Venous occlusion plethysmography (VOP) is a century-old technique used in the non-invasive measurement of blood flow [1]. The underlying rationale is that when venous outflow from an anatomical compartment such as the forearm or leg is occluded, any change in its volume must be due to (and proportional to) continued arterial inflow. This is held to be true in the early phase of occlusion (in the first few seconds): later, exhaustion of available vascular capacitance and limits to tissue compliance may limit both inflow and its associated volume change. Thus, measures of volume change early after venous occlusion are taken to reflect the rate of arterial inflow. Mercury-in-silastic strain gauges are typically used to measure such increases in limb volume.
VOP has been used extensively to study human vascular physiology and the effect of vasoactive drugs such as ACE inhibitors and calcium channel blockers [2, 3]. Additionally, the technique has been used in sport and exercise physiology to study the effects of training regimens [4], dietary supplements [5], and to characterise vascular adaptations in athletes [6], as well as in hypoxic research [7] for the assessment of how high altitude exposure affects peripheral blood flow [8–11]. In all these instances, whilst the underlying principle of VOP remains the same, the exact protocols and analytical methodologies applied somewhat vary. For example, exercise intensity, number of measurements taken and anatomical site (calf vs. forearm) may all vary between protocols.
A repeated measures protocol, with data recorded pre and post a metabolic stimulus, has been used on numerous occasions [4, 6, 9]. Exercise, such as the sequential squeezing of a rubber ring post baseline VOP measurements, has been used for this provocation [9]. The University College London’s Centre of Altitude, Space and Extreme Environment (CASE) Medicine [12] have adapted this simple methodology (further detailed below) and employed it in two studies to date [10, 11]. Data from these studies leads us to conclude that the classical analytical methods often described in the literature [4, 6, 9, 13–17] predominantly focuses on issues relating to the reproducibility and accuracy of VOP measurements but fails to consider what dissimilarities in the recorded traces may mean. In this methodological manuscript, following the description of our VOP protocol and a ‘classic’ method of trace analysis, we suggest six alternative methods of data analysis. Through employing these simple methodological approaches, one may be able to achieve a considerably greater insight into the physiology of, and biological mechanisms behind, peripheral blood flow.
Venous occlusion plethysmography protocol
The following rest/exercise protocol was utilised by CASE Medicine for their normobaric and hypobaric hypoxic studies. In these studies, the independent variable examined was hypoxic exposure. Ethical approval for the above studies was obtained through the University College London Research and Ethics Committee in accordance with the Helsinki Declaration and informed consent was gained from all participants.
Preceding VOP testing, participant’s dominant forearm circumference and volume were measured. Knowledge of forearm circumference allowed for selection of the correct size mercury-silastic strain gauge (maximum forearm circumference determined by using a tape measure minus 2 cm). Forearm volume measurement allows for the calculation of blood flow per cubic centimetre of tissue and the correction for differences in forearm size and muscle mass. Its measurement is straightforward and takes advantage of Archimedes’ principle: when the arm is slowly immersed into water until the elbow joint, the measured volume of displaced water represents the total forearm volume. This process is then repeated for the dominant hand. The volume of the arm minus the volume of the hand equals the forearm volume. For blood flow measurements, an inflating cuff (SC10D, Hokanson, Bellevue, USA) was placed around the participant’s bicep, to occlude venous blood flow, and connected to a rapid cuff inflator (E20, Hokanson), which was set above venous, but below arterial pressure (50 mmHg). A suitable strain gauge (Hokanson) was placed around the widest part of the forearm and connected to a plethysmograph (EC6, Hokanson). To exclude the hand circulation, which contains a large number of arterio-venous shunts, a segmental pressure cuff (TMC7, Hokanson) was placed around the wrist and inflated to supra-arterial pressure immediately before testing was commenced. Data were recorded on a laptop using the NIVP3 arterial inflow studies software (Hokanson).
Prior to testing, each subject was comfortably seated and allowed to adjust to the environment for 5 min. During this time, the procedure was explained, personal details verified and the software prepared for data capture. So as to prevent the strain gauge from touching the surface of the table during the measurement procedure, their dominant arm was supported by the wrist and elbow with foam pillows. To ensure that the occlusive pressures selected for the dominant arm’s bicep and wrist cuffs would be sufficient to prevent venous outflow and exclude the vasculature of the hand respectively, a baseline blood pressure measurement was taken at rest from participant’s non-dominant arm. On commencing testing, the wrist cuff was manually inflated to 250 mmHg, and the rapid cuff inflator was set to inflate the bicep cuff to 50 mmHg for 7 s at a time. During each 7-s occlusion interval, changes in forearm circumference were detected as changes in electrical resistance in the strain gauge. Five readings were taken at rest, at the end of which the wrist cuff was deflated. A standard 2-min forearm exercise protocol was then performed. This comprised of repeated handgrips of a foam tennis ball to maximum effort using their dominant hand in time with a metronome set at 60 Hz (alternating between squeeze and relaxation every second). Controlling the intensity of exercise in this way is important in ensuring accuracy in subsequent analyses. Once the exercise was completed, the wrist cuff was re-inflated to 250 mmHg and five post-exercise readings were obtained.
Data extraction
Using Hokanson’s proprietary software, the gradient of the five pre-exercise, and five post-exercise traces were measured at two time points: between 4–6 (Fig. 1) and 6–8 s. These timings refer to those on the X-axis of the graph, and it should be noted that the first 4 s on this axis are excluded, as full cuff inflation requires 2.5 s after which a non-specific jump in blood flow is observed relating to the distortion from external compression (movement artefact). As the software measures in straight lines, yet the traces are curved, the two time points are used to give more accurate readings of alterations in flow. The time points may subsequently be compared to give an insight into the dynamics of flow over time, and the flow over 4–8 s may be calculated as the mean of the two measured gradients. Notably, the gradients obtained are an expression of the percentage increase in flow per minute rather than an actual volume of blood over time. Whilst the latter could be calculated using participant’s forearm volume measurements, this may be considered unnecessary given that these values are directly proportional to one another.
