Test 6: Coagulation Testing Flashcards by Sarah King

What is the most common specimen of choice for coagulation laboratory testing?

Whole blood that is anti-coagulated with sodium citrate (3.2%) 9:1 ratio is required.

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Specimen is centrifuged for _____ minutes at _______ rpm in order to produce platelet-poor plasma (PPP)

20, 2000

(note: most labs will decrease the time and increase the rpm to save time)

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Why does the plasma need to be platelet-poor for PT and PTT testing?

-Plts. may release membrane phosphoilpids
-Plts. secrete fibrinogen, factors V, and VIII, and vWF
-Plts. may shorten PTs, PTTs, and clot-based assays

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do all same sized blood tubes require the same amount of blood?

No (be careful with this)

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If patient has a Hct of >____%, must use deceased amount of sodium citrate, otherwise it will cause prolonged coagulation results.

(ex: hemachromatosis or polycythemia vera)

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Any clot before testing is……

Unacceptable (consumes clotting factors, giving a prolonged result)

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EDTA contamination can ________ coagulation results.

increase

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why must specimens be assayed as soon as possible and the plasma kept cold?

to avoid factor deterioration

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What type of specimen is needed for platelet testing?

Platelet Rich Plasma (PRP)- low centrifugation or whole blood
(BT, platelet function, Plt aggregometry, flow cytometry, ELISA)

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What are the standard Coagulation screening tests?

-PT
-PTT
-Platelet count
-Fibrinogen
-D-dimers
-Bleeding time or PFA-100

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Tests for the extrinsic pathway

Prothrombin Time (PT)

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What are the reagents for Prothrombin Time (PT)?

Tissues factor, phospholipid, and calcium

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What is the principle of Prothrombin Time (PT) test?

Citrated PPP added to the reagent and the time for fibrin clot formation is measured.

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Prothrombin Time (PT) reference range?

11-13 seconds

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substitution of Russel’s viper venom instead of reagent, activates factor X directly (bypassing VII).

PT variation

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PT variation- substitution of Russel’s viper venom, instead of reagent, activates factor X directly (bypassing VII). This variation of the common PT is known as the _________ Time.

Stypven

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PT test is prolonged by…

-Coumadin therapy
-Vit. K deficiency
-Factor VII deficiency
-DIC
-liver disease

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Is Factor 13 monitored by PT test?

NO!*

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INR is for _________ info only!

dosage

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INR value of ____ means the dosage of anticoagulant is too high.

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The PT results that a lab would obtain if the test were performed using the standardized WHO reference thromboplastin reagent with an assigned International Sensitivity Index (ISI) value of 1.0

INR (International Normalized Ratio)

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INR =

(patient PT/mean normal PT)

*this is not performed in the lab

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Normal INR range?

2.0-3.0

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What is INR used to monitor?

anticoagulation therapy

Should never be used to assess hemostasis!

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Test for intrinsic pathway

APTT (Activated Partial Thromboplastin Time

Reagents for PTT test?

-phospholipid-rich preparation (previous partial thromboplastin) -activator (kaolin,ellagic acid, or Celite) -calcium chloride is used as an additional reagent to initiate clotting

Principle PPT test?

patient's PPP is pre-incubated with the phospholipid/activator reagent to activate contact factor activation. following incubation, calcium chloride is added as a separate reagent to intitate the clotting cascade. fibrin clot formation is timed.

reference range for PPT test?

25-35 seconds

Causes for prolonged PTT (bleeding patient)

-Heparin (UFH) -Von Willebrand disease -Hemophilia A or B -Factor XI deficiency (Cemophilia C)

Causes for prolonged PTT (non-bleeding or clotting patient)

*Lupus anticoagulant -contact factor deficiency (Factor XII, Pre-K, and HMWK deficiency)

What does TCT and TT measure?

conversion of fibrinogen to fibrin *NOT thrombin! Thrombin is added for this test

tests for deficiency or inhibition of fibrinogen

Thrombin Clotting Time (TCT or TT)

Prinicple of TCT/TT

bovine thrombin reagent is added to PPP and the time to clot formation is measured.

