(pt 1) exam #5 - heme II (cls 546)

secondary hemostasis testing + anti

end point detection methods in hemostasis (mechanical, photo-optical, nephelometric)

• Mechanical

  • Detects fibrin strand

• Photo-optical (turbidometric)

  • detects change in optical density of the plasma as the clot forms

• Nephelometric

  • Uses FSC and SSC to detect clot formation

end point detection methods in hemostasis (chromogenic, immunologic, viscoelastic)

• Chromogenic (amidolytic)

  • Measures activity of a specific factor (substrate based)

  • Spectrophotometry (405nm)

• Immunologic

  • Based on Ag-Ab reaction and light scatter end point detection

• Viscoelastic

  • Whole blood clotting – TEG

  • Measures whole clotting process – kinetics, strength, fibrinolysis

3 different levels of coagulation automation

1. manual

2. semiautomated

3. automated

what type of instrument is the fibrometer? end-point detection?

semi-automated ; mechanical detection

what tests do we use to evaluate disorders of secondary hemostasis?

• PT, PTT, TT, FIB

• 1:1 mixing study, specific coag factors, reptilase time (RT), prekallikrein, factor XIII, VWF assays, russell viper venom time (dRVVT), inhibitor assays

screening tests of secondary hemostasis

• Prothrombin time (PT)

• Activated partial thromboplastin time (APTT)

• Thrombin time (TT)

• Quantitative fibrinogen

what type of specimen is used for coagulation testing?

platelet poor plasma ; primarily sodium citrate

what is platelet poor plasma? why platelet poor plasma?

• plasma w <10 x 10^3 /uL platelets

  • Platelets have: PF4--neutralizes heparin

  • Phospholipids--affect factor assay

  • Proteases--affect vWF

specimen issues that can occur and their effect on coagulation testing

• short draw—affects dilution of blood in sodium citrate tube

• clot in specimen—unacceptable & cannot run the test

• hemolysis—in vitro activation of plts and coagulation, results unreliable

• lipemia/icterus—interfere w optical detection of clots

• prolonged tourniquet application—elevates conc of VWF & VII; shortens time on clot-based tests

• storage @ 1-6 C—cause ppt of large VWF & destroys platelet integrity

• storage @ >25 C—factor VIII deterioration

what are we detecting in the PT and PTT tests?

formation of a clot (fibrin strands)

what pathway is evaluated with the PT? PTT?

• PT = extrinsic + common

• PTT = intrinsic + common

what is the PT most commonly used for? what else can prolong a PT?

• Monitoring oral anticoagulant coumadin/warfarin

• Vitamin K deficiency

what is added in the PT testing system?

• Thromboplastin (TF/Ca2+)

• Comes from rabbit brains

what are the causes of prolonged PT's?

• Oral anticoagulant therapy

• Deficiencies in factor VII, X, V, II

• Fibrinogen inhibitors ; vit K deficiency

wow low does a factor activity have to be before it is detected as deficient in a PT or PTT?

25-40% of normal

what is added in the PTT testing system?

activated partial thromboplastin + Ca

activated partial thromboplastin time (APTT)

• Partial thromboplastin

  • Provides phospholipid surfaces

    • Stimulates activated platelet surfaces

  • Activator (kaolin, celite, micronized silica, ellagic acid)

    • Negatively charged surface for activation of FXII and PK

what are the causes of prolonged PTTs?

• Heparin therapy ; deficiencies in factor XII, XI, IX, VIII, X, V, II

• fibrinogen, PK, HMWK inhibitors

what does the thrombin time (TT) test?

conversion of fibrinogen → fibrin

what is added in the TT testing system? reference range?

thrombin ; 15-19s (TUKHS 12.6-17.0)

causes of prolonged thrombin times?

