Investigation of the influence of fluid dynamics on thrombus growth at the interface between a connector and tube

A swept-source OCT (Panasonic Healthcare, Japan) with a center wavelength and maximal energy of 1330 nm and 15 mW was employed to non-invasively examine the sequential process of thrombus formation. For testing, connectors with a 6-mm inner diameter and 8-mm outer diameter, without tapering at either end (Fig. 1a), or with taper (Fig. 1b) were manufactured. The inner and outer diameters of the connectors are similar to those of commercial connectors used in hemofiltration therapies. Due to the manufacturer, edge shapes of commercially available connectors are different. For example, one connector has a 10° taper to the tip and 300 μm width at the tip. Another connector has corner radium of 1 mm and 500 μm width at the tip. Another connector has no taper and 500 μm width at the tip. In this study, two connectors with 30° taper and tip width of 100 μm and without taper and tip width of 1.0 mm were manufactured. These connectors were made of polyurethane (SG7101 AT,SG7101 B, SID Co., Ltd., Saitama, Japan). The connectors were joined to polyvinylchloride (PVC) tubes with an inner diameter of 6 mm (Tygon^®, ACFJ00007, Saint-Gobain, Tokyo, Japan). The process of thrombus formation at the interface between the connectors and tubes was examined. The connectors and tubes were placed into an air-contactless closed circuit with a total volume of 50 mL. The circuit consisted of a roller pump (Masterflex 07528, Yamato Scientific Co., Ltd., Tokyo, Japan), a compliant reservoir tube made of blood-compatible segmented polyurethane (TM-5, Nipro Co., Ltd, Osaka, Japan), the PVC tubes, and a resistive unit (Hoffman pinchcock, AS ONE, Osaka, Japan) (Fig. 1c). A compliant reservoir tube with an inner diameter of 12 mm and length of 30 mm was made using a dipping method. Blood-contact surfaces of the PVC tubes and connectors were coated twice with a segmented polyurethane (Miractran^®, Nihon Unipolymer, Tokyo, Japan), and then coated twice with 2-methacryloyloxyethyl phosphorylcholine (MPC) (Lipidure-CM5206, NOF CO. Tokyo, Japan). The coating procedure and manufacture of the compliant reservoir tube were performed in a class 100 clean air room. All components of the circuit were sterilized by ethylene oxide gas before use in the tests.

This study was approved by the ethics committee of Waseda University (2015-212) and performed in accordance with the ethical standards stated in the 1964 Declaration of Helsinki and its later amendments. With informed consent, blood was sampled from 6 healthy volunteers. The blood bags (BB-T040CJ, TERUMO Corp., Tokyo, Japan) held 250 mL, and were coated with MPC twice in the clean air room and sterilized with ethylene oxide gas before use. A 22-G indwelling needle was inserted into a peripheral vein. Then, 250 mL blood was drawn by gravity drainage into the blood bag. To avoid the collection of activated platelets, the first 2 mL of blood was discarded. Blood sampling was performed aseptically. Prior to drawing blood, 0.4 units/mL heparin (Heparin Sodium, Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) was injected into the blood bag to expect the activated clotting time of approximately 150–180 s.

The activated clotting time was measured using a whole blood coagulation system (Hemocron^® Response, Accriva Diagnostics, representing ITC and Accumetrics, Edison, NJ, USA) before and after the test.

Blood pH was measured using a portable blood gas analyzer (i-STAT^®, Abbott Point of Care Inc., Princeton, NJ, USA) with a cartridge (G3+, Abbott Point of Care). The platelet count and the hematocrit were measured using an automated hematology analyzer (Celltac E MEK-7222, Nihon Kohden Corp., Tokyo, Japan) and hematocrit centrifuge (Model 3220, Kubota Corp., Tokyo, Japan). Blood viscosity was measured using a rotational viscometer (TPE-100, Toki Sangyo Co., Ltd., Tokyo, Japan) at 37 °C and the shear rate of 383/s. To avoid coagulation during the measurement of viscosity, blood was sampled to 3.2% sodium citrate blood collection tubes (VP-CA053 K, TERUMO Co., Tokyo, Japan).

Two identical circuits except for 2 connectors with different configurations were prepared. The circuits were filled with identical-source blood. The blood circulation tests were performed for 60 min. Using the roller pump, pulsatile flow was circulated under 27 rpm. The mean blood flow rate and pressure were adjusted to 100 mL/min and 70 mmHg using the roller pump and resistive unit (Fig. 1d). These flow and pressure conditions are prevalent in continuous hemofiltration therapy in Japan. Blood flow rate and pressure were monitored using an ultrasonic blood flow meter (ME4PXL/N, Transonic systems Inc., NY, USA) and pressure transducer (UK-801, Baxter, Irvine, CA, USA). The compliant reservoir tube and part of the PVC tube were placed in a 37 °C water bath to regulate circulating blood temperature. OCT images of the interface between the connecter and the tube were sequentially taken in a vertical direction at 10, 20, 30, 40, 50, and 60 min. Image acquisition was performed at 7 frames/s with 20 μm × 20 μm size. After the circulation tests, blood was immediately removed from the circuit, and the interfaces between the connectors and tubes were rinsed carefully with phosphate-buffered saline 3 times.

