Impedance whole blood electrical aggregometry: Measurements, evaluation and interpretation of aggregometry outcomes Cezary Watała Department of Haemostatic Disorders Medical University of Lodz cwatala@csk.umed.lodz.pl www.interhemostaza.pl
What is our object of study?
blood platelets
resting (non-activated) platelets
platelets activated with ADP
attachment of platelets to surfaces mediated by GPIIb/IIIa
Multiplate multichannel impedance aggregometer Multiplate - multiple platelet function analyzer multiple electrodes and multiple channels allow for multiple test procedures comprehensive assessment of platelet function 5 independent channels instrument integrated with computer optionally available with automatic pipetting
Technical details beyond Multiplate aggregometer pipetting inlet jack portion cup portion with sensor wires Multiplate test cell Test cell electrodes
Multiplate test cell how does it function? change of resistance between two electrodes immersed into the blood double determination during each test (two pairs of electrodes per cell) ensures monitoring of intravariability
how Multiplate cell works? electrode resistance enhanced by adhered platelet aggregates
when activated platelets adhere onto the metal sensor wires and rise the electrical resistance between the wires to rise the resistance plts have to adhere firmly
Multiplate mounting the cell pipetting inlet 2 sensors, each with 2 electrodes cup plug inlet teflon-coated stirrer
How to perform aggregation test? 1 2 put the test cell into the holder attach the sensor cable to the cell
How to perform aggregation test? 3 4 wait 3 minutes for warming and equilibration add 600 ml of 2-fold diluted whole blood 5 add the activator and monitor aggregation for 10-15 min
Random allocation of numbers in series seed: left blank for automatic seed
Multiplate aggregation curve Area under the curve (AUC) test cell Aggregation (AU) sensors time (min)
A number of specific test reagents are available various tests are available to allow triggering of different receptors / signal transduction pathways in platelets and detect changes induced by various drugs or congenital disorders
Typical aggregation curves in MULTIPLATE test TRAP test COL test COL test + ex-asa ASPI test control ASA responder ASA nonresponder
Which activation pathways are monitored with the Multiplate? using the different tests one can get the comprehensive information on platelet function and effectiveness of antiplatelet therapy
ASPI test
COL test
TRAP test
ADP test
Positive controls in MULTIPLATE test ASPI test: in vitro addition of ASA - selective inhibition of COX-1 GPIIb/IIIa antagonists - blockade of fibrinogen and GPIIb/IIIa interaction
COL test - examples no ASA intake treatment with ASA 100 mg / day COLtest COLtest + ASA COLtest COLtest + ASA normal aggregation no additional effect of in vitro ASA
calcium
How to collect blood for Multiplate analysis? in citrated blood > 98% free Ca ++ is bound Ca ++ deficiency affects platelet reactivity lower responses of blood platelets to activating agents, esp. ADP
blood anticoagulants role of free calcium Blood collection for Multiplate analysis recommended anticoagulants: a) citrate: addition of 3 mm CaCl 2 for the analysis (in the dilutent) to compensate for the chelated Ca ++ b) heparin: no interference with calcium in the sample, but certain interactions with platelets c) direct thrombin inhibitor (hirudin): no intereference with calcium; no intereference with platelets
how to compensate chelated Ca ++ in citrated blood? 1000 effect of the addition of CaCl 2 in Multiplate analysis of citrated plasma 800 AUC 600 400 200 0 COL COL Ca ADP ADP Ca
analysis of ASA effect in citrated blood 1200 1000 blood donors, no ASA patients treated with ASA Aggregation (AUC) 800 600 400 200 100 COL test COL test + ASA TRAP test
analysis of ASA effect in blood wityh TI control 1600 1400 blood donors ASA 100 mg/d ASA responder Aggregation (AUC) 1200 1000 800 600 ASA nonresponder 400 200 ASPI test 0 ASPI test
platelet concentrates quality control 150 µl platelet concentrate + 450 µl phosphate buffer * 3 min incubation + 20 / 40 / 60 µl collagen 3.2 / 6.4 / 9.6 µg collagen /ml final concentration * sodium phosphate (0.81% NaCl, 0.0067 M PO 4 ph 7.2)
platelet concentrates quality control aggregation [AUC] 1400 1200 1000 800 600 400 200 no agonis t collagen 1 collagen 2 collagen 3 0 0 7 time of storage [days]
Multiplate - advantages 5 channels and double sensor test cells allow for improved control of inter and intra-variability in the determinations various activators may be tested as in optical aggregometry (collagen, AA, ADP,TRAP, etc.) plasma vwf does not interfere with the outcome - effect of vwf visible only when testing ristocetininduced aggregation sensitive for monitoring of various antiplatelet drugs, incl. ASA, thienopyridines, GPIIb/IIIa receptor antagonists
mysteries of irreversible aggregation...