Reference range for TCT/TT?

14-19 seconds

What can cause prolonged TT?

-therapeutic heparin -pt with increased fibrin degradation products -pt with any disorder of hypofibrinogenemia

The best test to determine if a sample is contaminated with heparin

TCT/TT

If Heparin contamination is suspected, repeat TT with ___________ (neutralizes Heparin)

Hepzyme *can be used with most coagulation tests (PT, PTT, TT, fibrinogen, factor assays, etc) to eliminate the effects of Heparin from the sample.

An alternative method to test for fibrinogen activity (snake venom instead of thrombin reagent)

Reptilase Time

Reptilase Time methodlogy...

similar to TT except reptilase reagent is added to plasma instead of thrombin to activate proteolytic conversion of fibrinogen to fibrin.

RT is insensitive to __________ and is useful for detecting hypofibrinogenemia or dysfibrinogenemia

heparin

RT is prolonged in the presence of ______.

FDPs

Reference range for RT?

18-22 seconds

An expansion of the TT/TCT methodology

Fibrinogen Assay (Clauss Method)

Principle of Fibrinogen Assay (Clauss Method)?

a 1:10 dilution of patient PPP and control with Owren's buffer is prepared. Measured amount of thrombin reagent is added to the diluted PPP, and the clotting time is measured and compared with the clotting times of plasma fibrinogen standards containing known amounts of firbrinogen

Reference range for Fibrinogen Assay (Clauss Method)?

200-400 mg/dL

What test should be done before PTT Mixing Studies?

TCT (Thrombin clotting time) to rule out heparin as the cause.

PTT Mixing Studies: patients PPP is mixed 1:1 with ____________ Plasma.

Pooled Normal

If correction occurs after PTT Mixing Study then ________________ deficiency is suspected.

factor deficiency

PTT Mixing Studies: A second sample is incubated for 2 hours before testing to rule out _____ activity. If corrected, follow up with factor assays.

IgG

PTT Mixing Studies: If PTT is still prolonged after 2 hour incubation, _________ anticoagulant is suspected. Pt PPP is tested with a high-phospholipid reagent to rule out it out.

lupus

PTT Mixing Studies: IF correction occurs with incubated sample and incubated sample is prolonged, an __________ antibody may be present.

anti-factor

If there is correction after Mixing Study = If there is no correction in PT or PTT =

factor deficiency factor inhibition

What is a more accurate name for Lupus anticoagulants?

Antiphospholipid Syndrome (APS)

IgG immunoglobulins directed against phospholipid protein complexes (Antiphospholipid Syndrome (APS))

Lupus Anticoagulants -nonspecific inhibitors

Patients with Lupus Anticoagulats are prone to...

-thrombosis -excess bleeding -habitual abortion

What is necessary to detect lupus anticogagulants and to distinguish them from specific inhibitors and factor deficiencies, when the PTT is prolonged.

A PTT mixing study

What are the four systems of testing for Lupus Anticoagulants? how many are required?

2 are required -Dilute Russell Viper Venom time (DRVVT) -Kaolin clotting time (KCT) -Dilute thromboplastin time (DTT) -PTTs with low reagent phospholipid levels

Dilute Russell viper venom time (DRVVT) is a variation of what test?

PTT Mixing Studies

What are the reagents for DRVVT?

DRVV, CaCl2, and phospholipids

Russell viper venom triggers coagulation of factor _____, depending on factor V, prothrombin, and fibrinogen.

