Transcript Array Sales Training: Budapest

Hemostasis
Homeostasis
What are Platelets?
Disk-shaped cell
Mature
fragments produced
Platelet
in the
megakaryocytes
Megakaryocyte
Bone
Marrow
Storage and Circulation
Quantity - 200,000 - 400,000/mm3
Life Span - 10 days
33%
pooling
67%
in the
circulation
Spleen
Megakaryocyte
Types of Thrombi
Type
Components
Seen In
Treatment
Red
RBCS, WBCS,
PLTS, Fibrinogen
AMI
Therapy
Thrombolytic
White
Platelets Only
Unstable Angina
Hyperaggregable
Nitroglycerine
Yellow
Lipid Build Up
Unstable Angina
AMI
Laser Therapy
Angioplasty
Dynamics of Hemostasis
Platelets



Anucleate, multifunctional blood cells
First line of defense against injury to blood vessels
Adhere
Activate
Aggregate
Vascular
System
Vascular Responses
Platelet Function Defects
Adhesion
Shape Change Release
Aggregation
Coagulation
Delayed Fibrin Formation
1. Failure of platelets to adhere
2. Failure to release ADP
3. Failure to release TxA2
4. Failure to aggregate
5. Failure of surface binding of coagulation factors
Electron Micrograph
of the Cellular Components of Blood
Red Blood Cell
Activated Platelet
White Blood Cell
Anatomy of a Platelet
Delta Granules
ADP
Serotonin
ATP
Calcium
Dense Tubular
System
Microtubular
System
Plasma Membrane
PF3
Alpha Granules
PF4
Factor V
Factor VIII R:ag
(vWF:ag)
Glycogen
Fibrinogen
Fibronectin
b Thromboglobulin
Cytosol
Factor XIII
Exterior Coat
(Glycocalyx)
Mitochondria
Surface
Connecting System
Adhesion Surface Activated Platelet
Collagen
GPIa
GPIX
Arachidonic Acid
Ca+
Serotonin
CycloOxygenase
Endoperoxides
Thromboxane
Synthetase
Ristocetin
ADP
Receptor
ADP
Dense
Body
TxA2
GPIIIa
TxA2
GPIb
Released
vWF
GPIIb
Adhesion

Plaque rupture or
vascular damage
exposes
subendothelial
proteins to which
platelet receptors
bind, resulting in
platelet adhesion.
Aggregation: Activated Platelet
in Final Stage for Binding to Fibrinogen
Collagen
GPIa
GPIX
Arachidonic Acid
Ca+
Serotonin
CycloOxygenase
Endoperoxides
Thromboxane
Synthetase
ADP
Receptor
ADP
Dense
Body
Ristocetin
TxA2
GPIb
vWF
TxA2
GPIIb
GPIIIa
Released
GP IIb/IIIa Receptors:
The Final Common Pathway



Most numerous receptor on
platelet membrane surface
(40,000 to 80,000 per platelet)
Becomes receptive to
fibrinogen binding upon
platelet activation
Represents the final common
pathway for platelet
aggregation
Adapted from Charo et al. In: Colman RW et al, eds. Hemostasis and Thrombosis: Basic Principles
and Clinical Practice. 3rd ed. Philadelphia, Pa: JB Lippincott; 1994:495
Pharmacologic Intervention
in Platelet Function
Platelet Inhibition Mechanisms
GPIIb
GPIIIa
IIb/IIIa
Inhibitors
Arachidonic Acid
Aspirin
CycloOxygenase
Endoperoxides
Plasmin
Dense
Body
Thromboxane
Synthetase
GPIa
GPIb
ADP Inhibitors
ADP
Receptor
Acquired Functional Disorders







Drugs
Uremia
Cirrhosis/Chronic Hepatitis
Leukemia & Myelodysplastic Syndrome
Plasma Protein Abnormalities
Myeloproliferative Disorders
Cardio Pulmonary Bypass
Effect of Cardiopulmonary Bypass
on Hemostasis
Hemodilution
Platelets
Coagulation proteins
Activation
Platelets
Fibrinolysis
Coagulation
Drugs That Affect Platelets

