Design and Utility of a Point-of-Care Microfluidic Platform to Assess Hematocrit and Blood Coagulation

Abstract

Purpose—: To develop a small volume whole blood analyzer capable of measuring the hematocrit and coagulation kinetics of whole blood.

Methods and results—: A co-planar microfluidic chamber designed to facilitate self-driven capillary action across an internal electrical chip was developed and used to measure the electric parameters of whole human blood that had been anticoagulated or allowed to clot. To promote blood clotting, select chip surfaces were coated with a prothrombin time (PT) reagent containing lipidated tissue factor (TF), which activates the extrinsic pathway of coagulation to promote thrombin generation and fibrin formation. Whole human blood was added to the microfluidic device, and voltage changes within the platform were measured and interpreted using basic resistor-capacitor (RC) circuit and fluid dynamics theory. Upon wetting of the sensing zone, a circuit between two co-planar electrodes within the sensing zone was closed to generate a rapid voltage drop from baseline. The voltage then rose due to sedimentation of red blood cells (RBC) in the sensing zone. For anticoagulated blood samples, the time for the voltage to return to baseline was dependent on hematocrit. In the presence of coagulation, the initiation of fibrin formation in the presence of the PT reagent prevented the return of voltage to baseline due to the reduced packing of RBCs in the sensing zone.

Conclusions—: The technology presented in this study has potential for monitoring the hematocrit and coagulation parameters of patient samples using a small volume of whole blood, suggesting it may hold clinical utility as a point-of-care test.

Keywords: Biorheology; Coagulation; Electrical engineering; Hematocrit; Whole blood testing.

Figure 1

Design of a microfluidic platform for use with blood samples. (a) Illustration of the experimental setup. (b) Photograph of the assembled device. (c) Illustration of the sensing zone with two gold (Au) co-planar electrodes. (d) Basic circuit design in the absence of blood sample.

Figure 2

Microfluidic platform with anticoagulated whole human blood sample. Venous whole blood samples collected from a healthy human donor (Hematocrit of 43) were anticoagulated with bivalent direct thrombin inhibitor (40 µg/mL hirudin) and applied to the inlet of the microfluidic device. Real-time progression of blood sample within microfluidic device was (a) visualized with light microscopy with each pertinent transition point shown in panels (1–3) and (b) labeled on the voltage profile (1) dry sensing zone prior to blood entry, (2) entry of blood samples and (3) sedimentation of RBCs within sensing zone. Data representative of n > 30.

Figure 3

Estimation of microfluidic platform voltage sensitivity to hematocrit. (a) Model of a three component blood circuit between two co-planar electrodes. (b) Voltage profile of a RBC-capacitor contribution based on Ohm’s Law. (c) Illustration of RBC flow assumption for approximation of the time constant values (${\tau }_{H_x}$) as a function of hematocrit: Approximation 1 accounts for bulk flow of blood as a uniform colloid suspension, while Approximation 2 accounts for parabolic blood flow profiles and hematocrit-dependent viscosity. (d) Plot of approximated ${\tau }_{H_x}$ values as a function of hematocrit using Approximation 1 (grey line) or Approximation 2 (black line). The normal hematocrit range for men (40–54%) and women (36–48%) is highlighted with a grey box.

Figure 4

Sensitivity of the microfluidic platform to hematocrit. Venous whole blood anticoagulated with a bivalent direct thrombin inhibitor (hirudin) was subjected to a series of centrifugation steps to isolate RBCs and platelet-poor plasma (PPP) fractions. The RBC pellet was resuspended with autologous PPP to specified levels of hematocrit and samples were perfused through BSA-coated microfluidic devices. (a) Representative ‘voltage vs. reaction time’ curves of real-time progression of resuspended RBCs at select hematocrits within the microfluidic device are shown, with ‘PPP alone’, which has a hematocrit of zero, used as a control. The dashed arrows indicate calculation of empirical time constant values (${\tau }_{H_x}$) for samples with increasing hematocrits. (b) Plot of empirical ${\tau }_{H_x}$ values for samples with select hematocrit content is shown as mean ± SD, n ≥ 3. Single experimental data points are compared with approximated ${\tau }_{H_x}$ values from Fig. 3 (dashed lines: Approximation 1—grey, Approximation 2—black). The normal hematocrit range for men (40–54%) and women (36–48%) is highlighted in grey.

Figure 5

Sensitivity of the microfluidic platform to coagulation. Non-anticoagulated venous whole blood was pretreated with either vehicle buffer control or a bivalent direct thrombin inhibitor (hirudin) and added to either BSA-coated or PT reagent-coated microfluidic device. The real-time progression of blood cells within cartridge was recorded using voltage measurements. Graphs are representative of results from n ≥ 4 repeats.

Figure 6

Schematic of possible blood measurements and readouts.