Dilute Russel viper venom is neutralized by ___________ and the test is prolonged.

lupus anticoagulant -correction occurs when high-phospholipid reagent is added - confirmatory test

Prolonged DRVVT = normal DRVVT =

probable LA no LA

If a prolonged PT or PTT does NOT correct with mixing study, you must consider the presence of a ______________!

circulation anticoagulant

If a prolonged PT or PTT does correct with a mixing study, move on to doing _____________.

specific factor deficiency assays

What should be done before Mixing studies, or factor deficiency assays on a new patient?

call the floor to make sure the pt is not on anticoagulants

How are single factor assays done for the PTT?

serial dilutions are made with the patients PPP (1:10, 1:20, and 1:40) and control, then mixed with equal amount factor-depleted plasma and testing reagent

How are single factor assays done for the PT?

the pt's PPP is tested against factor-depleted plasma and testing reagent.

An old test that would be performed when pt presents with bleeding and the PTT, PT, Plt. count, and fibrinogen level is normal.

Factor XIII Assay: 5 mol/L Urea Solubility

Method that uses a synthetic substrate targeted to be enzymatically altered by a specific serine protease or serine protease inhibitor in a patient's PPP (i.e., Protein C and S)

Chromogenic Assays

principle of Chromogenic Assays?

specific substrate is cleaved by the targeted serine protease factor in the plasma sample to yield a chromogenic (colored) compound endpoint reaction yields a color the intensity directly proportional to the activity of the serine protease. This is measured by a spectrophotometer and quantified with a standard curve.

If the PTT is prolonged, PT and TT are normal, then?

Lupus anticoagulants, factor XI, XII, IX, VII, prekallikrien, HMWK.

If the Pt is prolonged, PTT and TT are normal, then?

coumadin (OACs), or factors VII

What are the two most common tests fro fibrinolysis?

FDP, and D-Dimer

Normally, FDP levels, including D-dimer, remain at plasma concentrations less than ______ng/mL

Increased FDP and D-Dimer levels are characteristic of....

-DIC -systemic fibrinolysis -DVT -PE

What is the principle of Fibrin Degradation Products (FDPs)?

Microlatex particles in saline are coated with anti-D and E fragment-specific polyclonal antibodies. A 1:5 and 1:20 dilution of patient plasma is prepared and mixed with the latex suspension Mixture is observed for agglutination after 2 minutes.

Generalized FDP assays are now mostly replaced by _________ assays.

D-dimer (Some facilities still use both. The only real value is to identify the rare case of first degree fibrinolysis)

What is the "Gold Standard" for Fibrin Degradation Products (FDPs)?

immunoturbidimetric assay: rapid (less than 7 min)and yields precise quantitative results.

D-Dimer principle

microlatex particles in saline are coated with anti-D-dimer antibodies. The coated particles are agglutinated by the patient plasma D-dimer: the turbidity is measured.

A positive D-Dimer test means....

there is some type of clot present

D-/dimer test is very sensitive , but not very _________.

specific

Negative D-Dimer has around a ____% negative predictive value for thrombotic events.

Negative __________ test can rule out with 95% certainty that PE, DVT, or DIC is present.

D-Dimer

Plasminogen or a2-antipasmin can be measured ____________ or with __________.

serologically, chromagenics

Plasminogen or a2-antipasmin principle...

Streptokinase is added to patient PPP, and activates plasminogen, which reacts with a chromogenic substrate releasing a color with intensity proprtional to original plasminogen concentration.

Tissue Plasminogen Activator (TPA) can be measured by ________ immunoassay.

enzyme

Tissue Plasminogen Activator (TPA) principle...

Reagent plasminogen is added to the patients plasma (plasma TPA activates the plasminogen) the resultant plasmin is measured with a chromogenic substrate. The color intensity is directly proportional to TPA activity.

Why is the specimen not chilled for Platelet Aggregation Studies?

chilling damages platelets.

Patient's PRP is warmed to 37 degrees C, stirred, and an agonist/stimuli is added. Plt aggregation is recorded on a graph (biphasic or monophasic curve)

Platelet Aggregation Studies

What are the agonist/stimuli that can be added for Platelet Aggregation Studies?

-ADP (biphasic?) -Epinephrine (biphasic?) -Collagen (monophasic?) -Ristocetin (depends upon vWF and GPIb/IX and targets agglutination (RIPA) -Arachidonic acid