhanced Online Sampling and Direct Optical Measurements.

3D printing has emerged as a robust technique to fabricate reliable and reproducible microfluidic devices. However, a limitation of 3D-printed devices has been the low transparency even when printed in a "clear" material. There are currently no reports regarding direct optical measurements through a 3D-printed device. Here, we present for the first time that the printing orientation can affect the transparency of a 3D-printed object. With the optimal orientation, we printed a microfluidic detector that was sufficiently transparent (transmittance ≈ 80%) for optical quantitation. This finding is significant because it shows the feasibility to directly 3D-print optical components for analytical applications. In addition, we created a novel microfluidic dialysis device via 3D printing, which enabled higher flow rates (for sampling with high temporal resolution) and increased extraction efficiency than commercially available ones. By coupling the microfluidic detector and dialysis probe, we successfully measured the release kinetics of indole from biofilms in a continuous, automated, and near real-time fashion. Indole is an intercellular signaling molecule in biofilms, which may regulate antibiotic resistance. The release kinetics of this molecule had not been quantitated likely because of the lack of a suitable analytical tool. Our results fill this knowledge gap.

From cells-on-a-chip to organs-on-a-chip: scaffolding materials for 3D cell culture in microfluidics.

It is an emerging research area to integrate scaffolding materials in microfluidic devices for 3D cell culture (organs-on-a-chip). The technology of organs-on-a-chip holds the potential to obviate the gaps between pre-clinical and clinical studies. As accumulating evidence shows the importance of extracellular matrix in in vitro cell culture, significant efforts have been made to integrate 3D ECM/scaffolding materials in microfluidics. There are two families of materials that are commonly used for this purpose: hydrogels and electrospun fibers. In this review, we briefly discuss the properties of the materials, and focus on the various technologies to obtain the materials (e.g. extraction of collagen from animal tissues) and to include the materials in microfluidic devices. Challenges and potential solutions of the current materials and technologies were also thoroughly discussed. At the end, we provide a perspective on future efforts to make these technologies more translational to broadly benefit pharmaceutical and pathophysiological research.

A Platelet Factor 4-Dependent Platelet Activation Assay Facilitates Early Detection of Pathogenic Heparin-Induced Thrombocytopenia Antibodies.

Heparin-induced thrombocytopenia (HIT) is a dangerous complication of heparin therapy. HIT diagnosis is established by recognizing thrombocytopenia and/or thrombosis in an affected patient and from the results of serological tests such as the platelet factor 4 (PF4)/heparin immunoassay (PF4 ELISA) and serotonin release assay (SRA). Recent studies suggest that HIT antibodies activate platelets by recognizing PF4 in a complex with platelet glycosaminoglycans (and/or polyphosphates) and that an assay based on this principle, the PF4-dependent P-selectin expression assay (PEA), may be even more accurate than the SRA for HIT diagnosis. Here, we demonstrate that the PEA detected pathogenic antibodies before the SRA became positive in two patients with HIT studied serially, in one case even before seropositivity in the PF4 ELISA. In one of the patients treated with plasma exchange, persistent dissociation between the PEA and SRA test results was observed. These results support a role for the PEA in early HIT diagnosis.

IVIg for Treatment of Severe Refractory Heparin-Induced Thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) complicated by severe thrombocytopenia and thrombosis can pose significant treatment challenges. Use of alternative anticoagulants in this setting may increase bleeding risks, especially in patients who have a protracted disease course. Additional therapies are lacking in this severely affected patient population. METHODS: We describe three patients with HIT who had severe thromboembolism and prolonged thrombocytopenia refractory to standard treatment but who achieved an immediate and sustained response to IVIg therapy. The mechanism of action of IVIg was evaluated in these patients and in five additional patients with severe HIT. The impact of a common polymorphism (H/R 131) in the platelet IgG receptor FcγRIIa on IVIg-mediated inhibition of platelet activation was also examined. RESULTS: At levels attained in vivo, IVIg inhibits HIT antibody-mediated platelet activation. The constant domain of IgG (Fc) but not the antigen-binding portion (Fab) is required for this effect. Consistent with this finding, IVIg had no effect on HIT antibody binding in a solid-phase HIT immunoassay (platelet factor 4 enzyme-linked immunoassay). The H/R131 polymorphism in FcγRIIa influences the susceptibility of platelets to IVIg treatment, with the HH131 genotype being most susceptible to IVIg-mediated inhibition of antibody-induced activation. However, at high doses of IVIg, activation of platelets of all FcγRIIa genotypes was significantly inhibited. All three patients did well on long-term anticoagulation therapy with direct oral anticoagulants. CONCLUSIONS: These studies suggest that IVIg treatment should be considered in patients with HIT who have severe disease that is refractory to standard therapies.

A Novel PF4-Dependent Platelet Activation Assay Identifies Patients Likely to Have Heparin-Induced Thrombocytopenia/Thrombosis.

BACKGROUND: Almost without exception, patients with heparin-induced thrombocytopenia/thrombosis (HIT) have antibodies that recognize platelet factor 4 (PF4) in a complex with heparin; however, many heparin-treated patients without HIT are also antibody-positive. A platelet activation test, the serotonin release assay (SRA), is useful for identifying a subset of antibodies that are platelet-activating and most likely to cause HIT. However, this "gold standard" assay for HIT diagnosis is technically demanding and is routinely available only through referral laboratories, limiting its availability for timely diagnosis and management. METHODS: We compared the diagnostic performance of the SRA with that of a technically simple platelet activation assay, the PF4-dependent P-selectin expression assay (PEA), which uses platelets pretreated with PF4 as targets for antibody detection. Archived serum samples from 91 patients for whom clinical information (HIT 4Ts [thrombocytopenia, timing of platelet count fall, thrombosis, and other causes of thrombocytopenia] score) was available were used. Patients with an intermediate 4Ts score and a PF4 ELISA (enzyme-linked immunosorbent assay) optical density ≥ 2.0, or a high 4Ts score and a PF4 ELISA optical density ≥ 1.0, were considered HIT positive; others were designated HIT negative. RESULTS: The PEA had higher diagnostic accuracy (area under the curve, 0.92 vs 0.82; P = .02) than the SRA, using this definition of HIT. Eleven of 16 serum samples that were PEA positive and SRA negative were HIT positive. Studies done with identical target platelets and serially diluted samples from patients with HIT showed that the PEA is inherently more sensitive than the SRA for the detection of platelet-activating antibodies. CONCLUSIONS: The PEA is technically less demanding than the SRA and may be more accurate for the diagnosis of HIT.