1. 1 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Part VII Laboratory Testing in Coagulation

2. 2 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE OVERVIEW O used to investigate a hemostatic disorder (1) screening tests - for defects of primary hemostasis: platelet count, bleeding time - for defects of secondary hemostasis: prothrombin time (PT), activated partial thromboplastin time (APTT) (2) more specialized tests O coagulation test results: depends on the blood specimen's appropriateness and integrity O grouping - primary hemostasis - secondary hemostasis - fibrinolysis - hypercoagulable states - anticoagulant therapy monitoring  Laboratory testing in coagulation

3. 3 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE SPECIMEN COLLECTION AND PROCESSING SPECIMEN COLLECTION O two-syringe or two-tube technique: with the blood form the second syringe or second tube used for the coagulation specimen - minimize contamination of the sample from tissue factor during phlebotomy O drawing blood through catheter: avoid heparin contamination O when to draw: certain fibrinolytic factor -> diurnal variability O 3.2% sodium citrate: anticoagulant for coagulation studies - problem: high hematocrits -> smaller volume of plasma O proper ratio of blood to anticoagulant: 9:1 - underfilling: much calcium can be bound -> falsely prolonged results - overfilling: clotting -> falsely prolonged results as occur in a consumptive coagulopathy  Laboratory testing in coagulation

4. 4 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE SPECIMEN PROCESSING O to obtain plasma: citrated whole blood -> centrifugation Platelet-Poor Plasma (PPP) O centrifuged for 15 min at 2,500 X g. O PPP: <10 X 10 9 /L platelets O use of PPP - platelet contain platelet factor 4 (PF4): in alpha-granule, neutralize heparin -> testing for the presence of heparin - platelet contain phospholipid -> lupus anticoagulant testing, factor assay testing - platelet contain protease -> von Villebrand factor testing O clotted sample: unacceptable  Laboratory testing in coagulation

5. 5 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O PPP: stored at 18-24 or 2-8 for up to 4 hours before testing stored at -20 for up to 1 week stored at -70 for up to 6 months - frozen sample - thawed rapidly at 37 - excessive heating (>5 min): loss of factor V and VIII - no freeze-thaw cycle Platelet-Rich Plasma (PRP) O for platelet function test O centrifuged for 10 min at 200 X g at RT O PRP: 200-300 X 10 9 /L O stored at RT, testing within 3 hours Citrated Whole Blood O for platelet function test O to check for clot: wooden applicator stick should be inserted after testing  Laboratory testing in coagulation

6. 6 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE LABORATORY INVESTIGATION OF PRIMARY HEMOSTASIS O tests for platelet concentration (count) and function - for function test: drugs, stress O screening test: bleeding time (BT), platelet function analyzer (PFA) O definitive testing: PRP and whole blood platelet aggregation O platelet aggregation: depend on the presence of Ca2+ - remaining Ca2+ after anticoagulation -> sufficient for aggregation  Laboratory testing in coagulation

7. 7 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE BLEEDING TIME (BT) O in vivo measurement of platelet function - affected by platelet number and vascular integrity O BT: meature time required for bleeding to cease form a superficial skin cut - affected by temp. amount of pressure, depth/location/direction of the incision, movement of the arm O Duke bleeding time (30 sec) - lancet, the ear lobe O Ivy bleeding time - lancet, forearm - constant venous pressure (40 mmHg) O modified Ivy BT - use template (spring-loaded blade: standard depth and width on the forearm) O reference interval: 1-9 mins - prolonged BT: platelet count < 100 X 10 9 /L O Table 40-1  Laboratory testing in coagulation

8. 8 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

9. 9 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE PLATELET FUNCTION ANALYZER O in whole blood at a high shear rate O procedure - citrated blood -> add to reservoirs with collagen/epinephrine add to reservoirs with collagen/adenosine disphosphate (ADP) - detect the formation of a platelet plug. O closure time: the time to occlude the aperture - a function of platelet count, platelet activity, vWF activity, hematocrit - sensitive to vWD, aspirin-induced platelet dysfunction, aggregation defect -> prolonged or abnormal closure times O expected results (text)  Laboratory testing in coagulation

