Dear George,
What is the effect of a frozen sample and frozen normal pooled plasma on factor VIII inhibitors. Is fresh normal pooled plasma a good option? Please comment.
Vilas Hiremath, Bangalore India
Hello, Vilas Hiremath, and thank you for your question. Frozen reagent pooled platelet poor plasma, such as Precision BioLogic’s CryoCheck Normal Reference Plasma is assayed for coagulation factor activity levels, including coagulation factor VIII. Provided the material is managed in accordance with the manufacturer’s instructions, it is reliable. Likewise, you can freeze test plasma specimens and thaw prior to performing a Bethesda titer without inducing deterioration of the suspected anti-factor VIII inhibitor.
There are two important principles for managing frozen plasma. First, it must be platelet-poor prior to freezing, which means it must be centrifuged so that the plasma platelet count is less than 10,000/mcL. Many manufacturers prepare their pooled normal plasma so that the platelet count is less than 5,000/mcL, and laboratory scientists typically double-spin their plasmas to bring their platelet count down. Second, frozen plasma can be thawed only once. Repeated freeze-thawing causes deterioration of factor VIII and von Willebrand factor.
Frozen reagent pooled normal plasma from distributors is assayed for accurate coagulation factor activity levels and tested for known plasma-borne pathogens. It incorporates plasma from at least 20 carefully selected healthy donors.
Fresh plasma from local donors costs less, of course, but because von Willebrand factor, factor V, and factor VIII levels deteriorate within a few hours, even at 4° to 6º C, it must be used immediately, limiting the possibility of pooling. There is also not enough time to verify the absence of infectious agents with serological screens.
Many local laboratories choose to prepare in-house pooled plasma. These laboratories freeze their plasmas, assay them for coagulation factor activity, and screen for infectious agents prior to using them, paralleling the process followed by reagent plasma distributors. I hope this discussion is helpful. Geo.
There is nothing wrong with using either frozen or fresh poo
There is nothing wrong with using either frozen or fresh pooled plasma per se for inhibitor assays. The important points are to make sure that the pool is reasonably large enough to normalise factor levels to as close to 100% as possible. Laboratory test samples, even those providing normal routine coagulation times, can individually vary greatly in individual factors such as FVIII. You do not only need to consider the effect of deterioration but also acute phase elevation. This needs to be considered when pooling of patient normal samples for potential use in inhibitor assays (factor inhibitors and lupus inhibitors). As noted previously by George, freezing requires consideration for removing platelets by double centrifugation. As FVIII is labile, freeze/thaws of normal pools should be limited, and so all frozen pools should be frozen in aliquots for single use and then discarded. FVIII inhibitors per se are fairly resistant to freeze/thaw events, so these samples can be tested fresh or frozen.
Recommended references:
1. Verbruggen B, van Heerde WL, Laros-van Gorkom BA. Improvements in factor VIII inhibitor detection: From Bethesda to Nijmegen. Semin Thromb Hemost. 2009 Nov;35(8):752-9.
2. Kershaw G, Jayakodi D, Dunkley S. Laboratory identification of factor
inhibitors: the perspective of a large tertiary hemophilia center. Semin Thromb Hemost. 2009 Nov;35(8):760-8.