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 repeated animal testing.

Although there exist many numerical models for predicting platelet deposition and thrombosis^3,4,5,6, only a few are suitable for simulating thrombus formation in macro-scale devices such as VADs^7,8,9. Moreover, existing models inevitably assume idealized surfaces and simplified "watertight" geometries, which do not accurately reflect the complexities and imperfections of real-world pump assemblies. When platelet-surface interactions are considered, these macro-scale models generally employ uniformly prescribed material properties (typically modeled as a coefficient in the surface-flux boundary conditions)^10,11,12. Consequently, numerical models cannot completely substitute for experimental testing with blood.

Both material choice and surface finish play critical roles in platelet adhesion on VAD surfaces^{13,14,15,16,17}. Imperfections such as rough spots or irregularities can promote platelet adhesion and thrombus formation. Additionally, crevices between components in the flow path can serve as a nidus for thrombosis, providing protected environments where clots can form and grow^18,19. The use of grease, lubricants, or sealants during assembly can also pose a risk, as these substances may seep into the flow path and interact with the blood, further increasing the risk of complications.

There is, therefore, a need for a well-defined in vitro testing protocol that can reliably assess the thromboresistance of VADs before they are subjected to animal testing or clinical use. While there is a widely adopted ASTM standard for the assessment of hemolysis²⁰, no such standard exists for thrombogenicity testing of VADs under clinically relevant operating conditions²¹. Despite seminal studies dating back three decades demonstrating the feasibility of in vitro thrombosis testing for blood pumps^22,23,24,25, animal testing has persisted as the de facto practice for evaluating thrombosis to date²⁶. The hindrance to wider adoption of in vitro methods has likely been the complex nature of coagulation, with the multitude of confounding factors that can influence test results, making it challenging to differentiate intrinsic pump thrombogenicity from artifacts arising due to methodological limitations and procedural errors.

This motivated us to share a detailed protocol as a guide for experimentalists to avoid pitfalls, hence promoting the use of in vitro testing and mitigating the reliance on animal studies. The protocol described herein, derived from Maruyama et al.²⁷, was refined and validated during the design of the 5^th generation PediaFlow (PF5) pediatric VAD^28,29. This testing method proved instrumental in systematically identifying and addressing potential thrombogenic risks in the VAD prototypes ahead of animal testing.

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-airing and allows the bag to be clamped with a hemostat across the fluid line^30,31. The location of the sealing line is chosen based on the priming volume of the VAD and the tubing to achieve a total priming volume of 150 mL.
   2. Introduce an air vent by making a 4-mm incision at the top of the bag. Insert a barbed female luer lock into the incision and seal the site with RTV silicone rubber adhesive. Attach a one-way stopcock to the luer lock.
2. Assemble the test loop using the modified IV bag, polyvinyl chloride (PVC) tubing, polycarbonate barbed connectors, and 3-way and 1-way stopcocks, as shown in Figure 1.
3. Connect the pressure manometer.
   1. Attach 6-inch extension lines to the inlet and outlet-side pressure ports. Attach a one-way stopcock to the other end of the extension line.
      NOTE: The stopcock allows disconnecting the manometer tubing to prime the extension line with fluid before starting the pump, as described in step 4.3.
   2. Connect one end of each 1/8" inner diameter (ID) tubing to a manometer barb and insert a male luer fitting to the other end.
   3. Connect male luer fittings to the one-way stopcocks. Open the stopcocks to enable pressure readings.
4. Apply a clamp-on ultrasonic flow probe to measure the flow rate.
5. Place a Hoffman clamp distal to the outlet pressure port to regulate flow resistance.

2. Preparation of the calcium chloride (CaCl₂) solution

1. Dissolve powdered CaCl₂ in 1x TRIS-buffered saline (pH 7.4) to prepare a 1 M solution. Avoid phosphate-containing buffers (e.g., PBS or DPBS) since calcium and phosphate react to form an insoluble precipitate.
2. If preparing CaCl₂ in large quantities, add the CaCl₂ powder incrementally in several steps, as its dissolution is an exothermic process.
3. Titrate the pH of the solution to 6-8 using hydrochloric acid (HCl) or sodium hydroxide (NaOH) if necessary.

