Tiffany Jones at Orlando Health writes: “Hi, George! We are trying to update our VWF disease panel and we use the VWF:RCo plasma testing on our Stagos using Siemens reagent. When researching other algorithms I noticed there was VWF:Ac as well. Can you help clarify to me if both these tests are VWF activity assays? If yes, besides the methodology of testing, what is the difference or are they saying the same thing?”
Hi, Tiffany, Dr. Jeanine Walenga and I are busy working on the upcoming sixth edition of Rodak’s Hematology: Clinical Principles and Applications, so we’ve recently pulled together a description of VWF testing methods. From the top, a VWD profile must include assays for VWF antigen, factor VIII activity, and VWF activity. The VWF antigen assay, VWF:Ag, is a quantitative immunoassay that employs a polyclonal antibody. VWF:Ag is analytically sensitive and reproducible, though it may miss a qualitative type 2 form of VWD. The factor VIII assay is the same clot-based assay we use to diagnose and monitor treatment of hemophilia A, which offers CVs of 20–30%, but is not necessarily definitive for VWD. Thirdly, we assay for VWF activity (VWF:Ac) using at least four available methods. These are…
- The ristocetin cofactor assay, VWF:RCo: the oldest and currently the most commonly used assay, We mix patient plasma with formalinized normal donor reagent platelets and add ristocetin, which enables patient VWF to bind platelet membrane GPIb receptor sites. The VWF:RCo only returns VWF activity levels > 10% and the results are semiquantitative with CV levels of 30%. Siemens Healthcare Diagnostics, Inc., Deerfield, IL, USA offers an automatable VWF:RCo reagent, the method that Tiffany has adapted to her Stago equipment.
An automated ristocetin-triggered non-platelet recombinant GPIb-based ligand immunoassay (LIA) or chemiluminescence immunoassay (CLIA) method, VWF:GPIbR, such as the HemosIL AcuStar VWF:GPIbR (IL-Werfen, Bedford, MA, USA). This assay relies on ristocetin, but is a quantitative immunoassay designed to measure VWF activity by its ability to bind GPIb. Both the LIA and CLIA methods provide analytical sensitivity below 10% and CV levels < 10%.
A new assay that incorporates a gain-of-function high affinity recombinant GPIb protein that resembles the GPIb of PT–VWD platelets, VWF:GPIbM. The GPIb binds patient VWF without the need for ristocetin. This is a LIA offered by Siemens Healthcare Diagnostics, Inc., Deerfield, IL, USA, and it appears to improve on ristocetin-based assays as it offers lower VWF detection limits and better reproducibility.
The VWF collagen binding assay, VWF:CB. This assay, available from the early 1990s, employs a carefully prepared mixture of types I and III collagen that is affixed to the microtiter plate wells. VWF binding is detected using enzyme immunoassay technology, yealding a CV of 10% and lower detection limit of 1–4%. The VWF:CB may be used in conjunction with one of the other activity assays, as it is sensitive to abnormalities other than those that affect GPIb. In the USA, hemostasis reference laboratories offer the VWF:CB.
When the ratio of the VWF:RCo, VWF:GPIbR, VWF:GPIbM, or VWF:CB assay value to the VWF:Ag concentration is < 0.7 (varies by laboratory), we infer qualitative or type 2 VWD and proceed with specialized type 2 VWD assays such as the low-dose RIPA and VWF mutimers.
I hope this helps. Several manufacturers are currently developing VWF activity assays that improvie on VWF:RCo analytical characteristics, so we may see additional comments appended to this entry in the next few days. Geo.