aPTT is a screening test (“first level”) that plays a key role in the evaluation of a patient presenting with bleeding symptoms. It is also used to monitor treatment with unfractionated heparin and argatroban (a parenteral direct thrombin inhibitor used in patients with heparin-induced thrombocytopenia and thrombosis). Finally, it is useful in screening and confirming the presence of the lupus anticoagulant ^[3][5].

3. Preanalytical Cause of Prolongation of aPTT and Other Coagulation Tests

3.1. Hemolysis, Hyperbilirubinemia, and Hypertriglyceridemia

Hemolysis interferes with both optical and mechanical measurement methods via optical interference due to the presence of cell-free hemoglobin absorbance and via biologic interference due to the release of molecules able to activate platelet and coagulation factors ^[5][9]. Hyperbilirubinemia and hypertriglyceridemia both interfere with optical methods. To overcome this interference, modern optical analyzers have the capability to increase the reading wavelength over 650 nm. Regardless, extreme hyperbilirubinemia and hypertriglyceridemia may make performing the coagulation test with optical analyzers unfeasible ^[5][9].

3.2. Blood/Anticoagulant Ratio and Hemoconcentration

As in the performance of other laboratory coagulation tests, for aPTT, an adequate amount of blood should be placed in the test tube; the optimal ratio of blood to anticoagulant should be 9:1. The importance of this ratio is confirmed by the fact that hemoconcentration (a hematocrit higher than 55%, which could be present in patients with heart disease and in newborns, for example) prolongs aPTT as the plasma volume per blood volume is decreased, leading to an excess of anticoagulant in the sample if no correction is applied (the necessary volume of the anticoagulant is consequently decreased) ^[6][8].

4. Isolated, Prolonged aPTT: Prevalence and Causes

4.1. Isolated, Prolonged aPTT: A Truly Unexpected Finding?

In an Italian retrospective study ^[7][26], 5.8% of 8.069 patients undergoing elective surgery had an abnormal aPTT; 2.9% (240 patients) had an aPTT ratio higher than 1.3 and for this reason, they were worthy of further investigation. An old review by Munro et al. ^[8][27] which considered 29 papers regarding the value of routine pre-operative coagulation testing showed an incidence of aPTT abnormalities of 15.6%. A recent large Danish study ^[9][24] showed a prolonged aPTT in 12% of 18.642 aPTT measurements performed on 10.697 patients (excluding those affected by known coagulation disorders); 79% of these abnormal aPTTs were reported to be moderately or severely prolonged (40–45 or >45 s, respectively).

4.2. Isolated, Prolonged aPTT: What Are the Causes?

4.2.1. Heparin Contamination

Heparin contamination can be a source of error and is often observed when blood is collected from a central venous line. The suspicion of this preanalytical problem should be an indication to repeat sampling from a peripheral vein ^[9][24]. However, it should be taken into account that the heparin contamination of samples destinated for use in coagulation tests could also happen if the blood is collected from a direct venipuncture that is performed after the venous lines are flushed with an amount of heparin solution containing enough heparin enough to induce an anticoagulant effect in the patient.

4.2.2. C-reactive Protein

C-reactive protein (CRP) interference with the measurement of aPTT is most likely phospholipid-dependent and depends on both the CRP concentration and aPTT assay type ^[10][11][12,13].

4.2.3. Lupus Anticoagulants

Lupus anticoagulants, a heterogeneous group of immunoglobulins that can bind β₂-glycoprotein I, prothrombin, or other proteins in a complex with negatively charged phospholipids, are able to prolong phospholipid-dependent coagulation tests, including aPTT tests ^[12][18]. The presence of lupus anticoagulants can increase the risk of (venous or arterial) thrombosis.

4.2.4. Drug Interferences

As mentioned previously, aPTT is used to monitor therapies using unfractionated heparin (UFH) and argatroban ^[13][14][28,29]: these drugs prolong aPTT. Low-molecular-weight heparins (LMWHs) are often reported not to affect aPTT, but some commercial brands can have this effect ^[15][11]. aPTT is also sensitive to direct oral anticoagulants (DOAC), which can cause an isolated prolongation of this clotting time, even if they often also prolong PT/INR, and aPTT is not recommended for monitoring DOAC therapy ^[16][25]. Anticoagulant therapy with vitamin K antagonists (VKAs) can also affect aPTT, but this type of therapy mainly prolongs PT, limiting the activation of factor VII.

4.2.5. Acquired Hemophilia A (AHA) and Acquired Von Willebrand Disease (AVWD)

These acquired deficiencies of clotting factors due to the presence of autoantibodies are very rare diseases (incidences of ~1–2 people per million per year) and are often associated with several pathological conditions, such as autoimmune disorders, malignancies, cardiovascular diseases, pregnancy, and drug administration. AHA and AVWD can lead to severe bleeding events which are sometimes life-threatening and can require immediate intervention with anti-hemorrhagic therapy (a factor concentrate or a bypassing agent) and underlying disorder treatment (e.g., immunosuppression) [17].

