Principle, Protocol, Instrumentation
The assay works by a basic, dual-color flow cytometric principle. In terms of sample type, the specimen is uncoagulated whole blood collected from patients who are taking anti-P2Y12 drug; the specimen is collected in a Vacutainer (BD) tube containing the anticoagulant sodium citrate. The specimen is stable at room temperature for up to 48 hours prior to testing. Only 30 microliters of specimen is needed to run the assay.
The sample is incubated with PGE1 in the presence and absence of ADP. Following incubation, the sample is fixed in formaldehyde. The fixed sample is then permeabilized and labeled with a primary antibody against the phosphorylated Ser-239 residue of VASP (mouse monoclonal anti-VASP-P). This is followed by staining with fluorescein isothiocyanate (FITC)-conjugated goat antimouse polyclonal antibody and counterstaining with antibody against platelet surface marker CD61. A negative isotype control is also run simultaneously to assess background reactivity of platelets with primary antibody.
All steps are performed at room temperature. All of these reagents are contained within the kit, and the protocol takes about 30 minutes to complete.
The fluorescence in each sample is measured by flow cytometry, which results in a statistic known as the platelet reactivity index (PRI). The following is the equation for calculating PRI:
PRI = [MFIPGE1 - MFIPGE1 + ADP/ MFIPGE1] X 100
MFI stands for mean fluorescence intensity. Based on these calculations: the higher the PRI percentage, the lower the responsiveness to anti-P2Y12 drug. In terms of instrumentation required for the assay, any flow cytometer can be used to measure the fluorescence, even benchtop models.
Use of VASP Assay in Clinical Trials
Like other platelet-function assays, the VASP assay has been incorporated in clinical trials to assess patient responsiveness to anti-P2Y12 drugs. In all trials, the mechanism of cardiovascular pathology was enhanced platelet reactivity, which was measured by in vitro platelet function assays such as the VASP assay.
For example, one small clinical trial has shown that, overall, the VASP assay is comparable to light transmission-based platelet aggregation assays, but has found that the VASP assay is better at identifying low responders to treatment with Cangrelor. In addition, several clinical trials have shown that clopidogrel, both alone and in combination with aspirin, reduced risk of atherosclerosis in patients with diabetes mellitus. In a more recent clinical trial, the VASP assay was used to determine responsiveness to Ticagrelor combination therapy.
Other potential clinical applications for the VASP assay include assessment of response to clopidogrel prior to angioplasty, assessment of treatment response prior to discharge following angioplasty, long-term assessment of treatment effectiveness, assessment of novel P2Y12 antagonists in preclinical and clinical development, and assessment of potential hemorrhaging following treatment with clopidogrel. Moreover, with its increasing use in clinical trials as a way of assessing responsiveness to clopidogrel (and other anti-P2Y12 compounds), there are discussions about using the VASP assay as a companion diagnostic.
In summary, traditional platelet aggregation tests are often not sensitive and specific enough to detect resistance or potential bleeding problems caused by anti-P2Y12 drug treatment. These limitations led to the development of the VASP assay kit, which is labeled for research use only and not for use in diagnostic procedures in the U.S. and Canada.