Ravi Sarode, MD Director, Transfusion Medicine and Hemostasis Reference Laboratory Professor of Pathology UT Southwestern Medical Center Dallas, TX Disclosure: Sponsored by Chronolog Corporation Platelet Disorders Bleeding - mucocutaneous • Quantitative - thrombocytopenia • Qualitative - thrombasthenia ¾ Congenital – rare ¾ Acquired - common • Drugs, herbs, fish oil, • Autoimmune Thrombosis – arterial • Thrombocytosis • Hyperactive platelets Investigation of Platelet Function Personal and family history Thorough drug and dietary history Screening tests • Platelet count • Peripheral Blood Smear • PFA-100® • Bleeding time obsolete Diagnostic • 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 Citrate Evacuated tube or plastic syringe 19-21G needle Gentle mixing Room temperature transport – upright Pneumatic tubing not recommended PRPPRP -- OpticalOptical MethodMethod FirstFirst introducedintroduced byby BornBorn inin 19621962 ConsideredConsidered ““thethe ((g)oldg)old standardstandard”” inin plateletplatelet functionfunction evaluationevaluation PlateletPlatelet aggregationaggregation isis measuredmeasured byby LTALTA ATPATP secretionsecretion measurementmeasurement--fireflyfirefly luciferinluciferin-- luciferaseluciferase usedused asas chemiluminescencechemiluminescence substratesubstrate forfor ATPATP releaserelease detection;detection; ATPATP secretionsecretion measuredmeasured inin nmolnmol LTALTA -- PRPPRP methodmethod WBWB centrifugedcentrifuged atat 170g170g xx 1515’’ forfor PRPPRP RemainingRemaining samplesample rere--centrifugedcentrifuged atat 1500g1500g xx 1515’’ toto obtainobtain PPPPPP PlateletPlatelet countcount inin PRPPRP shouldshould bebe inin 200200--300300 KK rangerange ¾¾ AdjustedAdjusted byby addingadding PPPPPP oror NSNS (preferred)(preferred) PRPPRP keptkept inin aggregometeraggregometer wellswells atat 3737°°CC forfor 55 minmin beforebefore studystudy MagneticMagnetic stirstir barbar inin PRPPRP cuvettecuvette maintainsmaintains plateletsplatelets inin suspensionsuspension PRPPRP -- OpticalOptical MethodMethod BaselineBaseline lightlight transmissiontransmission throughthrough PPPPPP (clear(clear plasma)plasma) isis setset atat 100%100% BaselineBaseline lightlight transmissiontransmission throughthrough PRPPRP (opaque(opaque plasma)plasma) isis setset atat 0%0% AddAdd anan agonistagonist (ADP,(ADP, EPI,EPI, AA,AA, Coll,Coll, Risto)Risto) PlateletsPlatelets aggregateaggregate -- allowsallows increasedincreased lightlight transmittancetransmittance throughthrough plateletplatelet suspensionsuspension ResultsResults reportedreported asas percentagepercentage (%)(%) ofof aggregationaggregation NormalNormal rangesranges areare establishedestablished inin eacheach lablab PRPPRP -- OpticalOptical MethodMethod PPP PRP ADP Light Baseline light transmission Increased light PRPPRP -- NormalNormal ControlControl Risto 0.25 mg = 2% AA 0.5 mM = 62% Coll 2 μg = 70% Coll 1 mg = 54% Risto 1.25 mg = 91% ADP 10 μM= 73% AA 0.5 mM = 1.5nm Coll 2 μg = 1.0nm Coll 1 mg = 54% WBAWBA-- ImpedanceImpedance MethodMethod