The centres in which the echo-colour-Doppler examinations were performed are:
Centre 1: Department of Public Health and Neurosciences, IRCCS "C. Mondino National Institute of Neurology" Foundation, University of Pavia, Italy
Centre 2: Barrie Vascular Imaging, Barrie Ontario, Canada
Centre 3: Hospital of Civitanova, Marche, Italy
Centre 4: Department of Neurosciences, Federico II University of Naples, Italy
Centre 5: Multiple Sclerosis Centre, IRCCS Don Gnocchi, Milan, Italy
Centre 6: CCSVI Project, "Policlinico di Monza", Italy
We collected data of 805 patients from the above centres, all patients signed a consent form. All had a diagnosis of MS according to McDonald's revised criteria  and all were submitted to Doppler sonography in the period between 2 January 2010 and 17 January 2011. No particular inclusion criteria were used, apart from a diagnosis of MS. In this way, we obtained six sets of 'consecutive' assessments of regularly monitored patients who had asked to undergo the procedure.
Of the original 805 patients, 95 were excluded because no data could be obtained regarding their form of MS. Therefore, the final study sample comprised 710 patients (466 females and 244 males).
The following clinical data were considered: sex, age at time of Doppler evaluation, age at disease onset, disease duration at time of Doppler evaluation, and Expanded Disability Status Scale (EDSS) score at time of Doppler evaluation. From these data the Multiple Sclerosis Severity Score (MSSS) score was computed, which is a validated measure of disease severity computed from disability (EDSS) and disease duration data .
The echo-colour-Doppler examinations of cerebrospinal venous drainage were performed by nine different trained ultrasound operators using three different ultrasound systems:
ECD Esaote-Biosound MyLabVinco 25 scanner equipped with a 3.5-10 MHz linear transducer and a 5.0-8.0 MHz microconvex transducer, both for extracranial measurements, and a 2.0-3.3 MHz phased array probe for transcranial analysis (Centres 1, 3,5,6)
P5 General Electric ultrasound machine equipped with a linear probe 3.5-10 MHz for extracranial examination and a 2.0-3.0 MHz sector probe for transcranial examination (Centre 2);
Philips iU22 system equipped with a 3.0-9.0 MHz linear wide-band transducer, a 5.0-8.0 MHz microconvex probe, and a 1.0-5.0 MHz phased array transcranial probe (Centre 4).
Each subject was investigated first in supine position and then in sitting position using a tilt chair. All were scanned following the Zamboni protocol for diagnosis of CCSVI, which is based on the detection of five parameters:
1 - Reflux in the internal jugular veins (IJVs) and/or vertebral veins (VVs) in sitting and supine posture.
2 - Reflux in the intracranial veins. Reflux is defined as a reversal of flow direction during the inspiratory and expiratory phase during normal breathing with mouth closed. The transcranial colour-coded duplex sonography (TCCD) studies were carried out using one of two different approaches: the classic transtemporal window or the transcondylar window.
The classic transtemporal window was used by one centre (Centre 4 - 118 MS patients), evaluating flow direction in the Rosenthal vein and transverse sinus, while all the other centres (592 MS patients) used the transcondylar window, which assesses the direction of flow in the cavernous and petrosal sinuses. The transducer was placed at the level of the mandibular condyle, sloping the tail approximately 10 degrees downwards. The insonation depth was set at 11 cm.
3 - B-mode evidence of abnormalities in the IJVs, such as stenoses, malformed valve, annulus, septums, etc.
4 - Flow not Doppler-detectable in IJVs and/or VVs despite numerous deep breaths.
5 - Reverted postural control of the main cerebral venous outflow pathways, detected by measuring the difference in IJV cross-sectional area (CSA) between the supine and upright positions.
ΔCSA in the IJV, obtained by subtracting the CSA measured in sitting position from that measured in supine position, is a positive value in normal subjects. A negative ΔCSA value indicates loss of postural control of the predominant outflow route in the supine position.
This parameter was assessed in B-mode in transversal access, at the J2 point which corresponds to the level of the thyroid gland, and carefully avoiding any compression of the vessel by the probe.
The Kolmogorov-Smirnoff test was used to check the distribution of quantitative variables for deviation from normal. Data were submitted to univariate analysis of variance (UNIANOVA), chi-square statistics (CROSSTABS) and logistic regression analysis using a stepwise forward procedure (NOMREG). The statistical analysis was conducted using SPSSPC+ software.