Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

Summary

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Abstract

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body's circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 °C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (β-thromboglobulin [β-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

Introduction

The in vivo application of implants and biomaterials, which interact with human blood, requires intense preclinical testing focusing on the investigation of various markers of the hemostatic system. The International Organization for Standardization 10993-4 (ISO 10993-4) specifies the central principles for the evaluation of blood-contacting devices (i.e., stents and vascular grafts) and considers the device design, clinical utility, and materials needed¹.

Human blood is a fluid that contains various plasma proteins and cells, including leukocytes (white blood cells [WBCs]), erythrocytes (red blood cells [RBCs]), and platelets, which carry out complex functions in the human body². The direct contact of foreign materials with blood can cause adverse effects, such as activation of the immune or coagulation system, which can lead to inflammation or thrombotic complications and serious issues after implantation^3,4,5. Therefore, in vitro hemocompatibility validation offers an opportunity prior to implantation to detect and exclude any hematologic complications that may be induced upon contact of the blood with a foreign surface⁶.

The presented flow loop model was established to assess the hemocompatibility of neurovascular stents and similar devices by applying a flow rate of 150 mL/min in tubing (diameter of 3.2 mm) to mimic cerebral flow conditions and artery diameters^2,7. Besides the need for an optimal in vitro model, the source of blood is an important factor in gaining reliable and unaltered results when analyzing hemocompatibility of a biomaterial⁸. The collected blood should be used immediately after sampling to prevent changes caused by prolonged storage. In general, a gentle collection of blood without stasis using a 21 G needle should be performed to minimize the preactivation of platelets and the coagulation cascade during blood drawing. Furthermore, donor exclusion criteria include those who smoke, are pregnant, are in a poor state of health, or have taken oral contraceptives or painkillers during the previous 14 days.

This study describes an in vitro model for the extensive hemocompatibility testing of stent implants under flow conditions. When comparing uncoated to fibrin-heparin-coated stents, results of the comprehensive hemocompatibility tests reflect improved hemocompatibility of the fibrin-heparin-coated stents⁹. In contrast, the uncoated stents induce activation of the coagulation cascade, as demonstrated by an increase in thombin-antithrombin III (TAT) concentrations and loss of blood platelet numbers due to the adhesion of platelets to stent surface. Overall, integrating this hemocompatibility model as a preclinical test is recommended to detect any adverse effects on the hemostatic system that are caused by the device.

Protocol

The blood sampling procedure was approved by the Ethics Committee of the medical faculty at the University of Tuebingen (project identification code: 270/2010BO1). All subjects provided written, informed consent for inclusion before participation.

1. Preparation of Heparin-loaded Monovettes

1. Mix the undiluted heparin (5,000 IU/mL) with sodium chloride (NaCl, 0.9%) solution and prepare a solution with a resulting concentration of 15 IU/mL of heparin.
2. Add 900 µL of the diluted heparin solution to each neutral monovette (9 mL) to obtain a final heparin concentration of 1.5 IU/mL after blood sampling. Prepare three monovettes per donor plus three reserve monovettes and store the heparin-loaded monovettes at 4 °C until blood sampling.

2. Blood Sampling

1. Take the heparin-loaded monovettes out of the refrigerator 30 min prior to blood sampling.
2. Collect a 27 mL blood sample from each healthy donor (n = 5) by venipuncture for the flow loop. Only apply a smooth tourniquet to avoid premature activation of the platelets and the blood clotting cascade.
3. Collect blood samples in three monovettes containing 900 µL of the heparin solution (1.5 IU/mL) and pool all three monovettes in one plastic container to ensure that all components are evenly distributed.
4. Directly transfer the pooled heparinized blood into three different monovettes containing either EDTA (1.2 mL), citrate (1.4 mL), or a mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD, 2.7 mL) to collect baseline values. Proceed with the samples as described in sections 5−8.
   NOTE: To guarantee uninfluenced clotting behavior, donors should avoid the intake of hemostasis-affecting drugs (e.g., acetylsalicylic acid, naproxen, and carbenicillin) within the last 14 days, as well as oral contraceptives and smoking.

