Mixing studies are tests performed onblood plasma used to distinguishfactor deficiencies from factorinhibitors, such as lupusanticoagulant, or specific factorinhibitors, such as antibodies directedagainst factor VIII.

Mixing studies can help determine theappropriate next steps to take todiagnose the cause of an abnormalAPTT or PT.

Plasma preparation Deficient factors

Fresh normal plasma nil

Plasma from patients on oral anticoagulants for 48-72 hrs

Factor VII,IX

Plasma from patients on oral anticoagulants for a week or more

Factors II,VII,IX,X

Aged plasma Factors V,VIII

Adsorbed plasma Factors II,VII,IX,X

Serum FactorsI,V,VIII

Pooled Plasma - platelet-poor plasma from 20 or

more healthy, male and female adult donors.

DO NOT use a single-sourced normal plasma.

Pooled plasma must be used to ensure approximately

100% of all factors are present.

Quality Control

The pooled plasma must be evaluated for the test to be

performed and results must fall within the reference range

or testing is repeated with a fresh pooled plasma.

The patient plasma is mixed 1:1 with

Normal pooled plasma that contains

100% of the normal factor level results

in a level ≥ 50% in the mixture (say

the patient has an activity of 0%; the

average of 100% + 0% = 50%).

Therefore,

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 (false positive

Nonspecific inhibitors like the lupus anticoagulant usually are not time dependent; the immediate mixture will show prolongation.

Many specific factor inhibitors are time dependent, and the inhibitor will not be detected unless the test is repeated after incubation (factor VIII inhibitors are notorious for this).

Factor deficiency/ abnormality

prothrom

Factor I Yes Yes Yes Yes

Factor II Yes Partial Yes Yes

Factor V Yes Yes No Yes

Factor VII Yes No Yes No

Factor X Yes No Yes Yes

Anticoagulants No No No No

Normal plasma

Adsorbed plasma

Aged serum

Coumarinplasma

Prothrombin time is corrected by mixing with

Factor deficiency/abnormality

Factor VIII yes yes no

Factor IX yes no yes

Factor XI yes yes yes

PTTK corrected by mixing with

Normal plasma Adsorbed plasma

Aged plasma

If the APTT or PT is corrected by normalplasma, a factor deficiency is indicated.

If the APTT or PT is not corrected by theaddition of nor-mal plasma immediately, astrong inhibitor is indicated.

A weak or time-dependent inhibitor isindicated by a prolonged APTT or PTfollowing incubation at 37°C for 1 to 2hours ( factor VIII inhibitor).

Factor deficiency No correction Correction

Immediate acting inhibitor

No correcttion No correction

Mixing study results

Not incubated correction

Incubated correction

Time/temperature dependent inhibitor

Correction(falsely)

No correction