Activated Partial Thromboplastin Time [APTT]
The APTT in contrast to the PT measures the activity of the intrinsic and common pathways of coagulation. The division of the clotting cascade into the intrinsic, extrinsic and common pathways has little in vivo validity but remains a useful concept for interpreting the results of laboratory investigations.
The term 'thromboplastin' in this test refers to the formation of a complex from various plasma clotting factors which then converts prothrombin to thrombin and the subsequent formation of the fibrin clot. The term 'Activated Partial Thromboplastin Time (APTT) derives from the original form of the test (devised in 1953) in which only the phospholipid concentration of the test was controlled (as opposed to the phospholipid and the surface activator concentrations) and the name 'partial thromboplastin' was applied at the time to phospholipid preparations which accelerated clotting but did not correct the prolonged clotting times of haemophilic plasma. Essentially the term 'partial' means phospholipid is present but no Tissue Factor.
The APTT is also known as:
-
Kaolin Cephalin Clotting Time (KCCT).
Do not confuse this with the Kaolin Clotting Time (KCT) which is a screening test for a lupus anticoagulant. -
Partial Thromboplastin Time with Kaolin (PTTK)
Kaolin is used as a surface activator. It binds directly to FXII resulting in its activation to XIIa. XIIa cleaves FXI to XIa but in the absence of calcium activation of the subsequent factors does not occur. Kaolin is rarely used when the APTT is automated as its opacity makes the optical detection of the endpoint (i.e. the formation of a fibrin clot) difficult. Commonly used activators for automated analysers include micronized silica and ellagic acid.
Cephalin is a phospholipid substitute that replaces platelet phospholipid in the test (remember the test uses platelet poor plasma and so requires a source of phospholipid for coagulation to occur.)
Principles
Patient platelet poor (PPP) plasma is incubated at 37°C then phospholipid (cephalin) and a contact activator (e.g. Kaolin) are added followed by the calcium (all pre-warmed to 37°C). Addition of calcium initiates clotting and timing begins. The APTT is the time taken for a fibrin clot to form.
Most laboratories use an automated method for the APTT in which clot formation is deemed to have occurred when the optical density of the mixture has exceeded a certain threshold (clot formation makes the mixture more opaque and less light passes through).
The diagram below shows the clotting cascade and the factors that affect the APTT.
Method
A schematic of the APTT is shown below:
Patient platelet poor (PPP) plasma is incubated at 37°C with phospholipid (cephalin) and a contact activator (e.g. Kaolin) are added followed by the calcium (all pre-warmed to 37°C). Addition of calcium initiates clotting and timing begins. The APTT is the time taken for a fibrin clot to form. Initial incubation times may vary from 2-10 minutes [- see comments.]
| Platelet poor plasma | See pre-analytical variables |
| Surface activator | Kaolin, Micronized silica, Celite, Ellagic acid |
| Phospholipid | For example Cephalin - to replace platelet phospholipid |
| Calcium | Calcium is required in molar excess for coagulation to occur. Calcium is removed (by chelation) when blood is collected into sodium citrate |
Interpretation
The APTT is frequently performed as part of a series of screening tests that comprise the PT, APTT and often the thrombin time and an estimation of the fibrinogen concentration.
1. Isolated Prolonged APTT |
Deficiencies of either XII, XI, X, IX, VIII, V, II and fibrinogen. 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 [but see comments below regarding APTT incubation times.] Contact factor deficiency e.g. pre-kallikrein deficiency [In multiple clotting factor deficiencies the APTT becomes prolonged with less severe reductions in factor levels] Acquired clotting factor inhibitors - these are most commonly directed against FVIII and may occur as either autoantibodies or alloantibodies (in patients with severe Haemophilia A). Inhibitors against other clotting factors are rare but do occur e.g. Factor V |
2. Prolonged APTT + Prolonged PT |
Vitamin K deficiency Liver disease due to: Direct thrombin inhibitors including Hirudin and Argatroban 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 In multiple clotting factor deficiencies the APTT becomes prolonged with less severe reductions in factor levels |
3. Increased APTT |
Unfractionated heparin |
4. Prolonged PT ± Prolonged APTT |
Warfarin [the 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 |
5. Short APTT |
1. An acute phase response leading to high FVIII levels - see also Comment 6 below. |
Reference Ranges
The clotting time for the APTT lies between 27-35 seconds. However, this varies widely between laboratories and is dependent upon a number of variables including whether automated or manual, the type of surface activator and the incubation time.
Comments
1.The coagulation activator should be selected to ensure that the reaction is sensitive to mild deficiencies of factors VIII:C, IX and XI (i.e. levels of 0.35-0.4 IU/ml). No activator is wholly sensitive to very mild factor deficiencies (i.e. levels of 0.4-0.5 IU/ml) and a normal APTT does not exclude these mild factor deficiencies. The coagulation activator can be selected to give different sensitivities to anti-phospholipid antibodies.
If an optical density method is used to monitor clot formation, falsely normal APTTs may occur is the patient’s serum is turbid (e.g. hyperbilirubinaemia, hyperlipidaemia).
