platelet function assays PFA-100 – quick screen test, sensitive to VWD also Light transmission platelet aggregometry on platelet-rich plasma (LTA)– platelet function evaluation, not suitable for antiplatelet therapy monitoring Impedance whole blood aggregometry- good for antiplatelet therapy monitoring TEG/PLTMAP – PLTMAP is designed for antiplatelet therapy monitoring

Which activation pathways are monitored for antiplatelet therapy

PFA-100

Occlusion of cartridge membrane aperture by platelet plug

a normal PFA-100 tracing (EPI cartridge) Flow rate Closure time induced by EPI

Falsely prolonged PFA-100 results Low HCT HCT is too low (< 35%). Plug tends to disassociate and causes “seesaw” tracing and prolonged closure time. When platelet count is too low (< 100 K), closure time could be prolonged too.

TEG Tracing and Clotting Process Parameters

TRG Parameters ~80% contribution from activated platelets ~20% contribution from Fibrin Not a reliable parameter since instrument couldn’t differentiate real clot lysis vs. artificial effect Not sensitive to fibrinolysis

TEG example 1– question

TEG example 1 – answer

TEG example 2– question

TEG example 2 – answer

TEG example 3– question

TEG example 3 – answer

TEG example 4– question

TEG example 4 – answer

TEG example 5– question

TEG example 5 – answer

TEG example 6– question

TEG example 6 – answer

TEG example 7

Bleeding Patient with Normal TEG Tracing

TEG result example

TEG result comments TEG is performed using both citrated whole blood and heparinase treated blood with commercially provided Kaolin. Results are reported as from CK (citrated whole blood + Kaolin activator) or CK/H (citrated whole blood + Kaolin activator + Heparinase) respectively. Results from CK and CK/H should be nearly the same if patient is not on heparin. When R is significantly prolonged due to heparin therapy, it could lead to decreased ANGLE and MA and increased K which doesn't accurately reflect patient's fibrinogen and platelet function.  The ANGLE and MA results from heparinase treated sample (CK/H) would be more suitable for reflecting patient's fibrinogen and platelet function in this case. TEG is NOT sensitive to Von Willebrand Disease and antiplatelet therapy. Normal results could be observed. DECREASED R: Shortened clotting time. May indicate hypercoaguable state or specimen activation. Clinical significance of shortened R interval is not well defined.   INCREASED R (CK) with NORMAL R (CK/H): Prolonged R corrected with heparinase, consistent with heparin effect.   INCREASED R (CK) with INCREASED R HEPARINASE (CK/H): Prolonged R, consistent with factor deficiency or anticoagulant effect such as Argatroban and Dabigatran.   INCREASED MA: Consistent with robust platelet/fibrinogen function.   INCREASED G: Consistent with robust platelet/fibrinogen function.   DECREASED MA: Consistent with decreased platelet/fibrinogen function or anticoagulated effect.   DECREASED G: Consistent with decreased platelet/fibrinogen function.   INCREASED K: Consistent with anticoagulant effect or factor deficiency.   DECREASED ANGLE: Consistent with Low Fibrinogen activity or anticoagulated effect.   DECREASED K: Consistent with elevated fibrinogen/platelet activity.   INCREASED ANGLE: Consistent with elevated fibrinogen/platelet activity.   INCREASED EPL: Consistent with increased fibrinolysis.

TEG tracing: Low FIB and Platelets Angel: 8.8 normal range: 53 – 72 MA: 11.5 normal range: 50 – 70 FIB: 35 mg/dL ↓ ↓ Platelet counts: 23K/uL ↓ ↓

PLTMAP tracing

PLTMAP is a personalized assay based on patient’s own baseline platelet function

Inhibition % = 1 – Activation % PLTMAP calculation formula MA(TT)- MA(FIB) = Full potential of platelet activation MA(ADP/AA) – MA(FIB) = Platelet activation by ADP /AA Inhibition % = 1 – Activation %

PLTMAP result example

Whole blood impedance (WBIA) method By Multiplate multichannel impedance aggregometer

how Multiplate cell works ? Electrical resistance is enhanced by aggregation of platelets on the sensor surface between the 2 sensor wires and reported as “area under curve” (AUC)

Multiplate – aggregation curve

ADP/AA assay on Multiplate aggregometer - protocol example Per disposable test cell: 300 μl NaCL/CaCL2 or 0.9%NaCL 300 μl citrated donor blood Incubation for 3 minutes Addition of 20 μl ADP or 20 μl AA  start test Monitoring for 6 minutes

Whole blood impedance (WBIA) method By Multiplate multichannel impedance aggregometer NOT a personalized assay Compare with a wide normal range Need consecutive tests: Baseline (before antiplatelet therapy) important! On antiplatelet drugs

WBIA Result example Result comments: The multiplate assay impedance aggregometry method may be used to assess platelet function in patients who have received platelet-inhibiting drugs such as Aspirin, ADP receptor inhibitors(e.g. Clopidogrel, Prasugrel,Ticagrelor), or GP IIb-IIIa inhibitors (e.g. Abciximab, Tirofiban and Eptifibatide). The platelet aggregation response to platelet activation agonists ADP and arachidonic acid are reported as AUC (area under the curve). The AUC is directly proportional to platelet function and will decrease with platelet inhibitory drugs, platelet functional defects or thrombocytopenia. The clinical utility of this test in therapeutic decision-making has not been established. Therapeutic ranges have not been established for platelet inhibitor drugs with this assay. This test may be useful in assessing compliance or in assessing response in individual patients. Results may be affected by platelet count <150 K/uL.

Antiplatelet drug monitoring Patient Date Multiplate AUC (impedance method) Antiplatelet /antocoagulant therapy   ADP nl( 49 - 107) AA nl (48 - 114) EJ 47938352 10/16/17 0910 107 114 PLAVIX 1mg/kg started on 10/26/17; Aspirin chew tab 81 mg twice a day started on 10/18/17 10/27/17 0910 84 15 11/9/17 1039 8 3 baseline On Aspirin (DAY 9) and PLAVIX (DAY 1) On Aspirin (DAY 21) and PLAVIX (DAY 13) The results from Multiplates on these two patients correlated well with clinical anti-platelet drug therapy.

Multiplate AUC (impedance method) Antiplatelet /antocoagulant therapy WBIA and LTA Patient Date Multiplate AUC (impedance method) PAP-8 (LTA method) % Antiplatelet /antocoagulant therapy   ADP nl( 49 - 107) AA nl (48 - 114) ADP nl ( > 63) AA nl ( > 74) YH 3/3/2016 15 7 42 disaggregated to 2 6 Plavix, ASA BK 80 91 98 >100 Heparin The results from Multiplates on these two patients correlated well with the results obtained by PAP-8 (LTA method). It also correlated well with clinical anti-platelet drug therapy.

Multiplate AUC (impedance method) Platelet mapping (mechanical method) WBIA, LTA and PLTMAP Patient Date Multiplate AUC (impedance method) PAP-8 (LTA method) % Platelet mapping (mechanical method) Antiplatelet Therapy   ADP nl( 49 - 107) AA nl (48 - 114) ADP nl ( > 63) AA nl ( > 74) ADP inhibition % AA inhibition % 97349219 11/28/2016 12 5 N/A 93.3 99.6 Plavix, ASA 97360254 11/29/2016 67 56 75 73 none The results from Multiplates on these two patients correlated well with the results obtained by PAP-8 (LTA method) and Platelet Mapping. It also correlated well with clinical anti-platelet drug therapy.