LABORATORY APPROCH TO THE BLEEDING PATIENT

Transcription

1 Anne Winkler, MD Emory University School of Medicine, Atlanta Georgia Assistant Professor, Pathology & Laboratory Medicine Medical Director, Transfusion Service, Grady Health System Assistant Director, Special Coagulation Laboratory, Emory University Hospital LABORATORY APPROCH TO THE BLEEDING PATIENT Annie Winkler MD, MSc Assistant Professor, Emory University Department of Pathology and Laboratory Medicine Medical Director, Grady Health System Transfusion Service Assistant Medical Director, Emory Special Coagulation Laboratory 6 th Annual Case-Oriented Symposium on Bleeding and Thrombosis October 5, 2013 Disclosures Conflicts of Interest: None Disclaimers: None 1

2 Introduction No matter how comprehensive and careful the clinical assessment is in patients with bleeding manifestations, the findings are often nonspecific Many disorders are asymptomatic until the individual is surgically or traumatically challenged While information from the history and physical exam may increase the clinical suspicion for a particular hemorrhagic disorder, laboratory confirmation is required to define the specific defect Basic Coagulation Screening Tests for Bleeding Patients Complete Blood Count with Platelet Count Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT) Fibrinogen Level Platelet Function Analyzer (PFA-100) History of Hemostasis Testing TT developed Born and by Jim and O Brien Goldfein to introduce Hartert describes assess modern light thromboelastography fibrinogen transmittance as a method to assess activity in platelet global hemostasis plasma aggregometry PT first described by Armand Quick PTT was first devised by Langdell, Wagner, and Brinkhous MacFarlane and Biggs record thrombin generation during plasma clotting Proctor and Rappaport develop the APTT modification INR introduced as a means of standardizing PT and improving management of patients receiving warfarin based oral anticoagulant therapy 2

3 Evolution of Hemostasis Testing PT PTT APTT Platelet Function Testing, Factors TEG /ROTEM Thrombin Generation Laboratory Approaches to Common Bleeding Disorders NASCOLA survey of coagulation diagnostic laboratory practices for the investigation of bleeding disorders 31 of 81 (38%) response rate Hayward CP. Am J Hematol2012: 87, S45-S50 Classic Extrinsic and Intrinsic Pathways Kottke-MarchantK. An Algorithmic Approach to Hemostasis Testing,

4 Cell Based Model of Coagulation Kottke-MarchantK. An Algorithmic Approach to Hemostasis Testing, 2008 Specific Coagulation Tests for Bleeding Patients Suspected Coagulation Factor Abnormalities Mixing Studies Clotting Factor Activity Assays Bethesda assay Thrombin time Suspected Platelet Disorders Platelet aggregation studies PT or APTT Mixing Studies In practice, 30-40% factor activity is sufficient to yield a normal 1:1 mixing study result A 4:1 APTT mixing study may help to detect mild inhibitors Only if an incubated mixing study is performed, will a time and/or temperature dependent inhibitor be detected Kottke-MarchantK. An Algorithmic Approach to Hemostasis Testing,

5 Mixing Study Interpretation Different targets are used to determine correction and there is no general consensus on how to interpret the results of mixing studies Commonly used targets include: Correction to within the reference range Correction to within 5 seconds of the control or 10% of the control value Lack of correction greater than 15% of the control plasma Algorithmic Approach to Bleeding Disorders Prolonged APTT Hepzyme Kottke-MarchantK. An Algorithmic Approach to Hemostasis Testing,

6 Prolonged APTT EQA UK NEQAS exercise to investigate a sample with a prolonged APTT Sample sent was a donor with severe hemophilia A Results were obtained from 110 centers 90% reported Hemophilia A Number of tests performed averaged 8 (3-13) Mixing Results 62% reported correction 15% partial or no correction Jennings I, IntJnlLab Hem 2013: 35, Prolonged PT Kottke-MarchantK. An Algorithmic Approach to Hemostasis Testing, 2008 Role of Basic Coagulation Tests to Predict Bleeding Risk CheeYL. HematolJ 2003: 4,