3. Preparation of blood

NOTE: The ovine blood used in this study was obtained from a commercial vendor listed in the Table of Materials. The blood was collected using a 14-G needle, with the animal restrained in a standard agricultural standing position. The collection process took 10-12 min from needle insertion to completion. The blood was anticoagulated with 14 parts CPD to 86 parts blood (CPD formulation: 26.3 g/L Na-Citrate, 25.2 g of dextrose, 3 g/L citric acid, and 2.2 g/L Na Phosphate in deionized [DI] water). The blood bag was shipped overnight in an insulated container with ice packs and was used for the experiment within 24 h of collection.

1. Prepare the donor blood for use.
   1. Place the blood bag on a tilting platform for 15-30 min to mix the blood gently and allow it to reach room temperature (RT).
   2. Place a magnetic stir bar into a polycarbonate beaker or container designated for the blood transfer.
   3. Transfer the blood into the beaker through a transfusion filter to remove macroaggregates. Handle the blood gently to prevent damage to cellular components. Pour the blood along the container walls to avoid free fall and minimize cellular trauma.
      NOTE: Discard the blood if large thrombi are present or if the filter clogs.
   4. Place the beaker or container on a magnetic stirrer and adjust the speed until the surface of the blood begins to spin gently without forming a large vortex. Stir continuously for at least 5 min.
   5. Measure the hematocrit and platelet count to ensure the values are within the normal range for the species.
2. Perform titrations to determine the target CaCl₂ concentration.
   NOTE: Citrated blood is recalcified to enable coagulation and thrombosis testing, with heparin added to prevent runaway coagulation. The target activated clotting time (ACT) for the start of the test is 300 s. Please note that the reference ACT values provided in this section were obtained using donor sheep blood and may vary depending on the species and the level of anticoagulation used during collection. Additionally, ACT measurements can vary between instruments^32,33,34. Some trial and error may be required to establish the optimal ACT range for a specific laboratory setup.
   1. Transfer 2 mL of the prepared 1 M CaCl₂ solution and 0.5 mL of heparin sodium (1000 U/mL) from the manufacturer's packaging into microcentrifuge tubes to avoid contamination of the stock solutions.
   2. Transfer 10 mL of blood into 14-mL polypropylene test tubes. Prepare four such tubes.
   3. To achieve 0.5 U/mL heparin concentration in blood, add 5 µL of heparin sodium to the 10 mL blood sample.
   4. To achieve a calcium concentration of 5 mM in blood, add 50 µL of 1 M CaCl₂ solution to the 10 mL blood sample. While dispensing, swirl the pipette tip to distribute the CaCl₂ over a wider area.
   5. Immediately, invert the tube 10 times, roll it horizontally (on a flat surface or between palms), then invert 10 more times to ensure thorough mixing.
   6. Measure the ACT using a point-of-care whole blood coagulation system following the manufacturer's instructions. The initial ACT will typically range between 400-500 s.
   7. While keeping the heparin concentration constant, increase the CaCl₂ concentration (to approximately 7-8 mM) to achieve an ACT of 300 s.
   8. Verify the final concentration and the resulting ACT in a separate tube of blood.

4. Pre-test procedures

NOTE: All the steps described in this section apply to Sections 5 and 6. Perform these steps before operating the pump with bovine serum albumin (BSA) or blood in the loop. Transfer blood between vessels using gravity-feeding to minimize mechanical stress. Avoid using the syringe plunger to drive blood, as this can create excessive pressure. Additionally, avoid throttling blood through narrow openings to prevent damage to cellular components.

