Factor VIII binding activity of von Willebrand Factor by flow cytometry

KEMPFER ANA C; SILAF MARÍA R; FARÍAS, CRISTINA; CARBALLO GONZALO A; AMARAL MARÍA M; LAZZARI, MARÍA A

Paris

Congreso; XVIII CONGRESS THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS; 2001

Von Willebrand Factor (VWF) is a multimeric glycoprotein found in plasma non covalently linked to factor VIII (FVIII). Type 2N von Willebrand disease (vWD) is caused by a mutation in the VWF gene that results in VWF with a normal multimeric pattern, but with reduced binding to FVIII. We describe a new sandwich enzyme-linked immunosorbent assay to study the ability of VWF to bind exogenous recombinant FVIII. Briefly, polystyrene beads were coated with polyclonal anti-VWF in carbonate buffer and incubated for 3h gently rocking. After washing, biotinylated polyclonal anti-FVIII antibody was added and incubated 1h at 37°. Finally, the beads were washed and incubated with phycoerythrin-conjugated streptavidin. The samples were analyzed on FACSCalibur flow cytometer. The machine was calibrated daily with Calibrate Beads and monitored for machine drift by channel targeting a low fluorescence bead control (beads incubated with plasma). A maximum of 30,000 events were acquired based on forward angle light scatter and side scatter in the logarithmic mode using Cell Quest software. Doublets were excluded by gating on the single bead population using the forward scatter histogram. Dose-response curves obtained using normal pool plasma VWF revealed a hyperbolic relationship between the optical density and the VWF concentration. The assay allows the quantification of FVIII binding with values expressed in U/dL; 100 U/dL was the amount present in normal plasma. To verify the accuracy of the assay, one patient with type 2N vWD, with characterized VWF gene mutations was studied using an existing chromogenic assay and our method. The patient who was heterozygous for the R91Q showed markedly decreased binding for the chromogenic method and yielded, in our method, values of 15 U/dL. Therefore, although the two methods produce qualitatively similar results, our method offers the advantage of allowing quantification of the FVIII binding function as in other ELISA methods. In our hands, the non cytometric ELISA method had variability of anti-VWF distribution in microwells carrying to less sensitivity. Besides we could quantify VWF in the same sample adding anti-VWF FITC. It allows that the data development of the curve were taken under the same conditions. In addition, this new method avoids the use of a carcinogenic substrate, i.e., the enzyme (peroxidase). It is simple, easy to produce, has a lower limit of detection and utilizes flow cytometry instrumentation available in laboratories diagnosing hematological diseases.