Routine AND Special Laboratory Assessment OF Coagulation PDF

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ROUTINE AND SPECIAL LABORATORYASSESSMENT OF COAGULATIONHEMOSTASIS SPECIMEN COLLECTION,PROCESSING, AND STORAGE Specimen: venous whole blood  9:1 with a 3% solution of sodium citrate o platelet function testing  well mixed whole blood o other tests  centrifuged to provide platelet-poor plasma (PPP...

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ROUTINE AND SPECIAL LABORATORY ASSESSMENT OF COAGULATION HEMOSTASIS SPECIMEN COLLECTION, PROCESSING, AND STORAGE  Specimen: venous whole blood  9:1 with a 3.2% solution of sodium citrate o platelet function testing  well mixed whole blood o other tests  centrifuged to provide platelet-poor plasma (PPP)  Centrifugation o Speed: 7200+/- 350(4227 rcf) o Platelet rich plasma: 10 min at 4000 rpm or 50xg for 30 min o Platelet poor plasma:  Double spin approach: 10 min at 4000 rpm then separate plasma then spin again at 4000 rpm for 10 min yields 5000 plts/ul  Single spin: 1500xg for 15 min or 44000 xg for 3 min  5000-6000 rpm

PATIENT MANAGEMENT SPECIMEN COLLECTION

DURING

HEMOSTASIS

   

avoid vigorous activities should rest quietly for 30 minutes prior to collection aspirin suppresses most platelet function Coumadin (warfarin) reduces the activities of factor II (prothrombin), factor VII, factor IX, factor X, protein C, protein S, and protein Z o Prolongs PT  Excessive bleeding  extend the time for observing the venipuncture site from 1 to 5 minutes

HEMOSTASIS SPECIMEN COLLECTION TUBES  plastic blue-stopper containing 0.105 to 0.109 M (3.2%) buffered sodium citrate anticoagulant.  UNSUITABLE o uncoated soda-lime glass  Negative charge activates platelets and factors o Siliconized (plastic-coated) glass  Potential breakage

ERRORS ENCOUNTERED IN SPECIMEN COLLECTION FOR HEMOSTASIS TESTING THAT REQUIRE COLLECTION OF A NEW SPECIMEN  Short draw o Whole-blood volume less than 90%  Clot in specimen o even a small clot requires that the specimen be recollected o visually inspected prior to centrifugation by gently rotating the capped sample end to end several times  Visible hemolysis o indicates in vitro activation o Results are unreliable o Hemolysate can act as tissue thromboplastin  Lipemia or icterus o Optical instruments may not measure clots in cloudy or highly colored specimens o Use mechanical instrument  Prolonged tourniquet application o Stasis elevates the concentration of von Willebrand factor and factor VIII o falsely decreases fibrinolytic parameters o falsely shortens clot-based test results  Specimen storage at 1° C to 6° C o precipitation of large von Willebrand factor multimers o activation of coagulation factor VII (extrinsic) o destroys platelet integrity

 Specimen storage at more than 25° C o Deterioration of factors V and VIII

ANTICOAGULANTS USED FOR HEMOSTASIS SPECIMENS  Sodium Citrate  Na3C6H5O7 ⋅ 2H2O o binds calcium ions o buffer stabilizes specimen pH o 0.3 mL of anticoagulant is mixed with 2.7 mL of whole blood o final citrate concentration of 10.5 to 10.9 mM

ADJUSTMENT OF SODIUM CITRATE VOLUME FOR ELEVATED HEMATOCRITS  Elevated hematocrits  polycythemia  Decreased hematocrits  same amount of calcium as normal blood so there is no need to adjust volume  decrease in plasma volume raises the anticoagulant-toplasma ratio causes falsely prolonged results Volume of citrate = (1.85*10-3)(100-HCT)(Volume of blood)

HEMOSTASIS SPECIMEN MANAGEMENT Application PT with no unfractionated heparin present in specimen PTT with no unfractionated heparin present in specimen PTT for monitoring unfractionated heparin therapy PT when unfractionated heparin is present in specimen Factor assays Optical platelet aggregometry using platelet-rich plasma Whole-blood aggregometry Storage in household freezer Storage for 6 months

Temperature

Time 24 hours

18–24° C

4 hours 18–24° C

18–24° C

Separate within 1 hour, test within 4 hours

18–24° C

Separate within 1 hour, test within 4 hours

18–24° C

4 hours

18–24° C

18–24° C -20° C -70° C

Wait 30 min after centrifugation, test within 4 hours of collection Test within 3 hours of collection 2 weeks 6 months

