5. Introduction
• PT was described by Quick in 1935
• Test was often referred to as 'Quick's Prothrombin
Time’
• PT was developed to measure Prothrombin
(Factor II)
• However, it subsequently became clear that it was
sensitive to abnormalities of factors VII, X, V, II and
fibrinogen
• PT measures the activity of the extrinsic and
common pathways of coagulation
7. Prothrombin Test(PT)
• Platelet Poor Plasma
• Citrated 9:1; 3.2%
• Thromboplastin
• Phospholipid
• Source of tissue factor (e.g. rabbit brain)
• Ca+2
• Time to clot detection (seconds)
• Sensitive to factor VII, but also V and X
• Standardization
• INR & ISI
8. Interpretation
Isolated
Prolonged PT
Factor VII deficiency
Prolonged PT in
association
with other
coagulation
abnormalities
Vitamin K deficiency
Vitamin K antagonists e.g. warfarin, phenindione, rodenticides
Liver disease
Malabsorption (leading to vitamin K deficiency)
High concentrations of unfractionated heparin
Direct thrombin inhibitors e.g. Lepirudin, argatroban
Afibrinogenaemia and dysfibrinogenemia
Dilutional coagulopathy e.g. massive blood transfusion
Multiple clotting factor deficiencies e.g. FV and FVIII deficiency
Abnormalities of the vitamin K cycle e.g. mutations within the
VKORC1 gene
Chromosomal aberrations - the F7 and F10 genes are located on
the long arm of chromosome 13 - deletions of which are
associated with reduced FVII and FX levels.
Shortened PT Following treatment with rVIIa
9. Reference Ranges
• The reference range depends upon a number of
variables including:
• Source of Tissue Factor e.g. human, rabbit etc
• The exact technique used e.g. manual or automated
• Method of end-point determination e.g. optical or
mechanical
• Each laboratory should establish its own normal
range
• In general the PT for a normal plasma sample, lies
between 13-15 seconds
11. Introduction
APTT is also known as:
• Kaolin Cephalin Clotting Time (KCCT)
• Do not confuse this with the Kaolin Clotting Time (KCT)
o screening test for a lupus anticoagulant but which contains no
Cephalin
• Partial Thromboplastin Time with Kaolin (PTTK)
• APTT measures the activity of the intrinsic and
common pathways of coagulation
13. aPTT
• Platelet Poor Plasma
• Citrated 9:1 3.2%
• Activator
• (e.g. silica, kaolin)
• Partial Thromboplastin
• No source of
tissue factor
• Phospholipid
• Ca+2
• Time to Clot Detection (seconds)
• Need to determine therapeutic range for heparin
• aPTT can be shortened due to elevated FVIII as an
acute phase reactant
14. Interpretation
Isolated Prolonged APTT
• Deficiencies of either XII, XI, IX & VIII
• However, the APTT can be normal with mild deficiencies of
these clotting factors
• In general the deficient factor has to be less than 20-40% of
normal before the APTT is prolonged
• Contact factor deficiency e.g. pre-kallikrein deficiency
• In multiple clotting factor deficiencies the APTT becomes
prolonged with less severe reductions in factor levels
15. Interpretation
Isolated Prolonged APTT
• Acquired clotting factor inhibitors
• Most commonly directed against FVIII
• May occur as either autoantibodies [e.g. acquired Haemophilia
A] or alloantibodies (in individuals with severe Haemophilia A
following exposure to exogenous factor VIII
• Use of factor VIII concentrate to treat a bleed
• Inhibitors against other clotting factors are rare but do occur
e.g. Factor V
• Lupus anticoagulant [LA]
16. Interpretation
• Prolonged APTT + Prolonged PT
• Vitamin K deficiency
• Liver disease due to:
• Malabsorption of vitamin K
• Leads to decreased gamma carboxylation of the vitamin K
dependent clotting factors
• Decreased synthesis of clotting factors
• An acquired dysfibrinogenemia due to changes in the sialic acid
content of the fibrinogen
• Direct thrombin inhibitors including Hirudin, Argatroban
and Dabigatran
17. Interpretation
• Prolonged APTT + Prolonged PT
• DIC - due to the consumption of clotting factors
• Massive blood transfusion leading to a dilutional coagulopathy
• In patients receiving thrombolytic therapy, the APTT may be
prolonged due to a reduction in fibrinogen levels
• In multiple clotting factor deficiencies the APTT becomes
prolonged with less severe reductions in factor levels
• Combined deficiency of clotting factors e.g. Factors V and VIII
• 'Common Pathway' clotting fasctor deficiences - FV, F, FII
[Prothrombin] and Fibrinogen
18. Interpretation
• Increased APTT ± Prolonged PT
• Unfractionated heparin [UFH]
• Significantly prolongs the APTT but the PT usually shows little
prolongation
• In cases of significant over anticoagulation with UFH the PT
will be prolonged
• Antiphospholipid antibodies
• Acquired clotting factor inhibitors e.g. FV, FX
19. Interpretation
• Prolonged PT ± Prolonged APTT
• Warfarin
• APTT may be only prolonged by a few seconds in patients who
are stably anticoagulated on warfarin
• But in patients who are overdosed the APTT may be significantly
prolonged
• Short APTT
• An acute phase response leading to high FVIII levels
• Difficulties in the collection of samples leading to activation
of coagulation within the collection tube
20. Reference Ranges
• Clotting time for the APTT lies between 27-35
seconds
• Varies widely between laboratories
• Dependent upon
• Whether the test is automated or manual
• Type of activator
• Incubation times employed in the test
22. Introduction
• Mixing studies are tests performed on blood plasma used
to distinguish
• factor deficiencies from factor inhibitors
• Mixing studies take advantage of the fact that factor levels
that are 50% of normal should give a normal Prothrombin
time (PT) or Partial Thromboplastins time
• Mixing studies can help determine the appropriate next
steps to take to diagnose the cause of an abnormal APTT
or PT
23. Test method
• The patient plasma is mixed 1:1 with Normal
pooled plasma
• That contains 100% of the normal factor level
results in a level ≥ 50% in the mixture (say the
patient has an activity of 0%; the average of 100%
+ 0% = 50%).
