Interpreting Mixing Studies – Hematology Highyield Concept I

Approaching Prolonged PTT and understanding Mixing Studies : 

 

ARCHER HEMATOLOGY REVIEW

Question :  On a mixing study, the PTT corrects initially but gets prolonged again after incubation for 2 hours. What is this condition?

This description of mixing studies is consistent with presence of a temperature- and time-dependent anti-VIII inhibitor. It just means that the inhibitor is a warm reacting IgG antibody that requires one or two hour’s incubation at 37°C to be detected. If the PTT corrects initially but prolongs again after incubation (meaning if thePTT is at least 15% longer than the normal reagent plasma’s incubated PTT ) –> anti-VIII should be suspected. This can be confirmed by obtaining a Factor VIII level which will be low in case of presence of inhibitor. Further coinfirmation of Factor VIII inhibitor can be obtained by Bethesda Titer. In such cases, you should also carefully review patient’s prior bleeding history – because patients with significant inhibitor will have prior bleeding history. 

 

Discussion : 

 

All about Mixing Studies :  

 Mixing studies involve mixing patient’s plasma with equal amount of normal plasma. If the PTT immediately corrects ( that is if mixture yields within 10% of normal value) – it indicates Factor Deficiency ( example : VIII, IX, XI, XII) . If the PTT does not correct – it means an inhibitor is present. Such an inhibitor is most likely to be Lupus anticoagulant (LA) if there is no bleeding history. Factor VIII inhibitor is also possible but such presence of factor VIII inhibitor would be associated with significant history of bleeding which will help you differentiate from LA . If the PTT corrects initially but prolongs again after 1 to 2 hr incubation, it means a Factor VIII inhibitor is present.

 

Approaching LA diagnosis :

After you know that there is possible LA on mixing studies, you should proceed with confirmatory tests for LA. At least Two test systems are necessary to confirm LA – to avoid false negative tests because LAs are so heterogeneous . PTT based tests can be used. Dilute Russell Viper Venom tests are sensitive and quite specific for LAs. So, Lack of correction in mixing studies of either abnormal test is presumptive evidence for LA. These results can be further followed with neutralization studies using high phospholipid reagents. Correction by the neutralizing reagents confirms the presence of LAC.Remember that Lupus anticoagulants are partof a family of antibodies called antiphospholipid antibodies. These are also detected using a series of immunoassays:· Anticardiolipin IgG, IgM, orIgA antibody· Anti-b2 glycoprotein I IgGor IgM antibody. 

 

Approaching Factor VIII inhibitor Diagnosis:

Once the mixing study corrects initially but prolongs again after 1 to 2 hour incubation such a result is more consistent with Factor VIII inhibitor ( acquired Factor VIII inhibitor). You should obtain a careful history regarding previous bleeding from the patient. Alternatively, acquired factor VIII inhibitors can develop transiently after pregnancy and/ or major surgeries and such patients will not have any history of bleeding. Also, obtain a Factor VIII level which will be low in case of presence of inhibitor. Further confirmation and levels of of Factor VIII inhibitor can be obtained by Bethesda Titer. If these patients have high Factor VIII titer and have significant bleeding, you can not treat them with Factor VIII replacement because remember the inhibitor is an antibody and giving factor viii may further induce antibody response and can be threatening. If significant titers of Factor VIII inhibitor are present and if the patient is bleeding , you must use FEBA ( Factor EIGHT BYPASS ACTIVITY) or Factor VII to treat this patient’s acute bleeding .  

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