10. 10 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE PLATELET AGGREGOMETRY O platelet aggregation study: foundation of platelet function test Platelet-Rich Plasma (PRP) Aggregation O citrated blood sample O adjusting platelet count with patient's PPP (usually 200,000/ul) O sample is stirred, warmed to 37 degree O aggregating reagent (agonist) added: ADP, epinephrine, collagen, ristocetin, arachidonic acid O change in optical density: detection using aggregometer O Fig 40-1 - depends on the agonist used and its concentration - primary wave direct response of the platelets to the aggregating reagent platelet shape change, formation of small aggregates - secondary wave complete aggregation response results of ADP being released from the activated platelet dense bodies - biphasic curve vs monophasic curve O Table 40-2  Laboratory testing in coagulation

11. 11 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

12. 12 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

13. 13 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O Ristocetin - depends on the interaction of plasma vWF and the GP Ib/IX) -> agglutination - type IIb vWD - restocetin-induced platelet agglutination (RIPA) abnormal in both vWD and Bernard-Soulier syndrome (BSS: defect in GPIb/IX) - plasma restocetin cofactor activity (vWF:A) decreased in vWD normal in BSS - adding normal plasma to the patient's PRP corrected in vWD (deficiency of plasma protein) not corrected in BSS (deficiency of the GP Ib/IX) O arachidonic acid (AA) - useful to screen for a patient's ingestion of aspirin - when the PRP produces no secondary wave with ADP or epinephrine - to differentiate from platelet release defects or storage pool disease O Fig. 40-2 O decreasing concentration of epinephrine - produce aggregation of patient PRP, not normal control PRP -> sticky platelet syndrome: PF4 and beta-thromboglobulin: normal plasma level  Laboratory testing in coagulation

14. 14 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

15. 15 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Whole blood Aggregation O need a smaller sample size, quicker O aggregation -> increased electrical resistance O agonist, thrombin - determination of the content of the dense granules - thrombin causes dense granules to release ATP - for delta-storage pool disease  Laboratory testing in coagulation

16. 16 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE ADDITIONAL TESTS EVALUATING PLATELET FUNCTION Flow Cytometry O using monoclonal Ab directed against GPIb/IX, GPIIb/IIIa Clot Retraction test( 혈병수축능검사 ) O performed on blood without anticoagulant O procedure - 1ml blood in a glass tube at 37 degrees for 1-3 hrs - retraction occurs 30-60 mins after collection except Glanzmann's thrombasthenia, severe hypofibrinogenemia, thrombocytopenia O PRP retraction - to remove variables of platelet count and fibrinogen level - PRP, APTT reagent, Ca2+ -> wooden stick, mixed, incubated at 37 for 1 hr - stick is gently removed: clotted and retracted platelets and fibrin are attached - quantitated by determining the serum remaining in the tube  Laboratory testing in coagulation

17. 17 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE ACQUIRED STATES OF THROMBOCYTOPENIA Heparin-Induced Thrombocytopenia (HIT) O functional heparin-platelet aggregation assay - normal platelet, patient's serum (HIT immunoglobulin), heparin -> activation of platelet -> aggregation - platelet 14 C serotonin assay - enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against heparin-PF4 complexes detect also antiheparin Ab in patients who do not have HIT -> false positive Neonatal Alloimmune Thrombocytopenia (NAIT or NATP) O NAIT: results from placental transfer of maternal alloantibodies directed against paternally inherited antigens present on the fetal platelets but absent from maternal platelets O confirmed by serologic or genotypic testing including immunophenotyping examined for the presence of antiplatelet antibody  Laboratory testing in coagulation

18. 18 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE LABORATORY INVESTIGATION OF SECONDARY HEMOSTASIS O to evaluate coagulation factors and inhibitors SCREENING TESTS O PT, APTT, thrombin time (TT), quantitative fibrinogen Prothrombin Time (PT) 1. for deficiencies in the extrinsic or common pathway of the coagulation cascade O thromboplastin (TF/phospholipid/calcium mixture): added to a citrated PPP -> time for fibrin formation (clot formation) -> general reference interval: 10-13 seconds  Laboratory testing in coagulation

