FVIII Linearity; LA Testing

From Mahnaz Sairi, Associate Director of Hematology,  MBHN: Hi George: What is the highest limit we can report For FVIII? And how we determine this? Is it by clinical significance or by instrument linearity?

Also, should we still do lupus anticoagulant (LA) testing if both the prothrombin time (PT) and partial thromboplastin time (PTT) are normal?

Hello, Dr. Sairi, and thank you for your question. I develop the concept of coagulation factor linearity in my audio module, Method Validation, Part 2, using protein S as my example. In short, begin with a calibrator of known factor activity such as CRYOcheck™ Normal Reference Plasma and prepare several ascending dilutions using the buffer required for your assay, often Owren-Koller buffer. Compute the anticipated activity for each dilution, then assay each dilution and prepare a linear graph of anticipated activity versus assay values. By inspection you will find where linearity is lost (where the lines diverge) at the top and bottom of the curve; select the points just inboard to establish your limits.

The limits of linearity vary among instruments and reagents, however, when preparing dilutions, just as a rule of thumb, develop a range of 1% (0.01 units)–150% (1.5 units). Some instruments use two curves, the second of which is designed to provide linearity at the low range, for instance, 0.1%–10%, in this case it is necessary to test linearity in both ranges by preparing and testing the appropriate dilutions. The instrument automatically selects dilutions and curve based upon the initial assay.

Specimens that are out of linearity, for instance, factor VIII activity levels over 150%, are diluted, dilutions are re-assayed, and the result of the assay is multiplied by the dilution factor. High-end coagulometers automatically make the appropriate dilutions, and it is not unusual for factor VIII levels to exceed 150%, as factor VIII is an acute-phase reactant. Because chronically elevated factor VIII is a thrombosis risk factor, laboratory directors may choose to report levels as high as 250%. Our participants may weigh in on the clinical limits they have selected, which will vary from institution to institution.

For your second question, the PTT reagents employed to monitor unfractionated heparin or to screen for coagulopathies are formulated with high phospholipid concentrations, rendering them relatively insensitive to LA. These are employed to reduce the frequency of detection of transient, clinically innocent LAs, a “laboratory nuisance.” See Fritsma GA, Dembitzer FR, Randhawa A, et al. Recommendations for appropriate activated partial thromboplastin time reagent selection and utilization. Am J Clin Pathol 2012;137:904–8. If there is clinical suspicion of an LA, for instance, an unprovoked thrombotic event, it is necessary to test using specially formulated, low-phospholipid PTT reagents such as PTT–LA, and to test in parallel using a separate platform such as CRYOcheck™ LA Check™ (dRVVT screening reagent). For current information on LA testing, refer to Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemostas 2009;7:1737–40. I hope this is helpful, and if you don’t mind another “product placement,” please check our modules, Laboratory Detection of Thrombosis Risk 1, 2, and 3, which are modules 14–16.