3. Preparation of the Flow Loop

1. Cut three heparin-coated polyvinyl chloride tubes with a length of 75 cm and inner diameter of 3.2 mm. Load the tubes with the neurovascular laser-cut implants with or without the fibrin-heparin coating. Remember to leave one tub unloaded as a control.
2. Place one end of the tube in a reservoir filled with 0.9% NaCl, connect the tubing to the pump head, and insert the other end into a measuring cylinder.
3. Adjust the settings of the peristaltic pump to achieve a flow rate of 150 mL/min by using a timer while checking the fill level in the measuring cylinder.

4. Performance of Hemocompatibility Testing

1. Use a 12 mL syringe to fill the tubes with blood. Let 6 mL of blood flow smoothly into each tube containing a sample or unloaded control.
2. Form a circuit and close the tubes tightly using a 0.5 cm length of silicone connection tubing. Place the tubes in a water bath of 37 °C and start the perfusion for 60 min.

5. Whole Blood Count Analysis

1. Put 1.2 mL of blood after sampling (baseline) or after perfusion into a monovette containing EDTA and carefully invert the tube 5x.
2. Insert the monovette into the blood analyzer and perform a blood count analysis for every sample. Then, incubate the monovettes on ice for 15−60 min after the blood count measurement for further analysis, as described in section 7.

6. Collection of Citrate Plasma

1. Fill the monovettes containing citrate with 1.4 mL of blood (freshly drawn or after circulation) and carefully invert 5x.
2. Centrifuge the tubes for 18 min at 1,800 x g at room temperature (RT). Aliquot three 250 µL samples of the plasma fraction into 1.5 mL reaction tubes and freeze the plasma samples in liquid nitrogen. Store them at -20 °C until analysis.

7. Collection of EDTA Plasma

1. Incubate the monovettes on ice for 15−60 min after the blood count measurement. Then, centrifuge the tubes for 20 min at 2,500 x g and 4 °C.
2. Aliquot three 250 µL samples of the plasma fraction into 1.5 mL reaction tubes after centrifugation and freeze the tubes in liquid nitrogen. Store them at -80 °C until analysis.

8. Collection of CTAD Plasma

1. Fill the monovettes containing the CTAD mixture with 2.7 mL of blood (freshly drawn or after incubation) and carefully invert 5x. Afterwards, incubate the monovettes on ice for 15−60 min. Then, centrifuge the tubes for 20 min at 2,500 x g and 4 °C.
2. Transfer 700 µL of the middle plasma fraction into a 1.5 mL reaction tube and centrifuge the filled reaction tubes for 20 min at 2,500 x g and 4 °C.
3. Aliquot two 100 µL samples of the middle fraction into 1.5 mL reaction tubes after centrifugation and freeze the tubes in liquid nitrogen. Store them at -20 °C until analysis.
   NOTE: The collection of EDTA plasma and CTAD plasma can be performed together because the operating conditions are the same.

9. Measurement of Human TAT from Citrate Plasma

1. Thaw the citrate plasma in a water bath of 37 °C.
2. Use the TAT enzyme-linked Immunosorbent assay (ELISA) kit according to the manufacturer's instructions. Reconstruct the plasma standards and control and dilute the washing solution, anti-human-TAT peroxidase (POD)-conjugated antibody, and chromogen solution. Leave all reagents and the microtiter plate at RT (15−25 °C) for 30 min before starting the test.
3. Pipet 50 µL of the sample buffer into each well of the microtiter plate and add 50 µL of the sample buffer (blank), plasma standard, plasma control, and undiluted plasma sample in duplicates to the well plate. Seal the plate and incubate at 37 °C for 15 min with gentle shaking. Then, wash the plate 3x with 300 μL of washing solution.
4. Add 100 µL of the POD-conjugated anti-human-TAT antibody to each well. Seal the plate and incubate at 37 °C for 15 min with gentle shaking. Then, wash the plate 3x with 300 μL of washing solution.
5. Add 100 µL of the freshly prepared chromogen solution to each well. Seal the plate and incubate at RT for 30 min.
6. Remove the seal film and add 100 µL of stop solution to each well. Read the optical density (OD) with a photometer at 490−500 nm. Fit the standard curve data as a trend line and calculate the concentration of the samples.

