Ravi Sarode, MDRavi Sarode, MDDirector, Transfusion Medicine and Director, Transfusion Medicine and Hemostasis Reference LaboratoryHemostasis Reference Laboratory

Professor of PathologyProfessor of PathologyUT Southwestern Medical CenterUT Southwestern Medical Center

Dallas, TXDallas, TX

Disclosure:

Sponsored by Chronolog Corporation

Platelet DisordersBleeding - mucocutaneous• Quantitative -

thrombocytopenia

• Qualitative -

thrombastheniaCongenital – rareAcquired - common• Drugs, herbs, fish oil, • Autoimmune

Thrombosis – arterial• Thrombocytosis• Hyperactive platelets

Investigation of Platelet Function

Personal and family historyThorough drug and dietary historyScreening tests• Platelet count• Peripheral Blood Smear• PFA-100®

• Bleeding time obsoleteDiagnostic• Aggregation study• Electron Microscopy

Platelet Aggregation Study

Light Transmission Aggregometry (LTA) by Optical method • Platelet Rich Plasma (PRP)

Impedance method • Whole blood aggregation (WBA)

CLSI published “Platelet Function testing by Aggregometry; Approved Guidelines” - Nov 2008

Specimen Handling

Non-traumatic venepuncture Minimum stasis 3.2% Sodium CitrateEvacuated tube or plastic syringe 19-21G needleGentle mixing Room temperature transport – upright Pneumatic tubing not recommended

PRP PRP -- Optical MethodOptical Method

First introduced by Born in 1962First introduced by Born in 1962Considered Considered ““the (the (g)oldg)old standardstandard”” in platelet in platelet function evaluationfunction evaluationPlatelet aggregation is measured by LTAPlatelet aggregation is measured by LTAATP secretion measurementATP secretion measurement--firefly firefly luciferinluciferin--luciferaseluciferase used as chemiluminescence used as chemiluminescence substrate for ATP release detection; ATP substrate for ATP release detection; ATP secretion measured in nmolsecretion measured in nmol

LTA LTA --

PRP methodPRP method

WB centrifuged at 170g x 15WB centrifuged at 170g x 15’’ for PRPfor PRPRemaining sample reRemaining sample re--centrifuged at 1500g x centrifuged at 1500g x 1515’’ to obtain PPP to obtain PPP Platelet count in PRP should be in 200Platelet count in PRP should be in 200--300 K 300 K range range

Adjusted by adding PPP or NSAdjusted by adding PPP or NS (preferred)(preferred)PRP kept in aggregometer wells at 37PRP kept in aggregometer wells at 37°°C for 5 C for 5 min before studymin before studyMagnetic stir bar in PRP cuvette maintains Magnetic stir bar in PRP cuvette maintains platelets in suspensionplatelets in suspension

PRP PRP --

Optical MethodOptical MethodBaseline light transmission through PPP Baseline light transmission through PPP (clear plasma) is set at 100%(clear plasma) is set at 100%Baseline light transmission through PRPBaseline light transmission through PRP(opaque plasma) is set at 0%(opaque plasma) is set at 0%Add an agonist (ADP, EPI, AA, Coll, Risto)Add an agonist (ADP, EPI, AA, Coll, Risto)Platelets aggregate Platelets aggregate -- allows increased light allows increased light transmittance through platelet suspensiontransmittance through platelet suspensionResults reported as percentage (%) of Results reported as percentage (%) of aggregationaggregationNormal ranges are established in each lab Normal ranges are established in each lab

PRP PRP --

Optical MethodOptical Method

Baseline light transmission Increased light

ADPPPP PRP

Light

ADP 10 μM = 73%

AA 0.5 mM

= 62%

Coll 2 μg = 70%

Risto 1.25 mg = 91%

Risto 0.25 mg = 2%

Coll 1 mg = 54%

AA 0.5 mM

= 1.5nm

Coll 2 μg = 1.0nm

PRP PRP --

Normal ControlNormal Control

Coll 1 mg = 54%

WBAWBA--

Impedance MethodImpedance Method

First introduced by Cardinal and Flower in First introduced by Cardinal and Flower in 19801980