• Afibrinogenemia (or hypo) ; dysfibrinogenemia

• Heparin therapy ; thrombin inhibitors

• DIC (FDPs

what do the thrombin time and reptilase time tests have in common?

thrombin time and reptilase times bypass both intrinsic/extrinsic pathways

• test for the polymerization of fibrin or conversion of fibrinogen to fibrin

prolonged thrombin time due to what? (detailed)

• Interference in conversion of fibrinogen to fibrin

  • Hypofibrinogenemia or dysfibrinogenemia

  • Presence of heparin, direct thrombin inhibitors (DTIs) or FDPs (interfere w fibrin formation)

  • Rare cases

    • Autoantibodies against thrombin, paraproteins (myelomas)

causes of extremelely long thrombin time (TT)?

• Usually indicates heparin effect

• Neutralized w hepzyme and repeat testing

what is a reptilase time?

used w TT to detect heparin contamination

• differentiates dysfibrinogenemia from FDP & paraproteins

reptilase time (general)

• Reptilase is a serine protease

  • Found in venom of Bothrops atrox snake

  • Cleaves fibrinopeptide A from fibrinogen

    • Thrombin cleaves both fibrinopeptide A and B

• Addition of reptilase to PPP

  • Initiates clot formation

  • Detected by optical or electromechanical methods

  • Manual, semi-automated, automated methods

• General reference interval

  • 18-22 seconds

increased reptilase times are due to what?

dysfibrinogenemia, hypofibrinogenemia, afibrinogenemia

• usually >25 seconds or longer

difference between thrombin time and fibrinogen assay?

• Both use thrombin reagent

• TT is functional ; fibrinogen is quantitative

what does the fibrinogen assay test?

amount of fibrinogen present

quantitative fibrinogen assay (general)

• Reference method: Clauss assay

  • Clot based functional measurement

  • Adds thrombin to various dilutions of known concentrations of fibrinogen

    • Clotting times are plotted on a log/log graph

      • X-axis--known concentrations

      • y-axis--clotting times

• Fibrinogen concentration inversely proportional to the clotting time

  • Determined from reference curve

• RR = 200-400 mg/dL ; critical <100 mg/dL

(quantitative fibrinogen assay) what dilution is used in the fibrinogen assay for the patient and controls? why?

1:10 ; prevents interference from FDPs

(quantitative fibrinogen assay) when you get a low fibrinogen result with a 1:10 dilution, what do you do?

make a smaller dilution --1:2 or 1:5

• divide by dilution factor!

what are the causes of decreased fibrinogen?

• DIC ; primary and secondary fibrinolysis

• Liver disease ; inherited diseases

what are the causes of increased fibrinogen?

• Inflammatory disorders

• Pregnancy ; oral contraceptives

• Cardiovascular disease

quantitative fibrinogen ANTIGEN assay

• Fibrinogen antigen tests

  • Immunoturbidimetric method

    • Immunoprecipitin analysis

    • Sample mixed with ABY to FIB

  • Detects dysfibrinogenemia vs afib or hypofib

results interpretation of quantitative fibrinogen ANTIGEN assay

• Activity & antigen low = hypofibrinogenemia

  • Absent = afibrinogenemia

• Activity low & antigen normal = dysfibrinogenemia

• normal range for a fibrinogen antigen = 196-441 mg/dL

tests to evaluate specific factor deficiency (general)

• Further testing performed if

  • PT and/or APTT is prolonged

• Mixing studies: Tell us if it is an inhibitor or a deficiency by adding in factors (pooled normal plasma)

• Specific coagulation factors

• Reptilase time

• Prekallikrein screening test

• Factor XIII screening test

• VWF tests

what is a mixing study or 1:1? what does it do?

• Add pt sample to pooled normal plasma (PNP) OR specific factor deficient plasma (specific coag factor assays)

• Function: differentiates factor deficiencies from inhibitors

(1:1 mixing studies) you add PNP to your sample; it corrects to a normal PTT. what is the problem with the patient sample? why?

Factor deficiency

• adding a 1:1 mix means that you are increasing the pt's deficiency to over the 50% mark where it can be detected

(1:1 mixing studies) you add PNP to your sample & the PTT is still abnormal. what is the problem with the patient sample? why?