19. 19 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O prolonged PT - deficiency of factors VII, X, V, II (prothrombin), fibrinogen - presence of an inhibitor O shortened PT - hemophiliac patients receiving treatment rF-VIIa concentrate 2. to monitor oral anticoagulant therapy (coumadin or warfarin:vitamin K antagonist) O reported using the INR (international normalized ratio) - ISI (international sensitivity index): 1.0 3. PIVKA (proteins induced by vitamin K-absence or antagonism) O F-II, X, VII, IX, protein C, protein S: vitamin K dependent O in the absence of vitamin K, dietary deficiency of K, liver dysfunction - immunologically similar, - dysfunctional: lack of gamma-carboxyglutamic acid for Ca2+ and PL binding O also termed non or descarboxylated proteins O prolonged PT  Laboratory testing in coagulation

20. 20 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Activated Partial Thromboplastin Time (APTT) 1. using O for deficiencies in the intrinsic or common pathway of the coagulation cascade O for the detection of circulating inhibitors of blood coagulation -> lupus anticoagulant O to monitor the effectiveness of standard (unfractionated) heparin therapy 2. reagents - activated partial thromboplastin (providing PL) - activator: kaolin, celite, micronized celite, ellagic acid) -> provide negatively charged surface for F-XII activation - Ca2+ - time for fibrin clot formation general reference interval for adults: 28-35 seconds 3. prolonged APTT O deficiency of F-XII, prekallikrein (PK), high molecular weight kininogen (HMWK) - absence of clinically significant bleeding  Laboratory testing in coagulation

21. 21 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Thrombin Time (TT) O measures the conversion of fibrinogen to fibrin by adding excess thrombin to undiluted plasma O three major interference with the conversion of fibrinogen to fibrin - the presence of hypofibrinogenemia or dysfibrinogenemia - the presence of heparin (extremely prolonged TT) - the presence of fibrin degradation products (FDP) O autoantibodies against thrombin myeloma proteins O general reference interval: 10-16 seconds - preterm and term infant: longer than adults  Laboratory testing in coagulation

22. 22 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Quantitative Fibrinogen O to determine the fibrinogen concentration O Clauss assay (reference method) - clot-based functional measurement - add thrombin to various dilutions of known concentrations of fibrinogen - thrombin-clotting time - Fig. 40-3 - using plasma att a 1:10 dilution - fibrinogen concentration is inversely proportional to the clotting time - FDP: do not affect the assay at the normal 1:10 dilution - reference interval: 200-400mg/dL  Laboratory testing in coagulation

23. 23 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

24. 24 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O interpretation (1) decreased level - disseminated intravascular coagulation (DIC) - primary and secondary fibrinolysis - liver disease - dysfibrinogenemia, hereditary afibrinogenemia (2) increased level - inflammatory disorders - pregnancy - oral contraceptives - fibrinogen: acute phase reactant protein  Laboratory testing in coagulation

25. 25 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE TESTS TO IDENTIFY SPECIFIC FACTOR DEFICIENCY O when the PT and/or APTT is prolonged Mixing Studies O known as circulating anticoagulant screen or screening test for circulating inhibitor O to differentiate a factor deficiency form the presence of a circulating inhibitor O repeat PT/APTT using several different dilutions of the patient's PPP mixed with a normal pooled plasma - factor level of about 50% of normal: sufficient to produce normal PT and APTT O clotting time is considered prolonged if it is longer than the normal plasma clotting time - testing: performed immediately or after incubation - loss of labile factors (F-V and F-VIII) O interpretation - if result is corrected: deficiency of one or more procoagulant factors - lack of correction: the presence of a circulating or a specific factor inhibitor slow acting inhibitor: certain F-VIII inhibitors - Table 40-3  Laboratory testing in coagulation

26. 26 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

27. 27 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Specific Coagulation Factors O factor assay - to confirm factor deficiency - to determine the actual activity of that factor within the plasma - based on the ability of the patient's plasma to correct a prolonged PT or APTT of a known factor-deficiency plasma (substrate) - measure clotting time of mixture of diluted test plasma and a substrate - construct standard curve using reference pooled plasma - Table 40-4, Fig. 40-4 - patient clotting time: usually using 1:10 and 1:20 diluent -> should be linear, showing that no inhibitory effect is seen - normal factor activity reference range: 50-150% O F-IX, XI, XII -> quite low at birth O for F-VIII: chromogenic kit  Laboratory testing in coagulation

