Coagulation assays part 1


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  • Probably the most commonly ordered test in the coagulation laboratory Most sensitivity to decrease in FVII Moderate sensitivity to FV and FX Insensitive to “intrinsic” factors Liver disease versus VKD  FV and FVII – FV is NOT VKDF, so if decreased then probably due to LD Sensitive to fibrinogen levels less than 100 mg/dL VDK – seen in malnutrition, broad-spectrum antibiotics (destroy gut flora), newborns (have decreased levels of VKDFs) Hemorrhage is likely in VKD – PT is probably the most sensitive indicator When FVIII and FIX are decreased – PT is not affected due to increased concentration of TF in the reagent – these factors are bypassed during fibrin polymerization Heparin may prolong the PT – most reagents contain polybrene to neutralize the effect of heparin Some thromboplastins are prolonged by LA  may be partially neutralized by the thromboplastin reagent How useful is the PT for screening asymptomatic surgical patient to predict intraoperative bleeding?  Current data doesn’t support UNLESS the patient is a member of a
  • During the 1 st week of warfarin therapy physicians should rely on the PT (seconds) rather than the INR. Warfarin works by slowing down the process in the liver that uses vitamin K to make certain proteins { clotting factors } that cause clotting. Warfarin interferes with the action of vitamin K. Vitamin K is essential for the production of prothrombin and other proteins necessary for blood to clot. If there is less vitamin K action, there will be less prothrombin action, and blood clotting will be inhibited.
  • 2 nd most commonly ordered test in the coagulation laboratory Partial thromboplastin LACKS TF DIC prolongs the PTT due to consumption of the clotting factors VKD leads to decreased levels of 2,7,9, and 10 eventually prolonging the PTT. Therefore the aPTT is NOT as sensitive to VKD or warfarin therapy as the PT. Sensitive to deficiencies of contact and intrinsic factors – however, a MILD deficiency may result in a normal aPTT – so we do factor sensitivity studies for all of the factors Clinical applications : Screen for coagulation factor deficiencies Monitor UFH therapy May detect both lupus anticoagulant and circulating anticoagulants
  • Anti-factor Xa measures the concentration of heparin rather than its anticoagulant effect . The heparin concentration is correlated to 1.5 – 2.5 “X” normal.
  • PTT is sensitive to deficiencies of contact and intrinsic factors – however, a MILD deficiency may result in a normal aPTT – so we do factor sensitivity studies for all of the factors
  • Procedure Uses dilutions of a reference plasma over a range of fibrinogen concentrations  these dilutions are correlated to individual clotting times Determine the clotting time of a 1:10 dilution of patient plasma (considered 100%) by adding a high concentration of thrombin (100U/mL). The 1:10 dilution minimizes the effect of heparin and elevated FDP’s. Bovine or human thrombin is added to dilute plasma. Catalyzes the conversion of fibrinogen to fibrin. Time to clot is compared to a reference curve. Diluted plasma is clotted with a high concentration of thrombin—note: a high concentration of thrombin (typically 100 U/mL) ensures that the clotting times are independent of the thrombin concentration over a wide range of fibrinogen levels . A calibration curve is constructed using reference plasma over a series of dilutions (1:5-1:40). Clotting time of each dilution is established and the results are converted to mg/dL of fibrinogen. The patient plasma clotting time is read from the standard curve. Diluting the PPP minimizes the antithrombotic effects of heparin, FDPs and paraproteins. Heparin levels <0.6 U/mL and FDP’s <100 mcg/mL do NOT affect the fibrinogen assay when the fibrinogen levels if >150 mg/dL. Hypofibrinogenemia is associated with severe LD and DIC. Hyperfibrinogenemia may be produced in moderately severe LD, pregnancy, chronic inflammation. Clauss—based on the time for a fibrin clot to form PT-derived—fibrinogen derived upon the PT that is derived by optical density change for a range of plasma dilutions with known fibrinogen levels. Immunological—measures fibrinogen antigen concentration rather than functional fibrinogen. Gravimetric—method based upon the weight of the clot—clot is compressed to extrude plasma and unused reagents, washed, dried and then weighed. Fetal fibrinogen and liver disease have abnormal sialic acid content.
