Journal Reading Arch Pathol Lab Med,

2021

Name : Shofia Widya Murti

Moderator : Leni Lismayanti
Day/Date : Thursday/April, 2022
Department of Clinical Pathology, Faculty of Medicine, Padjajaran University, Dr. Hasan
Sadikin Hospital Bandung

Evaluation of Activated Partial Thromboplastin Time Mixing Studies

Using Several Methods
Liu C., Ling L., Huang X., Mi J., Liao J., et al.

Introduction
Activated partial thromboplastin time (APTT) is a vital screening test for any disease
related to abnormal hemostasis or thrombosis. A prolonged APTT usually results from
deficiency of coagulation factors, the existence of coagulation factor inhibitors, or the
presence of antiphospholipid antibodies (APLs). However, not every hospital has a laboratory
that can run all the tests for coagulation factors, factor inhibitors, and APLs. This can cause a
delayed or even a wrong diagnosis and treatment, which could be life threatening in severe
cases.
Mixing studies of APTT are being optimized to explore the cause of a prolonged APTT.
Patient plasma is mixed with a normal plasma pool (NPP) to see if the prolonged APTT is
corrected. Some studies have demonstrated that a patient plasma:NPP ratio of 4:1 might be
better than a patient plasma:NPP ratio of 1:1 in detecting inhibitors because it gives a better
sensitivity in screening out factor inhibitors or APLs, but there is no consensus when and how
a 4:1 mixing study should be applied. Therefore, this study evaluated each existing standard
and explored when and how to combine the 1:1 and 4:1 mixing studies.

Methods
Patients with a prolonged APTT were enrolled in hospital from January 1, 2018, to
December 31, 2019. All samples were subjected to 1:1 mixing studies, while 134 were
subjected to 4:1 mixing studies. The inclusion criteria were patients with a prolonged APTT,
and patients with normal prothrombin time and thrombin time. The exclusion criteria were
abnormal liver function, anticoagulant medication (including APLs undergoing treatment),
and with any other abnormal coagulation tests (such as prothrombin time and/or thrombin
time). Samples were collected into vacutainer tubes with 3.2% sodium citrate and centrifuged
at 1500 g for 15 minutes to obtain platelet-poor plasma with a platelet count of less than 10 x
103/µL. NPP was from at least 20 healthy individuals. This study was approved by the Ethics
Committee of Sichuan University (Chengdu, China).
A Sysmex CS-5100 coagulation analyzer was used to test APTT, dilute Russell viper
venom time (dRVVT), and coagulation factors. A YHLO iFlash 3000-A chemiluminescence
analyzer was used to test the anti-cardiolipin antibody and anti-β2-glycoprotein I antibody.
This study compared the results between “mixing and then incubation samples” and
“incubation and then mixing samples” in 1:1 and 4:1 mixing study APTT. Factor deficiency
was identified with the extended time, the Rosner index, the percent-extended time, the
percent correction, and the incubated percent correction. Inhibitors were considered when the
patient was excluded from having factor deficiency, and when the extended incubation time
was more than 3, 7, or 10 seconds, the inhibitors were defined as time-dependent inhibitors.
Result
A total of 251 samples were involved, including 116 with factor deficiencies, 75 with
FVIII inhibitors, and 60 with antiphospholipid antibodies. The result of 1:1 Mixing study was
Rosner index less than 11%, the sensitivity and negative predictive value were up to 100%,
the accuracy and specificity value were 71.7% and 47.4%. For the percent extended time less
than 15%, the sensitivity and negative predictive value were 94.8% and 92.7%, the accuracy
and specificity were 74.1% and 56.3%. The cut off value of each existing standard was
calculated by ROC curve with the result Rosner index, extended time, percent-extended time,
and incubated percent correction were 9.1%, 3.3 seconds, 11.5%, and 53.8%. The sensitivity
and specificity of each standard for factor deficiency were 62.5% to 96.6% and 51.9% to
95.7%. It is obviously better to use the appropriate cut off value by the laboratory itself than
the established cut off value. The 1:1 mixing study is better than the 4:1 mixing study in
detecting coagulation factor deficiency, while the 4:1 mixing study is better than the 1:1
mixing study in detecting factor inhibitors or APLs. Therefore, using a combination of the 1:1
and 4:1 is a better choice than using only 1 of the mixing studies alone. The sensitivity of
diagnostic inhibitors by combining the 1:1 and 4:1 mixing studies was increased from 62.2%
to 87.4%, 83.0% to 86.6%, and 73.3% to 86.4% for the Rosner index, percent-extended time
and extended time, respectively, compared with those that used only the 1:1 mixing study.

Discusion
APTT mixing studies are useful in helping to find the specific cause of a prolonged APTT.
APTT mixing studies do not need extra reagents and instruments, and only the APTT test
itself can help to identify the cause of a prolonged APTT. Most of the cutoff values of
diagnostic standards were recommended based on a 1:1 mixing study. This is because 1-part
NPP mixed with 1-part patient plasma can cause the coagulation factor activity in the mix to
be more than 50%, which is enough for a “corrected” result. However, if there are factor
inhibitors or APLs, which can also block coagulation factors from both the patient’s plasma
and NPP, the APTT result “cannot be corrected”. If inhibitors were at low levels, a 1:1
mixing study would give a false “corrected” value and thus an incorrect diagnosis of
coagulation-factor deficiency. The 1:1 dilution by NPP will dilute factor inhibitors or APLs,
which will not be enough to cause a prolonged APTT. Therefore, Chang et al. conducted a
4:1 mixing study to improve the detection sensitivity of factor inhibitors or APLs, as only 1-
part NPP was diluted by 20% with 4 parts of the patient’s plasma. A 4:1 mixing study was
used to improve the detection sensitivity of factor inhibitors or APLs. The sensitivity for
detecting inhibitors was increased by using a combined 1:1 and 4:1 mixing study.
Moreover, approximately 85% of APLs are time-independent anticoagulants because they
interact very quickly with phospholipids, and thus can immediately cause a prolonged APTT;
incubation with APLs for more time will not make the prolonged APTT longer. In contrast,
FVIII inhibitors are time dependent and temperature dependent, and incubation for 1 to 2
hours at 37⁰C can lead to a better interaction between the FVIII inhibitor and FVIII, which
can make the prolonged APTT longer. It is inappropriate to generalize local extended
incubation time, because of the differences in instruments, reagents, and testing conditions
among laboratories.

Conclusion

2
 APTT mixing studies had overall good sensitivity and specificity in differentiating factor
deficiencies from inhibitors, or time-dependent from time-independent inhibitors. The
combination of 1:1 and 4:1 mixing studies can improve the diagnostic ability compared with
1:1 alone.