Catherine P. M. Hayward, MD PhD FRCPCProfessor, Pathology & Molecular Medicine, & Medicine, McMaster UniversityHead, Coagulation, Hamilton Regional Laboratory Medicine Program

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Slides showing figures with unpublished data and details of ISTH LTA recommendations have been removed from this presentation

Name diagnostic tests

▪ most useful for diagnosing platelet function disorders (PFD)

least two major challenges to ▪ external quality assurance (EQA) for platelet function tests

Give examples ▪ two types of EQA used for platelet function tests

Be familiar ▪ with the purpose of North American guidelines on platelet

aggregometry▪ why the guidelines include recommendations on

aggregometry interpretation

• Important cause of abnormal bleeding but challenging to diagnose

• Most recent Hamilton prospective cohort study• 2 von Willebrand disease - 30 inherited platelet disorders

• Heterogeneity in PFD pathogenesis

• Largely unknown, especially for common PFD

• Possibilities are enormous

• Platelets > 1000 proteins

• Megakaryocytes transcribe many more genes

Diagnostic tests

• No “simple” approach or kits

• Require rapid processing and testing of freshly collected blood samples

• Not optimally standardized, interpreted

• Only recently evaluated by EQA or workshops

Adhesive receptorsAgonist receptors

Established and emerging evidence:

Sometimes need platelet transfusion management

Bleeding risks similar to von Willebrand disease

Likelihood, as Odds Ratios ~34-132 for different bleeding problems

M. Pai, “CHAT” study to be presented at ISTH 2011

EssentialCBC warning: doesn’t always exclude thrombocytopenic PFD, e.g. RUNX-1 defects

Aggregometry

light transmission (LTA) and whole blood methods

Optional

PFA-100

Not Recommended

Bleeding time – can’t be controlled, minimally informative

Dense granule assays

Content release▪ often assess: adenosine triphosphate (ATP) release by lumi-aggregometry

▪ Contents include ATP, ADP, serotonin, calcium and phosphorous

Electron microscopy (EM) or meparine▪ quantify numbers of dense granules

▪ NASCOLA Platelet EM EQA

▪ all labs have properly diagnosed dense granule deficiency

Hayward et al, Am J Clin Pathol. 2009;131:671-5

Genetic tests▪ specific disorders, prevalent in some populations

Other assays?

Evaluate results according to reference intervals for:

1) maximal aggregation (MA) evaluated by light transmittance (or impedance if using whole blood)

2) ATP release (nM), evaluated by luciferin/luciferase method (using an ATP standard)

ATP release is delayed and less complete with epinephrine

Aggregation6 mM epinephrine5 mg/mL collagen

ATP release

% M

A

d-Luciferin +ATP

Oxyluciferin + AMP + light

Firefly luciferase

Strong agonist: immediate ATP releaseWeak agonist: ATP release with 2o aggregation

Strongest evidence of needs for PFD test standardization

• CLSI Guideline

Christie DJ, et al. Platelet function testing by aggregometry. CLSI document H58-A. Wayne, PA: CLSI, 2008

• NEW: North American guidelines (include LTA interpretation)

Hayward CP, et al. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry AJCP 2010;134:955-63

• ISTH Guidelines

1. Hayward CP, et al. Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function. J Thromb Haemost2006;4:312-319.

2. In progress: ISTH expert consensus guidelines on LTA practices• BSH (expected soon)

Used Rand method for when evidence is limited Int J Technol Assess Health Care 1986;2:53-63

Experts Selected

▪ Cattaneo, Italy; Hayward , Canada; Harrison, UK; Kenny, UK; Michelson, USA; Nugent, USA;Watson, UK; Cerletti, Italy; Nurden, France; Rao, USA; Schmaier, USA

Consulted, voted on statement appropriateness

Likert scales, comment boxes

Discard two extreme scores majority vote

LTA is

clinically useful for

▪ studying subjects with bleeding disorders

should NOT be used for

▪ identifying subjects at risk for thrombosis

▪ monitoring subjects on anti-platelet therapy▪ Dr. Kottke-Marchant will update information on this next

Agonist% clinical labs using for LTA

ISTH SurveyClinical labs

Median (range)