FirstFirst introducedintroduced byby CardinalCardinal andand FlowerFlower inin 19801980 MeasuresMeasures electricalelectrical resistanceresistance betweenbetween twotwo electrodeselectrodes immersedimmersed intointo wholewhole bloodblood samplesample SimultaneousSimultaneous ATPATP secretionsecretion measurementmeasurement-- fireflyfirefly luciferinluciferin--luciferaseluciferase usedused asas aa chemiluminescencechemiluminescence WBAWBA-- ImpedanceImpedance MethodMethod WBWB diluteddiluted 1:11:1 withwith 0.90.9 %% salinesaline (450(450 µµLL each)each) SamplesSamples withwith stirstir barsbars keptkept inin wellswells atat 3737°°CC forfor 55’’ ElectrodeElectrode assemblyassembly consistsconsists ofof twotwo wireswires ¾ Reusable = palladium and Disposable – gold plated ACAC voltagevoltage inin millivoltmillivolt rangerange appliedapplied toto circuitcircuit DuringDuring equilibriumequilibrium monolayersmonolayers ofof plateletsplatelets formform ¾ Stable impedance value established AddAdd agonistsagonists (ADP,(ADP, AA,AA, CollColl andand Risto)Risto) PlateletPlatelet aggregateaggregate onon wireswires -- increasesincreases electricalelectrical resistanceresistance toto flowflow ofof electricelectric currentcurrent AggregationAggregation measuredmeasured inin OhmsOhms ((ΩΩ)) WBAWBA-- ImpedanceImpedance MethodMethod ADP + - More aggregation Less current flow Increased resistance Expressed as Ω WBAWBA-- NormalNormal ControlControl Risto 0.25 mg = 0 Ω ADP 10 μM=12 Ω Risto 1 mg = 8 Ω AA 0.5 mM=24 Ω Coll 2 μg=24 Ω AA 0.5 mM=1.60 nm Coll 2 μg = 0.66 nm Thrombin 1 U = 0.75 nm ComparisonComparison BetweenBetween WBAWBA andand PRPPRP Impedance method Optical method Blood volume 6 cc >12 cc Test milieu Physiologic, RBCs & Platelets only WBCs present Centrifugation Not required Required ;activation, exhaustion of platelets Large platelets Present Lost Platelet count 100-1000K 200-300K Icteric, lipemic, Can be used Cannot be used hemolyzed ≈ 60 min ≈ 110-125 min Total test time CaseCase 11 AA 66--yearyear oldold boyboy onon valproicvalproic acidacid forfor seizuresseizures PresentedPresented withwith plateletplatelet typetype bleedingbleeding tendencytendency NormalNormal plateletplatelet countcount andand PFAPFA--100100® PRP=PRP= ArachidonicArachidonic acidacid (63%)(63%) andand ADPADP (64%)(64%) normalnormal maximummaximum aggregationaggregation WBAWBA == therethere waswas nono responseresponse toto thesethese agonistsagonists CaseCase--11 Patient = 0 Ω Patient =63% Control = 14 Ω Control 76% WBA = AA 0.5mM PRP = AA 0.5mM CaseCase--11 Patient = 0 Ω Control = 5 Ω Patient =64% Control 66% WBA = ADP 10 µM PRP= ADP 10 µM CaseCase--22 4949--WFWF--tennistennis playerplayer PresentsPresents withwith worseningworsening bruisingbruising SpontaneousSpontaneous largelarge bruisesbruises whilewhile onon fishfish oiloil SimilarSimilar largelarge bruisesbruises whenwhen onon 81mg81mg ASAASA ThreeThree vaginalvaginal deliveriesdeliveries withwith excessiveexcessive postpartumpostpartum hemorrhagehemorrhage MostMost significantsignificant