Calf blood flow and posture: Doppler ultrasound calibrated by plethysmography B. E. VAN
LEEUWEN,
J. LUBBERS,
Department of Medical Physics, Faculty 9712 KZ Groningen, The Netherlands
G. J. BARENDSEN,
of Medicine,
VANLEEUWEN,B.E.,J. LUBBERS,G.J. BARENDSEN,AND L. DE PATER.Calf blood flow and posture: Doppler ultrasound calibrated by plethysmography. J. Appl. Physiol. 72(5): 1668-1674, 1992.-A procedure was developed that enables measurement of rapid variations in calf blood flow during voluntary rhythmic contraction of the calf muscles in supine, sitting, and standing positions. During the exercise, maximum blood velocity is measured by Doppler ultrasound equipment in the popliteal artery. The Doppler signals are calibrated by plethysmography to enable calculation of blood flow during exercise in ml. 100 Knowledge of the cross-sectional area of the vesml-l min. sel and the angle of insonation is not required in this procedure. Evaluation of the calibration method with 10 healthy volunteers showed that for each subject a new calibration was necessary after a change in posture; the relationship between the blood flow and the maximum Doppler frequency averaged over one heart cycle was linear for each calibration. l
rhythmic emia
muscle contractions;
calf ergometry;
exercise hyper-
DURINGRHYTHMICEXERCISE oftheleginthesittingand
standing positions, the muscle pumps of the upper and lower leg contribute significantly to the systemic circulation (3). There are also effects on local hemodynamics, because these muscle pumps increase the perfusion pressure (arterial-to-venous pressure difference) in the leg by lowering the local venous pressure. As a result, leg blood flow may be higher during exercise in the sitting and standing positions than in the supine position. To investigate whether this is true, leg blood flow must be measured during exercise in the supine position as well as in the sitting and standing positions. The only method that has been used to compare calf blood flow during exercise in supine, sitting, and standing positions is the 133Xe clearance method (8). With this method, small depots of 133Xe are injected in the muscle and the clearance rate is used as a measure for muscle blood flow. Major limitations of this method, however, are that 1) 133Xe clearance rate is influenced by the trauma of injection and by inhomogeneous composition and perfusion of the muscle (14) and 2) the method has insufficient resolution in time to distinguish the blood flow during muscle contraction from that during relaxation. The Doppler effect of ultrasound has been used during leg exercise in the supine position to monitor changes in blood flow in the upper leg (15) and the calf (2). These measurements demonstrated that the method has suffi1668
0161-7567/92
$2.00
Groningen
AND
L. DE PATER
University,
cient resolution in time to distinguish the variations in blood flow that occur during muscle contraction and relaxation. For quantitative measurement of blood flow with ultrasound, one must either measure the cross-sectional area of the vessel and the angle of insonation (15) or apply a calibration procedure. In the procedure that was used by Lubbers et al. (12), continuous-wave (CW) ultrasonic Doppler equipment with a zero-crossing detector was calibrated immediately after calf exercise in the supine position by comparing the zero-crossing frequency with the blood flow measured by venous occlusion plethysmography. A major limitation of this procedure was the susceptibility of the zero-crossing frequency to displacements of the ultrasonic transducer that are likely to occur during exercise. Furthermore the procedure cannot be applied in the sitting or standing positions because venous occlusion plethysmography can be used only in the supine position. The objectives of the present study therefore were I) to develop and evaluate a new calibration procedure that can be used in sitting and standing positions and that enables measurement of blood flow during exercise in these positions, 2) to apply a signal-processing technique that minimizes the susceptibility of the Doppler frequency to displacements of the ultrasonic transducer, 3) to investigate whether a calibration performed during or after a certain amount of exercise remains valid during and after another amount of exercise, and 4) to investigate whether a calibration performed in the supine position remains valid in the sitting and standing positions. METHODS Principle of Doppler calibration. In normal subjects the calf blood flow can be considered equal to the blood flow in the popliteal artery. This is so because in normal subjects there are no significant functional collaterals at the knee (9) and, consequently, almost all the blood to the lower leg passes the popliteal artery. Furthermore, during (exercise) hyperemia, the blood flow through the foot is negligible. This is evident from measurements with venous occlusion plethysmography (7): during hyperemia there was no significant difference between calf blood flow obtained with and without a distal occlusion cuff around the ankle to exclude the foot blood flow. The purpose of the calibration procedure is to quantify the relationship between Doppler frequency and calf blood flow. The ultrasonic transducer was positioned
Copyright 0 1992 the American Physiological Society
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ULTRASOUND
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BY
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rn
