Assessment of Hypercoagulable State in Whole Blood in Sepsis Patients Using a Novel Microfluidic Dielectric Sensor

Abstract

Background: There is an intimate link between inflammation and thrombosis, and patients with pro-inflammatory/infectious disorders develop a hypercoagulable state. Extant coagulation assays are unable to distinguish the pro-coagulant state of a patient's blood, require 2-3 mL of blood, and take 2-3 hours for processing. These assays are also typically examined in plasma and do not represent the contribution of blood cellular elements that participate in thrombosis in vivo. Thus, a point-of-care device for rapid, comprehensive assessment of whole blood coagulation is crucial to ensure appropriate and timely evaluation in critically ill patients. We have introduced a microfluidic sensor (ClotChip) that uses dielectric spectroscopy to provide such an assessment in a handheld platform. We have shown in clinical studies in patients with a hypocoagulable state that ClotChip is sensitive to both coagulation factor and platelet defects, allowing for a global assessment of blood coagulation status using <10 µL of whole blood and in <30 min. In this study, we optimized ClotChip to assess the blood coagulation status in patients with a hypercoagulable state. Methods: Citrated blood samples from 12 patients with a diagnosis of sepsis and 11 healthy donors as controls were obtained under an IRB-approved protocol and tested with ClotChip within 2 hours of collection. ClotChip readout curve was calculated as the temporal variation of blood dielectric permittivity at 1 MHz, and the time to reach a permittivity peak (T peak) was taken as an indicator of coagulation time based on our prior studies. To increase the sensitivity of the ClotChip T peak parameter to a hypercoagulable state, we used two different anticoagulants, recombinant thrombomodulin (rTM) and activated protein C (APC). To optimize the anticoagulant concentration, whole blood samples from healthy donors were treated in vitro with lipopolysaccharide to mimic a pro-coagulant state of blood and tested with ClotChip after adding various concentrations of rTM and APC. We concluded that a concentration of 5 µg/mL for rTM and 10 µg/mL for APC would result in an optimal change in T peak for detecting the pro-coagulant state. Since heparin (or lovenox) is routinely used in hospitalized patients, sepsis and control samples were pretreated with hepzyme at a final concentration of 2 IU/mL to reverse the heparin effect. The T peak parameter was measured and compared in (i) hepzyme only-, (ii) rTM-, and (iii) APC-treated samples. Data are reported as mean ± standard deviation. Two-tailed t test is used to test for statistical significance between groups, and P < 0.05 is considered statistically significant. In box-and-whiskers plots, the box represents the range from the first to the third quartile, the horizontal line represents the median, plus sign (+) represents mean of the data; whiskers extend to the maximum and minimum data values, and dots represent individual subject data. Results: In hepzyme only-treated samples, T peak was significantly prolonged at 478±137 sec in sepsis samples, as compared to 357±58 sec in controls (Figs. 1A, 1B). rTM treatment resulted in T peak of 503±128 sec for sepsis samples and 443±81 sec for controls, whereas APC treatment resulted in T peak of 1,095±850 sec for sepsis samples and 477±71 sec for controls (Figs. 1A, 1B). Although T peak was prolonged at baseline in hepzyme only-treated sepsis samples, no further prolongation was noted with rTM treatment (difference in T peak of 24±94 sec; Fig. 1C), as compared to rTM-treated controls (difference in T peak of 85±40 sec; Fig. 1C). However, with a difference in T peak of 616±804 sec, the APC-treated sepsis samples exhibited T peak prolongation when compared to hepzyme only-treated sepsis samples, whereas the APC-treated controls did not (difference in T peak of 119±64 sec; Fig. 1D). A comparison between the APC- and rTM-treated samples revealed a significant prolongation of T peak in sepsis samples (difference in T peak of 591±815 sec) when compared to controls (difference in T peak of 30±66 sec; Fig. 1E). Conclusions: Our studies identify a unique coagulation profile in sepsis patient blood using a microfluidic dielectric sensor. These data suggest that the addition of rTM or APC can enhance the sensitivity of the ClotChip T peak parameter for detecting the pro-coagulant state in whole blood. Ongoing studies are examining the coagulation profile in other pro-inflammatory and infectious states. Figure 1 Figure 1. Disclosures Suster: XaTek Inc.: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Mohseni: XaTek Inc.: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Nayak: BioChip Labs: Current Employment.

ClotChip Portable Blood Clotting Sensor

“…Evidence for ClotChip remains early in its development, mostly limited to pilot and feasibility studies with small sample sizes, ranging from 12 to 104 study participants. 3,16,[21][22][23] Earliest investigations of the technology relied on ex vivo assessments, 3 with later studies using controlled methods and whole blood from study participants. 5 Evidence to date has demonstrated the feasibility of ClotChip in the assessment of coagulation and clinical risk of bleeding, as well as monitoring of coagulation factor replacement therapy, for patients with hemophilia (both with and without inhibitors, as well as for those receiving emicizumab).…”

“…18,19,21,22 Additional investigations have demonstrated the feasibility of ClotChip for identifying coagulation defects across a variety of clotting disorders, such as hemophilia and von Willebrand disease. 3,23,24 Further investigation has focused on the utility of ClotChip for monitoring DOAC therapy, demonstrating its sensitivity to detect the anticoagulant effects of these drugs. 16 While the accuracy of the ClotChip device has been compared against conventional technologies (including rotational thromboelastometry and thrombin generation assays) and produced comparable results, 19,21,22 no evidence was found that compared ClotChip to another PoC device.…”

“…22 In addition, ClotChip has been proposed and investigated as an intervention for measuring hypercoagulation in patients with conditions such as deep vein thrombosis, or infectious diseases that can cause dangerous clotting defects, such as sepsis and COVID-19. 23,34 These and related developments in PoC technology have the potential to improve care and care pathways for patients with bleeding disorders, and could introduce efficiencies and resource savings for health care facilities and systems.…”