The average of 6 consecutive time-difference images in 1 s just after the start of blood circulation was used to differentiate the still connectors and tubes from circulating blood. The still area of connectors and tubes was defined as the background image. Each of the 6 averaged consecutive time-difference images was analyzed at the start of circulation, and at 10, 20, 30, 40, 50, and 60 min. Otsu’s method [18] was used to calculate the optimal threshold separating the still area and the area of circulating blood. The background image was subtracted from the extracted images to define the area where thrombus formed.

The blood flow field at the interface between the connectors and tubes was examined using fluorescent particle imaging velocimetry (PIV). The circuit components, roller pump, a compliant reservoir tube, the PVC tubes, and a resistive unit were the same as in the blood circulation test. A straight flow tract with an inner diameter of 6 mm was manufactured using a block of silicone (KE-1603,Shin-Etsu Chemical, Tokyo, Japan), as an alternative to the PVC tube, in order to match the refractive index of the tube with that of working fluid as 1.4096 (Fig. 2).

Young’s modulus of the silicone model was set to 2.13 ± 0.08 MPa at room temperature, and the external size of the silicone block was set to 100 mm × 60 mm × 30 mm to prevent expansion of the silicone model. The expansion of the inner diameter in the circulation was less than 0.3% (0.018 mm). The circuit was filled with a mixed solution of glycerin and fluorescent particles (FLUOSTAR, EBM Co., Ltd, Tokyo, Japan) and the refractive index was adjusted to that of the silicone models. The viscosity of working fluid was adjusted to 4.80 ± 0.02 mPa s at 37 °C, which was the average value of human whole blood obtained in this study. Blood flow rate and pressure were regulated to the same conditions as in the blood circulation test. An Nd:YAG laser beam (DS20-527, Photonics Industries, Bohemia, NY, USA) was used to irradiate the interface between the connectors and the silicone model. The PIV images were taken using a high-speed camera (VC13-0192, Imager Pro XL, LaVision, Goettingen, Germany) through an optical filter with a cutoff frequency of 550 nm (CVI Melles Griot, NM, USA). Pixel size was 0.025 mm and frame speed was 400 frames/s.

The activated clotting time at the beginning of the test was 161 ± 20 s. The activated clotting times after 1 h of blood circulation for the connector without tapers and connector with tapers were 161 ± 20 and 160 ± 18 s, respectively. The platelet count at the beginning of the test was 19.7 ± 2.8 × 10⁴/μL. Platelet counts after 1 h of blood circulation for the connector without and with tapers were 16.6 ± 3.5 × 10⁴ and 16.0 ± 3.8 × 10⁴/μL, respectively. The platelet count after 60 min of blood circulation decreased regardless of the connector design. There were no significant differences in the platelet count between the connectors without and with tapers (p = 0.77). The hematocrit at the beginning of the test was 42 ± 2%. The hematocrit after 1 h of blood circulation in the circuits incorporating the connector without and with tapes was 41 ± 2 and 41 ± 2%, respectively. There were no differences in the hematocrit between connectors without and with tapers (p = 0.63), and before and after 1 h circulation.

The interface between the connector and the tube during 60 min of blood circulation was observed after a careful rinse with phosphate-buffered saline (Fig. 3).

The sites of thrombus formation during 10–60 min of circulation were visualized at 10-min intervals (Fig. 4) using OCT. The sequential changes in the thrombus formation area at the inlet interface between the connector without tapers and the tube as well as at the outlet interface between the connector without tapers and the tube were quantified (Fig. 5a, b). The thrombus formation area at the inlet interface between the connector without tapers and the tube increased from 0.32 ± 0.21 to 1.25 ± 0.66 mm² during circulation from 10 to 60 min (Fig. 5a). On the other hand, the thrombus formation area increased less at the outlet interface between the connector without tapers and the tube (from 0.30 ± 0.18 mm² at 10 min to 0.51 ± 0.31 mm² at 60 min) (Fig. 5b). Compared with the outlet interface, a larger amount of thrombus formed at the inlet interface of the connector without tapers in 30 min or more of circulation (p < 0.05) (Fig. 6a).

A similar thrombus formation process was found for the interface between the connector with tapers and the tube. The thrombus formation area increased from 0.27 ± 0.08 to 1.55 ± 1.12 mm² at the inlet interface during circulation from 10 to 60 min (Fig. 5c), and slightly increased from 0.29 ± 0.06 to 0.94 ± 0.14 mm² during circulation from 10 to 60 min at the outlet interface (Fig. 5d). The amount of thrombus was not significantly different between the inlet and outlet interfaces for the connectors with tapers (Fig. 6b).

There was no significant difference in the thrombus formation area between the connectors without and with tapers at the inlet interface during 60 min of circulation (p = 0.72) (Fig. 7a). On the contrary, a larger amount of thrombus formed at the outlet interface for the connector with tapers than for the connector without tapers in not less than 30 min of circulation (p < 0.05) (Fig. 7b).

Flow velocity distribution at the interface between the connector and tube was examined using particle imaging velocimetry. Flow distributions at the maximum and minimum flow phases during pulsatile flow were observed at the connectors without and with tapers, respectively (Fig. 8).

A larger stagnant flow region was observed at the inlet of the connector with tapers in the minimal flow phase.

A reattachment point of flow was observed approximately 1.5 mm downstream from the outlet junction of the connector and tube for the connector without tapers (Fig. 8, arrow).