Inhibitor present

• PNP has coag factors already, so there is something in the pt sample that is preventing clot formation

other names for mixing studies

• Circulating anticoagulant screen

• Screening test for circulation inhibitor

  • Factor VII inhibitor screen

  • Factor X inhibitor screen

• 1:1 Mix

results of mixing studies

• Patient's plasma corrected after incubation with normal plasma

  • Factor deficiency

  • Normal plasma replenishes the deficient factor in patient's plasma

  • Next step: perform factor assays

• Patient's plasma NOT corrected after incubation with normal plasma

  • Presence of circulating or specific factor inhibitor

  • Next step: perform test to verify type of inhibitor

mixing study results interpretation (chart)

• factor deficiency = immediate correction + correction after 37 C incubation

• lupus anticoagulant/heparin = no immediate correction + no correction after 37 C

• FVIII inhibitor = immediate correction + no correction after 37 C

• FV inhibitor = no immediate correction + no correction after 37 C

acquired blood clotting disorders occur in what conditions?

• Vitamin K deficiency

• Liver diseases

• Liver transplantation

• Disseminated intravascular coagulation

• Renal disease

• Primary pathological fibrinolysis

• During the course of anticoagulant therapy

specifc coagulation factor assays (function, principle, etc)

• Performed to:

  • Confirm specific factor deficiency

  • Determine actual activity of factor

• Principle

  • Ability of a patient's plasma to correct a prolonged PT or APTT of a known factor-deficiency plasma (substrate)

• One-stage assays based on the PT

  • Extrinsic (FVII) and common pathway (FII, FV, FX)

• One-stage assays based on the APTT

  • Intrinsic pathway (FVIII, FIX, FXI, FXII)

(coag factor tests) possible tests for factor X

• 1 stage PT-based assay (common pathway)

• 1 stage PTT-based Assay (Common pathway)

• Chromogenic Factor X assay

• Immunological Factor X assay

• Russell Viper Venom assay

  • Venom activates Factor X

procedure for factor assays

• Standard curve constructed from:

  • Clotting times of factor deficient substrate plasma containing varying dilutions of a reference pooled plasma

• Patient's clotting time

  • 1:10 and 1:20 and/or 1:40 dilutions of patient plasma with specific factor-deficient plasma

  • Perform PT or APTT on mixture (depending on the factor)

  • Times are converted to % activity from standard curve

  • Results should be linear

    • If not--suggests inhibitors

• Normal factor activity reference interval

  • ~50-150%

(factor assays) prekallikrein screening test

• Patients with PK (Fletcher factor) deficiency

  • Have a prolonged APTT

  • Correction of prolonged APTT to normal after:

    • 10 minute incubation period before adding calcium chloride

    • Longer incubation--increased contact activation of FXII

  • Confirmed with specific factor assay using PK-deficiency substrate

factor XIII screening test

aka 5m urea solubility test

• Factor XIII = fibrin stabilizing factor

  • Necessary for stable fibrin clot formation

  • Occurs by forming covalent bonds between fibrin monomers

• Screening test principle

  • Fibrin clot has increased solubility because of the lack of cross-linking of fibrin polymer in absence of FXIII

(factor XIII screening) 5M urea procedure

• Patient's PPP is clotted with 0.025M CaCl2

• Allowed to clot for 1 hr at 37 C

• Clot is placed in 5M urea of 1% monochloroacetic acid in 37 C water bath

• If Factor XIII deficient:

  • Patient's clot dissolves within 24 hour period

  • Indicates activity <1-2%

3 major categories of vWF deficiency? what does each category represent?

1. Type 1 VWD: partial quantitative deficiency of VWD ; most common

2. Type 2 VWD: qualitative deficiency of VWD

3. Type 3 VWD: complete absence of VWD

variables affecting VWF

• Endogenous release of adrenaline

  • Can result in transient FVIII and VWF increase (2-3x)

• Processing of citrated sample

  • Must be PPP with platelet count <10,000/uL

• Patient's blood type

  • Type O has lower VWF activity

• Variety of clinical disorders

  • Increased levels with inflammation, pregnancy, birth control pills

von willebrand’s disease (VWD)

• Requires a panel of quantitative and functional assays

  • VWF antigen (VWF:Ag) immunoassay

  • Clot based factor VIII assay

  • Functional VWF ristocetin cofactor (VWF:RCo) REAGENT PLT

  • Dilute ristocetin-induced platelet aggregation assay (RIPA)

    • Patient PLT

    • Aka--ristocetin response curve

    • Used for VWD subtype 2B

  • Immunoassays

    • VWF activity enzyme immunoassay

    • VWF collagen binding assay

definitive diagnosis of VWD requires what?