SPECIMEN PROCESSING FOR COAGULATION STUDIES PLATELET-POOR PLASMA (PPP)  15 minutes at 1500–2500 g at room temp  residual platelet count of less than 10,000/µl o more than this will affect clot based test result  use of PPP Tests affected Platelet constituent Action platelet factor 4 (PF4)

neutralizes heparin

Phospholipids (phosphatidylserine )

Activates coagulation factors and

testing for the presence of heparin falsely shortens PPT affect lupus anticoagulant testing and factor

neutralize coagulant Proteases

assay testing (especially if the sample is frozen and thawed) von Willebrand factor testing

 Storage: o 18–24°C or 2–8°C for up to 4 hours o -70°C for 6 months to 1 year o never be stored in self-defrosting freezers  Frozen samples o thawed rapidly at 37°C because excessive heating (75 minutes) can result in the loss of the labile factors V and VIII  freeze–thaw cycles o rupture any residual platelets in the plasma, activate coagulation factors, and compromise sample integrity and stability

CASE 25 y/o male with a complex congenital heart disease hgb: 23.1 hct:0.72 amount of whole blood required = {(60/100-HCT)*4.5}/2

PLATELET-RICH PLASMA (PRP)  for platelet function studies like light transmittance platelet aggregometry [LTA]

 residual platelet count of 200,000/µl

CITRATED WHOLE BLOOD  mixed 4 or 5 times  used for platelet function testing o platelet function analyzer [PFA] o whole blood aggregation o thromboelastography [TEG]  global coagulation test  sample should not be checked for a clot

ROUTINE AND SPECIAL LABORATORY EVALUATION OF COAGULATION  USES o Pre-operative assessment of hemostasis o Investigate Hemostatic bleeding disorder o Monitor anticoagulant therapy  clot formation or fibrin as the end point o proteolytic cleavage of fibrinogen  manual tilt-tube  electromechanical  optical density (turbidity) o more reproducible results  monoclonal antibodies  synthetic substrates for enzymatic degradation  Intrinsic pathway: activated by contact to collagen o HMWK, prekallikrein, XII, XI, IX, VIII  Extrinsic pathway activated by tissue thromboplastin o VII  Common pathway: X, V, II, I

LABORATORY INVESTIGATION OF SECONDARY HEMOSTASIS Test

Type

Prothrombin Time (PT) or Protime

 screening test  detect a factor deficiency when the factor decreases to a level of 25–40%

Normal Value

Used for

Principle

Reporting

inherited or acquired deficiencies of the extrinsic X-ase pathway

 thromboplastin (TF/calcium mixture) + CaCl2 will react with FVIIa to convert FX to Fxa  Fxa, Fva, phospholipid, and calcium will form the prothrombinase complex which will convert prothrombin to thrombin

 Patient time in seconds  Patient time with the control time in seconds  Prothrombin ratio (patient’s PT divided by the mean of the reference range then multiplied by 100)  Percent activity (outdated, not recommended)  The International Normalized Ratio (INR) – standard method of reporting worldwide

evaluates presence of FVIII, V, X, II, I 11 – 15 seconds 12.414.6

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Clinical significance Initiator: Tissue thromboplastin

Specific test to measure effectiveness of patients receiving Warfarin Therapy

Liver disease Vitamin K deficiency Warfarin therapy

2

Activated Partial Thromboplastin Time (APTT)

 screening test  detect a factor deficiency when the factor decreases to a level of 25–40%

Thrombin time (TT)

 screening test  measures the conversion of fibrinogen to fibrin

27–35 seconds

 inherited or acquired deficiencies of the intrinsic tenase pathway  tests for intrinsic and common  detection of circulating inhibitors  monitor the effectiveness of standard (unfractionated) heparin therapy

evaluating other parameters affecting the formation of fibrin

15-17 sec

 activated partial thromboplastin  simulates activated platelet surfaces by providing phospholipids  activator (kaolin, celite, micronized silica, or ellagic acid)  negatively charged surface for the activation of FXII and prekallikrein  CaCl2 provide calcium  adding excess thrombin to undiluted plasma  thrombin + citrated PPP  bypasses all coagulation reactions except fibrinogen polymerization  thrombin will convert fibrinogen to fibrin clot

 time required for a fibrin clot

 Prolonged in deficiency of FXII, PK, or HMWK, XII. XI, X, VII, V, II or fibrinogen  Deficiency is less than 2040%

 hypofibrinogenemia or dysfibrinogenemia  presence of heparin or direct thrombin inhibitors  presence of fibrin degradation products o because they inhibit fibrin polymerization  presence of autoantibodies against bovine thrombin  presence of paraproteins  extremely prolonged TT  heparin effect o neutralized with Hepzyme