Analgesics (aspirin, NSAIDs) affecting prostanoid synthesis
or action

Caffeine, theophylline, dipyridamole and drugs which
increase platelet cyclic AMP

Antimicrobials (penicillins, cephalosporins, nitrofurantoin)

Cardiovascular agents (quinidine, diurectics, vasodilators

Anticoagulants (coumadin, heparin) and Thrombolytics (t-PA,
streptokinase)

Psychotropics (tricyclics/phenothiazines) and anesthetics

Chemotherapeutic agents

Miscellaneous agents (dextrans, clofibrate, ETOH, Vitamin E,
onions, garlic, ginger, fish oil)
Platelet Aggregation
Platelet
Rich
Plasma
(PRP)
+
Baseline Light
Transmission
Aggregating
Reagent
Aggregate
Clumping
Increased Light
Transmission
Typical Biphasic Pattern
Platelet
Rich
Plasma
100
90
80
70
60
50
40
30
20
10
0
Platelet
Poor
Plasma
Secondary
Response
(Release)
Primary
Response
Lag
Injection
Point
Platelet Aggregation Testing


Important in assessing risk of
bleeding/need for transfusion in active
bleeding
Important in monitoring and managing
antiplatelet therapies (ReoPro, Aggrastat,
Integrilin, aspirin, etc.)


ReoPro use in Cath Lab
Emergency CPB surgery on Cath Lab patients
with ReoPro
Platelet Assessment


Quantitative - Platelet Count
Qualitative - Platelet Aggregation
ALL now can be done with
Plateletworks
®
Plateletworks Concept

The first and only test to measure platelets
both quantitatively and qualitatively

Uses cell counter as the instrument
platform (thus, also provides CBC,
including quantitative platelet count)

Addition of a platelet agonist (reagent)
causes a reaction (aggregation) indicative
of qualitative platelet function

Offer multiple agonists for optimal
sensitivity to different clinical scenarios
®
Plateletworks Procedure

Run CBC on EDTA. Note Platelet Count
(PC)

Run CBC on ADP using Plateletworks
tube. Note PC which will be reduced,
due to aggregation, from first sample.

EDTA - ADP x 100 = % of functional platelets
EDTA
Plateletworks Principle Overview

Baseline EDTA platelet count established using
normal CBC

All platelets in circulation quantified
Original Platelet count
(211 x103/mm3)
Plateletworks Principle Overview

Agonist (ADP) platelet count then performed
using Plateletworks agonist tube

Platelets remaining in circulation quantified
With agonist (ADP)
New count (8 x103/mm3)
Platelet Function = 96%
Result Calculation
Baseline (total plts)
Agonist (non-functional plts)
= 211,000
= 8,000
So…
Aggregated Plts
203,000
211,000
= 203,000
x 100
= 96% Aggregation
Platelet Aggregation Correlation:
Plateletworks vs. Chronolog
Collagen
100
R=0.91
80
ADP
60
40
100
20
y = 0.9401x
R = 0.88
P=.0001
0
0
20
40
60
80
% Aggregation Chronolog
R=0.88
100
120
90
% Aggregation Plateletworks
% Aggregation Plateletworks
120
80
70
60
50
40
30
20
y = 0.8753x + 17.656
R = 0.91
P=.0001
10
0
0
20
40
60
% Aggregation Chronolog
80
100
Platelets and CPB

About 20% of patients undergoing CPB
bleed enough to mandate intervention


Defined as multiple transfusions
About 2-3% of patients will undergo reexploration mandated by abnormal
bleeding


Surgical source
Severe acquired hemostatic defect source
Consequences of the Bleeding Patient







Re-operation
Delayed chest closure
Increased risk of mediastinitis or other
infection
Hypovolemia
Biventricular cardiac function
Blood product reactions (1.6% hep A, 11.6%
hep B)
Immunologic effects (anaphylaxis or acute
lung injury)
Plateletworks® Value in CPB







