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Does Actin FS Introduce a FVIII or FIX Assay Bias?

Here is an interesting question about Actin FS sensitivity for coagulation factors VIII and IX from Vanessa Chan at SickKids Hospital in Toronto:

I’m currently working on the validation of the Stago Sta-R Evolution. Throughout this process, I have run into a couple of problems with my FVIII and FIX assays. Using Siemens Actin FS, Stago Unical calibrator and Stago Deficient Plasma, I was finding a high bias for both factors starting at the 50% and up range. The folks at Stago sent me this article: Pouplard C, Trossaert M, Le Querrec LE, et al, Influence of source of phospholipids for APTT-based factor IX assays and potential consequence for the diagnosis of mild haemophilia B. Letter to the Editor, Haemophilia 2009; 15: 365–8. The authors concluded that Actin FS showed a high bias for factor IX compared to Stago CK Prest and Stago PTTA. The bias was limited to mild hemophilia B patients, those whose FIX levels were between 5 and 40 IU/dL, and to qualitative (type II) deficiencies in which immunoassay and activity results were discrepant.

Because all the trouble-shooting we tried did not solve this problem, we switched to CK Prest and so far (only one week), it has worked well. We ran the NIBSC standard on it and it recovered well. I know other labs use Actin FS as well but perhaps not in the exact combination of reagent, calibrator and control and have had problems. I was wondering if you know anything about matrix or pH interferences between reagents or lyophilized vs. frozen products.

Thank you in advance, George and I look forward to hearing from you.

Hi, Vanessa, thank you for your question. I confess, you drove me back to the books and also to check with several colleagues. I have a partial answer, and I hope to have some response to this post from Siemens experts who are members of the blog. Actin FS is, as you saw from the Pouplard article, a synthetic reagent whose phospholipids are produced from soy rather than the more traditional rabbit brain source. Its composition of fatty acids differs from the others, with a higher ratio of unsaturated to saturated acids and a lower concentration of phosphatidyl serine. This may somehow account for Actin FS having higher sensitivity to qualitatively abnormal (mutated) FIX.

The article only addresses FIX, not FVIII, and indicates the biased results only occur in qualitative FIX deficiency. Actin FS in this article compares closely to the two Stago reagents in hemophilia B sufferers with quantitative deficiencies or those with moderate or severe deficiency. I reviewed some FVIII data from recent CAP surveys and found no discrepancy between Actin FS and the other reported APTT reagents. Also a 2003 study, Shetty S, Ghosh K, Mohanty D. Comparison of four commercially available activated partial thromboplastin time reagents using a semi-automated coagulometer. Blood Coagul Fibrinolysis 2003;14:493–7, compared Actin FSL (slightly different) to three other reagents including CK Prest and found the four reagents matched well in measuring mild FVIII and FIX deficiency. So, the story is not complete.

I suspect you’ve done the right thing by switching to the Stago reagent, which matches up with Stago’s STA-R Evolution analyzer, and you may also want to review the effects of frozen versus lyophilized factor-deficient plasmas to learn if the discrepancy you are seeing relates to their matrix.

I hope to get responses from some others who are using the Siemens reagents, and please keep us informed as you proceed with your instrument validation. Geo.

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