• Quantitative VWF by both functional and antigenic methods

  • VWF:A—Activity (aka Ristocetin cofactor assay)

    • Functional assay

    • Uses a monoclonal ABY that targets GPIb binding

  • VWF:Ag—Antigen

    • Immunological assay

    • Quantitative measure of vWF in plasma

• Variables that affect accurate determination difference over time in VWF results in patients with VWD

  • Sometimes VWF:Ag and/or VWF:A levels will be normal

• Results of screening tests can be normal in type I VWD

  • Single evaluation cannot rule out VWD

  • Patients with history of bleeding--repeat testing is recommended

treatment for classic VWD

• Desmopressin (Desamino-D Arginine vasopression-DDAVP)

  • Causes two-fold to five-fold ↑ in patients VWF plasma level in type I patients

• Test dose of DDAVP is given to patient prior to surgery

  • Citrated plasma samples are drawn

    • Pre-infusion

    • 30 minutes post-infusion

    • 4 hours post-infusion

  • Assayed for FVIII activity, VWF:Ag, VWF:A

tests for VWD activity (VWF:A) (2)

• Ristocetin cofactor (RCoF) assay

  • In presence of ristocetin (reagent platelets)

    • VWF induces platelet agglutination

    • Measured on platelet aggregometer (ristocetin induced platelet aggregation – RIPA abnormal in BSS and VWD)

• Quantitative test for VWF activity (VWF:A)

  • Uses ristocetin to induce VWF to bind to glycoprotein IB/IX receptor on formalin fixed platelets

    • Eliminates variability of patient's GPIB/IX

  • Measured on platelet aggregometer

  • Reference standard curve

tests for VWF antigen (VWF:Ag)

• Laurell based assay

• sandwich ELISA

• LIA w latex particles

(VWF:Ag test) laurell based assay

• EIA that electrophoreses the plasma sample through an agarose gel

• Agarose gel contains rabbit anti-serum to FVIII

• Rocket-shaped immunoprecipitate forms

• Length is directly proportional amount of VWF:Ag

• Time-consuming and difficult to measure decreased levels

(VWF:Ag test) sandwich ELISA

• Microtiter plate wells coated with specific antihuman VWF antibody

  • Binds to VWF:Ag in test sample

• Second rabbit antihuman VWF:Ag antibody is enzyme labeled

  • Binds to initial antigen-antibody complex

• VWF protein is sandwiched between two specific Abs

• Substrate is added--colorimetric reaction

• Color intensity directly proportional to concentration

(VWF:Ag test) LIA w latex particles

• New LIA assay

  • Uses Latex particle-enhanced Immunoturbidometric Assay (LIA) to quantify VWF:Ag

  • Measures turbidity produced by agglutination of latex reagent

  • Specific anti-VWF monoclonal antibody to GPIb binding site on VWF is absorbed to latex reagent

    • Reacts with VWF in patient plasma

    • Degree of agglutination is directly proportional to VWF:A

  • Reliable, easy to automate, and timely

    • Can also detect low levels of antigen

  • Reference interval VWF:A 60–150%

what is VWF:FVIIIB? how is it measured?

lab test for factor FVIII binding assay VWF:FVIIIB

• Measures the binding of FVIII to VWF

  • 96 well plates coated with anti-human VWF

  • incubated with plasma samples

• Peroxidase-conjugated anti-vwf (sandwich) OR peroxidase conjugated anti-human F8

• Std curve with dilutions of PNP

• Color generation is detected

VWF multimer analysis

• Used to determine correct disease subtype

  • Gel electrophoresis

    • Low concentration of agarose (0.65%)

