Fundamentals of Mixing studies – PT & APTT

While investigating a patient with bleeding tendency the unexplained prolonged PT and/or APTT can be investigated with correction tests by mixing the patient’s plasma with normal plasma/adsorbed plasma/aged plasma etc.

First we look into Correction using Normal Plasma* (NPP)

Normal pooled plasma = NPP should be prepared from a minimum of 20 normal individuals (equal ratio of male to female), platelet poor (< 10 x 10⁹ /L), tested negative for LA and have coagulation factor activities > 80%.

Correction using Normal Plasma

• Factor Deficiency : PT or APTT of the mixture returns to within a few seconds of normal.
• Inhibitor**: Normal plasma fails to correct the APTT/PT

**There are three types of inhibitors to consider:

• Global inhibitors such as heparin and DTI’s (Direct Thrombin Inhibitors)
• Non-specific inhibitors such as lupus anticoagulants (LAC)
• Specific factor inhibitors (FVIII, FIX, etc.)

Coming onto Interpretation of APTT based mixing studies>>

[1] Prolonged APTT becomes normal after mixing study and stays normal after 2 hours: indicates factor deficiency; perform assays for factors VIII, IX, XI and XII; if PT also prolonged, consider assays for common pathway factors

[2] Prolonged APTT remains prolonged after mixing study: indicates inhibitor.

[3] Prolonged APTT becomes normal after mixing study, but prolonged after 1 – 2 hour incubation: Indicates “time dependent inhibitor” eg factor VIII inhibitor, rarely factor V inhibitor (Some inhibitors are time dependent. The clotting test performed immediately after the specimens are mixed may show correction because the antibody has not had time to inactivate the added factor however after incubation of 2 hours inhibitor has adequate time for action and hence APTT is prolonged after 1-2 hours of incubation.

• Mixing tests may be misleading in particular circumstances: Some lupus-like anticoagulants are relatively weak and may only be apparent if 25:75 mixes of normal and test plasma are used instead of 50:50 mixture. 

When we say that the PT or APTT has been corrected by Mixing study or not?

The are a number of different methods that can be used for determining if a mixing study has corrected. Some labs use a correction of anywhere from 2-5 seconds of the upper limit of the PTT normal range.

Others compare the PTT mix result with the PTT of the pooled normal plasma (PNP).

There are standardized methods in literature that are described below:

eg Rosner Index: This method involves subtracting the clotting time of the pooled normal plasma (PNP) from the clotting time of the 1:1 mix. Divide this result by the clotting time of the patient sample. (see example below)

Interpretation:

1. A high index value (~15) represents no correction (possible inhibitor).
2. A low index value (~10) would reflect a correction of a possible factor deficiency.
3. Index values falling within (10-15) should be repeated.

Other method for interpretation: Percent Correction Formula: Chang Percentage.

What is adsorbed plasma? How to interpret mixing studies using adsorbed plasma? 

Adsorbed plasma is used for mixing studies in coagulation disorders testing laboratory.

Barium sulphate adsorbs clotting factors II, VII, IX, and X and hence adsorbed plasma is deficient in clotting factors II, VII, IX, and X.

• When PT and/or APTT is performed on test plasma deficient of any of factor II, VII, IX, X these tests show prolonged results.
• When these tests are repeated after mixing Adsorbed plasma with this test plasma, PT and/or APTT are still prolonged i.e. not corrected, because adsorbed plasma has not any ability to correct the deficiency.
• If the test plasma is deficient in factors other than factor II, VII, IX, and X, on mixing with adsorbed plasma, it will replenish those factors from adsorbed plasma, thus the tests will be said to be corrected.

What is aged plasma? Aged plasma is deficient in factor V and factor VIII. Aged serum is prepared by incubating normal serum for 24 hours at 37°C.

The image below shows different types of plasma that can be used in mixing studies and specific factor/s in which they are deficient.

In absence of single specific factor deficient plasma a correction test using adsorbed plasma and or aged plasma can be done to zero down the probable factor deficiency as described in image below.

Lets see an example now?

A 10 years old boy developed Haemarthrosis of left hip joint with a history of recurrent swelling and pain in multiple joints and prolonged bleeding following minor trauma since childhood. Subsequent investigations revealed

PLT-250000/micoL, PT-13s, APTT-75s

• Mixing study with Normal pooled plasma > Corrected
• Mixing study with Adsorbed plasma > Not corrected
• Mixing study with aged plasma> Corrected.

What is your Diagnosis?

Answer = Factor IX deficiency (Hemophilia B)

As explained in table adsorbed plasma is deficient in II, VII, IX and X. Isolated APTT prolongation will be seen in factor IX deficiency among these and Mixing study with Adsorbed plasma will show no correction.

Now solve this MCQ based Q: Click here for Q