28. 28 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

29. 29 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

30. 30 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Reptilase Time O reptilase - serine protease, thrombin-like protease - cleave fibrinopeptide A from fibrinogen * thrombin: cleave both fibrinopeptide A and B O reptilase time - not affected by the presence of heparin in the sample - to detect heparin contamination of the sample - to help differentiate dysfibrinogenemia from the presence of FDP - Table 40-5 - general referrence interval time: 18-22 seconds - prolonged: dysfibrinogenemia, hypofibrinogenemia, afibrinogenemia  Laboratory testing in coagulation

31. 31 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

32. 32 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Prekallikrein (PK) Screening Test O PK deficiency (Fletcher factor deficiency): prolonged APTT O after 10 minute incubation before adding calcium chloride - correction of the prolonged APTT -> PK deficiency - longer incubation -> increases contact activation of F-XII in the absence of PK - activator of choice: kaolin, celite, silica O confirm by specific factor assay or quantitative chromogenic substrate  Laboratory testing in coagulation

33. 33 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Factor XIII (Screening Test) O F-XIII: necessary for the formation of a stable fibrin clot that occurs by forming covalent bonds between fibrin monomer O PT, APTT: do not detect X-XIII deficiency O delayed bleeding or a bruising disorder, screening test: normal O fibrin clot has increased solubility because of the lack of cross-linking of the fibrin polymer in the absence of F-XIII O procedure - patient's PPP -> mixed with 0.025M calcium chloride -> clot for 1 hr at 37 - clot: placed in 5M urea or 1% monochloracetic acid at 37 - patient's clot: dissolve within the 24 hrs (less than 1-2% of normal activity)  Laboratory testing in coagulation

34. 34 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE LABORATORY TESTS FOR VON WILLEBRAND FACTOR (VWF) O variables: affects its accurate determination - the difference over time - the results of screening tests (bleeding time, APTT): normal in type I vWD -> F-VIII:C, vWF:A, vWF:Ag: 45-55% - endogenous release of adrenaline -> transient increase in plasma F-VIII, vWF - Freezing plasma -> release of platelet proteases -> altering vWF multimeric structure -> increase in vWF antigen, decrease in vWF activity - reference standard - patient's blood type (Table 40-6)  Laboratory testing in coagulation

35. 35 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

36. 36 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE von Willebrand Factor Activity (VWF:A) O referred as the ristocetin cofactor (RCoF) assay: vWF:RcoF O RIPA (ristocetin-induced platelet agglutination) - presence of ristocetin - vWF induces patient's platelet agglutination - vWD, BSS -> decreased RIPA O vWF:A (vWF:RcoF) test - add ristocetin to formalin-fixed platelets suspended in patient plasma (the source of vWF) - use platelet aggregometer to measure the resultant platelet agglutination - reported as a percentage of activity of vWF:A - general reference range: 60-150% - decrease in vWD - normal in BSS  Laboratory testing in coagulation

37. 37 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE von Willebrand Factor Antigen Assay (VWF:Ag) O sandwich ELISA (Fig 40-5) O general reference range: 43-150% O immunoturbidimetric assay - rabbit antihuman wWF antibody-coated microlatex particles - mixed with plasma containing vWF - agglutination (turbidity) VWF Multimer Analysis O for differentiating type 1 and type 2 vWD O from dimer (600,000 Da) to 20 million Da O electrophoresis - using 0.65% agarose gel - staining with radiolabeled antibody to vWF O for diagnosing thrombotic thrombocytopenic purpura (TTP) - unusually large multimer of vWF - indicates reduced or absent ADAMTS-13 activity (vWF protease)  Laboratory testing in coagulation

38. 38 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

39. 39 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Collagen-Binding Assays for VWD O for discriminating vWD type 2A or 2B from type 2M ADAMTS-13 O ELISA O chronic TTP (childhood): due to mutations in the ADAMTS-13 gene acute TTP (adult): acquired deficiency of ADAMTS-13 resulting from autoantibody  Laboratory testing in coagulation

40. 40 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE TESTS TO EVALUATE CIRCULATING INHIBITORS O lupus-like anticoagulants (LA):antiphospholipid antibodies (aPL) O F-VIII inhibitors Lupus-Like Anticoagulants/ Antiphospholipid Antibodies O immunoglobulin directed against the protein component of protein-phospholipid complex O in vivo: thrombotic tendencies O in vitro: prolong phospholipid-dependent clotting assay O lack of correction in mixing studies O affected by quality of PPP - procoagulant phospholipids (originating from platelets) - can neutralize weak lupus-like anticoagulant activities - produce false negative results O APTT: test generally used to screen for LA/aPL - lower concentration of PL  Laboratory testing in coagulation