1. Filling and draining the test loop
   1. Attach a 30-inch extension line to the injection port and connect a 60-mL syringe barrel as a funnel. Open the reservoir stopcock to act as an air vent.
   2. Pour fluid into the funnel, raising it as needed to speed filing. Fill the loop to 1-2 cm below the air vent at the top of the bag. Close the air vent and injection port stopcocks.
   3. To drain, open the air vent and injection port stopcocks and lower the funnel below bench level. Raise the pump above the drainage port to evacuate the remaining fluid.
2. De-airing the test loop
   NOTE: Perform this procedure when filling the loop with BSA or blood prior to starting the pump.
   1. Ensure that there is no air trapped anywhere in the loop. Dislodge air bubbles on surfaces by gently tapping and squeezing the tubing and reservoir.
   2. If evaluating an axial flow blood pump, rotate it into a vertical position to release any air via buoyancy. If using a centrifugal pump, flip it upside down and rotate it to ensure no air is trapped in secondary flow paths.
   3. Closely inspect horizontal sections of tubing and the junctions between tubing and connectors to ensure no air bubbles are present.
   4. Squeeze the IV bag to bring the fluid level close to the top narrow portion and clamp the bag with a hemostat across the fluid line to eliminate the fluid-air interface. Close the air vent stopcock.
3. Priming the pressure lines and the sampling port
   1. Close the stopcock at the end of the pressure lines, remove the manometer tubing, and attach a 3-mL syringe. Open the stopcock and draw 1-2 mL of fluid into the extension line (approximately 4 cm).
   2. Close the stopcock, detach the syringe, and reconnect the manometer tubing. Open the stopcock to enable pressure readings.
   3. Prime the sampling port using a syringe.
4. Cleaning the injection and sampling ports
   1. Cut or tear a lint-free wipe into three equal fragments. Twist the corner of one fragment to form a tip and insert it into the port to absorb any residual blood.
   2. Twist the second fragment, moisten it with saline, and insert the wet tip into the port. Twist it around the port to remove all remaining blood.
   3. Use the third fragment to absorb any residual saline in the port.
      NOTE: Perform this cleaning procedure immediately after drawing a blood sample or whenever residual blood is present in the ports.

5. Passivating the blood-contacting surfaces

1. Fill the loop with 1% BSA solution and operate the VAD to circulate it for at least 30 min.
2. Inspect the loop for leaks and reassemble if necessary.
3. Drain the BSA solution completely to avoid hemodilution.