10. Measurement of PMN-elastase from Citrate Plasma

1. Thaw the citrate plasma in a water bath at 37 °C.
2. Use the polymorphonuclear (PMN)-elastase ELISA kit according the to manufacturer's instructions: reconstruct the PMN-elastase control and the PMN-elastase standard to prepare a standard curve using the kit's dilution buffer.
3. Dilute the washing solution according to the manufacturer's description. Leave all reagents and the microtiter plate at RT for 30 min before starting the test. Dilute the citrate plasma samples to 1:100 with the dilution buffer.
4. Add 100 µL of the sample buffer (blank), PMN-elastase standard curve (15.6−1,000 ng/ mL), PMN-elastase controls (high and low concentrations), and diluted plasma samples in duplicates to the well plate. Seal the plate and incubate at RT for 60 min with gentle shaking. Afterwards, wash the plate 4x with 300 μL of washing solution.
5. Add 150 µL of the enzyme-conjugated antibody to each well. Seal the plate and incubate at RT for 60 min with gentle shaking. Afterwards, wash the plate 4x with 300 μL of washing solution.
6. Add 200 µL of the 3,3',5,5'-tetramethylbenzidin (TMB)-substrate solution to each well. Seal the plate and incubate at RT for 20 min in the dark. Then, remove the seal film and add 50 µL of the stop solution to each well.
7. Read the OD with a photometer at 450 nm with a reference reading at 630 nm. Fit the standard curve data as a trend line and calculate the concentration of the samples.

11. Measurement of Terminal Complement Complex (TCC) from EDTA Plasma

1. Thaw the EDTA plasma in a water bath at 37 °C, and store on ice after defrosting.
2. Use the complement cascade SC5b-9 ELISA kit according to the manufacturer's instructions: dilute the washing solution as described in the manufacturer's protocol. Leave all reagents and the microtiter plate at RT for 30 min before starting the test. Dilute the EDTA plasma samples to 1:10 with the kit's dilution buffer.
3. Add 300 µL of washing solution to each well to rehydrate the surface and aspirate after 2 min. Add 100 µL of the sample buffer (blank), SC5b-9 standards, SC5b-9 controls (high and low concentrations), and the diluted plasma samples in duplicates to the well plate.
4. Seal the plate and incubate at RT for 60 min. Next, wash the plate 5x with 300 μL of washing solution.
5. Add 50 µL of the enzyme-conjugated antibody to each well. Seal the plate and incubate at RT for 30 min. Then, wash the plate 5x with 300 μL of washing solution.
6. Add 100 µL of the TMB-substrate solution to each well. Seal the plate and incubate at RT for 15 min in the dark.
7. Remove the seal film and add 100 µL of the stop solution to each well. Read the OD with a photometer at 450 nm. Fit the standard curve data as a trend line and calculate the concentration of the samples.

12. Measurement of β-thromboglobulin from CTAD Plasma

1. Thaw the CTAD plasma in a water bath at 37 °C.
2. Use the β-thromboglobulin (β-TG) ELISA kit according to the manufacturer's instructions: reconstruct the β-TG control and the β-TG standard and dilute the washing solution using distilled H₂O. Reconstruct the POD-conjugated antibody using the provided phosphate buffer. Leave all reagents and the microtiter plate at RT for 30 min before starting the test.
3. Prepare the standard curve and the control according to the manufacturer's instructions with the provided phosphate buffer. Dilute the CTAD plasma samples to 1:21.
4. Add 200 µL of the phosphate buffer (blank), β-TG standards, β-TG controls (high and low concentrations), and diluted plasma samples in duplicates to the well plate. Seal the plate and incubate at RT for 60 min. Afterwards, wash the plate 5x with 300 μL of washing solution.
5. Add 200 µL of the enzyme-conjugated antibody to each well. Seal the plate and incubate at RT for 60 min. Afterwards, wash the plate 5x with 300 μL of washing solution.
6. Add 200 µL of the TMB-substrate solution to each well. Seal the plate and incubate at RT for 5 min in the dark. Remove the seal film and stop the reaction by adding 50 µL of 1 M sulfuric acid (H₂SO₄) to each well.
7. Leave the plate for 15−60 min, then read the OD with a photometer at 450 nm. Fit the standard curve data as a trend line and calculate the concentration of the samples.

13. Sample Preparation for Scanning Electron Microscopy

1. Remove the implant from the tube using forceps and rinse the implant briefly by dipping it into 0.9% NaCl solution 3x.
2. Store in glutaraldehyde solution (2% glutaraldehyde in phosphate-buffered saline [PBS-buffer without Ca²⁺/Mg²⁺]) overnight at 4 °C.
3. Next, incubate the implants in PBS-buffer for 10 min. Dehydrate the samples by incubating in ethanol with increasing concentration for 10 min each: 40%, 50%, 60%, 70%, 80%, 90%, 96%, and 100%. Store the dehydrated samples in 100% ethanol until further analysis.
4. Perform critical point drying according to the instructions of the drying device or literature¹⁰ just before scanning electron microscopy (SEM).

