Biocompatibility Evaluation Testing of Devices

7/22/2019 Biocompatibility Evaluation Testing of Devices

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Blood compatibility Evaluation ofDevices

Part II

How to test a device


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ISO10993 Part 4 (2002):An Overview

Blood compatibility Evaluation of Devices

Hemocompatibilitydefines the ability of a biomaterial to stay in

contact with blood for a clinically relevant period without causingalterations of the formed elements (Cells) and plasma constituents

of the blood (Proteins) and without substantially altering the

composition of the material itself.


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What to test and Why to test

Blood-Material Interactions may lead to

Protein absorption

Cell adhesion

Plasticization/degradation of material and

Thrombi formation/embolism

Cell injury

Tissue damage

Hyperplasia in the in-vivosystem

Thus in-vitro testing of material is critical


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How to test

Screening of the blood

Thrombosis: thrombus mass, LM/SEM (adhered

platelets, leukocytes, aggregates, fibrin etc.) , Ab

labeling to thrombotic components,

Coagulation: Coagulometer (PT, APTT, TT)

Platelets: Activation by aggregomerty, flowcytometry

Haematology: % lysis, plasma Hb, Total Hb

Compliment System: Compliment pathway C3a, C5,

CH50 ELISA method


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12/4/2013 5

Platelet Aggregation

On activation platelets Adhered to each

other and form aggregate

This platelets aggregation can be

measured by using aggregometer

The method can be either optical orimpedence

In optical method PPP is used for

setting the base line considering 100%

transmittance and PRP at 0transmittance

As aggregates forms PRP get clear and

% transmittance increase


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Platelet Activation

After PLT adhesion

A change in PLT shapeGeneration of biologically active mediators

Degranulation

The specificity of PLT activation and signaltransduction is maintained by the presence of PLTreceptors that recognize the appropriate PLT agonists.

Thrombin

ADP

Archidonic Acid

Collagen

Epinephrin

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Platelet Plug Formation: Aggregation

Platelet-Platelet Interaction

Mechanism components

ATP

Ionized calcium

Fibrinogen

PLT receptor GPIIb/IIIa

Initial aggregation

REVERSIBLESecondary aggregation

IRREVERSIBLE = white clot,

platelet plug formed.


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Platelet Aggregometry

Platelet aggregation is an essential part of the investigation of

any patient with a suspected platelet dysfunction.

Principle

Aggregating agents to induce platelet aggregation or cause

platelets to release endogenous ADP, or both.

Platelet aggregation is studied by means of a platelet

aggregometer, Used Principle:

1. Photo-optical Method

2. Electrical Impedance Method

3. luminescence technology (Platelet Lumiaggregometry)


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Photo Optical

photometry: optical density of

PRP warmed to 37C is determinedbefore and after the addition ofvarious aggregating agents

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Graphics accessed URL http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.

Figure 1- Platelet-rich plasma in an optical aggregometer. Platelet count isapproximately 200 109/L, and platelets are maintained in suspension by a magnetic stirbar turning at 1000 rpm. (Courtesy of Kathy Jacobs, Chronolog, Inc., Havertown, PA.)
http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001


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Photo Optical Aggregometry

The Platelet-rich plasma, which is turbid in appearance,

is placed in a cuvette, warmed to 37C in the heating

block of the instrument, and stirred via a small magnetic

bar.

Baseline light transmittance through the platelet-rich

plasma is recorded. The addition of an aggregating agent

causes the formation of larger platelet aggregates with a

corresponding increase in light transmittance, because

of a clearing in the platelet-rich plasma. The change inlight transmittance is converted to electronic signals and

recorded as a tracing by the chart recorder.


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o Sample

o Platelet-Rich Plasma

(PRP)

o PRP is prepared and

adjusted, to a count of

200-300 X 109/L by

mixing with PPP.

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Graphics accessed URL http://www.mclno.org/webresources/pathman/BT_web/bt_paper.jpg , http://www.accumetrics.com/images/img_product_overview.jpg , & https://reader009.{domain}/reader009/html5/0308/5aa0e4013a21e/5a, 2009.
http://www.mclno.org/webresources/pathman/BT_web/bt_paper.jpghttp://www.accumetrics.com/images/img_product_overview.jpghttp://cmed-tech.com/graphics/platelet2.jpghttp://cmed-tech.com/graphics/platelet2.jpghttp://cmed-tech.com/graphics/platelet2.jpghttp://cmed-tech.com/graphics/platelet2.jpghttp://cmed-tech.com/graphics/platelet2.jpghttp://cmed-tech.com/graphics/platelet2.jpghttp://www.accumetrics.com/images/img_product_overview.jpghttp://www.mclno.org/webresources/pathman/BT_web/bt_paper.jpg


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Electrical Impedance Method

These types of analyzers may use citrated whole

blood, as the test sample. As platelets aggregate, the coat an electrode,

impeding the electrical current through the ana-

lyzer.