above the popliteal artery. From the ultrasonic signal an average Doppler frequency was derived in a way that \ B minimized the susceptibility to displacements of the I$ \ transducer. This Doppler frequency was compared with C the calf blood flow measured by strain gauge plethysmoFPhy. A linear statistical model was used to fit the re~~ sults. Calf ergometry. A series of different values of calf blood flow is needed for the Doppler calibration. In the sitting and standing positions these were obtained during calf exercise. In the supine position the Doppler calibration was performed immediately after exercise as the blood - \ flow decreased and returned to the value at rest. Exercise was performed on a calf ergometer. Details of this ergomete r have been published previously (12). The exercise consisted of 90 treads (rhythmic exten sions and flexions) of the right foot on a pedal, with a frequency of 0.5 Hz indicated by a metronome. The angle- through which the pedal rotated was 15". Work was performed only during the extension of the foot. The moment imposed to the pedal by a spring determined the work load of each tread. During flexion of the foot, the calf muscles were completely relaxed, and the pedal returned slowly 1. Experimental setup of supine position and details of transto the initial position by the combined action of the ducerFIG. assembly: calf ergometer (A); strain gauge plethysmograph (B); spring and a damper. occlusion cuff (C); metal strip (D) for fixation of transducer assembly The work that the calf muscles performed was varied (E). Details of transducer assembly: block (K) is fixed to leg with metal by application of various moments and variation of the strip (D); block (L) can rotate with respect to block (K) around axis (x); (M) can slide with respect to block (L) by means of screw (y); number of treads. The duty cycle (the duration of the block holder (N) can rotate with respect to block (M) around axis (2); holder muscle contraction divided by the total du ration of con- (N) contains ultrasonic transducer (T), which is pushed to skin (S) and traction and relaxation during on.e tread) was S0.5 and directed to blood vessel (V). such that it could be easily reproduced by the subject. This was checked during exercise by recording two elec- ment of 92 N m (work 2,170 J) was applied if the subject trical signals, one representing the angle of rotation of could manage it. the pledal and the other representi .ng th .e exerted mODoppler equipment. Commercially available bidirecment. tional pulsed Doppler equipment with a built-in spectral In the supine position, the subject lay on his back with analyzer (TC2-64B, Eden Medizinische Technik) and a his shoulders supported by stops. The position of the flat ~-MHZ transducer was used. This transducer was right leg was adapted to the application of venous occlu- manufactured from our specifications and contained the sion plethysmography: the right knee was slightly bent, same active element as the commercially available 4with the lower leg -15 cm above heart level. For each MHz pencil probe. To obtain a calibration that could be subject, calf exercise in this position consisted of two se- reproduced, an arrangement was applied that minimized ries of 90 treads and moments of 33 and 58 N. m (work the displacements of the ultrasonic transducer and the 780 and 1,370 J, respectively). Each series was followed changes in the angle of insonation in each position and by a 3-min period during which Doppler calibration was allowed us to (re)direct the ultrasonic transducer without performed. altering the angle of insonation. The arrangement conIn the sitting position, the subject was seated in a sisted of the flat transducer fitted in a small holder faschair. The trunk was vertical and the upper right leg tened to the upper leg with a strip of spring steel. The horizontal. The angle between upper and lower right leg position of the transducer was such that the popliteal was the same as in the supine position. The calf exercise artery was insonated at the back of the knee in the region in this position consisted of two or three series of 90 of the fossa poplitea. (Fig. 1). treads each with short breaks (duration -8 s) after the To minimize the susceptibility of the Doppler freZnd, 5th, 15th, and 30th tread. Moments of 33,58, and (if quency to displacements of the ultrasonic transducer, we the subject could manage) 80 No m (work 1,880 J) were used the maximum Doppler frequency [ f,,,(t)] of the applied. During each break and immediately after com- Doppler spectrum. This quantity is proportional to the pletion of the 90 treads, one blood flow value was availinstantaneous maximum blood velocity [umax(t)]. Disable for Doppler calibration. placements of the ultrasonic transducer can be allowed In the standing position, the subject was supported by so long as the part of the cross section that contains a bicycle saddle. The right leg was not stretched com- V max (t) is insonated. The f,,,(t) was averaged over each pletely, inasmuch as the angle between upper and lower heart cycle, and this quantity (D) was compared with the leg was the same as in the supine and sitting positions. calf blood flow (q) measured by plethysmography. Calf exercise and Doppler calibration were performed in Displacements of the transducer were monitored by the same way as in the sitting position, but an extra mo- the sound level of the (forward) Doppler signal. At the d
l
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DOPPLER
ULTRASOUND
CALIBRATED
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ECG trigger
start of each series of exercise, the sound level was maximized by directing
the ultrasound
beam to the center of
the vessel and by adjusting the depth range of the sample volume. A minimum signal level of 38 dB for reliable measurements was established from test bench measure-
ments. This minimum level should be considered typical for our equipment. When in the course of an exercise series the sound level fell below 38 dB, all signals were rejected and the transducer was redirected.
