Mixing Studies: A Key Tool in Diagnosing Coagulation Disorders Associated with Prolonged PT and PTT

Mixing Studies: A Key Tool in Diagnosing Coagulation Disorders Associated with Prolonged PT and PTT

Prothrombin Time (PT) and Partial Thromboplastin Time (PTT) are two cornerstone tests in the evaluation of the blood coagulation system, assessing the extrinsic/common and intrinsic/common pathways, respectively. Abnormal prolongation of these times may indicate various disorders, including deficiencies in coagulation factors, the presence of inhibitors, or conditions affecting hemostasis. In such cases, Mixing Studies emerge as a critical diagnostic tool. By combining patient plasma with normal plasma, these studies help differentiate the underlying causes of prolonged PT and PTT, determining whether the issue stems from a deficiency in coagulation factors or the presence of inhibitors, such as antibodies against specific factors or lupus anticoagulant.

Types of Coagulation Inhibitors

Coagulation inhibitors are classified into two main categories:

1. Inhibitors of Coagulation Factors (e.g., antibodies against Factor VIII or IX)
2. Lupus Anticoagulant (antiphospholipid antibodies)

Methodology of Mixing Studies

To perform Mixing Studies, equal volumes of patient plasma and normal pooled plasma (NPP) are typically combined, and PT and PTT tests are repeated. These tests are conducted at two time points:

1. Time Zero (immediately after mixing patient plasma with NPP)
2. Time 120 Minutes (incubation at 37°C)

Since a factor level of 25–40% is sufficient to normalize PT and PTT, even if the patient’s factor level is zero, mixing with NPP to achieve approximately 50% can result in normal test outcomes. Normal pooled plasma should be prepared from the plasma of at least six healthy individuals (a mix of males and females).

Interpretation of Mixing Study Results

Following the Mixing Studies:

• If the PT and PTT of the mixture normalize at both zero and 120 minutes, or deviate by no more than 5 seconds above the control value (laboratory normal PT/PTT), they are considered “corrected.”
• However, if the PT and PTT of the mixture remain prolonged by 6 seconds or more above the control value at both time points, they are deemed “uncorrected.”

To enhance test sensitivity, a 1:4 ratio (Mix PT/PTT 1:4) can be employed, where 4 volumes of patient plasma are mixed with 1 volume of normal plasma. This ratio is particularly useful when a stronger inhibitor is suspected.

Determining the Cause of Prolonged PT and PTT

Based on the Mixing Study results, the cause of prolonged PT and PTT can be elucidated:

1. Correction at Both Zero and 120 Minutes: Suggests a likely deficiency in one or more coagulation factors, ruling out the presence of inhibitors.
2. Prolongation at Both Zero and 120 Minutes: Raises suspicion of lupus anticoagulant (antiphospholipid antibodies). In this scenario, adding phospholipids neutralizes the antibody, leading to correction of PT/PTT.
3. Correction at Zero Minutes but Prolongation at 120 Minutes: Indicates the presence of an inhibitor (antibody) against coagulation factors. Prolonged incubation enhances the antibody’s effect, neutralizing the coagulation factors in the normal plasma.

Summary Table of Results

Next Steps

If a factor deficiency is suspected, Factor Assays should be performed for both the intrinsic pathway (related to PTT) and the extrinsic pathway (related to PT) to identify the specific deficient factor. Additionally, if lupus anticoagulant is suspected, confirmatory tests such as the Dilute Russell Viper Venom Time (dRVVT) are recommended.