Coagulation assays part 1

Coagulation assays part 1

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  1 Routine Assays in theCoagulation Laboratory Larry Smith, PhD Director, Coagulation/Hemostasis Laboratories Assistant Attending
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  2 Objectives • Review of the traditional coagulation cascade • Discuss routine assays frequently ordered • Describe how these assay work • Discuss interpretation and clinical utility
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  3 aPTT PT Fibrinogen D-Dimer
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  4 Prothrombin Time (PT) • Armand Quick 1935 • Measures the time required for fibrin clot to form when plasma is added to [TF + Ca2+] mixture Ca2+ • TF combines with FVII to form the “extrinsic” + tenase complex Thromboplastin • Measures (TF) a. FVII of the extrinsic pathway b. FX, FV, FII, FI of the common pathway Patient Plasma c. Measures 3 of the vitamin K-dependent factors • II, VII, X – does not measure IX • PT is prolonged in – Deficiencies of I, II, V, VII, X – Liver Disease – OAT – Increased sensitivity to reduction in VKDF’s – Increased FDP’s, antibiotics – High dose heparin therapy and DTI’s • PT is shortened following treatment with rVIIa • Most common use of PT  monitoring OAT
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  5 MonitoringOAT • Problems with the PT • INR developed by WHO using an IRP to ▫ Commercially available thromboplastins which all other thromboplastins can be vary in their sensitivity to VKDF’s compared ▫ PT values that result from using different ▫ Recommended that a PT value be expressed thromboplastins are not interchangeable as a ratio by normalizing it to the IRP (ISI) Reagent PT (sec) ISI INR INR = Patient’s PT GM Normal PT A 11 3.2 2.6 ▫ ISI = measure of the sensitivity and responsiveness of a particular B 20 2.4 2.6 thromboplastin reagent to warfarin- induced reduction of the VKDF’s C 12 3.0 2.6 ▫ ISI of the IRP = 1.0 Advantages: INR for monitoring patients on OAT D 18 2.2 2.6 1. Minimizes the variation in the PT assay 2. Allows comparability of PT results among E 31 different laboratories 0.9 2.6
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  6 Limitations of Warfarin Therapy • PT/INR Limitations Consequence Slow onset of action Need to overlap with a parenteral Narrow therapeutic index anticoagulant Early procoagulant effect Early prothrombotic effect Multiple drug/food Frequent monitoring required interactions Genetic variation in Variable dose requirement metabolism ADRs ~177,000/yr, ~30,000 ER visits/yr, ~3% major bleeds, ~0.6% fatal bleeds Narrow therapeutic index Bleeding or thrombosis • INR should ONLY be used for patients who stabilized on OAT • INR should NEVER be used as a substitute for the PT in patients who are NOT on OAT 1. Exponential nature of the INR may obscure interpretation of a mildly prolonged PT WHY??? that may be suggestive of a coagulopathy 2. Sufficient studies have NOT been done to demonstrate how well the INR correlates with diagnosis or outcome
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  7 aPTT • PTT – Langdell, Wagner and Brinkhous 1953 • aPTT – Proctor and Rappaport – 1961 Ca2+ • Measures a. Time required for fibrin clot to form when a plasma is incubated with an PL [activator + partial thromboplastin + Ca2+ ] + a. Activation of the contact factors activator PK, HMWK, FXII, FXI a. Intrinsic pathway factors Plasma FXII, XI, IX, VIII a. Common pathway FX, V, II, I • Prolonged in 1. Deficiencies of all factors except VII and XIII 2. Presence of inhibitors Specific inhibitors – FVIII and FIX Nonspecific inhibitors—LA, Heparin, DTI’s • Shortened ▫ Elevated FVIII • Monitor Heparin Therapy
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  8 LaboratoryMonitoring of UFH • aPTT ▫ 4-6 hours after bolus dosage and every 24 hours thereafter  A dose adjustment requires monitoring 6 hours after the dose adjustment ▫ Target range 1.5-2.5 x “normal”  ACCP, CAP  Correlated to 0.3-0.7 anti-Xa U/mL using a chromogenic anti-Xa Heparin Assay  Monitor platelet counts • Anti-factor Xa assay (UFH) ▫ 4 hours after administration ▫ Therapeutic target—0.3 - 0.7 anti-Xa U/mL Current therapeutic range ▫ Monitor platelet count daily 55 – 80 seconds
