Mixing Studies

Mixing Studies
Aug 6, 2020 7:47pm

A note from colleague Kathy Robinson that deserves a thorough discussion...

"I am updating our mixing study procedure and have some questions..."

1. What is the lower limit at which it is useless to perform studies for both PT & PTT [APTT ]?

There is no firm agreement about the limit. Some perform mixes whenever the PT or PTT exceeds the upper limit of the reference interval, others choose upper limit plus a selected number of seconds such as 5 seconds for the PTT , and some consult with the physician to learn if the patient has symptoms of either bleeding or thrombosis before proceeding with the mix.


2, At what range is it corrected? I have recently seen 5s from the upper reference range or 80%.

Some base the correction/no correction on the upper limit of the reference interval, or a percentage over the upper limit, for example 10%. So if the upper limit of the PTT reference interval was 35 seconds, the limit would be 38.5 seconds. Others base the limit on a percentage over the validated normal plasma value, so if the normal plasma returns 30 seconds, the limit would be 33 seconds. I recommend the second approach, as the value of the normal plasma may vary under certain conditions, for instance, incubation, so the limit reflects the conditions. Others choose to use the Rosner, where they set a limit of 10 or 11, or Chang index, where they set a limit of 75%, as explained in the attached PowerPoint, below.


3. If after incubation, you run the patient, normal plasma, and the 1:1 mix, should you also analyze an equal mixture of patient and normal plasma that is combined after incubation? If so, how is that reported?

If you use a simple percentage over the normal plasma value or the mean of the reference interval, you typically just incubate the 1:1 mix and the normal plasma and proceed as you would in the immediate mix. The Rosner and Chang formulas require you also incubate a patient aliquot, as its value after incubation enters the formula. Fewer people use these indices than use simple percentages. The formulas provide greater accuracy, but how much analytical accuracy can you expect from a mixing study?


4. Do most labs also perform a 1:1 patient/saline mix?

No , but my colleague Flo Newlin, technical director of Colorado Coagulation Consultants until she retired a few years ago, swears by the saline mix. In a handbook we prepared and last printed in 2002, she writes, "If the result from the immediate patient/normal plasma mix corrects and the saline mix is markedly prolonged, suspect a factor deficiency or a specific factor inhibitor. If the result from the immediate patient/normal plasma mix does not correct and the saline mix corrects or is only slightly prolonged, suspect a non-specific inhibitor such as lupus anticoagulant." I know of no laboratory director who currently uses the saline mix, but it may speed up the diagnostic process.

Lastly, click here for a PowerPoint presentation I prepared for Precision BioLogic in 2017 that provides case-based details on mixing studies.

3 Comments

A note from colleague Kathy Robinson that deserves a thorough discussion...

"I am updating our mixing study procedure and have some questions..."

1. What is the lower limit at which it is useless to perform studies for both PT & PTT [APTT ]?

There is no firm agreement about the limit. Some perform mixes whenever the PT or PTT exceeds the upper limit of the reference interval, others choose upper limit plus a selected number of seconds such as 5 seconds for the PTT , and some consult with the physician to learn if the patient has symptoms of either bleeding or thrombosis before proceeding with the mix.


2, At what range is it corrected? I have recently seen 5s from the upper reference range or 80%.

Some base the correction/no correction on the upper limit of the reference interval, or a percentage over the upper limit, for example 10%. So if the upper limit of the PTT reference interval was 35 seconds, the limit would be 38.5 seconds. Others base the limit on a percentage over the validated normal plasma value, so if the normal plasma returns 30 seconds, the limit would be 33 seconds. I recommend the second approach, as the value of the normal plasma may vary under certain conditions, for instance, incubation, so the limit reflects the conditions. Others choose to use the Rosner, where they set a limit of 10 or 11, or Chang index, where they set a limit of 75%, as explained in the attached PowerPoint, below.


3. If after incubation, you run the patient, normal plasma, and the 1:1 mix, should you also analyze an equal mixture of patient and normal plasma that is combined after incubation? If so, how is that reported?