41. 41 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O minimum diagnostic criteria 1. demonstration of an abnormality of phospholipid-dependent coagulation reactions - clotting assay using a phospholipid-based reagent: APTT, PT - tissue thromboplastin inhibition test (TTI) - dilute Russell's viper venom time (dRVVT) - kaolin clotting time (KCT) - PTT-LA 2. demonstration that the abnormality is due to the presence of an inhibitor rather than a factor deficiency - by mixing studies correction of prolonged APTT: factor deficiency lack of correction: presence of inhibitor without incubation->correction, with incubation for 1-2 hours->lack of correction: specific-factor inhibitor (F-VIII inhibitor): slow acting inhibitor 3. demonstration of the phospholipid-dependence of the inhibitor rather than a specific factor inhibition - accomplished by reducing or adding the amount of phospholipids to the test system  Laboratory testing in coagulation

42. 42 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Tissue Thromboplastin Inhibition (TTI) O modification of the PT test - dilute thromboplastin reagent is compared to the optimal strength reagent O PT of plasma with LA/aPL is more prolonged than normal plasma when diluted tissue factor is used O highly sensitive but not specific - presence of heparin Dilute Russell's Viper Venom (dRVV) Test O also known as the Stypven time O based on the premise that LA/aPL activity increases in the presence of reduced phospholipids: similar to the TTI test O Russel viper venom, calcium chloride, phospholipid -> activate F-X -> clot formation (PT test) O LA/aPL -> dRVVT: higher, prolonged - confirmatory test: with a higher phospholipid concentration  Laboratory testing in coagulation

43. 43 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Kaolin Clotting Time (KCT) O kaolin -> activate the contact factors and the intrinsic system O useful screening test for LA/aPL in patients with a normal or minimally abnormal APTT O more sensitive in detecting antibodies to prothrombin-phospholipid complexes Platelet Neutralization Procedure (PNP) O excess of phospholipids substantially shortens the prolonged APTT of aPL-containing plasma O ruptured (freeze-thawed) platelets -> source of phospholipids O presence of aPL: the clotting time with the added phospholipids is significantly shorter than the original APTT and the saline control APTT  Laboratory testing in coagulation

44. 44 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Hexagonal Phase Phospholipids (HPP) O egg phosphatidylethanolamine in an hexagonal phase configuration - based on the fact that many aPL antibodies specifically recognize the HPP configuration as an antigenic epitope O addition of HPP -> neutralize the inhibitory effect of the aPL antibodies O advantage - contains a heparin inhibitor - add normal plasma to the test system to correct any prolongation of clotting time due to factor deficiencies  Laboratory testing in coagulation

45. 45 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Specific Factor Inhibitor Assay (Bethesda Titer Assay) O monitoring the treatment of a severe hemophiliac with F-VIII concentrates) O by F-VIII:C assay - if the expected response level of F-VIII is not achieved after an infusion of F-VIII concentrate - preinfusion sample: <1% F-VIII - presence of inhibitor: be cleared quickly, producing a F-VIII level of <1% at 24 hrs or less O procedure - mix equal volumes of normal pooled plasma (containing known F-VIII activity) and patient plasma - various dilutions of the patient's plasma - incubated for 2 hrs at 37 degrees - presence of inhibitor: inactivate the F-VIII in the normal plasma -> any residual F-VIII can be measured - 1 BU = 50% residual F-VIII in the incubation mixture O Nijmegen modification - minimize pH shift - reduce F-VIII loss during the 2-hrs incubation  Laboratory testing in coagulation

46. 46 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE LABORATORY INVESTIGATION OF THE FIBRINOLYTIC SYSTEM O screening test: thrombin time (TT): FDP -> prolonged D-DIMER O excellent marker for disseminated intravascular coagulation (DIC) with secondary fibrinolysis O elevation: pulmonary embolism, deep vein thrombosis, arterial thromboembolism, recent trauma or surgery, cirrhotic liver disease, renal failure O utilize monoclonal antibodies against the D-dimer fragment - fibrinogen and fibrin do not cross-react with the D-dimer antibody  Laboratory testing in coagulation