Measures electrical resistance between two Measures electrical resistance between two electrodes immersed into whole blood electrodes immersed into whole blood samplesample

Simultaneous ATP secretion measurementSimultaneous ATP secretion measurement--firefly firefly luciferinluciferin--luciferaseluciferase used as a used as a chemiluminescence chemiluminescence

WBAWBA--

Impedance MethodImpedance MethodWB diluted 1:1 with 0.9 % saline (450 WB diluted 1:1 with 0.9 % saline (450 µµL each)L each)Samples with stir bars kept in wells at 37Samples with stir bars kept in wells at 37°°C for 5C for 5’’Electrode assembly consists of two wiresElectrode assembly consists of two wires

Reusable = palladium and Disposable Reusable = palladium and Disposable –– gold platedgold platedAC voltage in millivolt range applied to circuitAC voltage in millivolt range applied to circuitDuring equilibrium During equilibrium monolayersmonolayers of platelets formof platelets form

Stable impedance value establishedStable impedance value establishedAdd agonists (ADP, AA, Coll and Risto) Add agonists (ADP, AA, Coll and Risto) Platelet aggregate on wires Platelet aggregate on wires -- increases electrical increases electrical resistance to flow of electric current resistance to flow of electric current Aggregation measured in Ohms (Aggregation measured in Ohms (ΩΩ))

+ -

WBAWBA--

Impedance MethodImpedance Method

More aggregation

Less current flow

Increased resistance

Expressed as

Ω

ADP

Risto 1 mg = 8 Ω

Risto 0.25 mg = 0 Ω

ADP 10 μM=12 Ω

AA 0.5 mM=24 ΩColl 2 μg=24 Ω

Thrombin 1 U

= 0.75 nm

Coll 2 μg

= 0.66 nm

AA 0.5 mM=1.60 nm

WBAWBA--

Normal ControlNormal Control

Comparison Between WBA and PRP Comparison Between WBA and PRP

Impedance method Impedance method Optical method Optical method

Blood volume Blood volume 6 cc 6 cc >12 cc >12 cc

Test milieu Test milieu Physiologic, RBCs & Physiologic, RBCs & WBCs present WBCs present

Platelets only Platelets only

Centrifugation Centrifugation Not required Not required Required ;activation,Required ;activation,exhaustion of platelets exhaustion of platelets

Large platelets Large platelets Present Present Lost Lost

Platelet countPlatelet count 100100--1000K1000K 200200--300K300K

Icteric, lipemic, Icteric, lipemic, hemolyzed hemolyzed

Can be usedCan be used Cannot be usedCannot be used

Total test timeTotal test time ≈≈

60 min60 min ≈≈

110110--125 min 125 min

Case 1Case 1

A 6A 6--year old boy on year old boy on valproicvalproic acid for seizuresacid for seizuresPresented with platelet type bleeding Presented with platelet type bleeding tendencytendencyNormal platelet count and PFANormal platelet count and PFA--100100®®

PRP= Arachidonic acid (63%) and ADP PRP= Arachidonic acid (63%) and ADP (64%) normal maximum aggregation(64%) normal maximum aggregationWBA = there was no response to these WBA = there was no response to these agonistsagonists

CaseCase--11

Patient = 0 Ω

Control = 14 Ω

Patient =63%

Control 76%

WBA = AA 0.5mM PRP = AA 0.5mM

CaseCase--11

Patient = 0 Ω

Control = 5 Ω

Patient =64%

Control 66%

PRP= ADP 10 µMWBA = ADP 10 µM

4949--WFWF--tennis player tennis player Presents with worsening bruising Presents with worsening bruising Spontaneous large bruises while on Spontaneous large bruises while on fish oilfish oilSimilar large bruises when on Similar large bruises when on 81mg ASA81mg ASAThree vaginal deliveries with excessive Three vaginal deliveries with excessive postpartum hemorrhagepostpartum hemorrhageMost significant hemorrhage following Most significant hemorrhage following hysterectomy Hb dropped from 14 to 8 g/dLhysterectomy Hb dropped from 14 to 8 g/dLBleeding after neuroma removal from footBleeding after neuroma removal from footScheduled for extensive pelvic floor surgeryScheduled for extensive pelvic floor surgery

