08.11.11

1

Überprüfung der Funktion von Thrombozyten in

Thrombozytenkonzentraten

2011

Simon Panzer Clinical Department for Blood Group Serology and

Transfusion Medicine, Medical University Vienna, Austria

platelet biology

Aggregation! GpIIb/IIIa! Fibrinogen! vWF!

Inflammation! PF4! beta-TG! IL-1! CD40-L! Rantes! Interferon-gamma!!

Adhesion! GPIb/V/IX! GPIIb/IIIa! GPIa, GPVI! Fibrinogen! vWF! P-Selectin!

Coagulation! Plasminogen! FV! FXI! Fibrinogen! Protein S! PAI-1! vWF!

Proliferation! PDGF! TGF-beta! EGF! ECGF! Interferon-gamma!

Vasoconstriktion! ADP/ATP! Thromboxan A2! Serotonin!

08.11.11

2

bewirken die primäre Hämostase!!Blutungsneigung tritt ein bei!

!- Verminderung der Anzahl!- Funktionsstörung!

!

Thrombozyten

platelet adhesion

platelet aggregation (expression of the fbg binding site)

platelet release (P-Selectin, ADP, TX2,...)

08.11.11

3

platelet -activation, -function

Alan Michelson 2005

determine activation – infer function

1. level of activation 2. “activatability“

measure function directly

08.11.11

4

testing for platelet function

•  best: in vivo •  under close to physiologic conditions:

- in the natural enviroment (red cells, leukocytes, plasma)

- under high shear

factors that activate platelets

ADP, thrombin, collagen, thromboxane A2,

epinephrine, HOCl-oxidised LDL,

immunglobulin complexes, complement system, secretion products of leukocytes, mast cells, bacteria

5-hydroxytryptamine (serotonin) mechanical stimuli

and others

many stimuli several different receptors

multiple signalling pathways

08.11.11

5

measure metabolism (aspirin) thromboxane as the end point serum thromboxane B2 urinary 11-dehydro thromboxane B2

agonist induced activation Platelet aggregometry (turbidimetric) Flow cytometry Platelet aggregometry (impedance) Plateletworks TEG PlateletMapping System Impact Cone and Plate(let) Analyser Impact-R

PFA-100

methods

Light transmission aggregometry (Born, O‘Brien) (LTA)

Assesses platelet activation by the reduction of turbidity of platelet rich plasma

requires the preparation of PRP and PPP

low standardisation

no physiologic environment, no shear

08.11.11

6

Yee, 2005

Variable response to epinephrine in healthy individuals

flow cytometry very little blood required many aspects of platelet function can be studied

not universally avaliable

skilled operator required

no shear

08.11.11

7

CD45

CD

45

platelet-leukocyte aggregates

CD45 CD45 CD45

CD45

CD

61

CD

61

CD

61

CD

61

Granulocytes

Monocytes

Lymphocytes

Platelets

w/o agonist

08.11.11

8

CD

61

CD

61

CD

61

CD

61

CD45 CD45 CD45

CD45

Granulocytes

Monocytes

Lymphocytes

Platelets

after activation

ASA 500 mg

i.v.

whole platelet aggregometry (impedance) the Multiplate® analyzer

08.11.11

9

firm adhesion and aggregation of platelets on the sensor surface enhances the electrical resistance between the 2 sensor wires

impedance aggregometry

Calatzis 2007

Plateletworks Based on platelet counting

before after addition of agonist

no shear

08.11.11

10

TEG PlateletMapping System

Thrombelastography The platelet part is abolished by GPIIb/IIIa antibodies and clot strength refelcts only fibrin formation. Thus, comparison without abciximab with that containing abciximab allows calculation of the platelet contribution to the clot formation.

no shear

  citrated blood is passes through a small aperture in a collagen membrane

  platelets adhere to the collagen surface and lead to a cessation of blood flow

  parameter = Closure Time (CT)

  2 cartridges: -  EPI: costimulation by: shear stress,

collagen, epinephrin -  ADP: costimulation by: shear stress,

collagen, ADP

PFA-100 ®

Shear rate 5000 - 6000-1

08.11.11

11

Impact-R

High shear 1800-1

08.11.11

12

platelets shall improve hemostasis

donor testing

testing the platelet concentrate

testing the transfused patient

donor testing

screenig by PFA-100 more specific:

aggregometry, flow cytometry,…….

08.11.11

13

frequent donations may impair donors’ platelet function

testing the platelet concentrate

various aspects can be tested, like differencies between apheresis methods effects of storage effects of manipulations…..

however, not all methods suit, as whole blood assays need reconstitution!

08.11.11

14

TRANSFUSION 2008

platelet P-selectin increases during storage, while induced P-selectin declines

platelet P-selectin, comparison of

apheresis devices CS 3000 plus

MCS 3p

Transfusion 1998

08.11.11

15

TRANSFUSION 2008

platelet adhesion and size of aggregates decline during storage

TRAP-6 induced multiple electrode aggregometry(MEA)

inducible aggregation differs between Amicus and Trima/MCS+

Transfusion 2009

08.11.11

16

TRAP-6 induced LTA

inducible aggregation differs between all 3 apheresis devices

Transfusion 2009

Testing function after transfusion: the recipient’s response

A whole blood method and testing under high shear is preferred However, all methods are restricted by the need of “near normal” platelet counts

08.11.11

17

improved in vivo platelet function after PC transfusion

platelet function was determined under high shear conditions by Impact-R: adhesion increased, while the size of aggregates did not change

Summary (but not last slide!)

Vox Sanguinis (2011) 101, 1–9

08.11.11

18

concluding remarks

There is a variety of laboratory tools to monitor platelet function.

There is a broad variation of their specificity and sensitivity. We currently have no single “universial“ method.

There is no “gold standard“ to determine a best method.