Assess the extent of platelet dysfunction induced
by CPB
Monitor post-op recovery of platelet aggregation
Monitor recover/drop in platelet count
Guide transfusion decisions
Manage bleeding patients
Evaluate emergency surgery Cath Lab patients
with anti-platelet agents
FUTURE: Manage anti-platelet agents given
during beating heart procedures
CPB Clinical Highlights
Improving Patient Care: Predicting Bleeding Risk
Patient F - Bypass
90
80
70
 Negative Platelet
Function Recovery
60
50
% Aggregation
% Aggregation
Patient J - Bypass
60
50
40
30
20
10
0
40
30
20
10
0
Pre-Pump On-Pump 1 On-Pump 2 Post Pump
Pre-Pump On-Pump 1 On-Pump 2 Post Pump
Interval
Interval
Patient NG - site 5
100
90
90
80
70
60
70
80
% aggregation ADP
% aggregation ADP
Patient EC - site 5
50
40
30
20
10
0
60
50
40
30
20
10
0
baseline
heparin
CPB
Tim e
30 min CPB Leave OR
baseline
heparin
CPB
Tim e
30 min CPB Leave OR
 Potential Bleeding
Risk
 These patients did
bleed and received
PLT packs
(avg. 6 units each)
CPB Clinical Highlights
Improving Patient Care: Predicting Bleeding Risk
Patient HG - site 5
Patient CB - site 5
90
120
80
% aggregation ADP
% aggregation ADP
100
80
60
40
20
70
60
50
40
30
20
10
0
0
baseline
heparin
CPB
30 min CPB Leave OR
baseline
Tim e
80
70
% Aggregation
% Aggregation
90
70
40
30
20
10
0
CPB
30 min CPB Leave OR
Patient M - Bypass
80
50
heparin
 Low bleeding
risk predicted
Tim e
Patient E - Bypass
60
 Positive platelet
function recovery
60
50
40
30
20
10
0
Pre-Pump On-Pump 1 On-Pump 2 Post Pump
Pre-Pump On-Pump 1 On-Pump 2 Post Pump
Interval
Interval
 These patients did
not bleed
 “Normal” post
operative recovery
period
CPB Clinical Highlights
Improving Patient Care: Identifying the bleeding source
 Acceptable
“Working
Platelet
Number”
 If bleeding,
not because
of Platelets
 No platelet
transfusion
needed
CPB Clinical Highlights
Improving Patient Care: Identifying the bleeding source
 Acceptable
“Working
Platelet
Number”
 If bleeding,
not because
of Platelets
 No platelet
transfusion
needed
CPB Clinical Highlights
Improving Patient Care: Identifying the bleeding source
 “Working
Platelet
Number”
below
50,000!
 If bleeding,
most likely
due to
Platelets
 Platelet
transfusion
needed
CPB Clinical Highlights
Improving Patient Care: Treating the bleeding source
 6 units of
platelets given
6 units Plts given
 “Working
Platelet
Number” now
above 50,000!
 Bleeding
ceases, so
Platelet
transfusion
correct therapy
Anti-Platelet Monitoring –
Plateletworks - (contd)








Nine parameter
hematology profile
including…..
Red Cells
White Cells
Platelets
HCT
Platelet Function
Results in ~ 4 mins
Multiple clinical utilities
Clinical Experience – Plateletworks*

CPB – monitoring patients peri-operatively (help with
decision making re: platelet transfusion;re-operation etc.)

PCI - evaluate PCI patients on anti-platelet agents
(including tirofiban, eptifibatide, abciximab, clopidogrel and ASA)

Used to identify potential drug-drug interactions
(statins-clopidogrel??)