CaseCase--22

0.5 = Ø

Ω ↓

0.25 = 3 Ω ↓

0.35 = nm

0.20 = nm

AA

Thromb = 0.43 nm ↓

ATP

10 = 7 Ω20 = 7 Ω

ADP

2 μg

= 17 Ω

1 μg = 4 Ω

0.38 = nm

0.20 = nm

Coll

1 mg = 0 Ω0.5 mg = 0 Ω0.25 mg = 0 Ω

Risto

PFA-100

ADP = 90 (49-115)

EPI = 132 (83-164)

Case –

2

0.5 = 0 Ω ↓

0.5 = 0.31 nm ↓

1μg = 22 Ω

1μg

= 0.31 nm

2μg

= 0.31 nm ↓

2μg

= 28 Ω

Repeat Study 6 months later

AA Coll

Case –

2Risto 1 mg = 2 Ω

0.5 = 20 Ω

0.5

= 0.33 nm ↓

2 μg

= 25 Ω

2 μg

= 0.58 nm

20 μM = 6 Ω

10 μM = 3 Ω

1 mg = 10

Ω

0.5 = 4 Ω

0.25 = 0 Ω

AA Coll

ADP Risto

Post-

DDAVP

Case –

2

CaseCase--2277--33--0606 11--55--0707 11--99--0707

Thrombin Thrombin (0.73(0.73--1.75 1.75 nMnM))

↓↓

0.430.43 ↓↓

0.590.59 ↓↓

0.540.54

AA 0.5 mM(17AA 0.5 mM(17--26)26) ↓↓0/0.350/0.35 ↓↓0/0.310/0.31 ↓↓

88Coll 2Coll 2µµg (20g (20--30)30) ↓↓

17 /0.3817 /0.38 28/0.6128/0.61 25 /0.2525 /0.25

ADP 10 ADP 10 µµM (8M (8--18)18) ↓↓

77 ↓↓

00 ↓↓

66ADP 20 ADP 20 µµM (9M (9--20)20) ↓↓

77 ↓↓

22 ↓↓

33

Risto 1.0 mg (>5)Risto 1.0 mg (>5) ↓↓

00 ↓↓

22 1010Risto 0.25 mg (0)Risto 0.25 mg (0) 00 00 00

Post-DDAVP

Received 0.3ug/Kg DDAVP half an hour Received 0.3ug/Kg DDAVP half an hour before surgerybefore surgeryUnderwent 4 hour extensive pelvic Underwent 4 hour extensive pelvic reconstruction surgeryreconstruction surgeryNo significant bleeding!No significant bleeding!Diagnosis Diagnosis

? Mild global dysfunction? Mild global dysfunctionShe still bruises off and on She still bruises off and on –– much less than much less than fish oil or 81 mg ASAfish oil or 81 mg ASA

Case 2Case 2

3333--yryr--WF presented with flank pain, WF presented with flank pain, hematuria, painful lesions on Lhematuria, painful lesions on L--foot, foot, transient antiphospholipid antibodiestransient antiphospholipid antibodiesEpistaxis off and onEpistaxis off and onThese bleeding symptoms started recentlyThese bleeding symptoms started recentlyNo prior bleeding historyNo prior bleeding historyEmotionally disturbedEmotionally disturbedMedical Sales rep!Medical Sales rep!Had transient Had transient pancytopeniaspancytopenias

CaseCase--33

AA 0.25 = 0

ΩColl 1μg = 2 ΩADP 20 = 7

ΩRisto 1 = 6

Ω

AA = 0.17 nmColl = 0.12 nm

AA 0.5 = 1

Ω

AA 0.5 = 0.33 nm

Coll 2 = 7 Ω

Thro = 0.59 nm

Coll 2 = 0.26 nm

ADP10=6Ω

ADP 20 = 66%

AA 0.5 = 8%

Coll 2 = 39%

Risto 1.25 = 68%

PRP

WBA

AgonistAgonist WBAWBA PRPPRP

AAAA ↓↓ ↓↓

CollColl ↓ ↓↓

ADPADP NN NN

RistoRisto NN NN

ATP secATP sec ↓ ↓

Cas

e –

3

Interpretation: Aspirin like defectInterpretation: Aspirin like defect

CaseCase--33

Also called Also called ““sticky platelet syndromesticky platelet syndrome””Persistent elevation of FVIII/VWF and Persistent elevation of FVIII/VWF and fibrinogen fibrinogen -- thrombophilia risk factorsthrombophilia risk factorsSimilarly Similarly –– increased platelet reactivity could increased platelet reactivity could cause thrombosiscause thrombosis