14. Scanning Electron Microscopy

1. Attach the dried implants to a sample carrier for the scanning microscope and sputter the samples with gold palladium.
2. Introduce the sputtered implants into the sample chamber. Take pictures in 100-, 500-, 1,000- and 2,500-fold magnification of the areas with the representative surface and cell adhesion.

Results

Briefly summarized, human whole blood was collected in heparin-loaded monovettes then pooled and used to evaluate the baseline levels of cell counts as well as plasmatic hemocompatibility markers.

Subsequently, the tubing containing the neurovascular implant samples was filled, and the blood was perfused for 60 min at 150 mL/min and 37 °C using a peristaltic pump. Again, the number of cells was analyzed in all groups, and the plasma samples were prepared for ELISA analyses (

Discussion

The presented protocol describes a comprehensive and reliable method for the hemocompatibility testing of blood-contacting implants in accordance with ISO 10993-4 in a shear flow model imitating human blood flow. This study is based on the testing of laser-cut neurovascular implants but can be performed with a variety of samples. The results demonstrate that this method enables the broad analysis of various parameters such as the blood cell count, prevalence of several hemocompatibility markers, and microscopic visualiza...

Materials

Name	Company	Catalog Number	Comments
aqua ad iniectabilia	Fresenius-Kabi, Bad-Homburg, Germany	1088813
beta-TG ELISA	Diagnostica Stago, Duesseldorf, Germany	00950
Centrifuge Rotana 460 R	Andreas Hettich, Tuttlingen, Germany	-
Citrat monovettes (1.4 mL)	Sarstedt, Nümbrecht, Germany	6,16,68,001
CTAD monovettes (2.7 mL)	BD Biosciences, Heidelberg, Germany	367562
EDTA monovettes (1.2 mL)	Sarstedt, Nümbrecht, Germany	6,16,62,001
Ethanol p.A. (1000 mL)	AppliChem, Darmstadt, Germany	1,31,08,61,611
Glutaraldehyde (25 % in water)	SERVA Electrophoresis, Heidelberg, Germany	23114.01
Heparin coating for tubes	Ension, Pittsburgh, USA	-
Heparin-Natrium (25.000 IE/ 5 mL)	LEO Pharma, Neu-Isenburg, Germany	PZN 15261203
Multiplate Reader Mithras LB 940	Berthold, Bad Wildbad, Germany	-
NaCl 0,9%	Fresenius-Kabi, Bad-Homburg, Germany	1312813
Neutral monovettes (9 mL)	Sarstedt, Nümbrecht, Germany	2,10,63,001
PBS buffer (w/o Ca2+/Mg2+)	Thermo Fisher Scientific, Darmstadt, Germany	70011044
Peristaltic pump ISM444B	Cole Parmer, Wertheim, Germany	3475
Pipette (100 µL)	Eppendorf, Wesseling-Berzdorf, Germany	3124000075
Pipette (1000 µL)	Eppendorf, Wesseling-Berzdorf, Germany	3123000063
Plastic container (100 mL)	Sarstedt, Nümbrecht, Germany	7,55,62,300
PMN-Elastase ELISA	Demeditec Diagnostics, Kiel Germany	DEH3311
Polyvinyl chloride tube	Saint-Gobain Performance Plastics Inc., Courbevoie France	-
Reaction Tubes (1.5 mL)	Eppendorf, Wesseling-Berzdorf, Germany	30123328
neurovascular laser-cut implants	Acandis GmbH, Pforzheim	01-0011x
SC5b-9 ELISA	TECOmedical, Buende, Germany	A029
Scanning electron microscope	Cambridge Instruments, Cambridge, UK	-
Sealing tape (96 well plate)	Thermo Fisher Scientific, Darmstadt, Germany	15036
Syringe 10/12 mL Norm-Ject	Henke-Sass-Wolf, Tuttlingen, Germany	10080010
TAT micro kit	Siemens Healthcare, Marburg, Germany	OWMG15
Waterbath Type 1083	Gesellschaft für Labortechnik, Burgwedel, Germany	-