The PulsePen tonometer was validated versus
invasive method [Salvi et al. Validation of a new non-invasive portable tonometer for determining arterial pressure wave and pulse wave
velocity: the PulsePen device. J Hypertens. 2004; 22:2285-2293.] and versus MRI method [Joly et
al. Pulse wave velocity assessment by external noninvasive devices and phase-contrast magnetic resonance imaging in the obese. Hypertension.
2009;54:421-426].

No differences in PWV values measured by PulsePen compared to other devices were found in healthy children
and young adults [Kis et al. Measurement of pulse wave velocity in children and young adults: a comparative study using three different devices.
Hypertens Res. 2011;34:1197-1202.].

The reference values of pulse wave velocity (PWV) in healthy children and teenagers were published on
Hypertension in 2010. [Reusz et al. Reference values of pulse wave velocity in healthy children and teenagers. Hypertension. 2010;56:217-224.].
These reference values of PWV were obtained using the PulsePen tonometer. In this study PWV was assessed in 3 populations: (1) a Hungarian
population; (2) an Italian population; and (3) an Algerian population.

PulsePen tonometer is one of the 3 devices used by the Stiffness Collaboration to establish the
normal and reference values of PWV in healthy people [Determinants of pulse wave velocity in healthy people and in the presence of cardiovascular
risk factors: ‘establishing normal and reference values’ The reference values for Arterial Stiffness’ Collaboration. Eur Heart J. 2010;
31:2338-2350].

Comparison of the pressure waveforms and pulse pressure obtained with the PulsePen and with
an aortic and carotid intra-arterial catheter: the pressure waves assessed with the PulsePen to the carotid artery were compared with
intra-carotid and intra-aortic pressure waves. The intra-arterial catheter was placed at the origin of the ascending aorta and the
transcutaneous tonometer probe on the common carotid artery. At this point, using both available techniques, the pressure waves were
simultaneously recorded for a period of 10 s. Immediately after that, the intra-arterial catheter was placed at the initial section of the common
carotid artery and the transcutaneous tonometer probe at the bottom of the same artery. The pressure wave was then simultaneously recorded with
the two techniques for a period of 10 s. Pressure wave signals were then split into harmonics. The evaluation was limited to the first six
harmonics and was based on the Fourier analysis.

Pressure waveform obtained with the PulsePen matched with the waveform
obtained intra-arterially. The concordance between these waveforms was assessed with the Fourier analysis.
The comparison of the first six harmonics reveals that there is no difference between the two techniques. Variability of measurements
based on the SD for the two techniques was 2.5 mmHg (9.3%) and 1.6 mmHg (12.8%) concerning the first and the second harmonics. The same value
for the remaining harmonics was <1 mmHg. Similar results were found when both measurements were performed simultaneously at the carotid artery. The pressure
waveforms obtained non-invasively with the PulsePen at the site of the common carotid artery were very similar to the aortic waveforms obtained
invasively. This result enables a direct evaluation of central aortic pressure without the need for using models based on transfer function which
are necessary when the measurements are performed in a peripheral artery. The results of this study show
that the PulsePen is accurate in estimating central aortic pressure and pressure waveforms.

Reliability of Pulse Wave Velocity (PWV) recording at two intervals
in highly rapid succession (PulsePen method) compared with PWV recording in a single interval: one of the major questions concerning the
validity of the PulsePen device was whether or not PWV values measured with one probe (by rapid consecutive measurements) were comparable to those
measured with two simultaneous-probe recordings. The reliability of PWV recording at two intervals in highly rapid succession as opposed to
PWV recording in one single interval (simultaneous record of carotid and femoral pressure waves) was assessed. To do this, the carotid and femoral
pressure were simultaneously recorded; the peripheral wave delay to the central wave was considered as the pressure wave progression time.
Two Millar SPT 301 tonometers connected to Cardioline Delta3 ECG were used. The carotid pressure wave and femoral pressure wave were simultaneously
recorded, and then PWV values were determined using the standard procedures. The operators repeated this procedure a second time under the
same conditions. After completing this phase, PWV data obtained during the simultaneous recordings were compared with the PWV data obtained from the
first recording in carotid and the second one in femoral. The reliability of PWV
recording at two intervals in a rapid succession compared to PWV recording in a single interval was confirmed. The comparison does not
reveal any real difference between the two techniques. Reproducibility for PWV obtained with the Millar probe, R2=0.975, y=0.963x+0.305 (p<0.001),
with a coefficient of variation estimated at 6.59%; first acquisition versus two-interval acquisition, R2=0.975, y=0.933x+0.347 (p<0.001),
coefficient of variation: 6.79%; second acquisition versus two-interval acquisition, R2=0.968, y=0.953x+0.184 (p<0.001) with a coefficient of
variation of 7.43%; mean from the two acquisitions versus two-interval acquisition, R2=0.978, y=0.948x+0.217 (p<0.001) with a coefficient of
variation of 6.34%. The analysis of the two techniques according to the Bland-Altman method is shown in the figure. The difference in PWV
measurement using a standard method (first test and second test with two Millar tonometers) is comparable with the difference between the standard
method and the PWV measured using consecutive recordings, the first one in carotid and the second one in femoral.