North American Guideline

Recommendations

CLSI Recommendations

ADP (mM) 100% 5.0 (0.2-200) 2-10 0.5-10often 5 to start

Collagen (mg/mL) -78% use type I fibrillary

2.0 (0.08-100), low concentration that detects

NSAID abnormalities

1-5,often 2 to start

Epinephrine (mM) – 72% 7 (0.1-1500) 5 – 10 0.5-10Typically 5 to

startRistocetin (mg/mL) -Low dose – 80%High dose – 82%

0.5 (0.25-2.5)1.5 (0.6-5.0)

0.5-0.6 1.2-1.5

≤ 0.60.8-1.5

Arachidonic acid (mM) 82% 1.36 (0.125-16) 0.5 – 1.64 Optional0.5-1.6

Thromboxane analogue U46619 (mM) - 15%

1.0 (0.05-40) 1.0 (QMPLS) Optional:1-2

TRAP (mM) - 17% 10 (1-100) _ Only mentioned

Final agonist concentrations: practices versus recommendationsfrom JTH 2009;7:1029, CLSI guidelines, QMPLS Broadsheet; AJCP 2010;134:955-63

Options for evidence-based assessment

Association of test result with a clinical problem

▪ PFD or a Definite Bleeding Disorder (DBD)

▪ Often expressed as an Odds Ratio (OR)

Assess test sensitivity and specificity

▪ Area under Receiver Operator Curves (AUROC)1=perfect test

Non-inferiority/superiority analysis of tests

▪ Differences in AUROC

1.4

4.3 3.60

10.6

1.12

8.7

245419

6051000

3.50

32.00 39.00

78.00

1000.00

1

10

100

1000

BT with PFD LTA with Bleeding Disorder

LTA with Acquired PFD

LTA with Inherited PFD

Dense Granule Deficiency by

EM (one subtype of PFD)

Odds Ratio

Data from Hayward et al, JTH 2009;7;676-684

infinite

From Hayward et al, JTH 2009;7;676-684 A limited panel of agonists detects most common PFD (epinephrine, AA, thromboxane analogue, 1.25 mg/mL collagen)Larger panel is needed to detect rare disorders

better

worse

_∆ ∆0

Test A better Test B better

Difference is acceptable

Estimate differences in AUROC

Key findings: Quiroga et al, Br J Haematol2009;147:729-36

Normal : 93.3% confirmed

Abnormal: 90.4% confirmed

22

Abnormalities in maximal

aggregation (MA)

Odds Ratio (95% CI) for association with

PFD (subjects without thrombocytopenia

and VWD)

Inherited PFD Acquired PFD

All agonists combined

Only one agonist abnormality

Two or more abnormalities

2.3 (0.7-7.5)*

78 (11-605)

0.6 (0.0-11.1)*

39 (3.6-419)

From JTH 2009;7;676-684

Unpublished: Single agonist abnormalities (except with ristocetin, perhaps collagen) are often not confirmed by repeat testing

Serotonin release - few labs now perform (radioactive) Quiroga et al, Br J Haematol 2009;147:729-36

Most aggregation defects are associated with release

release occurs in some “bleeders” with normal aggregation (14.3%)

ATP release - measured by lumiaggregometry Principle: Uses a reagent containing D-luciferin, luciferase light emission triggered by released ATP▪ Glasson and Fritsma STH 2009 ;5:168-80

▪ Cattaneo STH 2009 ;5:158-67

Abnormality associations

Odds Ratio (95% CI)

Bleeding disorder

17 (6-46)

Inherited platelet disorder

128 (30-545)

Inherited platelet disorder, LTA normal

105 (20-545)

ROC for ATP release defects (abnormalities with one or more agonists)

Most abnormalities detected by a few agonists

6 μM epinephrine,

5.0 μg/mL collagen

1 mΜ U46619

Paterson et al Blood 2010;115:1264-1266.Test for PLAU (uPA gene) duplication mutation

Use → new cases diagnosed in both Canada and USA▪ in Quebec, prevalence of QPD > type 2B VWD

