Possibilities of Thrombogenicity testing by In Vitro Systems Wim van - - PowerPoint PPT Presentation

Possibilities of Thrombogenicity testing by In Vitro Systems

Wim van Oeveren Haemoscan Groningen The Netherlands Blood compatibility and biomarker detection

Topics to be adressed

• Pro's and Con's of in vitro models
• General overview of various available in vitro methods
• Flow loop model specifics
• In vitro assay validation
• Device geometry
• Investigate material changes in a marketed device

Blood compatibility and biomarker detection

Advantage small in vitro system

• Volume from 3,5 ml
• 1 Volunteer for all test and reference samples
• Reproducible per donor
• Puls, Flow and shear adjustable
• Low costs
• All immuno assays are commercially available (anti-human

antibodies)

• Results of a complete study in short time
• Small materials for testing (from 0,09 cm2)
• Blood compatibility and biomarker detection

Limitations small in vitro system

• Long duration of testing not possible
• Effects endothelial cells ignored
• No feedback functions (from organs)
• No surgical effects (incl release factors)
• Anatomy differences
• Aspecific blood activation (drawing, circuit)
• Anticoagulant needed
• Effects exagerated (ratio device/blood and accumulation)

Blood compatibility and biomarker detection

Blood circulation models

Blood compatibility and biomarker detection

Flow loop model specifics:

++ =good

Chandler Pump Hemobile Making ++ ++ + Handling ++ – ++ Replicates ++ – ++ Flow/shear – ++ + Pulse – – ++ Intrinsic activation ++ _ ++

Hemobile specifics

Blood compatibility and biomarker detection

Haemobile adjusted to heart beat frequency, Doppler flow measurement on tubing

Blood compatibility and biomarker detection (Tubing 8mm, position 3, 15V)

• 0,3
• 0,2
• 0,1

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 flowrate (L/min)

10 5 15

Time (sec)

Validation

• In vitro conditions (Repeatability, reproducibility, accuracy)

Blood compatibility and biomarker detection

• Clinical effects

PDMS induced Thrombus formation: Detachment is possible

1 2 3 4 5 6 7 8 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00

Platelet adhesion to PVC (duplicates)

donor nr Platelets (10E9/cm2)

1 2 3 4 5 6 7 8 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00

Platelet adhesion to PDMS (duplicates)

donor nr Platelets (10E9/cm2)

1 2 3 4 5 6 7 8 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00

Platelet adhesion to PTFE (duplicates)

donor nr platelets (10E9/cm2)

• Validation. In vitro conditions: reproducible

Blood compatibility and biomarker detection

1 2 3 4 5 6 0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70

Complement C5b-9 PVC

donor C5b-9 (ng/ml)

1 2 3 4 5 6 0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70

Complement C5b-9 PTFE

donor C5b-9 (ng/ml)

Validation: reproducibility of complement and donor varia

Blood compatibility and biomarker detection

• Validation. Clinical effects

Blood compatibility and biomarker detection In vitro findings correspond with clinical observations Examples: Heparin coated stainless steel Carmeda coated extracorporeal circuit

Stainless steel without coating: platelet adhesion and fibrin

Coated stainless steel, almost no deposition of blood elements

In vitro results of Carmeda correspond to clinical observations Terumo Duraheart stainless steel housing of LVAD after 150 days implantation in a patient

Uncoated stainless steel stent after 1 hour blood contact in vitro: Thrombus formation Blood compatibility and biomarker detection

1 2 3 4 5 6 7 8 9 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50

Complement activation in vitro (C5b-9)

coating A Carmeda

donor C5b-9 (ug/ml)

1 2 3 4 5 6 7 8 9 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0

Elastase release in vitro

coating A Carmeda

donor elastase (ug/ml)

• Validation. In vitro results of Carmeda correspond to clinical observations:

inhibition of complement activation and leukocyte activation

pre 15"CPB end CPB 30"prot day1 day2 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 uncoated Carmeda

C5b-9 (ng/ml)

pre end CPB day1 day2 0,05 0,1 0,15 0,2 0,25 0,3 uncoated Carmeda

Elastase (ug/ml)

Complement activation in patients Elastase release in patients

Blood compatibility and biomarker detection

Geometry

• Catheters, stents, vascular grafts

in PVC tubing

• Heart valves: special chamber
• Other shapes: attached device

Blood compatibility and biomarker detection

Geometry

Heart valves can be mounted in a special device with a circular inner volume.

Blood compatibility and biomarker detection

Blood compatibility and biomarker detection Testing of a left ventricle supporting device. Flow is generated with the Hemobile and is applied to the test chamber.

Geometry

Haemocompatibility testing ISO 10993-4

Five categories:

• 1. Thrombosis
• 2. Coagulation
• 3. Platelets
• 4. Hematology
• 5. Complement

Blood compatibility and biomarker detection

Choice of testing Thrombosis Inflammation

SEM/Platelet adhesion/P-selectin count/aggregation/function Release products BTG, TxB2,serotonin SEM/ Fibrin adhesion PTT/Thrombin generation TAT, FpA

Convertase activity C5b-9, C3a, C5a Elastase CH50/AP50

Scanning electron microscopy Platelet adhesion, Fibrin adhesion Thromboxane B2, Thrombin-antithrombin III, C5b-9, Elastase, Hemolysis Blood compatibility and biomarker detection Investigate material changes in a marketed device

Preferred direct surface examination

Thrombosis: Scanning electron microscopy Coagulation: Fibrin adhesion Platelets: Platelet adhesion, P-selectin expression Inflammation: Hematology: Leukocyte binding (CD11) Complement: C5-Convertase or C3b Separate experiments (24 hrs): Hemolysis

Blood compatibility and biomarker detection

To be determined

Circulation time 4 hours, or 1 hour, or shorter time (platelet and complement react optimally within 30 minutes.

Blood compatibility and biomarker detection

Conclusion

In vitro systems are excellent tools to determine the material properties. The small loop systems allow multiple testing incl references with blood from 1 (human) donor. Human blood is different from animal blood and all assays can be done on human blood samples. Differences between donors can be observed, which may lead to an estimate of number of donors needed. Thrombogenicity testing by in vitro systems creates new possibilities

Blood compatibility and biomarker detection