Mixing Studies

Prolongation of a coagulation test may be as a result of a deficiency in coagulation factors or because of the presence of in inhibitor of coagulation. Mixing studies are performed to differentiate between the two.

Principle of Mixing Studies

Inhibitors affecting activated partial prothrombin time (aPTT) are more common in clinical practice and the discussion that follows is in relation to aPTT. A mixing study with prothrombin time (PT) may be performed using the same principle. Prolongation of aPTT indicates decrease in the coagulation factors the test is dependent on. aPTT prolongs when the factor levels fall to less than 40% of normal.

When the test plasma is mixed with normal pooled plasma in a 1:1 ratio the factor levels are and average of that in the two samples. The coagulation factor levels in pooled plasma are close approximately 100%. Patients with severe coagulation factor deficiency typically have <1% of normal coagulation factors levels. When plasma from such patients is mixed with normal pooled plasma the factor levels in the plasma are will be 50%. For patients with a lesser degree of coagulation factor deficiency the coagulation factor levels in mixed plasma will be greater than 50%. As a normal aPTT needs 40-50% coagulation factor activity and mixing a factor deficient plasma with normal plasma  would result in normalization of a prolong aPTT even in the most severe form of coagulation factor deficiency.

Inhibitors of coagulation are antibodies directed against coagulation factors. Presence of the antibody is associated with diminished coagulation factor activity and a prolonged aPTT.  When the plasma from a patient carrying an inhibitor is mixed with equal amount of normal plasma the inhibitor present in the patients plasma inhibits the activity of the coagulation factors present in pooled plasma. The prolonged aPTT of patient’s plasma fails to correct because the activity of the mixture falls to below that needed for a normal aPTT. Some antibodies act with a lag period. The aPTT of these patients may correct immediately on mixing but will prolong after 2 hours of incubation.

 

Performance of the Mixing studies

Mixing studies involves performance of duplicate aPTT on the following samples

• Tube 1: Pooled plasma – Plasma from at least 20 normal individuals
• Tube 2: Patients plasma
• Tube 3: Patients plasma and pooled plasma incubated separately for 2 hours at 37°C. The two are mixed and the mixture immediately tested.
• Tube 4: Patients plasma and pooled plasma mixed and incubated for 2 hours at 37°C

 

 

Interpretation of Mixing Studies

Diagnosis	Normal Plasma (Tube 1)	Test Plasma (Tube 2)	Incubated separately and mixed (tube 3)	Mixed and Incubated (tube 4)
Normal	Normal	Normal	Normal	Normal
Immediate Inhibitor	Normal	Prolonged	Prolonged	Prolonged
Delayed inhibitor	Normal	Prolonged	Minimally prolonged if at all	Prolongrd