PWV measurements with two Millar tonometers. Upper panel: reproducibility of the
standard method (simultaneous recordings arterial pressure wave with two Millar tonometers). Left, the scatterplot shows a linear correlation
between the values of PWV determined when carotid pressure and femoral pressure waves were recorded simultaneously (Car1 and Fem1) with two probes
versus the values of PWV determined a few minutes later under the same conditions (Car2 and Fem2). Right, the Bland-Altman analysis shows
differences observed between PWV values obtained by the two measurements according to the mean values (Car1 Fem1 + Car2 Fem2)/2. Lower panel:
comparison of PWV obtained with the standard method with two Millar tonometers (Car1 and Fem1), with the PWV obtained using two consecutive
measurements, the first one in carotid (Car1) and the second one in femoral (Fem2). Left, the
scatterplot shows a linear correlation between the values of PWV determined when carotid pressure and femoral pressure waves were recorded
simultaneously (Car1 and Fem1) with two probes versus the values of PWV obtained using two consecutive measurements, the first one in carotid
(Car1) and the second one in femoral (Fem2). Right, the Bland-Altman analysis shows differences observed between PWV
values obtained by the two measures according to the mean values (Car1 Fem1 + Car1 Fem2)/2.

Therefore, changes in the transmission of electro-mechanical signals that occur between the two
measurements could provide erroneous PWV values. Typical examples of such changes are an important modification in heart rate (HR) and the
appearance of conduction anomalies. The PulsePen software always marks the changes in HR and blood pressure between the two measures, so the
operator can repeat the test when that difference is more than a predefined value. In any case, in practice, when the two measurements are
realized rapidly one after the other, this kind of modification is very unusual.

Comparison of the PWV values
obtained with the PulsePen and with a standard method:

The figure shows the individual PWV values measured with the PulsePen (one tonometer) and
with the Millar device (simultaneous measurements with two probes). A scatterplot on the left side shows a linear correlation between values of
PWV measured with the PulsePen and Millar probes. Results obtained with the two methods were closely correlated (R2=0.966), with a coefficient
of variation estimated at 7.78%. On the right-hand side, the Bland-Altman plot shows individual differences observed between PWV
values obtained with the two methods according to the mean PWV level calculated as (PulsePen and Millar)/2.

Pulse wave velocity: PulsePen
versus manual measures: the PWV determined by the PulsePen software was compared with the traditional manual measurement on paper.

The PWV variation
coefficient reached 4.2% (PWV determined by manual method versus PulsePen analysis, R2=0.991, y=0.969x+0.207, p<0.001), and decreased to 2.1%
if the delay of time were considered instead of pulse wave velocities (manual method versus PulsePen analysis, R2=0.994, y=1.010x+0.300, p<0.001).

Pulse Wave Velocity: intra-observer and inter-observer reproducibility: A strong intra-observer and inter-observer reproducibility were found
for the PulsePen PWV measurements. Over 95% of differences were included in the interval determined by repeatability coefficient (mean of
differences ± two SDs). The inter-observer coefficient of variation of our population reached 7.94% (first operator versus second operator R2=0.967,
y=0.999x+0.042, p<0.001), and the intra-observer coefficient of variation reached 7.20% (first acquisition versus second acquisition by the same
operator R2=0.971, y=0.984x+0.207, p<0.001).

Left panel: Pulse wave velocity
(PWV) measurements with PulsePen: inter-observer variability. Left, scatterplot shows a linear correlation between the values of PWV measured
with the PulsePen by two different observers (first observer and second observer). Right, the Bland-Altman analysis shows differences
between PWV values obtained by the two observers according to the mean values (observer 1 + observer 2)/2.
Right panel: Pulse wave velocity (PWV) measurements with PulsePen: intra-observer variability. Left, scatterplot shows a linear correlation between the values
of PWV measured with the PulsePen by the same observer at two different times (first test and second test). Right, the Bland-Altman analysis shows
differences between PWV values obtained by the two measures according to the mean values (test 1 + test 2)/2.

Augmentation Index: intra-observer and inter-observer reproducibility.
Concerning the Augmentation Index (A.I.) measurements, the inter-observer coefficient of variation of our population reached
15.80% (first operator versus second operator R2=0.976, y=0.974x+0.489, p<0.001), and the intra-observer coefficient of variation reached
15.15% (first acquisition versus second acquisition by the same operator R2=0.978, y=0.996x+0.326, p<0.001).

Left: Augmentation Index (A.I.) measurements with PulsePen: inter-observer variability. The Bland-Altman analysis shows differences between A.I. values obtained by the two observers according to the mean values (observer 1 +
observer 2)/2. Right: Augmentation Index (A.I.) measurements with PulsePen: intra-observer variability. The Bland-Altman analysis shows
differences between A.I. values obtained by the two measures according to the mean values (test 1 + test 2)/2.