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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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12/4/2013 6
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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12/4/2013 8
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_00017/22/2019 Biocompatibility Evaluation Testing of Devices
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12/4/2013 10
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.jpg7/22/2019 Biocompatibility Evaluation Testing of Devices
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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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12/4/2013 23
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