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Luminescence (Platelet Lumiaggregometry)

The lumiaggregometer may be used to simultaneously measure

platelet aggregation and secretion. The instrument recordsboth aggregation and secretion of dense-granule ATP.

The ATP is measured by its reaction with firefly luciferin to give

chemiluminescence. The resulting light emission is detected,amplified, and recorded by the instrument.

Performed by using whole blood or PRP.

This modification of aggregation is particularly sensitive to ATP

release, and is as sensitive measure of platelet activation.


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Control/ calibration

Instrument calibration has to be done by service

personnel or calibration cell. (speed/ tm)

Fresh PRP from healthy person is used as control

before running the test samples.

Agonist prepared and functionality is checked before

performing the test


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ASSESSMENT OF PLATELET ACTIVATION

TRANSLOCATION OF PLATELET GLYCOPROTEINS AND P-SELECTIN

DURING PLATELET ACTIVATION

ACTIVATION : - GPIb IX V : internalized- GPIIbIIIa : 1) membrane expression increased

2) complex occupied by fibrinogen, v. Willebrand Factor ...- P-selectin : translocated to the membrane

RESTING ACTIVATED

ACTIVATION

granules

P-selectin

GPIV

GPIIb-IIIa

GPIb/IX/V

P-selectin

GPIIb-IIIa

GPIV GPIb/IX/V

Fibrinogen


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Platelet activation by flowcytometry

Flow Cytometry is the technological process that allows for theindividual measurements of cell fluorescence and light

scattering. This process is performed at rates of thousands of

cells per second.

Flow cytometry integrates electronics, fluidics, computer,

optics, software, and laser technologies in a single platform.


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Fluorescence Activation Process

FITCFITC

FITC

FITC

Antibodies recognize specific

molecules in the surface ofsome cells

But not others

When the cells are analyzed by flow

cytometry the cells expressing the marker

for which the antibody is specific will

manifest fluorescence. Cells who lack the

marker will not manifest fluorescence

Antibodies are artificially

conjugated to fluorochromes

Antibodies


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Laser optics

Laser Beam

Flow

chamber

Sheath

Sample

Y

X

Z

Y Z

X

Cells are presented

to the laser using

principles of

hydrodynamic

focusing


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PE FL

FITC FL

488nm Sct

Laminar Fluidic Sheath

Core

Sheath

Outer

Sheath


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Each cell generates a quanta of fluorescence

PE FL FITC FL 488nm Sct

Confocal LensDichroic Lenses

Photomultiplier Tubes

(PMTs)

Discriminating

FiltersForward

Light

Scattering

Detector


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Negative cells are also detected

PE FL FITC FL 488nm Sct

Confocal Lens

Dichroic LensesForward

Light

Scatter


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Flow Cytometry Data

Smaller

Region,Live cells

mostly

Larger Region

includes all cells

Coagulation


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Coagulation Clotting of Blood

Factors Involved in the Process

PT

PTTVIIIa

Heparin

Hirudin,

Argatroban


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Two methodologies are available today:

1. Mechanical

2. Optical

Photo-optical:clot formation induceschange in the plasmas optical density.

P i i l


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Principle

Clotting determination is based on ball oscillation amplitudevariation recorded through an inductive displacement sensor

Constant Pendular swing of the ball at constant mediumviscosity is achieved on the two curvated rail tracks of thecuvettes through the application of:

An electromagnetic field created alternatively at oppositesides of each measurement well by two independent coils.

Intensity of the magnetic field can be varied depending on

test performed

Test


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Test

PT:Calcium Thromboplastin , Extrinsic pathway The

prothrombin time is the time it takes plasma to clotafter addition of tissue factors.

aPTT:Recalcification of the plasma in the presence

of cephalin and Kaolin

Fibrinogen:clotting time of plasma in the presence

of excess thrombin

Factors:Deficient Plasma

C lib ti / C t l


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Calibration/ Controls

Instrument calibration has to be done by service

personnel or calibration cell. (speed/ tm).

Commercially available controls Specialty Assayed

Ref Plasma and Specialty Assay Control as well in-

house stabilized plasma is used as internal control

Proficiency testing , inter-laboratory comparison to

maintain the quality system

ISO 10993-4 standard used for the blood material

interaction . Horzontal standard


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Haematology

Spectrophotometry Haematology Analyzer

Compliment ActivationELISA Method

Thrombosis

SEM/LM

Mass Analysis

Radioscintography

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Biocompatibility Evaluation Testing of Devices