How does an Activated Partial Thromboplastin Time (APTT) mixing study work?

How APTT Mixing Studies Work

APTT mixing studies are diagnostic tests that distinguish between coagulation factor deficiencies and the presence of inhibitors (such as lupus anticoagulant or factor-specific inhibitors) when a patient has a prolonged activated partial thromboplastin time (APTT). 1

Basic Principle and Methodology

• APTT mixing studies involve mixing patient plasma with normal plasma (typically in a 1:1 ratio) and measuring the APTT of this mixture 1
• The test is essentially a modified APTT mixing study using 50% patient plasma and 50% normal plasma 1
• The mixing study includes three key components: an activator, phospholipids, and calcium, which are the same components used in standard APTT reagents 1

Interpretation Process

Immediate vs. Incubated Testing

• Mixing studies are performed both immediately and after incubation (typically 1-2 hours at 37°C) 1
• This time-dependent aspect is crucial because certain inhibitors, particularly factor VIII inhibitors, are time and temperature-dependent 1
• Prolongation of the APTT in the mixture after incubation compared to the immediate mix is typical of factor VIII autoantibodies 1

Expected Results

• Factor deficiency pattern: The APTT will correct (normalize) when mixed with normal plasma, as the normal plasma provides the missing factors 1
• Inhibitor pattern: The APTT will remain prolonged or show incomplete correction, as the inhibitor in the patient's plasma interferes with the coagulation factors in the normal plasma 1
• Time-dependent inhibitors: Show greater prolongation after incubation compared to immediate testing 1

Interpretation Methods

Several methods exist for interpreting mixing study results:

• Normal range method: Simply determines if the mixed sample's APTT falls within the laboratory's normal range 2
• Percentage correction: Calculates how much the mixing corrected the prolonged APTT 2, 3
• Rosner index (Index of Circulating Anticoagulant): A formula that quantifies the degree of correction, with values >11% suggesting an inhibitor 3, 4
• Estimated Factor Correction (EFC) method: A newer approach that predicts factor levels in the sample and determines the expected correction needed 2

Clinical Applications

• Differentiating between factor deficiencies and inhibitors when a patient presents with prolonged APTT 1
• Diagnosing specific conditions like Acquired Hemophilia A (characterized by factor VIII inhibitors) 1
• Detecting lupus anticoagulant, which is important in diagnosing antiphospholipid syndrome 1
• Guiding further diagnostic testing and treatment decisions 5

Advanced Techniques and Considerations

• Some laboratories use derivative curves on automated analyzers to enhance interpretation 6
• In complex cases, a 4:1 (patient:normal plasma) mixing ratio may provide additional diagnostic information 3
• For patients with suspected lupus anticoagulant, performing both APTT and dilute Russell's viper venom time (dRVVT) tests is recommended for optimal detection 1

Potential Pitfalls and Limitations

• Immediate correction of APTT with normal plasma does not completely exclude inhibitors, especially in patients with suggestive clinical presentations 1
• Anticoagulant therapy (particularly heparin, direct oral anticoagulants, or vitamin K antagonists) can interfere with mixing study results 1
• Lupus anticoagulant can cause artifactual lowering of factor levels due to inhibition of phospholipid in the assay 1
• Multiple factor deficiencies may complicate interpretation and require different thresholds for defining correction 2

Follow-up Testing

• Regardless of mixing study results, specific factor assays should be performed to facilitate accurate diagnosis 1
• For suspected inhibitors, quantification using assays like the Bethesda assay may be necessary 1
• In cases of suspected lupus anticoagulant, specific confirmatory tests should be performed 1