47. 47 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE 1. macroscopically latex agglutination (semiquantitative) - lack of agglutination: <0.5ug/ml - agglutination in undiluted sample, no agglutination in 1:2 diluent: 0.5-1.0ug/ml - noagglutination both in undiluted and 1:2 diluted sample: >1.0ug/ml 2. ELISA (more sensitive, quantitative, but time consuming) 3. microscopic latex agglutination (automated) - detect increased turbidity O interpretation - no elevation: no thrombotic process, no venous thromboembolism (VTE) - elevation: clotting process due to VTE, other conditions (Table 40-7) O not used in anticoagulant therapy - anticoagulant -> decrease circulating D-dimer  Laboratory testing in coagulation

48. 48 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

49. 49 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE FIBRIN DEGRADATION PRODUCTS O increased FDP-> increased fibrinolytic activity O collection tube - containing thrombin: to clot the sample and ensure that all fibrinogen has been removed - containing fibrinolytic inhibitor: to prevent in vitro fibrino(geno)lysis O procedure - mix sample and latex particles coated with antibodies specific for FDP - presence of agglutination - sample 1:2 and 1:8 dilution agglutination in both diluent: >20ug/ml agglutination in 1:2 diluent no agglutination in 1:8 diluent: 5-20ug/ml no agglutination in both diluent: <5ug/ml O do not distinguish between fibrin degradation products and fibrinogen degradation products - not fully clotted, fibrinogen presence -> falsely high levels O this test: nonspecific - increased FDP: liver disease, alcoholic cirrhosis, kidney disease, cardiac disease, postsurgical complication, carcinoma, myocardial infarction, pulmonary embolism, DVT, eclampsia, DIC  Laboratory testing in coagulation

50. 50 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE EUGLOBULIN CLOT LYSIS O euglobulin protein fraction -> precipitated form plasma in an acid solution (1% acetic acid) fibrinogen, plasminogen, plasminogen activator, no fibrinolytic inhibitors O procedure - precipitation - precipitate: redissolved, clotted with thrombin - observed for clot lysis at 37 degree O euglobulin lysis time: longer than 90 mins -> normal - shortened lysis times indicate increased fibrinolytic activity secondary to intravascular coagulation liver disease (poor clearance of activator) thrombolytic therapy (administration of plasminogen activator) fibrinogen: false positive  Laboratory testing in coagulation

51. 51 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE LABORATORY INVESTIGATION OF HYPERCOAGULABLE STATES O repeated thrombotic episodes -> in a hypercoagulable state O determine whether the thrombosis is due to hereditary or acquired abnormalities of the hemostatic system O infants in general are prone to thrombotic events O functional assay (activity) vs antigenic assay ANTITHROMBIN (AT) O antiprotease(antithrombin) action in the presence of heparin - decreased level of AT -> the poor clinical response to heparin O chromogenic assay (1) plasma (AT) +known excess of thrombin +heparin -> incubation - AT neutralizes a proportional amount of the thrombin (2) determine the residual thrombin activity - substrate: fibrinogen tagged with a chromophore such as p-nitroanilin (pNA) released -> yellow color -> measured at 405 nm - residual amount of thrombin -> inversely proportional to the AT level  Laboratory testing in coagulation

52. 52 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O clot-based assay (1) plasma (AT): defibrinogenated, incubated with heparin and thrombin (2) transfer to a standardized fibrinogen solution -> clotting time measured - the higher the AT level -> the higher the amount of thrombin neutralized -> a lower level of residual thrombin -> a prolonged clotting time O immunological mehtod - ELISA, microlatex particle immunological assay -> measure AT protein conc. O inherited deficiency: decreased protein level or dysfunctional protein O acquired deficiency - DIC - liver disease due to decreased synthesis - nephrotic syndrome due to urinary protein loss - health newborn: about half the normal adult concentration of AT O administration of heparin: decrease plasma AT level - by accelerated clearance of the heparin-antithrombin complex O occasionally elevated into the normal range by warfarin in individuals with AT deficiency  Laboratory testing in coagulation