Millions of patients on antiMillions of patients on anti--platelets drugsplatelets drugsAspirin and clopidogrel resistance (20Aspirin and clopidogrel resistance (20--30%) is 30%) is well described well described

Detected by increased aggregation Detected by increased aggregation responses to submaximal concentrations of responses to submaximal concentrations of agonistsagonists

Hyperactive PlateletsHyperactive Platelets

““Thrombosis and bleeding in MPD: Thrombosis and bleeding in MPD: identification of at risk pts with WBAidentification of at risk pts with WBA””

ManoharanManoharan et al, Br J et al, Br J HematolHematol 19991999

““Aggregometry detects platelet hyperactivity Aggregometry detects platelet hyperactivity in healthy individualsin healthy individuals””

Paul BrayPaul Bray’’s group s group –– Blood 2005Blood 2005

Hyperactive PlateletsHyperactive Platelets

Spontaneous aggregationSpontaneous aggregationADP = 10, 5, 2.5 and 1.25 ADP = 10, 5, 2.5 and 1.25 µµMMAA = 0.5, 0.25, 0.125 and 0.0625 AA = 0.5, 0.25, 0.125 and 0.0625 mMmMColl = 2.0, 1.0, 0.5 and 0.25 Coll = 2.0, 1.0, 0.5 and 0.25 µµg/mLg/mL

Establish normal ranges Establish normal ranges Do not use ATP secretion Do not use ATP secretion

WBA WBA --

Hyperactive Platelet StudyHyperactive Platelet Study

Case Case --

4 4

A 47 year-old female with history of SLE, MI and multiple CVAs despite being on daily 81mg ASADiagnosed with Hyperactive Platelet Syndrome in the past

CaseCase--44

ADP

5 µM; 8 ΩAA

0.25 mM; 26 Ω

ADP

10 µM; 22 Ω

AA

0.5 mM; 21 Ω

AA

0.125 mM; 24 Ω

ADP

2.5 µM; 0 Ω

Collagen

2 µg/ml; 23 Ω

AA

0.063 mM; 0 Ω

Collagen

1 µg/ml; 20 Ω

Collagen

0.5 µg/ml; 0 Ω

(normal = 3-14 ) (normal = 0-10 )

(normal = 0-3)

(normal = 9-14 )

(normal = 7-15 )

(normal = 10-17 )

Hyperactivity

with AA and

Collagen

Case Case --

55

55 yr male with a diagnosis of Essential 55 yr male with a diagnosis of Essential ThrombocythemiaThrombocythemiaPlatelet counts between 500Platelet counts between 500--700K on 700K on chemotherapychemotherapyDevelops sudden SOB and chest painDevelops sudden SOB and chest pain

Chest CT Chest CT –– PEPEThrombophilia workThrombophilia work--up negativeup negativeHyperactive platelet studyHyperactive platelet study

Case -

5

32 Ohms

30 Ohms

5 Ohms

36 Ohms

Aspirin and Clopidogrel EffectAspirin and Clopidogrel Effect

WBA detects antiplatelet drug effects WBA detects antiplatelet drug effects reliably and quicklyreliably and quicklyAgonists used Arachidonic acid and Agonists used Arachidonic acid and collagen for ASAcollagen for ASAADP for clopidogrel (ticlopidine and ADP for clopidogrel (ticlopidine and dipyridamoledipyridamole))