If you use a simple percentage over the normal plasma value or the mean of the reference interval, you typically just incubate the 1:1 mix and the normal plasma and proceed as you would in the immediate mix. The Rosner and Chang formulas require you also incubate a patient aliquot, as its value after incubation enters the formula. Fewer people use these indices than use simple percentages. The formulas provide greater accuracy, but how much analytical accuracy can you expect from a mixing study?


4. Do most labs also perform a 1:1 patient/saline mix?

No , but my colleague Flo Newlin, technical director of Colorado Coagulation Consultants until she retired a few years ago, swears by the saline mix. In a handbook we prepared and last printed in 2002, she writes, "If the result from the immediate patient/normal plasma mix corrects and the saline mix is markedly prolonged, suspect a factor deficiency or a specific factor inhibitor. If the result from the immediate patient/normal plasma mix does not correct and the saline mix corrects or is only slightly prolonged, suspect a non-specific inhibitor such as lupus anticoagulant." I know of no laboratory director who currently uses the saline mix, but it may speed up the diagnostic process.

Lastly, click here for a PowerPoint presentation I prepared for Precision BioLogic in 2017 that provides case-based details on mixing studies.

By George Fritsma
Aug 6, 2020 7:57pm
Thank you for your question, Kathy!
By Coag Research Dave McGlasson
Aug 9, 2020 12:32pm
George, are any laboratories performing the 4:1 mixing study? At one time it was thought to be the most sensitive for picking up low positive LA presence. Thoughts?
I don't know of any currently, I think the tendency is to employ LAC sensitive reagents.
By Dr Emmanuel Favaloro
Aug 16, 2020 10:05pm
The topic of mixing studies is perennially raised. I have 2 papers specifically in the area that I highly recommend [1,2]. The take home messages: 1. No process or formula is 100% correct; 2. Most processes or formulas have similar levels of ‘correctness’ providing the selected cut-offs are appropriate – this includes ‘% correction’ (‘Chang method’), ‘index of circulating anticoagulant’ (‘Rosner index’), or correction into the reference range. Our lab now uses correction into the reference range, since it was easiest to develop within our expert auto-validation rules [3], and also aligns to the CLSI guidance on LA [4]. For this purpose, the initial prolonged result needs to be a few seconds above the upper limit of the normal reference range (NRR) to be effective. This is since slight prolongations above the NRR will invariably deliver an interpretation of ‘correction’, even if correction can be questioned. For example, an APTT of 40 sec for a sample with a NRR of 24-38 sec will likely correct into the NRR irrespective of an inhibitor (perhaps yielding 37s for a weak inhibitor or LA, or 32s for a factor deficiency). We have settled on an APTT 5s above the NRR for triggering a mix with our new version of our rules [3]. We don’t generally perform incubated mixing tests, as this is difficult to manage across our large lab network, instead simply reflexing to factor assays (especially FVIII) if there is a suggestion of a temperature dependent inhibitor based on clinical information. We only perform 1:1 mixes, since other mix formulas are also difficult to manage in a lab network. Irrespective, labs need to verify whatever they decide to do, and whichever process they decide to adopt.

References

1: Kershaw G. Performance and Interpretation of Mixing Tests in Coagulation. Methods Mol Biol. 2017;1646:85-90. doi: 10.1007/978-1-4939-7196-1_6. PMID:28804820.

2: Kershaw G, Orellana D. Mixing tests: diagnostic aides in the investigation of prolonged prothrombin times and activated partial thromboplastin times. Semin Thromb Hemost. 2013 Apr;39(3):283-90. doi: 10.1055/s-0033-1336832. PMID: 23457048.

3. Mohammed S, Ule Priebbenow V, Pasalic L, Favaloro EJ. Development and implementation of an expert rule set for automated reflex testing and validation of routine coagulation tests in a large pathology network. Int J Lab Hematol. 2019 Oct;41(5):642-649. doi: 10.1111/ijlh.13078. PMID: 31271498.

4. Clinical and Laboratory Standards Institute (CLSI). Laboratory Testing for the Lupus Anticoagulant; Approved Guideline. CLSI document H60-A. Wayne, PA: CLSI, 2014.

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