Perspectives on Platelet Functional Disorders in 2007

Transcription

1 latelet Functional Disorders and Testing Catherine. M. Hayward, MD hd rofessor, athology & Molecular Medicine McMaster University, Hamilton, Ontario, Canada Head, Coagulation Hamilton Regional Laboratory Medicine rogram Disclosures for Catherine. M. Hayward Research Support/I Employee Consultant Major Stockholder Speaker s s Bureau Scientific Board Advisory o support from industry Supported by: Heart and Stroke Foundation of Ontario Canadian Institutes for Health Research Canada Research Chair /A /A /A /A /A /A = ot applicable (no conflicts) Session Objectives Important tests for evaluating platelet functional disorders Common forms of platelet functional disorders Why platelet function testing is difficult to standardize and issues important to test quality Understand, through case-based examples, the importance of diagnostic testing for platelet functional disorders erspectives on latelet Functional Disorders in 27 Disorders - common and important Uncertainties about best test practices for evaluating these conditions and about test sensitivity, specificity, positive predictive value, negative predictive value Also lack tools for standardizing the clinical part of the diagnostic assessment Current Concepts on latelet Functions Implications for Types of otential Defects tethering activation translocation stable adhesion collagen, von Willebrand factor, fibrin, other matrix elements propagation of coagulation platelet recruitment thrombus growth & stabilization scab temporary bandage Disorders with Impaired latelet Function Congenital or acquired defects in Receptors for: Adhesive proteins Signaling/Activation roblems Receptors for important agonists* Signaling/secretion pathways that enhance activation (including release of dense granule contents)* latelet procoagulant activity Some conditions that affect platelet numbers also impair platelet function * common 1

2 Adhesion Receptor Defects AD (2Y12) AD (2Y1) Thrombin Thromboxane AF vwf GIb BSS leckstrin cam Gi AC KC TxA2 AT TS DG I2 GG2/GH2 Gq LC CO Arachidonic Acid I LA2 TK hospholipids Ca MLC MLC Ca vwd Fibrinogen Haemophilia 26;12 (Suppl( ): VIIIa Ca IXa GIIb-IIIa Aggregation Thrombasthenia Afibrinogenemia Secretion Disorders of Secretion/ Signal Transduction IIa latelet Coagulant Activities C Va a II Xa X Acquired Qualitative Defects Drugs antiplatelet agents are the most common Uremia Liver disease Cushing s s Syndrome Cardiopulmonary bypass Inhibitory antibodies Bone marrow disorders Diverse e.g. storage pool defects, membrane glycoprotein deficiencies latelet Disorders Lack data from population surveys Secretion defects are more common than dense granule deficiency Dense granule deficiency is almost as common as von Willebrand disease about -5% of referred patients at our center Screening Tests for latelet Functional Disorders Bleeding time Sensitivity limited, performance issues Use predicting response to DDAV therapy? Closure Time measured by FA-1 TM Rapid, simple, test of shear-dependent platelet adhesion Sensitivity not perfect for screening 24% to >9% sensitivity for congenital platelet disorders oorer for studies prospectively evaluating platelet disorders oorer for common platelet disorders Hayward, Harrison, Cattaneo, Ortel and Rao; the latelet hysiology ogy SSC of ISTH. latelet function analyzer (FA)-1 closure time in the evaluation of platelet disorders and platelet function. JTH 26; 4: FA-1 Closure Times in Congenital latelet Disorders JTH 26; 4: * - common disorders - associated with thrombocytopenia Glanzmann Thrombasthenia Aspirin-Like Defect AD Receptor/Signaling Defect Dense Granule Deficiency* Hemansky udlak Syndrome rimary Secretion Defects* latelet rocoagulant Defect Bernard-Soulier Syndrome latelet-type type von Willebrand Disease Gray latelet Syndrome Hereditary Macrothrombocytopenia Associated with onmuscle Myosin Heavy Chain IIa Syndromes Undefined Autosomal Dominant Thrombocytopenia CAD CT - or to CEI CT or to or or CT in Diagnostic Testing for latelet Disorders otential advantages JTH 26; 4: JTH 25: ;19-11 Early clues about a defect if abnormal Abnormal results may trigger a referral More evidence is needed on its most appropriate use in clinical practice related to platelet disorders Sensitivity is better for VWD than for platelet disorders Diagnostic Screening: FURTHER TESTIG EEDED, REGARDLESS Role in drug monitoring needs further evaluation 2