2. Biphasic waveforms. The use of photo-optical coagulation allows additional qualitative and quantitative information on the transmittance waveform that is not obtained from the clotting time alone. The most evaluated of these is the Biphasic APTT waveform seen in DIC. This abnormal waveform is seen using the MDA series of automated, photo-optical coagulation analysers and is due to the formation of a macromolecular complex between C Reactive Protein [CRP] and Very Low Density Lipoprotein [VLDL.] The biphasic waveform appears to be sensitive and specific for DIC and although a biphasic waveform may be seen in other conditions that do not fit the diagnostic criteria for DIC, invariably such cases shows evidence of a coagulopathy.
A normal waveform is shown in the illustration below. As the clot develops, the decrease in optical transmittance is clearly visible.
In contrast the following illustration is from a patient with DIC.
If you click HERE you can see a QuickTime movie which shows sequential waveforms from a patient who developed with a Gram Negative septicaemia and DIC. The transition from a normal to a biphasic waveform is seen.
For more information on the biphasic waveform see 'References' below.
3. A deficiency of factor XIII does not prolong the APTT or the PT.
4. The APTT may be used for the detection of antiphospholipid antibodies [i.e. lupus anticoagulant] but in these cases reagents that are known to be sensitive to a lupus anticoagulant and frequently employing low concentrations of phospholipid, must be used.
5. The APTT can be made more or less sensitive to specific clotting factors by varying the incubation time. A short incubation time e.g. 2 minutes makes the test very sensitive to the levels of contact factors whereas a long incubation period make it very insensitive to the levels of contact factors. Many laboratories employ a 5 minute incubation period. If a contact factor deficiency is suspected, shortening the incubation times may be useful.
6. The APTT is frequently used to monitor patients receiving unfractionated heparin [UFH.] However, the APTT is very sensitive to the levels of FVIII which is an acute phase protein. If the FVIII levels are raised then the APTT may be misleadingly short and not accurately reflect the degree of anticoagulation. In these cases, anti-Xa assays should be performed to monitor anticoagulation.
7. The APTT forms the basis for a number of factor assays including Factors VIII, IX, XI and XI. Factors II, V and X can also be assayed using an APTT-based system although they are more commonly assayed using a 1-stage PT-based assay.
8. The APTT is used to screen for the presence of a number of clotting factor inhibitors including FVIII and FIX.
9. In patients receiving very high concentrations of unfractionated heparin e.g. during cardiopulmonary bypass, the APTT will be unclottable. In these cases, monitoring of the degree of heparinisation is undertaken using a different assay - usually the Activated Clotting Time (ACT).
10. In patients receiving unfractionated heparin and in whom the APTT is used to monitor the degree of anticoagulation, it is important to separate the plasma from the cells within 60 minutes of collection. Platelet factor 4 (PF4) released from platelets neutralises heparin and can lead to an artefactual reduction in the APTT and therefore, an underestimate of the degree of heparinisation.
11, When selecting a phospholipid for the APTT it is important to choose a reagent that is sensitive to deficiencies in clotting factors. Some laboratories may choose APTT reagents that are insensitive to a Lupus Anticoagulant to prevent it (if present) in interfering with factor assays. In such cases, an alternative reagent may be necessary if the APTT is used as a screening test for a lupus anticoagulant.
What Test Next?
1. Mixing studies: A mixing study in which patient plasma is mixed with normal plasma [ratio 1:1] may help to distinguish between a clotting factor deficiency and an inhibitor. If the mixture fails to correct the APTT with 3-4s this is strongly suggestive of:
- a coagulation factor inhibitor e.g. an acquired FVIII antibody
- an anti-phospholipid antibody i.e. a lupus anticoagulant. In practice in many modern laboratories, mixing studies have been replaced by factor assays and a lupus anticoagulant screen as the process is almost fully automated.
2. The table above outlines the various causes of a prolonged APTT and suggests, therefore, what tests to carry out next.
Useful Links & References
1. Chopin, N., et al., Activated partial thromboplastin time waveform analysis: a new tool to detect infection? Crit Care Med, 2006. 34(6): p. 1654-60.
2. Dempfle, C.E., et al., Utility of activated partial thromboplastin time waveform analysis for identification of sepsis and overt disseminated intravascular coagulation in patients admitted to a surgical intensive care unit. Crit Care Med, 2004. 32(2): p. 520-4.
3. Smith, E.Y., L.A. Charles, and E.M. Van Cott, Biphasic activated partial thromboplastin time waveform and adverse events in non-intensive care unit patients. Am J Clin Pathol, 2004. 121(1): p. 138-41.
4. Toh, C.H. and A.R. Giles, Waveform analysis of clotting test optical profiles in the diagnosis and management of disseminated intravascular coagulation (DIC). Clin Lab Haematol, 2002. 24(6): p. 321-7.
5. Toh, C.H., et al., Biphasic transmittance waveform in the APTT coagulation assay is due to the formation of a Ca(++)-dependent complex of C-reactive protein with very-low-density lipoprotein and is a novel marker of impending disseminated intravascular coagulation. Blood, 2002. 100(7): p. 2522-9.
6. Toh, C.H., et al., Early identification of sepsis and mortality risks through simple, rapid clot-waveform analysis. Implications of lipoprotein-complexed C reactive protein formation. Intensive Care Med, 2003. 29(1): p. 55-61.
Data Interpretation
Click HERE to go to the Data Interpretation Exercise.