INR  Developed due to discrepancies in varied responsiveness of the thromboplastin  Different sources of thromboplastin o rabbit brain o recombinant human tissue factor o cow lung o human placenta  reagent responsiveness is now determined by comparison with World Health Organization reference thromboplastin  calibration number is known as the International Sensitivity Index (ISI)  degree of anticoagulation achieved by warfarin therapy may be compared regardless of thromboplastin

 If aPTT is abnormal but PT is abnormal  no XII, XI, IX, VIII and PK  If PT is abnormal but aPPT is abnormal  no VII and X

PROCEDURE  Activated Partial Thromboplastin Time (APTT) o citrated PPP and APTT reagent are incubated at 37°C for approximately 3 minutes. Activation of the contact factors occurs during the incubation. Next, calcium chloride is added, and the time required for a fibrin clot to form is measured  Thrombin time (TT) o citrated PPP is incubated at 37°C. Thrombin reagent is added, and the time for a clot to form is measured

 used to monitor patients taking oral anticoagulants  not intended to be used for initial evaluation of the hemostatic system or thrombotic conditions  INR greater than 1.0 means the clotting time is elevated  Normal patient  1-1.5 or 1-1.2  Oral anticoagulant therapy  target INR of 2.0 to 2.5/3.0

QUANTITATIVE FIBRINOGEN ASSAY AKA CLAUSS ASSAY  thrombin is added to various dilutions of known concentrations of fibrinogen (reference plasma or calibrator) to produce a thrombin-CT in seconds  Decreased levels o DIC o , primary and secondary fibrino(geno)lysis o liver disease o congenital disorders

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 dysfibrinogenemia and hereditary afibrinogenemia  Increased levels o inflammatory disorders o cardiovascular disease o pregnancy o use of oral contraceptives

fibrinogen and factors V, VIII, XI, XII, and XIII  Prekallikrein and HMWK are slightly reduced fibrinogen, II (prothrombin), VII, IX, X, XI, XII, and XIII

Barium sulfate or Aluminum hydroxide adsorbed plasma

CLOTTING TIME  Lee and White (7 to 15 min) o Three tubes at 37 deg celsius with 1 mL of blood each o Tilt tube at 45 degree angle after 5, 6 (90 deg) and 7 add 30 seconds o Report as time it takes to finish clotting o Dispense blood at tube 3 first then 2 then 1  Slide Ct (2-7 min) o Perform finger puncture  add 3 drops to slide o Check clotting by fishing out fibrin thread with lancet for 2 min  Activated clotting time (75-120) o Place glass beads in test tube then dispense 1 or 3 mL of blood ** tests for intrinsic pathway***  Plasma Recalcification time (100-150 for PRP and 130-240 for PPP) o Anticoagulated blood  transfer plasma to glass tube  add 1mL of CaCl2 o Too long  15 minutes

Aged plasma

"labile" factors V and VIII

fibrinogen, prothrombin, and factors V, VIII, and XIII Modification of mixing studies by substitution using PT, APTT, and TT to detect coagulation factor deficiencies Adsorbed plasma  II, VII, IX, X Serum  I, II, V, VIII, XIII VII, IX, X, XI, and XII

Serum

Adsorbed Plasma

Ab n Ab n Ab n Nor

Ab n Nor

C

C

C

Nor

C

N C C

Nor

C

Ab n Ab n Ab n Ab n Nor Ab n

Nor

C

Nor

C

Nor

C

Nor

C

N C N C C

Nor Ab n

C N C

C N C

VIII (hemophilia A)

Nor

IX

Nor

X

Ab n

XI OR XII

Nor

XIII (hyperfibrinolysis) Circulating inhibitor or Heparin therapy

Nor Ab n

N C C

Serum

PNP (citrated plasma)

VII

TT (15-17)

V

Ab n Ab n Ab n Ab n

(27-

II

APTT 35)

I

PT (11-15)