Aggregation curve
How to define the optimal conditions for monitoring? what is the optimal assay? what is the optimal anticoagulant? what is the optimal agonist?
Comprehensive score multiparametric measure of platelet response to activating / inhibiting agents used to standardize to platelet response regardless of the experimental conditons based upon the procedure of normalization and natural variability of measured parameters weighs the contributions of analyzed factors on the measured parameters of overall platelet function
Comprehensive score example 1 platelet responsiveness to ASA in CABG patients using various methos for monitoring of platelets response: PFA-100, WBEA collagen, WBEA AA accepted threshold values of reduced sensitivity to ASA: 150 s for CT CEPI, 0 Ω for WBEA AA, 14 Ω for WBEA coll three-parametric responsiveness to ASA CS = [Std (x/sd): 150 CT CEPI ] + [Std (x/sd): WBEA collagen - 14] + [Std (x/sd): WBEA arachidonate - 0]
Comprehensive score how to calculate? after CABG
Comprehensive score how to calculate? after CABG
Comprehensive score how to calculate? after CABG
Comprehensive score how to calculate? before CABG
Comprehensive score how to calculate? before CABG
Comprehensive score comparison
Comprehensive score example 2 platelet aggregation monitored in two groups: treated and non-treated with ASA three parameters of aggregation curve monitored: maximal aggregation (A max ), averaged rate of aggregation (V avgr ), and area under curve (AUC) reference values recorded in control group (not treated with ASA) three-parametric responsiveness to ASA CS aggregation =[Std (x/sd): A max (ASA) A max (reference) ] + [Std (x/sd): V avgr (ASA) V avgr (reference) ] + [Std (x/sd): AUC (ASA) AUC (reference) ]
Comprehensive score how to select the best parameters? aggregation with arachidonic acid
Comprehensive score benefits of CS calculus real conditions plt in some samples do not respond to ASA
Comprehensive score benefits of CS calculus ideal conditions plt respond to ASA in > 90%
Comprehensive score how to select the best parameters? each of the parameters included in the equation is characterized by individual variability with regards to platelets response to ASA these individual changes in response to ASA depend on hypo- or hyperreactivity of platelets agonized with a given agonist CS allow to weigh the contribution of each measured parameter more reliably and to quantitatively assess the overall effect of ASA on platelet aggregation parameters of aggregation any other confounding factors, like anticoagulant or agonist, may be utilized in this approach
Comprehensive score what is optimal agonist?
Comprehensive score what is optimal agonist?
Comprehensive score what is optimal agonist?
calcium
Comprehensive score what is optimal agonist?
Aggregation - examples
Effect of 75 mg/d ASA (10 days) ) on platelet aggregation in a healthy volunteer good response to ASA AA 0.5 mm collagen 1 ug/ml ADP 5 um ADP 10 um ADP 20 um
Effect of 75 mg/d ASA (10 days) ) on platelet aggregation in a healthy volunteer weak response to ASA AA 0.5 mm collagen 1 ug/ml ADP 5 um ADP 10 um ADP 20 um
Effect of in vitro ASA on platelet aggregation in statin-treated treated hypercholesterolemic patient ADP 5 um; ; AA 0.5 mm, collagen 1 ug/ml ASA 0 um ASA 10 um ASA 50 um ASA 100 um ADP 5 um collagen 1 ug/ml AA 0.5 mm
Optimal ADP concentration for platelet aggregation in rat blood subthreshol aggregation at 1 um ADP higher ADP concentrations delays disaggregation ADP 1 um ADP 2 um ADP 5 um
Effect of AA concentration for platelet aggregation in rat blood AA 0.1 mm AA 0.3 mm AA 0.5 mm
Effect of low polyphenol concentrations on platelet aggregation ADP 5 um; ; AA 0.5 mm, collagen 1 ug/ml polyphenol 0 polyphenol 10 ug/ml polyphenol 50 ug/ml ADP collagen AA
Platelet stimulation with collagen in rat blood subthreshold maximal collagen 0.5 ug/ml collagen 1 ug/ml
Concentration effect of pertenolid on platelet aggregation in rat blood ADP 5 um partenolid 100 um partenolid 250 um partenolid 500 um
Effect of pertenolid on platelet aggregation in rat blood
Thank you