Week 4: Laboratory Evaluation

anticoagulant:blood ratio for coagulation testing

1:9

needle gauge for routine coagulation testing

21

needle gauge for platelet testing

19

Routine coag. should be performed within ____- hours of collection no longer than _____ hours

2, 4

which test is most sensitive to the timing of collection?

aPTT

patients on heparin should be tested after ________ of collection

1 hour

specimen should be centrifuged for ____________ at 2000 g’s for platelet poor plasma (PPP)

20 minutes

a hematocrit of ________ is too high and will cause a prolonged result if collected with a normal collection tube

greater than 50%

formula for correcting for high HCT in collection tube

1. citrate (mL) = ( (100-HCT) / 60 ) * 0.3

2. usually normal 2.7 mL draw

with too low of a hematocrit, too little anticoagulant may cause…

activation of factors and a clotted specimen

physiological activators in clotting assays

tissue extracts

non physiological activators in clotting assays

silica, kaolin, celite

therapeutic window of PT for coumadin therapy

2.0-3.0

therapeutic range for INR

1. standard dose = 2.0-3.0 → prophylaxis, VT, PE

2. high dose = 2.5-3.5 → mechanical heart valve

the russell viper venom (RVV) time or stypen time activates the pathway at ___________

Factor X

RVV time does not require __________

Factor VII

what does RVV time help to evaluate?

X or V deficiency

normal RVV time

12 seconds

partial thromboplastin mimics PLF3, and _______ acts as a contact activator to speed up the reaction and provide a negative charge

kaolin

what is aPTT most sensitive to?

VIII and IX

which test is affected by lupus anticoagulant?

aPTT

activated clotting time (ACT)

1. similar to aPTT, but with whole blood

2. without PL and CaCl2

3. uses a contact activator

4. monitors very high doses of unfractionated heparin

5. associated with open heart surgery

normal time for ACT

300 seconds

ACT is not useful for _____

LMWH

lee white whole blood clotting time

1. old and insensitive test

2. overall coagulation

3. bedside

4. 4-8 minutes at 37C

5. 8-15 at RT

clauss quantity of functional fibrinogen

1. normal = 200-400 mg/dl

2. high conc. of thrombin added

3. standard curve from known fibrinogen and sequential dilutions

4. clotting time is recorded

what does not interfere with clauss method?

heparin

radial immunodiffusion assay (RIA)

1. antigenic amount of molecule

2. not for function

thrombin time

1. screening for fibrinogen activity

2. low concentration of thrombin

3. sensitive to < 100 mg/dl of fibrinogen

what can thrombin time indicate?

1. fibrinogen deficiency

2. heparin therapy/contamination

heparin + ____ rapidly neutralizes thrombin, which prolongs the TT

AT

reptilase test

1. thrombin-like enzyme

2. it cleaves fibrin peptide

how is the reptilase test useful?

1. heparin does not interfere

2. a prolonged TT and normal RT → confirms heparin presence

protamine sulfate neutralizes __________

heparin

use of toluidine blue/hepzine

1. when TT is abnormal, check for heparin contamination

2. sub toluidine blue for the buffer and repeat the test

3. if corrected → there is heparin contamination

4. if still prolonged → lack of fibrinogen in patient

protamine sulfate

used to detect the presence of fibrin monomers, which are early FDP (X and Y)

when can protamine sulfate yield a positive result?

• DIC

• cirrhosis

• pulmonary emboli

what specimen is used with protamine sulfate and ethanol gelation?

plasma

ethanol gelation

detects the presence of fibrin monomers, and a screener to rule out DIC and primary fibrinolysis

what specimen can be used for FDP tests?

serum or plasma

FDP tests for which fragments?

X and Y

D-dimer tests for which fragments?

D and E (D-dimer)

the D-dimer test is what type of assay?

monoclonal antibody-based

euglobulin and its components

• a group of plasma proteins that precipitate out when blood plasma is diluted and made acidic

• fibrinogen, plasminogen, tissue plasminogen activator (tPA), and plasmin

euglobulin lysis is sensitive to increased _____________________

plasminogen activators

euglobulin lysis in less than __________ is an indication of increased fibrinolytic activity

2 hours

clot lysis test

• clot should be intact for 48 hours

• measures all fibrinolytic activity

• could detect any fibrinogen deficiency

when is a mixing study done?

after a prolonged PT or aPTT

before mixing studies be sure there are no ______ in the sample or the presence of __________

clots, heparin

how is a mixing study performed?

1. add equal parts normal plasma + patient plasma

2. repeat the PT and aPTT

3. correction → factor deficiency

4. no correction → inhibitor

what is a factor assay?

specific assay to figure out what is causing a prolonged PT and aPTT

how is a factor assay performed?

1. patient’s plasma has dilutions made

2. substrate is added with only 1 factor missing

3. read off standard curve from a known sample

what does a factory assay evaluate?

the percent activity of the factor to determine mild, moderate, or severe deficiency

how is XIII evaluated?