Used to monitor platelet function in HIT
*Independent studies
Clinical Experience – Plateletworks*
 A Rapid Platelet Function Assay Used to Regulate Platelet
Transfusion Prophylaxis Following Cardiopulmonary Bypass
Surgery JECT 2004; Vol 26: 2, pp 145-148.
 Plateletworks Platelet Function Test Compared to the
Thromboelastograph for Prediction of Postoperative Outcomes
JECT 2004; Vol 36: 2, pp 149-152.
 Point of Care Testing Shows Clinically Relevant Variation in the
Degree of Inhibition of Platelets by Standard-Dose Abciximab
Therapy During Percutaneous Coronary Intervention Cath
Cardiovascular Interventions 2004; Vol 62: pp 150-154.
 Whole Blood Point of Care Platelet Testing During Cardiac
Surgery Predicts Perioperative Bleeding
* Reprints available from www.helena.com or [email protected]
Clinical Experience – Plateletworks
 Efficacy of High-Dose Bolus Tirofiban Compared to RegularDose Glycoprotein IIb/IIIa Inhibitors on Platelet Aggregation
Inhibition in Myocardial Infarction Patients Treated with Primary
Angioplasty ESC, August 2003.
 Impact of Angina Class on Inhibition of Platelet Aggregation
Following Clopidogrel Loading in Patients Undergoing Coronary
Intervention Cath Cardiovascular Interventions, 2003, Vol 59: pp
21-25.
 Monitoring Platelet Function During Cardiopulmonary Bypass in
the Presence of Tirofiban Soc. Cardiovasc Anest., April 2003
Clinical Experience – Plateletworks

Clopidogrel Poor Responders Discovered During Pointof-Care Platelet Aggregation Testing ACC, March 2003

Atorvastatin Reduces the Ability of Clopidogrel to
Inhibit Platelet Aggregation: A New Drug-Drug
Interaction Circulation, 2003; 107: pp 32 - 37.

Contribution of Hepatic Cytochrome P450 3A4
Metabolic Activity to the Phenomenon of Clopidogrel
Resistance Circulation, 2004; 109: pp r1 – r6.

Evaluation of Platelet Count and Function in Patients
Administered Tirofiban or Eptifibatide Undergoing
Percunateous Coronary Intervention Point of Care: The
Journal of Near Patient Testing and Technology
Recent Clinical Experiences –
 Platelet protection with tirofiban and


monitoring using Plateletworks
The regulation of platelet transfusion
prophylaxis following cardiopulmonary
bypass surgery
Issues regarding potential clopidogrel
resistance – how to monitor and manage
Case Study
Case Study: Platelet Protection Using the
Glycoprotein IIb/IIIa Inhibitor Tirofiban in
a Patient with Heparin Induced
Thrombocytopenia Undergoing Aortic
Valve Replacement Requiring
Cardiopulmonary Bypass.
Kirk E. Guyer BS, Wei C. Lau MD, David G.M. Carville PhD
Heparin induced thrombocytopenia - HIT


HIT is a serious clinical scenario that
occurs with the administration of
unfractionated heparin (UFH) and to a
lesser extent with low molecular weight
heparin (LMWH)
HIT has a high incidence of morbidity
and mortality particularly in patients
undergoing cardiac surgery
HIT - continued
 HIT patients spontaneously generate
antibodies against heparin bound to platelet
factor 4 (PF4) with the subsequent formation
of antibody-antigen complexes which result in
platelet activation and thrombin generation
 This physiological sequence of events elicits
a significant decrease in platelet count with a
simultaneous increase in thromboembolic
events
 Such thromboembolic events may result in
either mesenteric and/or peripheral infarctions
of which necrosis and amputation may arise
Case Report
 84-year-old female admitted for aortic valve




replacement (U of M Medical Center)
Previously diagnosed with HIT
To protect the patients platelets and prevent HIT,
the short-acting GPIIb/IIIa antagonist tirofiban
was administered (10mg/kg loading &
0.15mg/kg/min infusion)
UFH @ 300 units/kg with an ACT of ≥ 480 secs
Tirofiban infusion was stopped 30 minutes prior
to the termination of CPB
Patient Monitoring

The patient was evaluated for both platelet count and
function using Plateletworks

This platform utilizes traditional electronic
impedance principles
 A reference platelet count is performed and
compared with a platelet count in the presence of
20mM ADP

The ratio of the platelet count between the ADP and
reference tubes is calculated as percent platelet
aggregation (TAT four mins.)
Peri-Procedural Platelet Count
Platelets
Platelets transfused (@ five packs)
Peri-Procedural Platelet Function
Post-procedural recovery
Summary

