Coagulation Testing Flashcards
What does PT measure ?
- measures the extrinsic clotting system and the common pathway
What does PTT measure ?
- measures the intrinsic pathway and common pathway
What does thrombin time measure ?
- TT measures conversion of fibrinogen to fibrin polymer.
What is a requirement of
coagulation testing ?
- must have citrated platelet poor plasma
- this will help prevent coagulation from being initiated because you remove the phospholipid and the calcium
read p. 71 principles of performance
Many coagulation assays are clot-detection based,
what are some of the down sides of using an optical detection method ?
- optical instruments suffer from more interference by lipemia, icterus and hemolysis
What factor deficiencies is PT sensitive to ?
- decreased levels of factors VII, X, V, II and fibrinogen
- Frequently used to monitor Wafarin therapy
- Started by adding Thromboplastin
- Tissue factor plus phospholipid
What is the time frame that PT/INR testing
may be performed ?
- may be done up to 24 hours from the time of collection
- sample must be uncentrifuged or centrifuged with plasma remaining on top of the cells
- must be stored at 2-4 deg C or at room temp
- if testing can’t be done in 24 hours
- plasma must be removed from cells and frozen at -70 deg C
What factors is the PT most and least
sensitive to ?
- Most sensitive: Factor VII
- Least sensitive: Factor II (prothrombin) and and fibrinogen
Note: most instruments have a Heparin neutralizing substance for the PT (such as polybene) in order to decrease the effect of Heparin. Also want minimal sensitivity to lupus anticoagulants.
When there is a strong lupus
anticoagulant that interferes with the PT in a patient on
oral anticoagulation, what test is appropriate to monitor response to the oral anticoagulant ?
- Chromogenic factor X assay
- should be used to monitor the response to the oral anticoagulation assay.
What is the ISI value in terms of determination
of the INR ?
- it is an experimentally determined value indicating the sensitivity of the thromboplastin to Warfarin induced factor deficiencies.
- Vitamin K dependent factors
- this value may be specific for instrument and reagent combinations
- use the geometric mean when the population is skewed
What factor levels would significantly
prolong the PT ?
- Factors VII, V, or X : < 40%
- Fibrinogen : < 100 mg/dL
- Prothrombin : < 30%
What is the starter for aPTT and what
does the test monitor ?
- partial thromboplastin
- cephalin or phosphatide
- measure the intrinsic and common coagulation pathways
- screen/monitor inherited or acquired coagulation disorders
- monitor unfractionated heparin therapy
What factor deficiencies is aPTT less sensitive to
and what can it detect ?
- less sensitive to VII and XIII (cannot detect deficiencies of these)
- aPTT usually normal if all coagulation factors have a concentration of 30%
- aPTT is prolonged in:
- Heparin therapy
- Direct thrombin inhibitors
- Specific factor inhibitors
- Lupus-like anticoagulants
How are aPTT values in newborns different
from adult values ?
- newborns have a prolonged aPTT
What can cause a shortened aPTT?
- pre-analytical activation of clotting factors (difficult venipuncture)
- hypercoagulability
- cancer, MI
- hyperthyroidism
- diabetes
- pregnancy
- acute phase reactants
What are some pre-analytical variables that
can affect aPTT and sometimes PT ?
- prolonged aPTT
- blood drawn from catheter that was flushed with Heparin
- underfilled collection tube
- prolonged PT and aPTT
- polycythemic samples - excess citrate to plasma ratio
What does thrombin time measure ?
- assesses fibrinogen activity in plasma
- measures the time for clot formation when thromin is added to citrated plasma
What can prolong the aPTT ?
- Heparin therapy or sample contamination (Hep lock, IV line)
- Lupus anticoagulant
- Hereditary or acquired deficiency
- Severe liver disease
- Factor or assay inhibitors
- Contact system deficiency (not associated with bleeding), Factor 12, HMWK, Pre-Kallikrein
What can prolong the thrombin time (TT)?
- test is sensitive to heparin and fibrinogen
- Prolonged in:
- heparin therapy or contamination
- severe fibrin or fibrinogen degradation (DIC)
- dysfibrinogenemia
- thrombin inhibitor:
- lepirudin
- drugs
- antibodies
How does the
Platelet function analyzer (PFA) work?
- whole blood is drawn through a drug coated opening
- Collagen/Epinephrine
- Collagen/ADP
- measure the time to occlusion of the orifice
What can increase the PFA
closure times?
- Thrombocytopenia
- Anemia
- Antiplatelet drugs
- von Willebrand disease
- other hereditary or acquired platlet dysfunction
What is the mechanism of the antithrombin
activity assay and what is it measuring ?
- measures antithrombin activity
- Chromogenic substrate assay (anti-IIa assay)
- plasma gets diluted in a heparin buffer and excess thrombin (IIa) is added
- residual thrombin is measured with a chromogenic substrate
- difference between original and final is equal AT activity
What conditions can decrease
antithrombin activity ?