1. urea solubility → not soluble in 5M

2. normal clot = intact 24 hours

3. XIII deficiency = clot dissolves

anti phospholipid syndrome (APS) is _____________

acquired

with anti phospholipid syndrome, the aPTT is ____________, but the patients tend to ___________

prolonged, thrombosis

lupus anticoagulant

1. IgG, IgM, and IgA types

2. an antibody with activity against the PL in the cascade

anti-cardiolipin antibodies (ACA)

1. IgG and IgM types

2. they are directed against cardiolipin and other negatively charged phospholipids

tests performed to evaluate a circulating inhibitor

1. aPTT

2. platelet neutralization procedure

3. tissue thromboplastin inhibitor TTI

4. dilute RVV

5. hexagonal phase

6. bethesda titer

platelet neutralization procedure

1. confirms/rules out lupus anticoagulant

2. run after prolonged aPTT

3. add freeze-thawed platelets to sample (PL source)

4. correction → LA is present

the dilute RVV uses dilute ______________

thromboplastin

normal time for dilute RVV

35-37 seconds

hexagonal phase confirm test

• confirms the presence of lupus anticoagulants

• uses the addition of phospholipids

• correction → LA is present

what does the bethesda titer measure?

amount of factor inhibitors

how is the bethesda titer assay performed?

1. patient plasma is mixed with normal plasma

2. incubate at 37C for 2 hours → any inhibitor will neutralize the factor

3. remaining factor activity is compared to control

bethesda units

1 unit = the amount of inhibitor that neutralizes 50% activity in normal plasma

what questions should be raised if the aPTT is prolonged?

1. was the anticoagulant : blood ratio correct?

2. what is the HCT?

3. was the sample drawn through a line?

4. is there heparin contamination?

5. do we need a mixing study?

aPTT abnormal → mixing study → no correction → ?

specific factor inhibitor or lupus anticoagulant

aPTT abnormal → mixing study → correction → ?

deficiency, perform a factor assay

aPTT abnormal → toluidine blue → correction → ?

heparin contamination

aPTT abnormal → hepzyme → no correction → ?

test for abnormal fibrinogen

laurell rocket is an agar that contains what?

antibody specific to antigen

what does the laurell rocket electrophoresis measure?

• protein C

• protein S

• VIII:vWf

• measures total factor present, not activity

ELISA for factors

sandwich technique to measure total protein, where increased color is increased concentration

chromogenic methods measure the _____________ levels of AT and protein C and S

functional

how does solid-phase EIA work?

1. well is coated with antigen

2. add patient’s plasma (it may contain Ab)

3. add HRP-conjugated AHG (this binds Ab)

4. add enzymatic substrate (TMB)

5. measure absorbance

how does the chromogenic assay work?

1. peptide + chromophore (para-nitroaniline)

2. protease cleaves peptide → free chromophore

3. change in absorbance = enzyme activity

4. pNA = 405 nm

when performing a chromogenic assay for protein C, what is first added to the patient’s plasma?

the activator (southern copperhead venom)

in a chromogenic assay after protein C is activated, what happens when the substrate is added?

protein C cleaves the substrate and pNA is released → yellow color read at 405 nm

what is the purpose of an anti-Xa assay?

measures the amount of heparin or other anti-factor Xa drugs in your blood to make sure your dose is safe and correct

what type of heparin can be evaluated by the anti-Xa assay?

• unfractionated

• LMWH

  • lovenox

  • fragmin

  • fondaparinux

  • aristra

electromechanical and electromagnetic methods are both __________________

mechanical

turbidity and nephelometry methods are both ___________

photometric

what are 4 main automation methods?

1. mechanical

2. photometric

3. chromogenic

4. immunologic

what methodology is described?

• 2 metal electrodes measure a change in conductivity or electrical impedance of an electrical current

• change is caused by formation of fibrin

• after clotting, the current is interrupted → timer stops

electromechanical

what methodology is described?

• change in viscosity is measured

• metal ball oscillates in a magnetic field

• fibrin clotting increases viscosity and inhibits the ball → timer stops

electromagnetic

what methodology is described?

• initial light transmission is measured

• increased optical density → electrical signal

• fibrin strands decrease light transmission

turbidity

increased optical density = increased light ______________

absorbance

what methodology is described?

• variation of photometric end point detection

• 90 degree side scatter or 180 degree forward angle

• measures scattered light

• clot curve generated

• quantitative → not functional

nephelometry

what methodology is described?

• latex particles are coated with monoclonal Abs

• light is passed through suspension

• increased agglutination → increased light absorbance → increased antigen level

immunologic

D-dimer testing is an example of an ______________ methodology

immunologic

what methodology is described?

• POC analyzer

• evaluates in vivo hemostasis from clotting → clot lysis

• measures time at the start, strength, and dissolution of the clot

• used for bleeding during surgery, hypercoagulability, and dysfunctions of hemostasis

thrombelastography (TEG)