Blood compatibility Evaluation of Devices Part II How to test a device

ISO10993 Part 4 (2002):An Overview

Blood compatibility Evaluation of Devices

Hemocompatibility defines the ability of a biomaterial to stay in contact with blood for a clinically relevant period without causing alterations of the formed elements (Cells) and plasma constituents of the blood (Proteins) and without substantially altering the composition of the material itself.

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-vivo system

Thus in-vitro testing of material is critical

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

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 or impedence In optical method PPP is used for setting the base line considering 100% transmittance and PRP at 0 transmittance As aggregates forms PRP get clear and % transmittance increase
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Platelet Activation
After

PLT adhesion A change in PLT shape Generation of biologically active mediators Degranulation

The

specificity of PLT activation and signal transduction is maintained by the presence of PLT receptors that recognize the appropriate PLT agonists. Thrombin ADP Archidonic Acid Collagen Epinephrin
Islamic Unversity of Gaza 10/11/2010

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Platelet Plug Formation: Aggregation

Platelet-Platelet Interaction Mechanism components ATP Ionized calcium Fibrinogen PLT receptor GPIIb/IIIa aggregation REVERSIBLE Secondary aggregation IRREVERSIBLE = white clot, platelet plug formed.
Initial
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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 37 C is determined before and after the addition of various aggregating agents

Figure 1 - Platelet-rich plasma in an optical aggregometer. Platelet count is approximately 200 109/L, and platelets are maintained in suspension by a magnetic stir bar turning at 1000 rpm. (Courtesy of Kathy Jacobs, Chronolog, Inc., Havertown, PA.)

Islamic Unversity of Gaza

10/11/2010

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

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 in light transmittance is converted to electronic signals and recorded as a tracing by the chart recorder.

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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.
Islamic Unversity of Gaza 10/11/2010

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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, & http://cmed-tech.com/graphics/platelet2.jpg, 2009.

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 analyzer.

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Luminescence (Platelet Lumiaggregometry)

The lumiaggregometer may be used to simultaneously measure platelet aggregation and secretion. The instrument records both 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

ASSESSMENT OF PLATELET ACTIVATION TRANSLOCATION OF PLATELET GLYCOPROTEINS AND P-SELECTIN DURING PLATELET ACTIVATION

RESTING
granules GPIIb-IIIa P-selectin GPIV GPIb/IX/V

ACTIVATED

Fibrinogen

P-selectin GPIIb-IIIa GPIV

GPIb/IX/V

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

Platelet activation by flowcytometry

Flow Cytometry is the technological process that allows for the individual 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.

Fluorescence Activation Process

Antibodies recognize specific molecules in the surface of some cells
FITC FITC Antibodies

Antibodies are artificially conjugated to fluorochromes

FITC
FITC

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

But not others

Sample

Y
Sheath

X
Flow chamber

Cells are presented to the laser using principles of hydrodynamic focusing

Y
Laser optics

X
Laser Beam

Laminar Fluidic Sheath Core Sheath

PE FL
FITC FL Outer Sheath

488nm Sct


(PMTs)

Each cell generates a quanta of fluorescence

Photomultiplier Tubes

PE FL

FITC FL

488nm Sct

Discriminating Filters
Dichroic Lenses Confocal Lens

Forward Light Scattering Detector

Negative cells are also detected

PE FL

FITC FL

488nm Sct

Dichroic Lenses Confocal Lens

Forward Light Scatter

Flow Cytometry Data

Smaller Region, Live cells mostly

Larger Region includes all cells

Coagulation

Clotting of Blood Factors Involved in the Process

PT PTT
VIIIa

Heparin

Hirudin, Argatroban

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Two methodologies are available today: 1. Mechanical

2. Optical Photo-optical: clot formation induces change in the plasmas optical density.

Principle

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

Constant Pendular swing of the ball at constant medium viscosity is achieved on the two curvated rail tracks of the cuvettes through the application of:

An electromagnetic field created alternatively at opposite sides of each measurement well by two independent coils. Intensity of the magnetic field can be varied depending on test performed

Test

PT: Calcium Thromboplastin , Extrinsic pathway The prothrombin time is the time it takes plasma to clot after 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

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 inhouse 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

Haematology

Spectrophotometry Haematology Analyzer

Compliment Activation
ELISA Method

Thrombosis
SEM/LM
Mass Analysis Radioscintography