HIT is of major clinical concern in patients undergoing CPB
who receive high-dose heparin

Alternative anti-coagulation strategies must be considered
(prostacyclins, prostaglandins, DTIs)

Anti-GPIIb/IIIa agents (especially the shorter acting agents
tirofiban and eptifibatide may prove most suitable)

In this study tirofiban was used to protect against HIT
during CPB

Also when the platelet count dropped to approximately
53x103/mL the Plateletworks test platform was still capable
of monitoring platelet function

Other systems cannot evaluate platelet function in the
hemodilute/thrombocytopenic patient
Case Study
Case Study: A Rapid Platelet Function Assay
Used To Regulate Platelet Transfusion
Prophylaxis Following Cardiopulmonary
Bypass Surgery
Kathy E. Shaffer MT, (ASCP), David T. Pearman, BS, Robert
S. Galen MD, MPH, David G.M. Carville PhD
Transfusion Triage
 The concept of blood conservation in cardiac


surgery is under continuous scrutiny
Need for a reduction in institutional expenditures
due to rising blood costs
Also there are increasing availability issues (need
for cross-matched and leukocyte-reduced products)
 A major goal for the regulatory bodies and
healthcare providers should be to establish a
universal transfusion algorithm (based on “all”
available current technologies)
Role of the Platelet
 Open-heart surgery activates platelets
 Initiates a cascade of events including:


- generation of thrombin
- expression of GPIIb/IIIa receptor complexes
- involvement of other factors (complement, PAF etc.)
End result is a significant activation of platelets
with subsequent loss and dysfunction
Suggested that platelets (and other factors)
need to be monitored (near patient)
Platelet Function Testing
 Gold standard platelet rich plasma (PRP) aggregometry
not suitable for acute settings


Platelets transfused as per NIH/WHO guidelines
Other tests having utility in the assessment of the
coagulation profile require specialized equipment and
training and are, in some cases, laborious (flowcytometry, immunoassays etc.)

Other platforms provide valuable overall information
and when used in conjunction with other systems
prove to reduce transfusion requirements (TEG)
Objective of Study
 The purpose of the current study was to
determine if the use of a *point-of-care platelet
analyzer to evaluate platelet function and
count, as part of a complete blood count
(CBC), in post-operative CPB patients would
enhance transfusion triage
* System has been described elsewhere:- reprints of papers
and this presentation are available [email protected]
Patients and Study Protocols






Aultman Blood Center, Canton Ohio between Aug
‘01 - Jan ‘02 (c.f. same period previous year)
310 CPB cases were performed
Post-operatively patients were evaluated “POC”
for platelet count and function (only difference)
Samples were drawn from an arterial line
Protocol for platelet transfusion was based upon the
AABB/WHO guidelines (and platelet evaluation)
Decision to transfuse FFP or CRYO was based on
observational bleeding and/or the platelet function
Comparative Data
08/00-01/01
08/01-01/02
Difference
Total CPB
Procedures
238
310
72
Platelet
Packs
2676
1770
906
Units of
Platelets/patient
11.75
5.5
6.25
A Comparison of Prospective and
Retrospective Platelet Transfusion Data
700
Units of Platelets Transfused
Units of Platelets Transfused
600
500
400
2001/02
2000/01
300
200
100
0
Aug
Sep
Oct
Nov
Month
Dec
Jan
Number of PLT transfusion per case as
illustrated on a monthly basis
20
Number of Platelets Transfused per Case
18
16
14
12
2001/02
2000/01
10
8
6
4
2
0
Aug
Sep
Oct
Nov
Month
Dec
Jan
FFP (circles) and CRYO (triangles)
transfused per case
Number of Platelets Transfused per Case
25
20
15
2001/02
2001/02
2000/01
2000/01
10
5
0
Aug
Sep
Oct
Nov
Month
Dec
Jan
Summary

CPB affects platelet count and function that may
contribute to significant post-operative bleeding

This may result in significant morbidity and mortality
without the appropriate intervention