3 Drug CT with anti-platelet drugs inhibitors of α IIb β abciximab, tirofiban, eptifibatide COX-1 1 inhibitors (aspirin and other SAIDs) Thienopyridines: Ticlopidine or clopidogrel Ticlopidine or clopidogrel plus aspirin JTH 26; 4: CAD CT or or CEI CT or or Tests for Drug Resistance Assay Aggregation Verifyow Aspirin & 2Y12 (mod. aggreg.. with fibrinogen coated beads; RFA) lateletworks (count platelets post-activation) FA-1 1 (high shear adhesion test with activation: CEI or CAD) Thromboxane Assays Serum (ex vivo generation) or urine (in vivo generation) Impact (Cone and late device) Research Assays of latelet Activation (e.g. flow cytometry) VAS (vasodilator stimulated phosphoprotein phosphorylation influenced by 2Y12) Advantages Gold standard oint-of of-care, simple, rapid, semi- automated Simple, rapid, uses platelet counter Simple, Rapid, Semi-automated With both: samples can be stored for batch analyses More complex than oint-of of-care Simple, Rapid, Semi-automated modifiable Endpoint for 2Y12 function Disadvantages All: need more data on relationship to outcomes, sensitivity and specificity for resistance Tech challenging, time consuming, not standardized between labs, some variability, choice of procedures (light transmission, electrical impedance; use of platelet rich plasma, whole blood) relationship to outcomes? influenced by other variables (hematocrit( hematocrit,, platelet count, von Willebrand factor level) methods currently not in widespread use possible problems re specificity relationship to outcomes? ot in wide use, complex, time consuming Flow cytometry-based test; not in wide use Complexity of Diagnostic Testing for latelet Disorders Include an assessment of/for: latelet number and size, platelet and leukocyte morphology ~17% of referrals for testing are thrombocytopenic Option immunostaining for some conditions e.g. MHY9 related disorders latelet function, evaluated by aggregation tests latelet dense granule deficiency Aggregation, BT, FA-1 1 CT - may be normal latelet secretion, evaluated by release of dense granule contents ts? More sensitive endpoint for defective function Adhesion testing apart from tests such as the FA-1 1 CT, this remains in research domain Optional Tests for procoagulant defects (appear rare, testing rarely done -?Serum protein consumption to screen) Others transmission electron microscopy, glycoprotein analysis, thromboelastography (platelets contribute to properties of clots), etc What do we find with our standardized testing? Data for 91 Unselected atients rospectively Evaluated for von Willebrand Disease & latelet Disorders platelet function abnormality &/or dense granule deficiency von Willebrand disease no laboratory abnormalities found abnormalities of uncertain signficance 17% 9% 9% 5% Hamilton Registry Data March 22 Setting General Diagnosis of latelet Dysfunction Monitoring of Antiplatelet Therapy Quality Assurance and latelet Tests Hayward CM, Eikelboom J. latelet function testing: Quality assurance. a STH Characteristic Convenient Accurate & recise Standardized Sensitive Specific opulation norms to guide interpretation roven utility Specific Clinically relevant Modifiable Cost effective Additional comments Simple (no operator expertise required), rapid, inexpensive The test measures what it is supposed to measure. Reproducible, different observers agree on interpretation Test procedure is well described, standards are available, existing quality control program egative test rules out disease ositive test rules in disease Test has been evaluated in full range of subjects (mild & severe, treated and untreated disease) and in subjects with other conditions that fall within the differential diagnosis atients are better off as a result of undergoing the test Measures the effect of the drug on its target Results independently correlated with clinical outcome Altering antiplatelet treatment based on the results of the test improves clinical outcome Benefits of testing outweigh the direct and indirect costs of testing and follow up All Labs are ot the Same.. Variability Between Clinical Laboratories in Diagnostic Testing for Disorders of latelet Function Moffat et al, Thromb Haemost. 25;9: Goals identify common practices and problems in the testing for disorders of platelet function Enthusiastic participation! 47 participating labs