53. 53 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE PROTEIN C (PC) / ACTIAVTED PROTEIN C (APC) O chromogenic assay O clot-based assay O immunologic assay * normal PC Ag reduced PC anticogaulant activity in clot-based assay normal amidolytic activity in chromogenic assay -> defect in the ability of APC to interact with platelet membranes or the F-Va or F-VIIIa substrates O clot-based assay (modified APTT) - reagent: snake venom (contact factor activator) protac (a particulate activator for the activation of protein C platelet factor 3 (phospholipid) - patient plasma +PC-deficient plasma (to compensate for any factor deficiency) + APTT reagent (PL, activator) +calcium chloride -> clotting time measured - APC -> inactivation of F-Va and F-VIIIa -> prolongation of the modified APTT the longer APTT, the more functional PC  Laboratory testing in coagulation

54. 54 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O chromogenic assay - plasma (PC) +PC activator -> enzymatic activity on chromogenic substrate -> released pNA, measured at 405 nm -> direct proportional to amount of APC O diagnosis is complicated for patients on oral anticoagulant therapy - warfarin -> reduce functional measurement of PC - heparin therapy -> do not alter plasma PC level O acquired PC deficiency - individuals with uremia: normal PC amidolytic activity and antigen decreased PC anticoagulant activity in clot-based assay -> dialyzable moiety in uremic plasma interferes with most clotting assay for PC activity  Laboratory testing in coagulation

55. 55 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE PROTEIN S (PS) O two forms: free (40%) -> serves as a cofactor for APC bound to C4b binding protein (60%) O functional assay (clot-based assay) - based on the ability of PS to serve as a cofactor for the anticoagulant effect of APC - require four reagents PS-deficient plasma purified activated protein C purified activated F-Va calcium chloride - patient PPP +PS-deficient plasma +activated PC and F-Va -> incubation -> adding calcium chloride - clotting time: proportional to the PS activity  Laboratory testing in coagulation

56. 56 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O in PC or PS clot-based assays - factor V Leiden, APC resistance - elevated F-VIII level -> false positive results O nephrotic syndrome - total PS antigen: increase - functional assay: decrease due to loss of free PS in the urine and elevations in C4b-binding protein levels  Laboratory testing in coagulation

57. 57 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE ACTIVATED PROTEIN C RESISTANCE (APCR) O one of the most common risk factors for thrombosis - factor V Leiden: resistance of F-Va to degradation by APC O clot-based assay - APTT with and without the addition of APC - APCR ratio: clotting time with APC / clotting time without APC -> decrease O acquired APCR: pregnancy, oral conceptive use, elevated F-VIII, stroke congenital condition: F-V Cambridge O PCR test  Laboratory testing in coagulation

58. 58 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE PROTHROMBIN G20210A O prothrombin level: up to 30% higher than normal O PCR ADDITIONAL TESTING FOR THROMBOSIS O abnormalities in the fibrinolytic system -> thrombosis Plasminogen O plasminogen level - measured by chromogenic assay conversion of plasmingen to plasmin by an excess of streptokinase (SK) chromogenic substrate -> release of p-NA -> measured at 405 nm the pNA absorbance is directly proportional to the plasminogen quantity O inherited deficiency: quantitative or qualitative O acquired deficiency: DIC, liver disease, leukemia O useful in monitoring hepatic regeneration of plasminogen controling and adjusting the rate of infusion of FFP  Laboratory testing in coagulation

59. 59 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Alpha 2 -antiplasmin activity O chromogenic method - patient plasma (alpha2-antiplasmin) + excess of plasmin - the amount of plasmin activity inhibited -> proportional th the amount of plasmin inhibitor in the patient plasma O congenital deficiency: delayed bleeding acquired decrease: liver disease, DIC increase: postoperative period Plasmin-alpha 2 -antiplasmin (PAP) complex O using ELISA methed: detect elevated levels - during thrombotic events - in cases of endogenous hyperfibrinolysis - during thrombolytic therapy O sample collection: special tube containing citrate, aprotinin, benzamidine  Laboratory testing in coagulation

60. 60 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Plasminogen activator inhibitor (PAI-1) O primary inhibitor of tPA O varies diurnally O increased level -> impaired fibrinolytic function -> thrombosis - thrombolytic disease, acute myocardial infarction, DVT, pregnancy, sepsis Tissue plasminogen activator (tPA) O tPA -> plasminogen to plasmin -> fibrin to FDP O special blood collection tube - provide mild acidification and stabilization of the sample - block the effect of PAI-1  Laboratory testing in coagulation