Deficiency

LABORATORY INVESTIGATION TO IDENTIFY A SPECIFIC FACTOR DEFICIENCY  Mixing Studies AKA circulating anticoagulant screen or screening test for circulating inhibitor  performed to differentiate a factor deficiency from the presence of a circulating inhibitor  uses several different dilutions of the patient’s PPP mixed with a pooled normal plasma (PNP)  Pooled platelet-free normal plasma  provides all the coagulation factors at or near 100 IUs/dL  corrects the prolonged patient result due to deficiency of one or more procoagulant o because at least 50% factor levels gives normal PT and APTT  lack of correction of the prolonged test by normal plasma indicates the presence of a circulating inhibitor or a specific factor inhibitor Testing after 2 Immediate hours with Condition incubation at Testing 37oC Factor Correction Correction deficiency Immediate No correction acting inhibitors (e.g. heparin, FIX No correction inhibitor, most lupus anticoagulants ) FVIII inhibitor Correction No correction FV inhibitor Not corrected Not corrected

vitamin Kdependent factors II, VII, IX, and X

N C N C N C C N C C C C C N C

REPTILASE TIME (RT)     

not neutralized by heparin more sensitive to dysfibrinogenemia than Thrombin time specimen: PPP stability: 6 hours at 37 deg cel, 5 days at ref temp used in conjunction with the thrombin time (TT) to detect heparin contamination  Reptilase o serine protease from the venom of the Bothrops atrox snake o cleaves fibrinopeptide A  thrombin cleaves both fibrinopeptide A and B  NV: 18–20 seconds  25 seconds or longer  indicates dysfibrinogenemia, hypofibrinogenemia, or afibrinogenemia

COMPARISON OF RESULTS USING THROMBIN TIME AND REPTILASE TIME AT DIFFERENT CONDITIONS

SUBSTITUTION STUDIES

Condition

 modification of the mixing studies  pooled samples  20 samples from blood donors o collect serum, citrated plasma (CP and BaSo4/Al(OH)3), and aged plasma o Join all samples in one tube Removes Pooled sample Provides type

Heparin contamination Dysfibrinogenemia Hypofibrinogenemi a FDPs Immunologic

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Thrombin Time Prolonged Prolonged Prolonged Greatly prolonged Prolonged

Reptilase Time Normal Greatly prolonged Prolonged Prolonged Normal

4

antithrombins

LABORATORY INVESTIGATION FOR THE IDENTIFICATION OF INHIBITORS  inhibitor’s action may be o confined to a specific factor such as FVIII inhibitor o nonspecific such as the lupus anticoagulant (LA) or the antiphospholipid antibodies (aPL) o global, affecting several factors simultaneously such as heparin  mixing studies Normal Principle Clinical Reporting Test Value significance  dRVV has FVIIa like  ratio of the patient’s CT to Incre ased activity the CT of the normal  Reagent  dilute if control is determined LAs/a  after confirmatory test: Russell viper venom Dilute Russell ratio of the high (dRVV), calcium PLs Viper Venom and low chloride, and Or Time (dRVVT) less phospholipid phospholipids antic Test concentration than 1.2  platelin + PPP + dRVV ardiol + CaCl2  activation of (Stypven Time compared with the ipin Tes) values of a Ab FX reference  LA/aPL activity is population enhanced in the presence of reduced phospholipids Specific Factor Inhibitor Assay (Bethesda Titer Assay)

 quantify inhibitory antibodies against infused FVIII  can be adapted  to treat patients with hemophilia A o Have inhibitory antibodies to FVIII

5M UREA SOLUBILITY TEST    

Additional comments  confirmatory test  higher phospholipid concentration  if

Evaluate FXIII activity Control: plasma recalcified and add NaCl Test: plasma recalcified and add 5M urea NV: clot should not dissolve in both test and control tubes after overnight (24-48 hours) incubation

LABORATORY INVESTIGATION OF THE FIBRINOLYTIC SYSTEM  plasminogen, plasmin, TPA, and PAI-1 binds to lysine amino acids in fibrin via lysing binding sites (LBSs) located in the “kringle domains”

 euglobulin protein fraction (containing fibrinogen, plasminogen, and tissue plasminogen activators [TPA], but no fibrinolytic inhibitor [PAI-1])  precipitated from plasma in a weak acid solution  time in minutes required for complete degradation of the clot o greater than 90 minutes  shortened in o hyperfibrinolysis o DIC o liver disease o therapeutic administration of a plasminogen activator  not reliable for assessing thromboembolic states

PROTAMINE SULFATE TEST D-DIMER  marker for DIC with secondary fibrinolysis because you cannot test using mixing studies or PT and APTT  present after thombolysis  test to indicate plasmin  reference range:...