4 Aggregation methodologies 7% of ASCOLA sites used >1 method ASCOLA Survey Data agonists used for clinical aggregation minority (15%) compared arachidonic acid & thromboxane analogue responses % sites latelet Rich lasma Whole Blood Luminescence AD Arachidonic Acid Ristocetin Epinephrine Thromboxane Analogue Thrombin TRA Spontaneous AT % sites Survey 1 Survey 2 Final Agonist Concentration for Testing latelet Function by Aggregation Range Survey 1 Median Range Survey 2 Median Sources of reference intervals 26% of sites used >1 method 29% if sites had not determined their own reference range ( tests/yr) Only 1 site did qualitative, without quantitative, interpretations ns Only 1 site formally evaluated data for normality in distribution See Moffat abstract this meeting AD µg/ml.5 1 µm 5 µg/ml 5 µm µg/ml 1 2 µm 2.5 µg/ml 5 µm Determined mean +/- 2 S.D. Epinephrine.1 1 µm 18 µm.1 1 µm 1 µm ublished literature Arachidonic Acid Ristocetin mm mg/ml.5 mm Low Dose:.5 mg/ml High Dose: 1.2 mg/ml mm mg/ml 1.6 mm Low dose:.5 mg/ml High dose: 1.25 mg/ml Vendor of instrument or reagent qualitative interpretation % sites Concerns Raised About latelet Aggregation Testing there were many... Labor intensive Lack of evidence-based guidelines Uncertainties how to: evaluate thrombocytopenic patients Tam Abstract - this meeting interpret epinephrine aggregation Challenging to obtain reliable drug histories, uncertainties about the effects of different drugs Influence of pre-analytical errors proper sample procurement & transport Aggregation Testing What is Best? Agonist Concentrations Medians some conformity Are these appropriate concentrations? Review of published literature The medians are probably good concentrations for testing Useful strategies e.g. comparing arachidonic acid/thromboxane responses ASCOLA Study: 15% of labs used this comparison to sort out possible ASA/SAID-like defects at the time of this survey 4

5 Control (red, green) vs. atient () with Secretion Defect : also reduced aggregation with arachidonic acid and thromboxane e analogue AD 2.5 and 5 μm Ristocetin 1.2 &.5 mg/ml Illustration of Aggregation Findings % aggregation with 4 μm AD 1.2 and 5 mg/ml and C: no agg. with low dose, which screens for VWD type 2B & lt-type Epinephrine 6 & 1 μm % subjects higher concentration of agonist: shown in red (C) & black () patient control % aggregation (interval mean) data is bimodal in distribution likely due to an influence of 2Y12 polymorphisms o 2 o only 1 o Epinephrine Aggregation Variability in Aggregation Tests? data from repeat tests done on 115 patients in Hamilton abnormal on one or more occasions concordant results control Healthy Controls % aggregation (interval mean) % subjects % AD AA HD Rist all agonists Illustration: Usefulness of Aggregation Tests AD 5 um 5 ug/ml 1.25 ug/ml Epinephrine 6 um Arachidonic Acid 1.6 mm Thromboxane analogue 1 um Ristocetin.5 mg/ml Ristocetin 1.25 mg/ml Reference Interval % aggregn Glanzmann Thrombasthenia 47 Secretion Defect Dense Granule Deficiency (1/ have normal results) Secretion absent or reduced with these agonists but normal with thrombin Thromboxane Generation Defect δ-granules: ~ 2-5/platelet2 α-granules: ~ 8/platelet Diagnostic Evaluation of latelet Luggage Defects Types of Luggage alpha (α)( protein storage container delta (δ) ( electron dense* δ-granule deficiency: Fairly common ~ 4% prevalence in our patients Aggregation, BT, CT may be normal α-granule deficiency: GRAY platelets - rare Clue from evaluation of blood film combined αδ deficiency: Rarer than δ-granule deficiency 5