  • 1. Patients exposed to bovine thrombin develop antibodies that cross-react with human thrombin which prolongs the thrombin time but does NOT affect the reptilase time. Fetal fibrinogen (in neonates) consists of increased sialic acid content leading to temporary dysfibrinogenemia. Hyperfibrinogenemia may interfere with fibrin assembly due to the excess fibrinogen. Hypofibrinogenemia may interfere with fibrin polymerization but may be more an in-vitro phenomenon.
  • What would the D-dimer and FSP levela be in a person who is FXIII deficient? D-dimer and FSP levels be in individuals with FXIII deficiency due to DIC?
  • What would the D-dimer and FSP levela be in a person who is FXIII deficient? D-dimer and FSP levels be in individuals with FXIII deficiency due to DIC?
  • Patient plasma is mixed with latex particles which are coated with monoclonal anti-FDP antibodies. 3 If FDP are present in the patient plasma, the latex particles agglutinate as FDP bind to the antibodies on the particles. These large agglutinated clumps are detected visually by the technologist. Various dilutions of patient plasma can be tested to provide an estimation of the FDP titer (semiquantitative result). D-dimer is a specific FDP that is formed only by plasmin degradation of fibrin, and not by plasmin degradation of intact fibrinogen. Thus, the presence D-dimer is a specific FDP that is formed only by plasmin degradation of fibrin, and not by plasmin degradation of intact fibrinogen. In contrast, a positive FDP assay indicates that fibrin and/or fibrinogen is being degraded by plasmin, because the FDP assay detects fibrin degradation products, including D-dimers, and fibrinogen degradation products.
  • Coagulation assays part 1

    1. 1. 1Routine Assaysin the Coagulation LaboratoryLarry Smith, PhDDirector, Coagulation/Hemostasis LaboratoriesAssistant Attending
    2. 2. 2Objectives• Review of the traditional coagulation cascade• Discuss routine assays frequently ordered• Describe how these assay work• Discuss interpretation and clinical utility
    3. 3. 3aPTT PT Fibrinogen D-Dimer
    4. 4. 4Prothrombin 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
    5. 5. 5 Monitoring OAT• 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
    6. 6. 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 PTWHY??? 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
    7. 7. 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
    8. 8. 8 Laboratory Monitoring 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
    9. 9. 9Therapeutic aPTT versus Anti-Xa RangesTherapeutic 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 toPentasaccharide 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)
    10. 10. Reagent Factor Sensitivity • aPTT is sensitive to deficiencies of contact and intrinsic factors • MILD deficiency may result in a normal aPTT
    11. 11. 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
    12. 12. 12Fibrinogen• Low levels suggest 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!!!
    13. 13. 13Thrombin Time / Reptilase TimeThrombin 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 gluesReptilase time• Cleaves fibrinopeptide A only • Unaffected by Heparin • Unaffected by bovine thrombin glues • Same as above
    14. 14. 14D-Dimer Specific degradation product 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
    15. 15. 15D-Dimer• Monoclonal antibody raised 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
    16. 16. 16Clinical Utility• Diagnosis of 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!
    17. 17. 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?
    18. 18. 18Algorithm of Mixing Tests Prolongation of the Coagulation Time of the Screening Test Mixing: Patient Plasma + Normal Plasma Correction No Correction Factor Deficiency Lupus Anticoagulant Specific Factor Inhibitor
    19. 19. 19Mixing Studies• Look for 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
    20. 20. 20Interpreting 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
    21. 21. 21Mixing 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*
    22. 22. 22aPTT 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
    23. 23. 23Lupus 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
    24. 24. 24ISTH Criteria for Lupus 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
    25. 25. 25ISTH Criteria for Lupus 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
    26. 26. 26 Detection of 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
    27. 27. 27dRVVT Screen (Normal plasma) X dRVVT Xa Prothrombin Xa Phospholipid Va Ca2+ (PF3) Thrombin Fibrinogen Fibrin
    28. 28. 28dRVVT Screen (Lupus Anticoagulant) X dRVVT Xa Prothrombin Xa Va Ca2+ Low Phospholipid Thrombin Content Fibrinogen Fibrin
    29. 29. 29dRVVT Confirm (LA) X dRVVT Xa Prothrombin Xa Va Ca2+ High Phospholipid Thrombin Content Fibrinogen Fibrin
    30. 30. 30 Assays to 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
    31. 31. 31 Patients with 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%)]
    32. 32. 32The End