4.2.6. Hemophilia A and B (HA, HB)

Hemophilia A and B are rare (incidences of ~1 in 5.000 and 1 in 30.000 male births, respectively) X-linked inherited clotting diseases caused by the deficiency of factors VIII and IX; affected patients present a prolonged aPTT and a clinical phenotype with several bleeding symptoms, whether spontaneous (in mild and moderate forms) or provoked by traumas or surgery ^[18][14].

4.2.7. Von Willebrand Disease (VWD)

Von Willebrand Disease is the most common inherited bleeding disorder (affecting up to 1% of the general population) and is characterized by a reduced or abolished synthesis or an altered function of Von Willebrand factor, which plays a role in both primary and secondary hemostasis.

4.2.8. Factor XI Deficiency

Factor XI deficiency has a prevalence of 1 in 1 million persons in most populations and is more prevalent in Ashkenazi Jews and French Basques. Its clinical picture is very heterogeneous, without a correlation between factor level and bleeding symptoms. Patients with a severe disorder are at a higher risk of bleeding, but some of them may remain asymptomatic, and patients with a partial deficiency may bleed after trauma or surgery.

4.2.9. Contact Pathway Factor Deficiency

Deficiencies in high-molecular-weight kininogen, prekallikrein, or factor XII cause a (sometimes remarkable) prolongation of aPTT but cannot provoke bleedings; these disorders are frequently incidentally diagnosed from screening before surgery and are important for differential diagnosis with the other causes of isolated prolongation of aPTT ^[19][15].

5. Isolated, Prolonged aPTT: Differential Diagnosis

How can We Identify the Cause of Isolated, Prolonged aPTT?

First step—possible pre-analytical interferences? First, evaluate the appropriateness of the sample; exclude that the sample is jaundiced, hemolysis, or hyperlipemic. Verify the correct blood/anticoagulant ratio, namely, the adequate sample filling and hematocrit ^[5][6][20][8,9,10]. Second step—presence of anticoagulants? Then, the presence of interfering drugs (heparin and DOAC) should be excluded; performing thrombin time and anti-Xa assays will provide information about the type of drug:

• Heparin will be detected by both tests;
• Direct anti-IIa inhibitor will be detected only via thrombin time;
• Direct anti-Xa inhibitors will be detected only by an anti-Xa assay.

Reptilase time is a functional test based upon the enzymatic activity of a snake venom, Batroxobin, that is able to cleave fibrinogen in a different site compared to thrombin. For this reason, this clotting time is not sensitive to heparin, direct anti-IIa inhibitors, or direct anti-Xa inhibitors. Third step—inhibitor or factor deficiency? In presence of prolonged aPTT, after the exclusion of plasma contamination by anticoagulant drugs, a mixing test should be performed to discriminate whether the prolongation of aPTT is due to the deficiency of one or more coagulation factors (factor XII, prekallikrein, high-molecular-weight kininogen, factor XI, factor IX, or factor VIII) or to the presence of a circulating anticoagulant directed against one or more of the same factors or against membrane phospholipids ^[4][7]. Taking these aspects into consideration, in different situations, based on appropriate clinical–anamnestic considerations, the aPTT assay can be performed on the test mixture immediately or after incubation:

• For an immediate mixing test, the plasma is prepared using equal volumes (1:1) of NPP and patient plasma, and aPTT is performed on it at room temperature ^[4][7]. In parallel, as a control, aPTT is performed on the NPP. Chang et al. ^[21][34] suggested that the aPTT correction in a mixing test with a 4:1 ratio of NPP and patient plasma can achieve better sensitivity and specificity, mostly in the situations in which the antibody power is relatively weak.
• An incubated mixing test is realized by incubating the mixture at 37 °C for 120 min before performing aPTT (and also incubating the NPP and patient plasma separately before performing aPTT as a control) ^[3][22][5,33].

One of the critical points of the mixing test is the interpretation of the results. Two different methods have been proposed:

• The normality range method, in which the aPTT value measured from the mixture must fall within the normal range (a range determined by the laboratory that is effective for the specific combination of the reagent and coagulation analyzer used for the measurement). The advantages of this method are its easiness and immediacy; it has an important diagnostic power in the situations in which the patient plasma aPTT is markedly prolonged.
• The index of circulating anticoagulant (ICA) method, ICA (or Rosner index), is defined by the following formula:

The higher the ICA value, the higher the probability that the mixing will not correct aPTT. The cut-off value is usually fixed at 10–15% ^{[3][22][23][24][25]}[5,32,33,38,39], but it depends on the combination of the reagent/analyzer (which should be determined in every single laboratory). ICA has been demonstrated to be helpful in predicting the presence of lupus anticoagulants with great accuracy ^[25][39].

6. Conclusions

After about seventy years from its first description in medical journals, aPTT is still a milestone in coagulation screening. The modern automated procedures offer prompt and reliable results; in combination with thrombin time and the mixture test, it is essential in the diagnostic workup of many alterations of the intrinsic coagulation pathway.