Impedance whole blood electrical aggregometry:

Transcription

1 Impedance whole blood electrical aggregometry: Measurements, evaluation and interpretation of aggregometry outcomes Cezary Watała Department of Haemostatic Disorders Medical University of Lodz

2 What is our object of study?

3 blood platelets

4 resting (non-activated) platelets

5 platelets activated with ADP

6 attachment of platelets to surfaces mediated by GPIIb/IIIa

7 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

8 Technical details beyond Multiplate aggregometer pipetting inlet jack portion cup portion with sensor wires Multiplate test cell Test cell electrodes

9 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

10 how Multiplate cell works? electrode resistance enhanced by adhered platelet aggregates

11 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

12 Multiplate mounting the cell pipetting inlet 2 sensors, each with 2 electrodes cup plug inlet teflon-coated stirrer

13 How to perform aggregation test? 1 2 put the test cell into the holder attach the sensor cable to the cell

14 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 min

15 Random allocation of numbers in series seed: left blank for automatic seed

16 Multiplate aggregation curve Area under the curve (AUC) test cell Aggregation (AU) sensors time (min)

17 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

18 Typical aggregation curves in MULTIPLATE test TRAP test COL test COL test + ex-asa ASPI test control ASA responder ASA nonresponder

19 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

20 ASPI test

21 COL test

22 TRAP test

23 ADP test

24 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

25 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

26 calcium

27 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

28 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

29 how to compensate chelated Ca ++ in citrated blood? 1000 effect of the addition of CaCl 2 in Multiplate analysis of citrated plasma 800 AUC COL COL Ca ADP ADP Ca

30 analysis of ASA effect in citrated blood blood donors, no ASA patients treated with ASA Aggregation (AUC) COL test COL test + ASA TRAP test

31 analysis of ASA effect in blood wityh TI control blood donors ASA 100 mg/d ASA responder Aggregation (AUC) ASA nonresponder ASPI test 0 ASPI test

32 platelet concentrates quality control 150 µl platelet concentrate 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, M PO 4 ph 7.2)

33 platelet concentrates quality control aggregation [AUC] no agonis t collagen 1 collagen 2 collagen time of storage [days]

34 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

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38 mysteries of irreversible aggregation...

39 Aggregation curve

40 How to define the optimal conditions for monitoring? what is the optimal assay? what is the optimal anticoagulant? what is the optimal agonist?

41 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

42 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]

43 Comprehensive score how to calculate? after CABG

44 Comprehensive score how to calculate? after CABG

45 Comprehensive score how to calculate? after CABG

46 Comprehensive score how to calculate? before CABG

47 Comprehensive score how to calculate? before CABG

48 Comprehensive score comparison

49 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) ]

50 Comprehensive score how to select the best parameters? aggregation with arachidonic acid

51 Comprehensive score benefits of CS calculus real conditions plt in some samples do not respond to ASA

52 Comprehensive score benefits of CS calculus ideal conditions plt respond to ASA in > 90%

53 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

54 Comprehensive score what is optimal agonist?

55 Comprehensive score what is optimal agonist?

56 Comprehensive score what is optimal agonist?

57 calcium

58 Comprehensive score what is optimal agonist?

59 Aggregation - examples

60 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

61 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

62 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

63 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

64 Effect of AA concentration for platelet aggregation in rat blood AA 0.1 mm AA 0.3 mm AA 0.5 mm

65 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

66 Platelet stimulation with collagen in rat blood subthreshold maximal collagen 0.5 ug/ml collagen 1 ug/ml

67 Concentration effect of pertenolid on platelet aggregation in rat blood ADP 5 um partenolid 100 um partenolid 250 um partenolid 500 um

68 Effect of pertenolid on platelet aggregation in rat blood

69 Thank you