In the recommendations of ISTH, frozen samples for AT testing can be stored for up to 24 months at ≤-24 °C [4]. In PC tests, frozen samples can be stored below − 20 °C for up to 2 weeks or up to 18 months below − 70 °C [5]. The same as PC tests, samples for PS tests can be frozen below − 20 °C for up to 2 weeks or up to 18 months below − 70 °C [6]. However, the recommendation does not indicate whether this stability is for antigen assay or activity assays. In part of clinical laboratories in China, the frequency of AT, PC, and PS activity tests was to be 2–3 times a week. So, the plasma will be packaged and frozen first for centralized testing.

In our study, four evaluation methods were used to assess the effect of storage time and temperature on the activity of antithrombin, protein C, and protein S.

According to our research, the percentage deviation of AT and PC activity assay was<10%. It can be seen that the activity of AT and PC was stable when stored at -20 °C or -80 °C for at least a week. However, the activity of PS was unstable in frozen plasma. The results showed significant negative bias, the percentage deviations of PS activity assay were all > 10%.

TCL had been calculated to assess the stability of common biochemical analytes [11,12,13], which takes into account the acceptable imprecision based on the analytical imprecision of the assay and the within-subject variation. In our study, interestingly, although PS activity was considered unstable, they did not exceed the TCL. We were considering it was associated with the analytical imprecision of PS assay. From a research by Mark T. Cunningham et al. [14], the all-method accuracy and precision of PS assays were lower than AT, and PC assays in laboratories in United States. The causes of lower accuracy and precision of PS assay, especially PS activity assay may be related to its reagents and detection method.

When we used repeated measures ANOVA to find whether there was a statistical difference between the baseline and frozen-thawed results, most values of AT and PC frozen samples had statistic differences at P < 0.05 compared to baseline. The results of PS activity were of course the same. We considered that this method was not suitable for the stability evaluation of coagulation parameters.

Furthermore, 6.9% (2/29) tests using ACL TOP 750 and 27.6% (8/29) tests using STA Compact Max had classification changes in frozen plasma. Their baseline PS values were within the reference range, but were below the limit in some or all of the storage conditions. In this case, if PS activity is postponed, and samples are frozen after acceptance by laboratory in advance will result in a false-reduction of PS activity and overdiagnosis of PS deficiency.

The recommendations for clinical laboratory testing for PS deficiency of ISTH has emphasized that activity assays may result in overdiagnosis of PS deficiency, as PS activity assays have a higher potential to generate falsely decreased PS values than the antigenic-based assays [6]. While the PS activity assay is sensitive in identifying all types of inherited PS deficiency, both qualitative and quantitative. However, activity assay has a specificity of only 40–70% [6, 15, 16]. Therefore, despite the possibility of the free PS antigen assay missing the diagnosis of type II PS deficiencies, the recommended initial assay for PS deficiency should be the free PS antigen assay rather than PS activity assays. Currently, there is a limited availability of registration certificates for free PS antigen reagents in the Chinese market, and the screening test for protein S deficiency is replaced by PS activity assays.

In previous studies, the effect on the results varied due to differences in freezing conditions and duration. In a former study by Woodhams et al [17], PS assay can achieve a ≤ ± 5% variation for 8 and 6 months when stored at -74 °C and − 24 °C, respectively. In the study of Zander [18], Free protein S showed slightly decrease when stored at ≤-20 °C, ≤-80 °C, ≤-130 °C for 7 days. Gupta et al. study confirmed that AT, PC and free PS all declined significantly after a fortnight freezing at -25 °C [19]. After a 24 h freezing, only free PS observed a slightly decline [19]. Data comparing differences between PS activity assays and PS antigen assays are lacking. A previous study revealed that PS activity and free PS increased during the first week after freezing, moreover PS activity decreased after 2 month [20]. Our study confirmed that PS activity decreased significantly after only 3 days of freezing storage. In conclusion, PS is labile in cryopreservation, especially the activity assay. To obtain the most accurate results possible, the test should be performed as soon as possible after blood collection by venipuncture, and it is inappropriate to use frozen specimens for the test.

There were also several limitations in our study. Firstly, our sample size is small and the duration of storage is short in our study. However the storage duration in the design can cover the needs of clinical laboratories. Secondly, we weren’t able to test free PS antigen in parallel to analyze this reduction whether qualitative or quantitative.