Académique Documents
Professionnel Documents
Culture Documents
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
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
12/4/2013 5
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
12/4/2013
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)
12/4/2013
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.)
10/11/2010
12/4/2013
Graphics accessed URL http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.
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.
12/4/2013
10
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
11
12/4/2013
11
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.
12/4/2013
12
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.
This modification of aggregation is particularly sensitive to ATP release, and is as sensitive measure of platelet activation.
13
12/4/2013
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.
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
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
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.
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
Sample
Y
Sheath
X
Flow chamber
Y
Laser optics
X
Laser Beam
PE FL
FITC FL Outer Sheath
488nm Sct
(PMTs)
Photomultiplier Tubes
PE FL
FITC FL
488nm Sct
Discriminating Filters
Dichroic Lenses Confocal Lens
PE FL
FITC FL
488nm Sct
Coagulation
PT PTT
VIIIa
Heparin
Hirudin, Argatroban
12/4/2013
23
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
Compliment Activation
ELISA Method
Thrombosis
SEM/LM
Mass Analysis Radioscintography