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

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 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 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 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE O PPP: stored at 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: 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 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 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 COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE  Laboratory testing in coagulation

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

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

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

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 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 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 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 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: seconds  Laboratory testing in coagulation

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): 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 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: 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 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: seconds - preterm and term infant: longer than adults  Laboratory testing in coagulation

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 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: mg/dL  Laboratory testing in coagulation

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

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

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 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: % O F-IX, XI, XII -> quite low at birth O for F-VIII: chromogenic kit  Laboratory testing in coagulation

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

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

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 general referrence interval time: seconds - prolonged: dysfibrinogenemia, hypofibrinogenemia, afibrinogenemia  Laboratory testing in coagulation

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

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 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