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  9 Therapeutic aPTT versusAnti-Xa Ranges Therapeutic aPTT Ranges Therapeutic Ranges with anti-Xa Drug Range Drug Ratio UFH 0.3 – 0.7 U/mL 1.5 – 2.5 UFH 2x/day 55.0 – 80.0 sec 0.5 – 1.1 U/mL LMWH 1x/day N/A 1.1 – 2.0 u/mL LWMH Not sensitive to Pentasaccharide LMWH Prophylaxis of VTE Trough – 0.14 – 0.19 Argatroban 1.5 – 3.0 Fondaparinux Peak – 0.39 – 0.50 Treatment of VTE Trough – 0.46 – 0.62 Lepirudin Peak – 1.20 – 1.26 1.5 - 2.5 (Refludan)
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  Reagent Factor Sensitivity • aPTT is sensitive to deficiencies of contact and intrinsic factors • MILD deficiency may result in a normal aPTT
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  11 Fibrinogen • Clauss Technique ▫ Functional assay ▫ Fibrinogen concentration is inversely High conctr proportional to the thrombin time of thrombin diluted plasma ▫ A reference (standard) curve is prepared 1:10 dilution using known fibrinogen concentrations plasma versus their respective thrombin times • Detects ▫ Quantitative  Hypofibrinogenemia  Hyperfibrinogenemia ▫ Qualitative deficiency  Dysfibrinogenemia • Acute phase protein  elevated in ▫ Inflammation ▫ Trauma ▫ Infection ▫ Increases with age ▫ Associated with CVD and thrombosis
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  12 Fibrinogen • Low levelssuggest bleeding ▫ DIC ▫ Thrombolytic therapy  Results in increased levels of FDP’s (>190 ug/mL)  Interfere with fibrin monomer polymerization ▫ Liver disease 1. Decreased synthesis of fibrinogen 2. Abnormal fibrinogen may be seen due to abnormal/increased sialic acid content 3. May result in elevated levels of fibrinolytic activators and decreased levels of fibrinolytic inhibitors ▫ Some patients following treatment with l-asparaginase ▫ Heparin (UFH) may lead to underestimation • Increased levels ▫ Increasing age ▫ Pregnancy ▫ OCT ▫ Disseminated malignancy DTI’s falsely decrease the fibrinogen level — DON’T ORDER!!!
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  13 Thrombin Time /Reptilase Time Thrombin Time • Measure the conversion of fibrinogen to fibrin • Cleaves fibrinopeptides A and B • Screen for heparin contamination • Prolonged ▫ Heparin therapy (UFH) ▫ Hypofibrinogenemia, Dysfibrinogenemia ▫ Paraproteins (Amyloidosis, Myeloma) ▫ Severe LD ▫ Elevated FDP’s ▫ DTI’s ▫ Bovine thrombin glues Reptilase time • Cleaves fibrinopeptide A only • Unaffected by Heparin • Unaffected by bovine thrombin glues • Same as above
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  14 D-Dimer  Specific degradationproduct of fibrin clots that results from the action of 1. Thrombin  Converted fibrinogen into fibrin clots 1. FXIIIa  Cross-linked fibrin monomers clots 1. Plasmin  Cleaved the cross-linked fibrin clot D e D D D
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  15 D-Dimer • Monoclonal antibodyraised against specific epitopes on D- dimer that react with cross-linked fibrin ▫ Does not react with  Fibrinogen degradation products  Non-cross-linked fibrin degradation products ▫ Ensures high specificity for D- dimer as a biomarker of fibrin formation and stabilization
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  16 Clinical Utility • Diagnosisof VTE in combination with pretest clinical probability ▫ High negative predictive value for exclusion of DVT ▫ Poor positive predictive value for DVT  Elevated in conditions unrelated to thrombosis  Almost all patients with acute disease will have elevated D-dimer levels • Clinical use ▫ Identification of individuals at increased risk thrombotic events (arterial and venous) ▫ Identification of individuals at increased risk of recurrent VTE  Elevated levels following discontinuation of anticoagulant therapy ▫ Establishing of optimal duration of secondary prophylaxis after a first episode of VTE ▫ Pregnancy monitoring ▫ Diagnosis/monitoring of DIC  Sensitive but NOT specific marker for DIC 1. A positive D-dimer is NOT specific for VTE 2. Negative D-dimer is highly unlikely for VTE 3. The greatest utility of D-dimer is its negative predictive value for VTE!
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  17 Fibrin(ogen) Degradation Products • Patient plasma mixed with latex particles coated with monoclonal anti-FDP antibodies • Positive FDP assay indicates ▫ Fibrin and/or fibrinogen is being degraded by plasmin • Elevated FDPs ▫ Dysfibrinogenemia ▫ LD, DIC, DVT, PE, MI ▫ Thrombolytic therapy ▫ Primary and secondary fibrinogenolysis • What would the D-dimer and FSP levels be in a person who has a congenital FXIII deficiency? • What would the D-dimer and FSP levels be in individuals with FXIII deficiency due to DIC?