Recent recommendations are to reduce the use of
transfusion products (have an unnecessary risk to patients
who do not require them)

Also there is concern is that transfusion practices are
quite variable (institution dependent)

Recent cost V’s benefit analyses have highlighted the
significant expense, associated with transfusion
Summary - continued

In the current study 25% more cases were
performed (c.f. same period previous year)

All patients had platelet count and function
evaluated “near patient” (in addition to a central
lab. CBC)
 A reduction in 906 platelet transfusions was noted
 Units of platelets transfused/patient fell from 11.75
in 2000/01 to 5.75 in 2001/02

FFP and CRYO usage was reduced by greater than
80% in the study period (c.f. same period previous
year)
Conclusion






Need a rational approach to the management of
coagulation disorders during CPB
Most methods are laborious, require highly trained
personnel and have long turn-around times
Recent development of near-patient systems permits
“more accurate” and timelier intervention
Studies have demonstrated that those patients managed
“bed-side” received significantly less blood products
This study supports the suggestion that establishing a
transfusion protocol/algorithm is the penultimate tool for
successful transfusion triage
Additional, prospective, studies to support the findings
presented here are warranted
Clinical Study
Clinical Study: St. John’s Wort
Enhances the Platelet Inhibitory
Effect of Clopidogrel in Clopidogrel
“Resistant” Healthy Volunteers
Wei C Lau, David GM Carville, Kirk E Guyer,
Charlene J Neer, Eric R Bates
Patients and Study Protocols



Background: It has been reported that the variability in platelet
aggregation inhibition with clopidogrel correlated with the metabolic
activity of cytochrome P450 (CYP)3A4.
St John’s Wort (SJW), an herb, genetically induces CYP3A4 expression
mediated by nuclear pregnane X receptor response element to increase
hepatic CYP3A4 enzymatic activity. This study was designed to
determine whether SJW would enhance the platelet inhibitory effect of
clopidogrel in non-responder and low-responder (clopidogrel “resistant”)
subjects.
Methods: 5 healthy clopidogrel “resistant” subjects (relative decrease in
platelet aggregation < 30% using Plateletworks™ whole blood
aggregometry and 20µM ADP at 0, 2, 4, and 6 hours after clopidogrel 450
mg).
After a 14 day washout period, subjects were treated with SJW 300 mg,
three times daily for 14 days. Platelet aggregation was again measured
at 0, 2, 4, and 6 hours after clopidogrel 450 mg. With each subject as his
own controls, the platelet aggregation profile of clopidogrel was
compared before and after SJW.
Patients and Study Protocols – contd.
P = 0.004
P = 0.03
100
P = 0.004
95 + 2 97 + 2
79 + 11
84 + 7
84 + 7
90
Platelet Aggregation (%)
64 + 17
80
51 + 16
70
46 + 19
60
50
40
30
20
Baseline
St. John's Wort
10
0
Baseline
2 Hours
4 Hours
Time
6 Hours
Patients and Study Protocols – contd.
 Results: After co-administration with

SJW, clopidogrel significantly inhibited
platelet aggregation. Similar results were
obtained with light transmission
aggregometry.
Conclusion: This herb-drug interaction
increases CYP3A4 activity, converting
clopidogrel non- and low-responders to
clopidogrel responders
Conclusions
 Platelets are integral for normal haemostasis
 Inter-patient variability in platelet response to


anti-platelet agents are recognized
As such platelet function (count) monitoring
is required
Each institution should recognize this - to
improve clinical outcome and reduce cost
Summary
 The ability to assess platelet function has been
elusive, especially in the clinical environment
 Nonetheless, it is believed that platelet
dysfunction is an important contributor to CPB
bleeding and other hemorrhagic problems
 Results can be used to “rule in/rule” out source
of bleeding
 Results can help to guide transfusion decisions
and identify presence of anti-platelet therapies
Summary - continued


It uses the Ichor cell counter and
provides both quantitative (platelet
count) and qualitative (platelet
aggregation) information to the
clinician
Plateletworks is a new, rapid, easy test
for measuring platelet function