- Heparin therapy
- ongoing or extensive thrombosis
- systemic coagulation activation (DIC)
- nephrotic syndrome
- Hereditary deficiency
- Aspariginase therapy
What is the methodology for measuring
Protein C activity and what can
decrease Protein C activity ?
- Chromogenic assay
- Protein C activity decreased in :
- Warfarin therapy, Vitamin K deficiency
- Liver disease
- Ongoing extensive thrombosis
- DIC
- Hereditary deficiency
How is protein S measured ?
- Screen with Free Protein S antigen
- Quantitative immunoassay
- Protein S activity
note:
- there is interference from elevated levels of Factor VIII (falsely low S), and thrombin inhibitors like Lepirudin (falsely high S)
What situations can decrease the protein S levels
as well as the protein S activity ?
- Warfarin therapy
- Vitamin K deficiency
- Liver disease
- ongoing or extensive thrombosis
- systemic coagulation (DIC)
- pregnancy
- Inflammatory syndromes due to increase in C4B biniding protein
- Hereditary deficiency
How is the Lupus inhibitor assay
performed ?
- perform a lupus sensitive PTT with and without addition of excess hexagonal phase phospholipid
- calculate the difference in clotting time with and without hexagonal phospholipid
How is the activated protein C
resistance assay performed ?
- PT based assay
- using Factor V activation and inhibition by APC
- Calculate the ratio with and without APC added
- Reduced ratio indicates APC resistance
- Factor V Leiden
- Reduced ratio indicates APC resistance
How is the vWF antigen assay performed ?
- quantitative automated latex immunoassay
How is vWF factor function evaluated ?
- enzyme immunoassay
- antibody is directed to the functional site on vWF
- Highly correlated with Ristocetin cofactor assay
- Type 1: reduction in parallel with vWF antigen
- Type 2: greater reduction than vWF antigen
How is the vWF collagen binding assay performed ?
- collagen coated plate, plasma vWF binds to collagen
- bound vWF detected with anti-vWF antibody
- Type 1: reduction in parallel with vWF antigen
- Type 2: greater reduction than vWF antigen
How are the vWF multimers evaluated ?
- uses a western blot assay of the von willebrand factors
- Type 2A: loss of intermediate and high MW multimers, abnormal forms
- Type 2B: loss of high MW multimers, normal pattern
What lab values should make you think that the
sample has been heparinized ?
- PTT and TT are prolonged
- this is also the pattern seen with a direct thrombin inhibitor
What can cause a prolonged PT and normal PTT ?
- deficiency or inhibitor of Factor 7
- early Warfarin therapy (Factor 7 falls first)
- Atypical Lupus inhibitor
What can cause a prolonged PTT and a normal PT ?
- Lupus inhibitor
- deficiency or inhibitor of Factor 8, 9, 11, or 12 or contact phase
What can cause prolongation of both PT and PTT ?
- Warfarin therapy, Vitamin K deficiency, liver disease
- Deficiency or inhibitor to: Factor 2, 5, or 10
- Thrombin inhibitor
- Fibrinogen deficiency
What causes a prolonged Thrombin time ?
- Hypofibinogenemia (usually <90)
- Dysfibrinogenemia
- The presence of fibrin or fibrin split products that block the conversion of fibrinogen to fibrin
- unfractionated Heparin
- LMW Heparin usually does not cause prolonged TT
- Presence of thrombin inhibitors
- Presence of high immunoglobulins which can prevent fibrin formation
Note: if PTT is prolonged, can obtain a TT to rule out or rule in Heparin contamination
What are the uses for Fibrinogen assays ?
- diagnosis of dysfibrinogenemia and hypofibrinogenemia
- diagnosis of DIC, liver failure and primary fibrinolysis
- guiding transfusion therapy with cryoprecipitate or NovoSeven
What is the definition of a Bethesda unit ?
- quantity of inhibitor that neutralizes 50% of the Factor VIII in normal plasma in 2 hours
- quantification of the Bethesda assay –> serial dilutions to help identify an inhibitor level to a factor
- low titer: 0.5 to 5 BU
- high titer: > 5 BU
What are the specimen transport and handling conditions
often required for coagulation testing?
- transported at temps 15-22 deg C
- too cold of temperatures can activate platelets and the clotting cascade
- too hot of temperatures can degrade the clotting factors
- For plasma based testing:
- plasma should be separated within 4 hours of sample draw
- For Heparin activity testing
- 1 hour requirement for plasma separation
- Plasma should be separated for most coagulation tests
- platelets can activate coagulation
- must double centrifuge for Lupus anticoagulant testing
- Repeat freeze-thaw events may result in loss of Factor VIII and Factor V testing
