We present a microfluidic approach that allows simultaneous interrogation of RBC properties in physiological flow conditions at a single cell level. With this method, we studied healthy hemoglobin A (HbA) and homozygous sickle hemoglobin (HbS) containing RBCs using whole blood samples from twelve subjects. We report that HbS-containing RBCs are heterogeneous in terms of adhesion and deformability in flow.
Oral squamous cell carcinoma (OSCC) claims the lives of thousands in the U.S. and hundreds of thousands worldwide annually. The gold standard for diagnosing OSCC, a biopsy followed by pathology review, is painful, invasive, costly, and often performed unnecessarily, particularly in primary care settings. Therefore, there is an unmet clinical need for a non-invasive, cost-effective test for OSCC that informs the clinician when a biopsy is warranted. We previously reported that human beta-defensin 3 (hBD-3), an epithelial cell-derived antimicrobial peptide (AMP), is pro-tumorigenic and over-expressed in early-stage OSCC when compared to hBD-2, another epithelial cell-derived AMP. In the present study, we validated this dichotomy of expression in carcinoma-in-situ (CIS) and OSCC lesions using immunofluorescence microscopy and demonstrated, by flow cytometry, that cytobrushed cells collected from OSCC lesions maintain this characteristic expression profile. We designed an ELISA-based assay to test our hypothesis that the ratio of hBD-3 and -2 protein expression in lesional cells, compared to the contralateral normal site in the same patient, could distinguish OSCC from non-cancerous lesions. We refer to this ratio as the Beta-Defensin Index (BDI). Prospective proof-of-principle and blinded discovery phase studies demonstrated that the BDI was able to discriminate OSCC from benign lesions. In addition, a multi-center validation study, consisting of three non-overlapping patient cohorts, determined sensitivity and specificity values of 98.2 (95% CI 90.3 to 99.9) and 82.6 (95%CI: 68.6 to 92.2), respectively. Finally, an additional proof-of-principle study showed promise in transforming the BDI laboratory-based assay into a microfluidic intact cell assay. In conclusion, our data demonstrate that the BDI could objectively and non-invasively determine patients that need a tissue biopsy, thus augmenting the gold standard biopsy. Additionally, it offers a way to monitor oral premalignant lesions in real-world practice and fulfill a major unmet need, particularly in low-socio economic countries where oral cancer is highly prevalent, yet laboratory/pathology services are lacking.
Abstract Sickle cell disease (SCD), which afflicts 100 000 Americans, as well as millions worldwide, is associated with anemia, lifelong morbidity, and early mortality. Abnormal adhesion of sickle red blood cells (RBCs) to activated vascular endothelium may contribute acutely to the initiation of painful vaso-occlusive crises and chronically to endothelial damage in SCD. Sickle RBCs adhere to activated endothelium through several adhesion mechanisms. In this study, using whole blood from 17 people with heterozygous SCD (HbS variant) and 55 people with homozygous SCD (HbSS) analyzed in an in vitro microfluidic assay, we present evidence for the adhesion of sickle RBCs to immobilized recombinant intercellular adhesion molecule 1 (ICAM-1). We show that sickle RBC adhesion to ICAM-1 in vitro is associated with evidence of hemolysis in vivo, marked by elevated lactate dehydrogenase levels, reticulocytosis, and lower fetal hemoglobin levels. Further, RBC adhesion to ICAM-1 correlates with a history of intracardiac or intrapulmonary right-to-left shunts. Studies of potential ICAM-1 ligands on RBC membranes revealed that RBC–ICAM-1 interactions were mediated by fibrinogen bound to the RBC membrane. We describe, for the first time, RBC rolling behavior on ICAM-1 under high shear rates. Our results suggest that firm adhesion of sickle RBCs to ICAM-1 most likely occurs in postcapillary venules at low physiological shear rates, which is facilitated by initial rolling in high shear regions (eg, capillaries). Inhibition of RBC and ICAM-1 interactions may constitute a novel therapeutic target in SCD.
Summary Individuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin‐III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient‐derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin‐activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule‐1 (VCAM‐1) mediated by thrombin. Our findings suggest that, by attenuating thrombin‐mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.
Manipulation and assembly of cell-encapsulating hydrogels offer unique opportunities for regenerative medicine, microphysiological system engineering, pharmaceutical research, biological research, and space sciences. Utkan Demirci and co-workers show on page 1137 that temporal and spatial control over hydrogels in microscale can be exerted by exploiting their paramagnetic properties without using magnetic nanoparticles.
Severe hemorrhage associated with trauma, surgery, and congenital or drug-induced coagulopathies can be life-threatening and requires rapid hemostatic management via topical, intracavitary, or intravenous routes. For injuries that are not easily accessible externally,
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