In Vitro Thrombosis Test for Ventricular Assist Devices

Summary

We present a benchtop protocol to induce thrombosis in ventricular assist devices (VAD) within a recirculating test platform. This method serves to identify thrombogenic hotspots in the blood flow path and can help improve thromboresistance ahead of preclinical testing in animal models.

Abstract

The risk of thrombosis remains a significant concern in the development and clinical use of ventricular assist devices (VADs). Traditional assessments of VAD thrombogenicity, primarily through animal studies, are costly and time-consuming, raise ethical concerns, and ultimately may not accurately reflect human outcomes. To address these limitations, we developed an aggressive in vitro testing protocol designed to provoke thrombosis and identify potential high-risk areas within the blood flow path. This protocol, motivated by the work of Maruyama et al., employs a modified anticoagulation strategy and utilizes readily available components, making it accessible to most laboratories conducting in vitro blood testing of VADs. We demonstrated the utility of this method through iterative testing and refinement of a miniature magnetically levitated pediatric VAD (PediaFlow PF5). The method has been effective in identifying thrombogenic hotspots caused by design and manufacturing flaws in early VAD prototypes, enabling targeted improvements before advancing to animal studies. Despite its limitations, including the absence of pulsatile flow and the influence of donor blood characteristics, this protocol serves as a practical tool for early-stage VAD development and risk mitigation.

Introduction

Ventricular assist devices (VADs) have become a standard of care in the management of patients with advanced heart failure, yet the risk of thrombosis and stroke remains a significant challenge^1,2. Thrombosis within VADs is typically assessed during preclinical animal studies, which, while valuable, present substantial costs and logistical challenges. These studies are resource-intensive, time-consuming, and are susceptible to a single defect compromising the entire test and necessitating additional trials. This not only increases the financial burden but also raises ethical concerns due to the need for repeat....

Protocol

Ovine whole blood used in this study was obtained from a commercial vendor and, therefore, did not require a review by Cornell University's Institutional Animal Care and Use Committee.

1. Construction of the test flow loop

NOTE: See the Table of Materials for a detailed list of loop components and all other materials used in this protocol.

1. Modify the intravenous (IV) bag.
   1. Use a plastic heat sealer to modify an IV bag to adjust its volume and shape, as depicted in Figure 1.
      NOTE: The shape of the bag facilitates de-....

Results

Successful execution of this protocol enables the identification of localized areas of platelet deposition, revealing problematic spots within the pump's flow path. Consistent application of this protocol allows for incremental improvements by addressing these identified "hotspots".

For example, during the development of the PediaFlow PF5 VAD, we encountered challenges in manually polishing the pressure side of the stator vanes due to the miniature size of the components. In vi.......