    • Staining w Ab to VWF

    • Type is determined by electrophoretic pattern

  • Also useful for diagnosing TTP

    • Presence of unusually large VWF (UL-VWF) multimers indicates decreased ADAMTS-13 activity

  • Performed in specialized reference labs or coagulation centers

collagen binding assays for VWD (VWF:CB)

• Specialized assays

  • Differentiate VWD type 2A and 2B from 2M

  • ELISA assay

  • Perform both collagen binding assays and VWF:A assays

    • Increases ability to differentiate VWD type 2 variants

      • Only 2A and 2B have abnormal VWF:CB

(VWF) ADAMTS-13

• VWF cleaving protease

  • Methodologies

    • ELISA, fluorescence resonance energy transfer (FRET), and other direct or indirect assays available

    • Measure ADAMTS-13 activity, antigen, or autoantibody levels

    • Use citrated PPP (platelet poor plasma – blue top)

what are the VWF tests used to differentiate the different types of VWD? (see chart)

circulating anticoagulants (inhibitors)

• Acquired pathologic plasma proteins that inhibit normal coagulation

• Primarily immunoglobulins

• Produced in response to variety of stimuli

  • Blood or blood products

  • Release of tumor substances into circulation

  • Autoimmune disorders

types/action of inhibitors in hemostasis

• Inhibitor's action may be:

  • Confined to a specific factor (ex VIII inhibitor)

  • Nonspecific (ex: lupus anticoagulant, which is anti-phospholipid)

  • Global, affecting several factors simultaneously (ex: heparin

immediate acting vs time dependent hemostasis inhibitors

• Immediate acting

  • Heparin, lupus anticoagulant, factor IX inhibitor

• Time dependent

  • Factor VIII inhibitor

conditions associated with inhibitors

• Hemophilia A (VIII) and hemophilia B (IX)

• DIC ; pregnancy

• SLE, Waldenstrom's macroglobulinemia, plasma cell dyscrasias

• Elderly patients

ID of common circulating inhibitors

• Most common circulating inhibitors (2)

  • Lupus-like anticoagulants (Las) or antiphospholipid antibodies (aPLs)

  • FVIII inhibitors

• Various procedures used for detection

  • No single definitive assay

  • Unexpected prolonged PT/aPTT

  • 1:1 would be the next step

    • No correction with PNP

what are the 2 most common inhibitors?

1. lupus anticoagulant/antiphospholipid antibodies

2. specific factor inhibitors

lupus anticoagulant (LA) / antiphospholipid anitbodies (aPL)

• Originally described as lupus anticoagulants

• Now referred to as Antiphospholipid antibody or aPL

• Usually IgG

  • Directed against the protein component of protein-phospholipid complexes

• In vivo—thrombotic tendency

• In vitro—prolong phospholipid-dependent clotting assays

(lupus inhibitor) LA, LAC, anti-PL

• Circulating immunoglobulins (IgG, IgM, or both)

• Specific activity against phospholipids

• Interferes with phospholipid-dependent complexes that involve factors V & VIII

  • Tests that may be affected: PT, PTT, DRVVT

• More often associated with incidents of thrombosis than hemorrhage

(lupus inhibitor) antiphospholipid antibodies

• Seen in a wide variety of autoimmune conditions

• Can occur spontaneously

• Not all patients with SLE have the inhibitor

• Associated with both arterial & venous thromboembolitic events as well as recurrent abortions

  • These patients RARELY bleed unless there is some additional abnormal hemostasis (ex: prothrombin deficiency or decreased platelets)

(lupus inhibitor) diagnosing LA/aPL

• Diagnosis requires demonstration of:

  • Abnormal PL dependent clotting test

  • Presence of and inhibitor (clotting test does not correct in mixing study)

  • Phospholipid-dependent inhibitor

• Sample (PPP) integrity important

  • Procoagulant phospholipids can be in

    • Patient plasma

    • Normal plasma used for mixing study

  • Can neutralize weak lupuslike anticoagulant activities

    • May give false negative results

lupus anticoagulant vs antiphospholipid screening tests

• Two different screening tests recommended

  • Dilute RVVT (dRVVT)—initial screening test

  • APTT—second test for screening

• If either test is prolonged

  • Proceed with designated testing

• When to suspect a LA or aPL?