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  18 Algorithm of MixingTests Prolongation of the Coagulation Time of the Screening Test Mixing: Patient Plasma + Normal Plasma Correction No Correction Factor Deficiency Lupus Anticoagulant Specific Factor Inhibitor
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  19 Mixing Studies • Lookfor the presence of: ▫ Factor Deficiency versus Inhibitor Step 1 Mix and Run TUBE 1 TUBE 2 TUBE 3 300 uL PNP 1 mL PNP 300 uL patient 1 mL Run immediately plasma patient plasma Incubate @ 37oC Step 2 Mix and Run TUBE 1 TUBE 2 TUBE 3 300 uL PNP Run after 1 hour incubation PNP 300 uL Patient patient plasma plasma
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  20 Interpreting Mixing Studies If the 50/50 corrects after the immediate and remains corrected after the 60 minute incubation  Factor Deficiency  Follow-up with specific factor assays  If the 50/50 corrects after the immediate, but prolongs after the 60 minute incubation  Time-dependent inhibitor – usually a specific factor inhibitor  ~ 15% of LA’s may be time-and-temperature dependent  Follow-up with a specific factor assay and specific factor inhibitor assay  If the 50/50 prolongs after the immediate and remains prolonged after the 60 minute incubation  Nonspecific inhibitor such as a lupus anticoagulant  Follow-up with Lupus anticoagulant assay  Mixing studies on samples minimally prolonged (<3 sec) may produce confusing results  Addition of normal plasma sometimes dilutes a weak inhibitor  lead to a “false” correction
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  21 Mixing Study Panel •Look for the presence of: ▫ Factor Deficiency versus Inhibitor • Thrombin Time ▫ Rule-out the presence of aPTT Mixing Study Panel heparin 1.aPTT Patient • PTT-FS 2.50/50 mix immediate ▫ Lupus-insensitive reagent for 3.Normal Plasma the aPTT 4.aPTT Patient  Not prolonged in the 5.50/50 mix 60 minutes presence of LA 6.Normal Plasma  Sensitive to FD 7.Thrombin Time 8.PTT-FS*
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  22 aPTT Mixing Studies Patient 65.3 Patient 51.0 NP 31.4 NP 31.4 50/50 49.2 50/50 31.1 Patient (inc) 65.9 Patient (inc) 52.7 NP (inc) 31.4 NP (inc) 31.4 50/50 (inc) 48.1 50/50 (inc) 32.2 PTT-FS 29.3 PTT-FS 47.4 Thrombin T 18.6 Thrombin T 19.1 aPTT 24.4 – 33.2 sec PTT-FS <30.0 sec TT 16.8 - 24.4 sec
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  23 Lupus Anticoagulant/APAs • Paradox ▫ LA is a riddle wrapped in a mystery inside an enigma  Prolonged clotting time in vitro  Thrombosis in vivo • Lupus Anticoagulant ▫ Auto-antibodies directed against phospholipid-binding proteins ▫ Targets  β2GPI—thrombosis  Prothrombin—bleeding  PC, PS, Annexin V— thrombosis
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  24 ISTH Criteria forLupus Anticoagulant Testing  The ISTH has defined the minimum diagnostic criteria for lupus anticoagulants to include 1. A prolonged clotting time in a screening assay such as the aPTT 2. Mixing studies indicating the presence of an inhibitor 3. Confirmatory studies demonstrating phospholipid dependence of the inhibitor a. Screen – decreased amount of phospholipids  prolonged clotting time b. Confirm—increased amount of phospholipids  shortened clotting time 4. No evidence of other inhibitor-based coagulopathies  Specific factor assays if the confirmatory step is negative or there is evidence of a specific factor inhibitor
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  25 ISTH Criteria forLupus Anticoagulant Testing • Updated ISTH guidelines (2009) ISTH ▫ Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7: 1737–40 ▫ Choice of tests 1. Two tests based on different principles 2. dRVVT should be the first test considered 3. Seconds test should be a sensitive aPTT (low phospholipids and silica as activator) 4. LA should be considered as positive if one of the two tests gives a positive result
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  26 Detectionof LA • Assays  dRVVT* Clot-based assays SCT dPT  SCT* Why do we see so few LA’s on the extrinsic side???  HEX  Kaolin CT  dPT DRVVT
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  27 dRVVT Screen (Normalplasma) X dRVVT Xa Prothrombin Xa Phospholipid Va Ca2+ (PF3) Thrombin Fibrinogen Fibrin
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  28 dRVVT Screen (LupusAnticoagulant) X dRVVT Xa Prothrombin Xa Va Ca2+ Low Phospholipid Thrombin Content Fibrinogen Fibrin
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  29 dRVVT Confirm (LA) X dRVVT Xa Prothrombin Xa Va Ca2+ High Phospholipid Thrombin Content Fibrinogen Fibrin
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  30 Assaysto Detect APA’s • All LA’s are antiphospholipid antibodies, BUT not all APA’s are LA’s • Antiphospholipid antibodies ELISA-based assays  Anticardiolipin antibodies (IgG, IgM, IgA) β2-Glycoprotein I antibodies • Systematic reviews have consistently reported ▫ LA is a stronger risk factor than aCL aβ2GPI for both arterial and venous thrombosis and obstetric complications ▫ aCL and aβ2GPI only show some significant association with thrombosis and obstetric complications at high titer ▫ Independent of LA and aCL neither aβ2GPI nor antiprothrombin antibodies are associated with arterial or venous thrombosis
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  31 Patientswith LA on Warfarin • LA can influence PT/INR  can lead to INRs that do not accurately reflect the true level of anticoagulation a. Use of the INR (to standardize PTs) may be invalid for some patients with LA b. To prevent supratherapeutic or subtherapeutic anticoagulation  these patients must be individually monitored with a test that is insensitive to LA Ann Intern Med. 1997;127:177-185 • Chromogenic Factor X • Therapeutic Range = 23-47% • [INR 4.0 (23%) – INR 2.0 (47%)]
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  32 The End