Discussion

First-in-human trial of a new pump is always a precarious endeavor, as preclinical studies cannot reliably predict the thrombogenicity of VADs in humans²⁶. Notably, some VADs that demonstrated freedom from thrombosis in animal trials have later exhibited significant thrombogenicity in clinical use³⁶. An aggressive in vitro testing regimen specifically designed to provoke thrombosis provides a valuable opportunity to identify potential design or manufacturing flaws .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
14-mL test tubes	Falcon	352059	Round bottom polypropylene test tubes with snap-cap
1-way stopcock	Qosina	99759	Female Luer Lock, Male Luer with Spin Lock
3-way stopcock	Qosina	99771	2 Female Luer Locks, Rotating Male Luer Lock
ACT+ cuvettes for Hemochron	Werfen	000JACT+	45/Box
All-purpose cleaner/degreaser	Simple Green	2710200613005	Simple Green Cleaner and Degreaser. Use 1% solution.
Barbed connectors	Qosina	73311	Material: polycarbonate; ¼” x ¼” straight connector
Barbed connectors w/ luer lock	Qosina	73316	Material: polycarbonate; ¼” x ¼” straight connector with luer lock
Bovine Serum Albumin (BSA)	Thermo Scientific Chemicals	AAJ6465522	Or equivalent
Calcium chloride, CaCl2	Thermo Scientific Chemicals	AA89866-30	Anhydrous, ≥96.0% ACS
Dissecting scope (recommended)	Olympus	https://www.olympus-lifescience.com/en/technology/museum/micro/1984/	Olympus SZH10 (continuous zoom magnification 7x - 70x) or similar
DPBS (w/o calcium and magnesium)	Gibco	14200075	Dulbecco's phosphate-buffered saline, no calcium, no magnesium, 10X (must be diluted to 1X before use)
EDTA	Quality Biological	351-027-721EA	0.5 M, pH 7.0–8.0 (Ethylenediaminetetraacetic acid)
Endoscope/borescope/otoscope camera (optional)	Bebird	https://bebird.com/products/earsight-pro-ear-wax-removers	3–4 mm probe diameter
Enzyme-active powdered detergent	Alconox	1304-1	Alconox Tergazyme. Use 1% solution.
Extension Line, 30"	Qosina	36218	30" length,  female luer lock to male luer lock
Extension Line, 6"	Qosina	36212	6" length,  female luer lock to male luer lock
Female luer lock, barbed	Qosina	11548	Fits 1/8 inch ID Tubing; material: polycarbonate;
Flow meter	Transonic	https://www.transonic.com/t402-t403-consoles	Transonic TS410 module
Hemostat	Fisherbrand	13-820-004	Locking hemostat with at least 5 cm tip length
Heparin Sodium	McKesson Packaging Services	949513	1000 U/mL concentration
Hoffman clamp	Humboldt	H8720	Fine-threaded clamp
IV bag (compliant blood reservoir)	Qosina	51494	Material: PVC, 2 Tube ports 0.258” ID. The 100-ml bag is modified using a heat sealer
Lint-free wipes	Kimberly-Clark Professional	34120	Kimtech Science Wipers
Magnetic stirrer	INTLLAB	MS-500	Or similar
Male luer lock, barbed	Qosina	11549	Fits 1/8 inch ID Tubing; material: polycarbonate;
Manometer (digital)	Sper Scientific	840081	SPER-840081 or similar
Nylon filtering mesh	McMaster-Carr	9318T21	100-μm (0.0039") opening size
Ovine blood	Lampire	7209004	Donor whole blood, anticoagulated with ACD 14:86, shipped overnight
Plastic bag heat sealer	Uline	H-190	Uline H-190 or similar (without cutter)
Silicone rubber adhesive	Smooth-On	B00IRC1YI0	Sil-Poxy or similar
Syringe w/ luer lock, 1 mL	Fisher Scientific	14-955-646	Fisherbrand manual syringe without needle for research purposes
Syringe w/ luer lock, 3 mL	Fisher Scientific	14-955-457	Fisherbrand manual syringe without needle for research purposes
Syringe w/ luer lock, 60 mL	Fisher Scientific	14-955-461	Fisherbrand manual syringe without needle for research purposes
Transfusion filter	Haemonetics Corporation	SQ40S/SQ40NS	Haemonetics Corporation SQ40S pall blood transfusion filter
TRIS Buffered Saline	Thermo Scientific Chemicals	AAJ62938K2	TBS 10x (must be diluted to 1X before use), pH 7.4
Tubing	Tygon	ADF00017	Tygon ND-100-65 tubing (medical grade)
Ultrasonic flow sensor	Transonic	https://www.transonic.com/hqxl-flowsensors	Select appropriate flow sensor model for the tubing size used. ME6PXL clamp-on sensor fits the 3/8” OD tubing. The sensor is calibrated by Transonic for the test fluid (e.g., blood at  24C) and tubing grade (e.g. Tygon ND-100-65)
Ultrasonic sonicator (optional)	Branson Ultrasonics	CPX952238R	Branson CPX2800H or similar
VAD system	PediaFlow	PF5	The VAD system to be tested; includes the pump and the controller
Whole Blood Coagulation System	Werfen	https://www.werfen.com/na/en/point-of-care-testing-devices/ACT-machine-hemochron-signature-elite	Hemochron Signature Elite or Signature Jr