M.G. Macey et al., Cytometry, 1999 ADVIA 120 에서 PLT 활성도 측정 Use of Mean Platelet Component to Measure Platelet Acitvation on the ADVIA120 Hematology System M.G. Macey et al., Cytometry, 1999 혈액학계 : 심정은

PLT Activation (I) Normally circulate in a resting state Stimulation  platelet adhesion & aggregation Weak agonist (ADP) conformational change in the PLT GPIIb/IIIa complex  fibrinogen binding  aggregation Strong agonist(thrombin, vessel collagen) shape change, swelling, and degranulation

PLT activation (II) WBC activation Inflammatory mediator Nitric oxide Platelet factor 4 Beta-thromboglobulin Platelet-derived growth factor Histamine-releasing factor Inflammatory mediator Oxygen free radical Platelet activating factor Thromboxane Nitric oxide

혈소판 활성도 측정의 임상적 유용성 Coronary artery disease Alzheimer’s disease Myeloproliferative disorders Diabetes Pre-eclampsia Inflammatory bowel disease Glomerular disease

2-D Platelet Parameters 혈소판 활성(Activation)에 관여하는 ADVIA 120 platelet parameters Mean platelet component (MPC) concentration (platelet density) MPC(g/dl)=[(RI-1.333)/0.0018 dl/g] = 100 * platelet mass(pg)/platelet vol. Platelet component concentration distribution width (PCDW) (platelet density distribution) Mean platelet mass (MPM)

PLT Flow Cytometric Analysis High angle detector (5o - 15o) Low angle detector (2o-3o) 670nm Laser Diode

PLT Scatter Cytogram

PLT Activation Studies (I) I. Immunofluorescent flow cytometry : CD62P - Cell marker study II. ADVIA 120 - PLT Activation - Volume and Refractive index - parameter (MPC, MPM)

Method I. Thrombin 에 의한 PLT 활성도 측정 : EDTA Whole blood 1mL 에 Bovine thrombin (0-0.12 u/mL) dose II. Thrombin 에 의해 활성화된 PLT 에 대한 Ridogrel 의 효과 측정 10-8, 10-7 , 10-6 , 10-5 M 농도의 Ridogrel을 넣고 incubation III. EDTA blood 에서 CD62P 와 MPC 의 측정 EDTA & SC samples immediately & at 3hr

Result (I) Activation of PLTs by Thrombin ADVIA 120 MPC and CD62P-PE MFI vs. Thrombin Concentration 25 25.5 26 26.5 27 27.5 0.015 0.03 0.06 0.12 Thrombin concentration (U/mL) MPC (g/dL) 10 20 30 40 50 60 70 % CD62P

Result (II) Activation of PLTs by Thrombin Change in PLT CD62P expression and MPC over 30 min in EDTA blood stimulated with bovine thrombin(0.12 U/ml)

Result (III) Effect of Ridogrel on the Activation of PLTs by Thrombin Ridogrel dose response changes in PLT CD62P expression and MPC

Correlation of MPC and CD62P ADVIA 120 MPC vs. CD62P y = -0.0251x + 25.603 R 2 = 0.97 r = 0.98 17 20 23 26 50 100 150 200 250 300 350 % CD62P MPC (g/dL) The correlation between the change in platelet CD62P expression and MPC in EDTA anticoagulated blood from mormal controls at 3h.

Result (IV) 실온에서 3시간 incubation 한 EDTA Blood 에서의 시간별 분석

CD62P 와 MPC 의 항응고제에 따른 변화 분석

Discussion MPC의 감소는 PLT activation 되었음을 보여준다 MPC와 MPM은 PLT degranulation 정도를 반영한다 Bovine thrombin 으로 자극시 thrombin 양에 비례하여 CD62P 증가, MPC는 감소한다(Ridogrel 에 의해 억제) MPC (platelet density)는 CD62P(marker of platelet activation)와 역상관성을 보임 Sodium citrated blood 가 EDTA blood 에 비해 시간경과에 따른 변화에 대해 더 안정적이다 Flow cytometry 에 비해 특별한 장비와 시약이 필요치 않고 비용 감소와 시간 단축의 장점이 있다