7 Role of Basic Coagulation Tests to Predict Bleeding Risk CheeYL. HematolJ 2003: 4, Role of Basic Coagulation Tests to Predict Bleeding Risk 5 day audit of coagulation samples in a 522 bed hospital Additional testing was performed for abnormal results PT: 1:1 mix APTT: 1:1 mix, factor XII, PTT-LA and DRVVT McHugh J. Blood CoagulFibrinolysis 2011: 22, University of Washington Performance Improvement Project 28,737 coagulation screens (PT, APTT, fibrinogen, TT) were performed in a 6 month period Of those results, 39% normal with 15% bleeding symptoms 12% prolonged PT with 25% bleeding symptoms 13% prolonged APTT with 10% bleeding symptoms 21% prolonged PT and APTT with 5% bleeding symptoms 7

8 University of Washington Performance Improvement Project Removal of Coagulation Panel Physician Education AmukeleTK. Blood CoagFibrinolysis 2011: 22, Normal APTT and PT Kottke-MarchantK. An Algorithmic Approach to Hemostasis Testing, 2008 Overview of VWD Testing Specific testing for VWD generally involves assessment of FVIII Activity VWF protein (VWF:Ag) VWF function (VWF:RCo) Other specialty testing includes VWF multimeranalysis Ristocetininduced platelet aggregation (RIPA) VWF collagen binding or other functional assays FVIII or platelet binding assays Genetic evaluation 8

9 VWF Antigen Assays Detects all forms of VWF equally whether it be functional or dysfunctional/ defective and irrespective of molecular weight The most popular methodology is currently LIA based, but ELISA methods are also used ECAT Proficiency Testing Ristocetin Cofactor Activity Assays First utilized in the 1970s and historically described the first VWF activity assay, detecting VWF adhesive activity associated to GPIb binding Dependent upon the multimericstructure of VWF with the largest multimersshowing the greatest adhesive capacity VWF:RCo as an agglutination assay originally used platelet aggregometers or visual slide agglutination However, both methods are laborious and typically not used in most laboratories As a result, most laboratories have automated the platelet agglutination assay although other methods using flow cytometry and ELISA have been published Limitations and Alternatives to VWF:RCo Main limitations include Assay time Imprecision Poor sensitivity to low levels of VWF Ongoing problems with the VWF:RCo assay have brought attention to newer functional VWF assay as potential replacements for VWF:RCo Collagen binding assay (VWF:CB) Various VWF activity assays ECAT Proficiency Testing

10 Ristocetin Binding Site Polymorphisms DNA sequencing revealed a trio of SNPS that appeared frequently in the AA controls I1380V N1435S D1472H 3 SNP haplotypewas present in 22% of AA compared to 3% of Caucasians An additional 39% of AA had the D1472H SNP alone Total 63% Flood VH. Blood2010:116, Ristocetin Binding Site Polymorphisms The D1472H polymorphism affects in vitro ristocetin binding without affecting other VWF functions Collagen binding assays are unaffected since it is a separate binding site Previously published case of a P1467S sequence variation causing similar findings Flood VH. Blood2010:116, Clinical Significance of D1472H 30 Flood VH. Blood2013:121,

11 Other Functional VWF Assays Several VWF:Actassays have been developed over the past few years LIA based method which uses a specific monoclonal antibody directed against the GPIbbinding site Not truly a functional assay, but more like a VWF capture assay Did not perform to the same level as VWF:RCo or VWF:CB Cross laboratory evaluation of samples with loss of HMW multimers(favaloro EJ J Thromb Haemost 2012) Chen D. J ThrombHaemost2011: 9, VWD Testing Summary NHLBI The Diagnosis, Evaluation, and Management of von WillebrandDisease, 2007 PFA-100 PFA-100 was introduced in 1994 based on the prototype developed by Kratzer and Born in 1985 Test simulates primary hemostasis through the interaction of platelets with the aperture of a membrane coated with collagen and ADP or epinephrine FranchiniM. Hematology2005: 10,

12 PFA-100 Performance Published meta-analysis of 6 studies to determine the diagnostic performance of the PFA in detecting a disorder of primary hemostasis 75% of patients with a positive PFA-EPI/PFA-ADP have a disorder of primary hemostasis, whereas 79% of patient with negative results do not KargerR. Platelets2007: 19, LTA Performance Hayward CP. J ThrombHaemost2009: 7, ATP Release Performance PaiMAm J ClinPathol2011:136,

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