  • If patient presents with a positive history of thrombophilia and APTT is prolonged

  • Should proceed with testing for LA/aPL

dilute russell’s viper venom test (dRVVT)

• Venom from the russell viper (daboia ruesselii)

• Reagent

  • Dilute russell's viper venom, CaCl2, phospholipids (low level)

  • Added to patient's PPP

• Activated FX to produce clot

• Used to confirm LA/aPL

dRVVT ratio & confirmations

• Determine ratio of patient's CT to CT of normal control (normal plasma)

  • LA/aPL is present if patient's dRVVT is longer than normal control

    • Ratio of patient's CT to normal control CT is <1.2 normally

• Confirmatory tests

  • Use higher PL concentration RVV

  • Report ratio of high and low PL tests

dilute russell’s viper venom (info)

• Has thromboplastic activity & can be substituted for tissue thromboplastin in the PT

• Does NOT require Factor VII for activity

• Activity is dependent on Factors X, V, prothrombin, fibrinogen

• If these factors are present & provide normal levels of activity, the DRVVT is sensitive to lupus anticoagulant

hexagonal phospholipids (HPP)

• Substitutes egg phosphatidylethanolamine in a hexagonal phase configuration (HPP)

  • aPL Abs recognize the HPP configuration

  • Addition of HPP

    • Neutralizes inhibitory effect of the aPL antibodies

    • Does not neutralize factor-specific antibodies

• Incubate test plasma with and without HPP

  • Perform APTT on both mixtures

  • Test with HPP will have a shortened clotting time if LA or aPL is present

testing algorithm for suspected LA/aPL

1. Coag screening (PT/PTT)

2. Mixing study 1:1 (did not correct at initial)

3. dRVVT or hexagonal PL

4. Confirm w increasing PL in screening test

(circulating anticoagulants) acquired inhibition of factors

• Heparin—most common

• VIII—most common specific factor inhibitor

  • 5-20% of hemophilia A patients

• IX—5% of hemophilia B patients

• Detection of inhibitors is by 1:1 mix using PT or PTT protocol

specific factor inhibiton assay

aka Bethesda titer assay

• Developed to measure FVIII inhibitors

  • Develop in 3-52% of patients with severe hemophilia A

• Can be used for other inhibitors to coagulation proteins

• Specific Inhibitors

  • Occur in 10-15% of hemophiliacs at any time after first factor concentrate infusion

  • Monitor patient's response to treatment by ordering FVIII:C assays

  • If bleeding does not stop--could be presence of inhibitor

bethesda inhibitor assay (procedure)

• Mix patient's plasma with equal volume of PNP of known FVIII activity and incubate for 2 hours

  • Allows inhibitor to neutralize FVIII in PNP

  • Perform FVIII assay on incubation mixture to measure residual activity

  • Convert activity to Bethesda units (BU) using standard Bethesda chart

    • 1BU = 50% residual FVIII

• Testing is always done on pre-infusion trough sample

anti-Xa activity assay

• Designed to measure plasma heparin (unfractionated & low molecular weight) levels and to monitor anticoagulant therapy

• Reference ranges:

  • Therapeutic ranges of heparin

    • LMWH: 0.5-1.2 IU/mL

    • UH: 0.3-0.7 IU/mL

  • Prophylactic ranges of heparin

    • LMWH: 0.2-0.5 IU/mL

    • UH: 0.1-0.4 IU/mL

anti-Xa activity TESTING

• When a person is not taking heparin, anti-Xa levels should be zero or undetectable

  • Activity of both UFH and LMWHs is dependent upon binding to antithrombin (AT)

    • Binding induces a conformational change in the molecule which accelerates its inhibitory activity

  • LMWHs have primarily anti-Xa activity while UFH has both anti-Xa and anti-IIa activity

  • Chromogenic assays are most commonly used