6. Thrombosis testing

1. Prepare the test loop.
   1. Fill the loop with 150 mL of blood.
   2. De-air the loop and prime the pressure lines and sampling port as described in steps 4.2-4.4.
   3. Start the pump at a low speed and operate it for about 5 s to dislodge any trapped air bubbles within the pump, then stop the pump.
   4. If any bubbles appear in the bag, repeat step 4.2 to perform de-airing again.
   5. Restart the pump at a low speed to circulate the blood in the loop.
2. Add heparin to the blood in the loop.
   1. Transfer 1 mL of heparin sodium (1000 U/mL) and 1.5 mL of ethylenediaminetetraacetic acid (EDTA; 0.5 M) from manufacturer's packaging into separate tubes for ease of handling during the test.
   2. Use the transverse port of the 3-way stopcock to administer substances into the loop using a syringe. Rotate the male luer lock on the stopcock so the injection port faces upward to trap air bubbles at the top.
   3. Achieve a heparin concentration of 0.5 U/mL in the loop by adding 75 U of heparin sodium to 150 mL of blood. Aspirate 75 U of heparin (75 µL if using 1000 U/mL concentration) into a micropipette and dispense it into a 3-mL syringe by simultaneously dispensing the pipette and drawing the syringe plunger. Ensure all liquid is transferred without spilling.
   4. Attach the syringe to the injection port via the luer lock, with the port facing up and the syringe tip pointing vertically down. Draw the syringe plunger to fill it with blood and mix the heparin with the blood while aspirating any air into the syringe. Allow the air bubble to rise to the top of the syringe. Inject the mixture into the loop, ensuring no air enters.
   5. Shuttle blood in and out of the syringe 4-5 times to ensure that the heparinized blood does not stay confined to the space in the port. Ensure that no air enters the loop.
3. Titrate the ACT of blood in the loop.
   1. Achieve an initial calcium concentration of 5 mM in the loop by adding 750 µL of 1 M CaCl₂ solution to 150 mL of blood using the same procedure as heparin.
      1. Blood exposed to high calcium concentrations may start coagulating in the syringe. Swiftly inject the fluid after removing residual air. Optionally, dilute the CaCl₂ in the syringe with 1 ml of TRIS-buffered saline to reduce the risk of premature coagulation.
   2. Allow the injected CaCl₂ to circulate in the loop for at least 2 min before drawing a blood sample to measure the initial ACT.
   3. Attach a 1-mL syringe to the sampling port. Draw and discard 0.5 mL of waste blood to clear stagnant blood from the port.
   4. Attach a new 1-mL syringe, draw 0.5 mL of blood for analysis, and measure the ACT. Clean the sampling port using the procedure in step 4.4.
   5. Refer to the titration values in step 3.2 to inform the target CaCl₂ concentration. Incrementally increase the calcium concentration to achieve an ACT of 300 s. Avoid adding the full amount of CaCl₂ at once to prevent rapid clotting.
      NOTE: In the testing used in this study, the initial calcium concentration of 5 mM resulted in ACT of 450 ± 50 s, and the final concentration of 7-8 mM yielded ACT of 300 ± 20 s. While this response may vary, aim to bring the ACT to or below 300 s for the start of the test.
4. Commence the test.
   1. Once the ACT of 300 s is achieved in the loop, begin the 1-h test. Adjust the pump to the desired flow rate and pressure by modifying the rotor speed and regulating the resistance using the Hoffman clamp.
   2. Measure the ACT every 15 min following steps 6.3.3-6.3.4.
   3. If the ACT drops below 200 s, inject an additional 25 U of heparin sodium into the loop.
5. End the test.
   1. At the end of 1 h, inject EDTA into the loop to inhibit further coagulation, targeting a final concentration of 5 mM in blood. (Inject 1.5 mL if using a 0.5 M EDTA solution.)
   2. Allow the EDTA to circulate and mix for 2 min, then stop the pump.
6. Disconnect and flush the pump.
   1. Clamp the tubing connected to the pump inlet and outlet using hemostats, positioning the clamps 3-4 cm away from the inlet and outlet barbs.
   2. Carefully disconnect the tubing to release the pump.
   3. Drain the blood from the pump and flow loop into a container.
   4. Wash any residual blood from the pump by gravity-feeding or pipetting saline through the pump inlet and outlet.
      NOTE: Steps 6.7 and 6.8 are performed concurrently.
7. Inspect the pump flow path.
   1. Capture images of the pump inlet and outlet using an endoscope/borescope camera.
   2. Disassemble the pump (if applicable). Wash components in a saline bath and inspect for thrombi using a dissecting scope or macro lens.
   3. Photograph blood-contacting surfaces for documentation.
      NOTE: Consistent inspection and thorough documentation are critical for comparative testing.
8. Filter the used blood.
   1. Cut a piece of 100-µm nylon mesh fabric large enough to cover the opening of a catch container.
   2. Position the mesh over the container with a slight drape to allow blood to flow through it effectively. Secure the edges of the mesh to prevent it from slipping during filtration.
   3. Pass the used blood through nylon mesh. Dispose of the blood according to the institution's biological waste handling guidelines.
   4. Examine the mesh for captured thrombi and document the findings.
9. Prepare for histological analysis.
   1. If histological analysis is planned, fix any thrombi found in the formalin solution following appropriate safety procedures.
      CAUTION: Always use a fume hood when handling formaldehyde.

7. Cleaning procedure

1. Disassemble the flow loop. Discard the PVC blood bag and tubing.
2. Soak all other blood-contacting components (connectors, stopcocks, luer locks, etc.) in a 1% enzyme-active powdered detergent solution overnight. Rinse with warm tap water, followed by DI water, and air-dry.
   1. If clots remain, sonicate components in the detergent solution. Rinse thoroughly and air dry.
3. Clean the pump.
   1. If the pump can be disassembled, soak and sonicate the blood-contacting components with a 1% all-purpose cleaner/degreaser solution, followed by a 1% enzyme-active powdered detergent solution.
   2. If the pump cannot be disassembled, connect it to a new flow loop filled with cleaning solutions and operate it at high speed for at least 30 min. Repeat as necessary.
4. Rinse the pump thoroughly with warm tap water, followed by DI water, and air-dry.

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

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