hemorrhagehemorrhage followingfollowing hysterectomyhysterectomy HbHb droppeddropped fromfrom 1414 toto 88 g/dLg/dL BleedingBleeding afterafter neuromaneuroma removalremoval fromfrom footfoot ScheduledScheduled forfor extensiveextensive pelvicpelvic floorfloor surgerysurgery 0.5 = Ø Ω ↓ 0.25 = 3 Ω ↓ 1 μg= 4 Ω 1 mg = 0 Ω 0.5 mg = 0 Ω 0.25 mg = 0 Ω 2 μg = 17 Ω AA Coll Risto 0.35 = nm 0.38 = nm 0.20 = nm 0.20 = nm Case – 2 20 = 7 Ω PFA-100 10 = 7 Ω ATP ADP ADP = 90 (49-115) Thromb = 0.43 nm ↓ EPI = 132 (83-164) Repeat Study 6 months later 0.5 = 0 Ω ↓ 1μg= 22 Ω AA Coll 2μg = 28 Ω 2μg = 0.31 nm ↓ 0.5 = 0.31 nm ↓ 1μg = 0.31 nm Risto 1 mg = 2 Ω Case – 2 0.5 = 20 Ω 2 μg = 25 Ω AA Coll 2 μg = 0.58 nm Case – 2 0.5 = 0.33 nm ↓ Post- DDAVP 20 μM= 6Ω 0.25 = 0 Ω 10 μM= 3 Ω 0.5 = 4 Ω 1 mg = 10 Ω ADP Risto CaseCase--22 Post-DDAVP 77--33--0606 11--55--0707 11--99--0707 ThrombinThrombin ↓↓ 0.430.43 ↓↓ 0.590.59 ↓↓ 0.540.54 (0.73(0.73--1.751.75 nMnM)) AAAA 0.50.5 mM(17mM(17--26)26) ↓↓0/0.350/0.35 ↓↓0/0.310/0.31 ↓↓ 88 CollColl 22µµgg (20(20--30)30) ↓↓ 1717 /0.38/0.38 28/0.6128/0.61 2525 /0.25/0.25 ADPADP 1010 µµMM (8(8--18)18) ↓↓ 77 ↓↓ 00 ↓↓ 66 ADPADP 2020 µµMM (9(9--20)20) ↓↓ 77 ↓↓ 22 ↓↓ 33 RistoRisto 1.01.0 mgmg (>5)(>5) ↓↓ 00 ↓↓ 22 1010 RistoRisto 0.250.25 mgmg (0)(0) 00 00 00 CaseCase 22 ReceivedReceived 0.3ug/Kg0.3ug/Kg DDAVPDDAVP halfhalf anan hourhour beforebefore surgerysurgery UnderwentUnderwent 44 hourhour extensiveextensive pelvicpelvic reconstructionreconstruction surgerysurgery NoNo significantsignificant bleeding!bleeding! DiagnosisDiagnosis ¾ ? Mild global dysfunction SheShe stillstill bruisesbruises offoff andand onon –– muchmuch lessless thanthan fishfish oiloil oror 8181 mgmg ASAASA CaseCase--33 3333--yryr--WFWF presentedpresented withwith flankflank pain,pain, hematuria,hematuria, painfulpainful lesionslesions onon LL--foot,foot, transienttransient antiphospholipidantiphospholipid antibodiesantibodies EpistaxisEpistaxis offoff andand onon TheseThese bleedingbleeding symptomssymptoms startedstarted recentlyrecently NoNo priorprior bleedingbleeding historyhistory EmotionallyEmotionally disturbeddisturbed MedicalMedical SalesSales rep!rep! HadHad transienttransient pancytopeniaspancytopenias AA 0.25 = 0 Ω AA 0.5 = 1 Ω Coll 1μg= 2Ω ADP10=6Ω ADP 20 = 7 Ω Coll 2 = 7 Ω Risto 1 = 6 Ω WBA AA 0.5 = 0.33 nm Thro = 0.59 nm AA = 0.17 nm Coll = 0.12 nm Coll 2 = 0.26 nm Case – 3 Agonist WBA PRP AA 0.5 = 8% AA ↓ ↓ Coll 2 = 39% PRP ADP 20 = 66% Coll ↓ ↓ ADP N N Risto 1.25 = 68% Risto N N ATP sec ↓ ↓ CaseCase--33 Interpretation:Interpretation: AspirinAspirin likelike defectdefect HyperactiveHyperactive PlateletsPlatelets AlsoAlso calledcalled ““stickysticky plateletplatelet syndromesyndrome”” PersistentPersistent elevationelevation ofof FVIII/VWFFVIII/VWF andand fibrinogenfibrinogen -- thrombophiliathrombophilia riskrisk factorsfactors SimilarlySimilarly –– increasedincreased plateletplatelet reactivityreactivity couldcould causecause thrombosisthrombosis ¾ Millions of patients on anti-platelets drugs ¾ Aspirin and clopidogrel resistance (20-30%)