• Therefore, correction with mixing indicates factor
deficiency; failure to correct indicates an inhibitor.
24.
25. Test method
• Some inhibitors are time dependent.
• The clotting test performed immediately after the
specimens are mixed may show correction
because the antibody has not had time to
inactivate the added factor (false positive).
• A test performed after the mixture is incubated
for 2 hours at 37°C will show prolongation
28. Interpretation
• The first step when evaluating unexpected prolonged PT
or PTT results is to rule out preanalytical interference, e.g.,
presence of contaminating heparin.
• If the APTT or PT is corrected by normal plasma, a factor
deficiency is indicated.
• If the APTT or PT is not corrected by the addition of nor-
mal plasma immediately, a strong inhibitor is indicated.
• A weak or time-dependent inhibitor is indicated by a
prolonged APTT or PT following incubation at 37°C for 1
to 2 hours ( factor VIII inhibitor)
29. Differentiation of Factor Deficiency
and Inhibitors By Mixing Studies
1:1 Mixing Study Results
Not incubated Incubated
Factor deficiency Correction Correction
Immediate acting inhibitor No correction No correction
Time/temperature
dependent inhibitor
Correction
(Falsely)
No correction
30. Possible Interpretations
Coagulation Screen Results: PT prolonged
PT mixing study results: PT corrects
Most likely interpretation: Factor VII deficiency
Probable cause(s):
Early response to warfarin, early vitamin K
deficiency
Rare cause: Congenital factor VII deficit
Coagulation Screen Results: PTT prolonged
PTT mixing study results: PTT corrects
Most likely interpretation: Factor deficit
Probable cause(s):
Factor VIII or IX (male) deficiency, or von
Willebrand Disease (female)
Possible cause Factor inhibitor
Coagulation Screen Results:
PTT markedly prolonged (>200
seconds)
PTT mixing study results: PTT corrects
Most likely interpretation: Severe Contact Factor deficit
Probable cause(s): Factor Prekallikrein, HMWK, XI, or XII
31. Possible Interpretations
Coagulation Screen Results: PT and PTT prolonged
PT & PTT mixing study results: PT and PTT correct
Most likely interpretation: Acquired, multiple factor deficiency
Probable cause(s): DIC, Liver Disease, Vitamin K deficiency
Possible cause: Warfarin therapy
Coagulation Screen Results: PTT slightly – moderately prolonged
PTT mixing study results: No correction
Most likely interpretation: Immediately reacting antibody inhibitor
Probable cause(s): Lupus anticoagulant
32. References
• Rohrer MJ, Natale AM. Effect of hypothermia on the coagulation cascade. Crit
Care Med. 1992 Oct. 20(10):1402-5
• Nakano Y, Kondo T, Osanai H, Murase Y, Nakashima Y, Asano H, et al. Clinical
usefulness of measuring prothrombin time and soluble fibrin levels in
Japanese patients with atrial fibrillation receiving rivaroxaban. J Cardiol. 2014
Sep 2
• Kamal AH, Tefferi A, Pruthi RK. How to interpret and pursue an abnormal
prothrombin time, activated partial thromboplastin time, and bleeding time in
adults. Mayo Clin Proc. 2007 Jul. 82(7):864-73
• Levy JH, Szlam F, Wolberg AS, Winkler A. Clinical Use of the Activated Partial
Thromboplastin Time and Prothrombin Time for Screening: A Review of the
Literature and Current Guidelines for Testing. Clin Lab Med. 2014 Sep.
34(3):453-477
• Ng VL. Prothrombin time and partial thromboplastin time assay
considerations. Clin Lab Med. 2009 Jun. 29(2):253-63.
• Tripodi A, Lippi G, Plebani M. How to report results of prothrombin and
activated partial thromboplastin times. Clin Chem Lab Med. 2015 Aug 6
• Coagulation Test Handbook
http://mghlabtest.partners.org/coagbook/co003400.htm
• Mixing Study, Incubated APTT, Cleveland Clinic Laboratories pdf