Detection of Clopidogrel EffectDetection of Clopidogrel Effect

17 volunteers given 75 mg/day x 10days17 volunteers given 75 mg/day x 10daysPlatelet function at baseline and on day 11Platelet function at baseline and on day 11PRP, WBA and PFAPRP, WBA and PFA--100100WBA more sensitive to detect clopidogrel WBA more sensitive to detect clopidogrel inhibition by ADPinhibition by ADPFollowFollow--up showed PRP normalizes before up showed PRP normalizes before WBA WBA RBCs known to modulate platelet function RBCs known to modulate platelet function due to its ADP contentdue to its ADP content

Korpanty et al, Platelets, 2007

PostPost--clopidogrel Platelet Inhibitionclopidogrel Platelet Inhibition

MethodMethodADP (n=16)ADP (n=16)

PrePre PostPost %%↓↓

WBA WBA 10 10 μμMM((ΩΩ))

18 18 ±±

66 5 5 ±±

66 70 70 ±±

3535**

PRP PRP 10 10 μμMM(%)(%) 80 80 ±±

1111 53 53 ±±

2020 36 36 ±±

2525

PFAPFA--100100®®(Sec.)(Sec.) 86 86 ±±

1313 101 101 ±±

2525 17 17 ±±

2323

*P = <0.001Korpanty et al, Platelets, 2007

Detection of Aspirin EffectDetection of Aspirin Effect

Compared WBA with VerifyNow AspirinCompared WBA with VerifyNow Aspirin50 volunteers given 325 mg x 3days50 volunteers given 325 mg x 3daysBaseline and day 4 evaluation by WBA Baseline and day 4 evaluation by WBA (AA and Coll) and VerifyNow Aspirin(AA and Coll) and VerifyNow Aspirin12/50 (24%) non12/50 (24%) non--responders by WBAresponders by WBAAll good responders (<550 ARU) by All good responders (<550 ARU) by VerifyNowVerifyNow

Seems the cut off should be much lower Seems the cut off should be much lower than 550 ARU.than 550 ARU.

Korpanty et al, Thromb Res, 2006

5555--yryr--WF with a diagnosis of basilar artery WF with a diagnosis of basilar artery aneurysm of brainaneurysm of brainScheduled for stent assisted coiled Scheduled for stent assisted coiled embolizationembolizationTo prevent thrombosis at the site of coil To prevent thrombosis at the site of coil embolization platelet function has to be embolization platelet function has to be inhibited completelyinhibited completelyGiven ASA 325 mg and loading dose of Given ASA 325 mg and loading dose of clopidogrel 300 mgclopidogrel 300 mgAntiAnti--platelet effect 4 hours later platelet effect 4 hours later –– no effect no effect Doubled ASA and Clopidogrel Doubled ASA and Clopidogrel –– adequate adequate effecteffect

Case Case --

66

10 μM ADP

= 13

Ω

Coll 1mg = 14 Ω

AA 0.5 = 6 Ω All = 0

Ω

I. ASA/Plavix -

No Effect 2. ASA/Plavix –

Adequate

Case –

6

Summary

PRP PRP --

LTA LTA

PRP method isPRP method isLaborLabor--intensiveintensiveTime consuming Time consuming Injurious to platelets (centrifugation)Injurious to platelets (centrifugation)

Giant, hyperGiant, hyper-- or hypoactiveor hypoactive--plateletsplateletsLost during centrifugation Lost during centrifugation Excluded from evaluationExcluded from evaluation

PRP milieu devoid ofPRP milieu devoid ofRBCs andRBCs andWBCs that influence platelet function WBCs that influence platelet function

Offers Offers Rapid evaluation of platelet functionRapid evaluation of platelet functionNo sample processingNo sample processingNo centrifugation injury to plateletsNo centrifugation injury to platelets

More physiologic milieu, RBCs & WBCs includedMore physiologic milieu, RBCs & WBCs includedSmaller quantity of blood requiredSmaller quantity of blood required

Advantage in young childrenAdvantage in young childrenPatients with difficulties in drawing bloodPatients with difficulties in drawing blood

More suitable and sensitive method forMore suitable and sensitive method forAssessment of antiAssessment of anti--platelet therapy platelet therapy Platelet hyperactivityPlatelet hyperactivity

WBWB--ImpedanceImpedance--based Aggregationbased Aggregation