Future? - CHIPs to test for a variety of inherited PFD

Engage participants

Discussions, lectures, etc

▪ can include wet or dry test exercises

Examples

Development of North American LTA Guidelines

▪ In person (QMP-LS), virtual (NASCOLA)

Wet workshop on PFD

▪ e.g. 7th ECAT Participants’ Meeting, 2010

Hackeng, Eikenboom, Verbruggen, Meijer

▪ 7th ECAT Participants’ Meeting, 2010

• 20 participants, 5 Groups• 8 Patients: 4-5 tested per group

• Platelet function measurements in whole blood with:

• PFA-100

• Chronolog Impedance and Luminescence

• Multiplate

• VerifyNowSt. Antonius Center for

Platelet Function Research

Virtual “Dry” workshopsResults of initial NASCOLA EQA challengesSee tremendous improvements: poster #811 by Moffat et al

Case Diagnoses, First EQA Challenges% correct

interpretations

Normal ~92%

Rare but well characterized disorder(e.g. Glanzmann thrombasthenia, Bernard Soulier Syndrome)

~85%

Common defect with multiple aggregation abnormalities(e.g. Secretion defect , dense granule deficiency)

~33%

False positive/non-diagnostic findings ~18-44%

Normal variant ~16%

Tracings, results and RI for interpretation were distributed to NASCOLA and ECAT labs

LTA Finding Recommended Interpretation

QMPLS and NASCOLA

Follow-up Investigation

Aggregation absent or marked with

AA, normal with U46619, with low

dose collagen.

Aspirin-like defect (drug induced or

inherited).

Repeat testing when subject not on

aspirin or other NSAIDs.

Aggregation is present with only

ristocetin.

Possible Glanzmann thrombasthenia

(inherited or acquired).

Glycoprotein analysis of fibrinogen

receptor alphaIIb-beta3.

Aggregation absent with high conc. of

ristocetin and the patient has

thrombocytopenia with very large

platelets.

Possible Bernard Soulier Syndrome

(inherited or acquired). VWD should be

excluded.

Glycoprotein analysis to assess

glycoprotein IbIXV, the VWF

receptor.

Aggregation with high conc. of

ristocetin without thrombocytopenia.

Possible von Willebrand disease. VWF levels.

Aggregation abnormally with low

conc. of ristocetin.

Possible type 2B or platelet-type VWD. VWF levels. Consider genetic

testing.

Aggregation abnormal with multiple

agonist. Markedly with ADP with

significant deaggregation.

Possible platelet ADP receptor defect

(P2Y12). Drug induced defect should

be excluded.

Repeat aggregation testing.

Other abnormalities with two or more

agonists.

Suggest a platelet function disorder is

present. Confirm on repeat testing.

Platelet ATP release and/or EM for

dense granule deficiency (DGD).

Abnormalities with only one agonist

(excluding collagen or ristocetin).

Non-diagnositic and could represent a

false positive.

Repeat aggregation testing, ATP

release and/or EM for DGD.

Examples of some

pitfalls if testing is

limited

Case 1

No challenges, very

large bruises, heavy

periods

Case 2

Bruises, serious

bleeding with

surgery

Case 3

No major challenges

but relatives have

bleeding problems

including joint bleeds

ADP 2.5 mM Normal Normal Normal

ADP 5.0 mM Normal Normal Normal

Collagen 1.25 mg/mL Normal Normal

Collagen 5.0 mg/mL Normal Normal

Epinephrine 6 mM Normal Normal

Arachidonic acid 1.6 mM Normal Normal

Thromboxane analogue

U46619 1 mM (few labs test) Normal

Ristocetin 0.5 mg/mL Normal (<7% aggregation)

Normal (<7% aggregation)

Normal (<7% aggregation)

Ristocetin 1.25 mg/mL Normal Normal Normal

LTA Interpretation Findings suggest a

platelet function

disorder (aspirin-like

defect excluded)

Non-diagnostic Non-diagnostic

Diagnosis after other tests Dense granule

deficiency

Secretion defect

based on abnormal

ATP release

Quebec platelet

disorder based on

genetic tests

Colleagues and Collaborators

HRLMP Special Coagulation

NASCOLA

QMPLS

ISTH SSC