61. 61 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE Thrombin activatable fibrinolysis inhibitor (TAFI) O induce hypofibrinolysis by decreasing fibrin's ability to bind tPA and plasminogen O high TAFI -> elevated risk of thrombosis Lipoprotein(a) O interfere with fibrinolytic functions of plasminogen and plasmin - promote thrombotic event O ELISA assay  Laboratory testing in coagulation

62. 62 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE LABORATORY EVALUATION OF ANTICOAGULANT THERAPY ORAL ANTICOAGULANT THERAPY AND THE PROTHROMBIN TIME-INR VALUE O oral anticoagulant: coumadin, warfarin O international normalized ratio (INR) - determined from the PT results. - the PT ratio equivalent to using the WHO international reference preparation as the source of thromboplastin in the performance of a PT - formula (Fig. 40-6) O international sensitivity index (ISI): calculated by manufacturer - for commercial thromboplastins - specific correction factor for manufacturer's standard against the WHO reference - instrument dependent - try to produce ISI values close to 1.0  Laboratory testing in coagulation

63. 63 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

64. 64 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O for monitoring long-term oral anticoagulant therapy - report both the PT and the INR - INR: independent of the reagents and methods used to determine PT O INR: useful in defining the therapeutic range - INR between 2.0 and 3.0 for hypercoagulable state (thromboembolism) HEPARIN THERAPY O variation in an individual patient's response to heparin due to - a resistance to a specific species of heparin (bovine or porcine) - variability in heparin-binding proteins and physiologic clearance mechanisms - dramatic decrease in the patient's platelet count caused by HIT  Laboratory testing in coagulation

65. 65 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) O to monitor standard (unfractionated) heparin therapy - therapeutic level (0.3 to 0.7 U/ml) - 0.5 U/ml or above: do not give measurable clotting time - prolongation of APTT: variable due to reagent, equipment - variation in standard heparin preparation - variation in patient response to heparin O factors impacting heparin monitoring (Table 40-9) THROMBIN TIME O less commonly used to monitor heparin therapy O advantage: not influenced by plasma factor deficiencies  Laboratory testing in coagulation

66. 66 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

67. 67 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

68. 68 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE ANTI-Xa ASSAY O APTT: not demonstrate a response to heparin during initial heparin treatment - presence of acute phase reactant proteins (Table 40-9) - F-Xa inhibition assay O low molecular weight heparin (LMWH) - more reliable phamacokinetics and bioavailability - reduce the risk of heparin-induced thrombocytopenia - reduce the risk of osteoporosis with long-term heparin use - F-Xa inhibition assay O F-Xa inhibition assay (anti-Xa assay) - chromogenic assay - patient PPP (heparin) +excess activated F-X (F-Xa): inhibition - residual F-Xa: cleavage of chromogen  Laboratory testing in coagulation

69. 69 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE ACTIVATED CLOTTING TIME (ACT) O during cardiac surgery to monitor heparin O similar to the APTT - activates whole blood with a contact activator - clot is detected using a POC (point of care) instrument - result: seconds - influenced by heparin concentration coagulation factors, inhibitors, lysed platelets, increased hemodilution, hypothermia MOLECULAR MARKERS OF HEMOSTATIC ACTIVATION MAKRERS OF FIBRIN FORMATION AND FIBRINOLYSIS O RIA, ELISA - the activation peptides released on activation of the zymogen - enzyme-inhibitor complex that forms as a result of zymogen activation O Table 40-10  Laboratory testing in coagulation

70. 70 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

71. 71 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE LABORATORY MARKERS OF PLATELET ACTIVATION O for detecting platelet hyperreactivity or circulating activated platelets O ELISA assay - plasma platelet factor 4 (PF4) - beta-thromboglobulin (B-TG) - soluble P-selectin - urine assay for thromboxane A2 metabolites (TX-B2) O flow cytometry - use fluorescent -conjugated monoclonal antibodies - Ab specific for activation-induced conformation change in GPIIb/IIIa - Ab specific for P-selectin (CD62-P, GMP-140, PADGEM) a component of the alpha granule membrane of resting platelets and is expressed on the platelet surface after alpha granule secretion - Ab specific for GPIb/IX/V: platelet activation -> decrease redistribution of the GPIb/IX/V complex to the membrane of the surface-connected canalicular system with platelet activation - Table 40-10  Laboratory testing in coagulation