6 Whole Mount Most popular method for assessing dense granule deficiency in orth America Controls: average of 4 or more electron dense granules per platelet EDS Electron Dispersion Spectral Analysis analysis of the different dense granule constituents control Ca αδ-storage pool defect Ca Diagnostic Evaluation of latelet Secretion Secretion Defects aradox or knowledge translation gap most common form of platelet disorder, yet secretion testing isn t commonly done otential implications of OT evaluating secretion? Diagnostic label issue Reduced detection of some platelet disorders? Methods to evaluate secretion Radioactive: e.g. serotonin release onradioactive: : e.g. luminescence, other assays for nucleotides nm AT Release Hamilton latelet Secretion Testing Second Line Investigation - Luminescence rocedure ~56% of patients are abnormal - half of these have normal aggregation studies bars - lower limit of reference range (determined using 48 controls) local testing done with 8 parameters, 6 agonists Thrombin AD Testing latelet Function in Thrombocytopenic atients 17% of patients tested in Hamilton Data from 22 21% 8% 4% % 2%1% latelet function defect IT asymptomatic liver disease known/probable MDS VWD other 61% Reference Interval for samples with platelet count of 25 X 1 9 /L % aggregation Bernard Soulier Syndrome R: 29 X 1 9 /L (less than 5% G IbIXV by flow) Control tested at same platelet count (R: 29 X 1 9 /L) Special Diagnostic Evaluations Illustration of Glycoprotein Analysis for Glanzmann Thrombasthenia AD 5 um ug/ml ug/ml Epinephrine 6 um Arachidonic Acid 1.6 mm Thromboxane analog. 1 um Ristocetin.5 mg/ml -7 Ristocetin 1.25 mg/ml

7 Testing for Rare Disorders - Quebec latelet Disorder clues: family history, delayed bleeding responsive only to fibrinolytic inhibitors, absent epinephrine aggregation, reduced to low normal platelet counts latelet u-a u Western Blot known controls Quebec family members u-a Q C J Thromb Haemost 26;4: Thromboelastography platelets contribute to clot strength blood samples recalcified, added TF & low concentration of t-a lysis affected? Control 12 QD 12 Clots prepared with or 12 X 1 9 platelets/l J Thromb Haemost 26;4: Mystery Case VWD screen 55 year old male, severe bleeding after renal biopsy First sample (referred in) FVIIIC 2.4 U/mL (24 U/dL dl) VWF:Ag 1.1 U/mL (11 U/dL dl) VWF:RCo.29 U/mL (29 U/dL dl) Interpretative comment: The von Willebrand factor ristocetin cofactor activity is significantly reduced. The discrepancy between een this value and the normal VWF antigen suggest a form of type 2 von Willebrand disease. An analyses of von Willebrand factor multimers would be helpful to further evaluate. Is there a family history of von Willebrand disease or a bleeding history that suggests acquired von Willebrand disease? Repeat testing, including ristocetin-induced induced platelet aggregation would be helpful to confirm and further evaluate the von Willebrand factor abnormalities. AD 5 um 5 ug/ml 1.25 ug/ml Epinephrine 6 um Arachidonic Acid 1.6 mm Thromboxane analog. 1 um Ristocetin.5 mg/ml Ristocetin 1.25 mg/ml Further Investigations RI for samples with 25 X 1 9 platelets/l % aggregation atient Control tested same day at same platelet count atient CBC: lt 64 X 19/L, MV 8.5 fl Within RI for sample platelet count Additional Investigations VWD screen done on day of aggregation testing FVIIIC 1.4 U/mL VWF:Ag 2.9 U/mL VWF:RCo neat -.26; 1/2 -.6; 1/8.85 U/mL Multimers ormal Further RIA testing (1.25 mg/ml ml) ) done after a minute incubation of patient or control, with control R ( added to adjust platelets from 44 down to 25 X 1 9 /L) atient Mixture: 2% aggregation Control Mixture: 86% aggregation Acknowledgments: Colleagues and Collaborators Clinical and Research Lab Staff ASCOLA latelet Study K. Moffat, M. Ledford-Kraemer, W. L. ichols ISTH latelet hysiology SSC FA-1 Working Group Diagnosis? Further tests that you would do? 7