The quantity f,,,(t) was available only for blood flow in the forward direction. This is not a limitation, because there is no blood flow in the backward direction during exercise, at least not between contractions (2) and for some time after exercise. To detect the reoccurrence of blood flow in the backward direction after exercise, we used the zero-crossing frequency (the number of times the Doppler signal crosses its baseline per unit of time) of the bidirectional Doppler signal. Plethysmography. Strain gauge plethysmography has become an established method for noninvasive physiological or clinical studies of blood circulation in extremities (4) since its introduction by Whitney (16) in 1953. Our strain gauge plethysmograph
consisted of two mercury-
filled tubes of silicon rubber mounted mechanically parallel and electrically in series. This assembly was stretched around the thickest part of the calf. With relaxed muscles, relative changes in calf volume dV/V were equal to
I I I I I I i I I I I I I I 4r
fd W-M
321IP'I
measurement of calf blood flow. We therefore developed an alternative method of strain gauge plethysmography that could be used in the sitting
and standing positions.
With this method the blood flow was calculated from the increase in calf volume that occurred after one or more
relative changes in resistance dRlR of the mercury con-
treads of the foot.
ductor. These changes in resistance were measured with the circuitry proposed by Hokanson et al. (11): a con-
Essential for this method of strain gauge plethysmography is a properly functioning calf muscle pump, so
gauge, and
that the venous system of the calf is emptied during the
changes in resistance were measured as changes in voltage. Voltage and current terminals were separated (by
treads of the foot; i.e., the venous valves must be competent. Moreover, there had to be no outflow of blood from
the d-lead method) to rule out errors caused by the resis-
the venous system for some interval
tance of lead wires. For calibration of the plethysmograph, the electrical method described by Brakkee and
treads were completed. A special procedure was applied to 11 exercise series selected at random to confirm that
Vendrik
these conditions
stant current
was sent through
the strain
(5) was used.
were fulfilled
of time after the
(see RESULTS).
Because
In the supine position, the calf blood flow was determined by venous occlusion plethysmography (VOP)
all the blood that flowed into the venous system during that interval was used for refilling, the rate of volume
after exercise. Venous occlusion was achieved by inflat-
increase was equal to that of the calf blood flow.
ing a C&cm-wide occlusion cuff around the upper leg, just above the transducer assembly, to 6.6 kPa (50 mmHg).
The procedure of Doppler calibration in the sitting and standing positions was as follows. During the breaks after the Znd, 5th, 15th, and 30th tread and directly after completion of the 90 treads, the blood flow was measured for each heartbeat. As the amount of work increased,
The lower leg was positioned above heart level, and the cuff was alternately inflated and deflated for periods of three heartbeats to keep the venous pressure relatively low. This ensured that the increase in calf volume during
the occlusions was proportional to arterial blood flow. To enable short occlusion periods, and beat-to-beat calculation of blood flow, the occlusions were triggered by an
blood flow also increased. During each of the breaks and
after each series of exercise the venous system is filled rapidly, so we selected the heartbeats for which the blood flow was maximal.
The blood flow during these selected
the blood flow through the foot is negligible during exercise hyperemia (7).
heartbeats was then compared with the corresponding averaged Doppler frequencies, D. Data acquisition and processing. A personal computer system (Olivette M280 with 40-Mbyte hard disk and Labmaster analog-to-digital board) was used for data ac-
When the cuff was inflated, the position of the ultrasonic transducer relative to the vessel sometimes differed from that when the cuff was empty. The value of D was therefore calculated between occlusions. The corresponding blood flow value was obtained by linear interpolation (Fig. 2). The calibration was terminated when blood flow in the backward direction started to occur. VOP can only be applied in the supine position for
quisition and semiautomatic data processing. The following signals were sampled: plethysmogram and calibration pulse, ECG trigger, exerted moment and angle of rotation, forward f,,,(t), and sound level of forward and reverse Doppler signals. These signals and the forward and reverse zero-crossing frequencies of the Doppler signal were recorded on a strip chart recorder (SiemensElema Mingograf 800). This registration was used during
electrocardiogram (ECG) (1). The first occlusion began within two heartbeats after the exercise period. A distal occlusion cuff around the ankle was not applied, because
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DOPPLER
ULTRASOUND
100
100
supr
100
ne
r
r
80.
80.
60.
60.
40.
40.
20.
20.
o 33Nm D SBNm o 80Nm x
0’ 0
. I
’ 2 D
(kHz)
’ 3
’ 4
0’ 0
’ I
’ 2 D (kHz)
3
’ 4
oL 0
*
*
I
2 D
.
3
92Nm
’
4
(kHz1
3. Calibration diagrams of flow (q) against Doppler frequency for subj I, in supine, sitting, and standing positions.
FIG.
(D)
1671
BY PLETHYSMOGRAPHY
the calibration line did not change significantly after a change in position. The intercept a, for zero Doppler frequency differed significantly from zero in most calibrations (Table 1).
uprlghl
slllnO
c
CALIBRATED
the experiments to check the signal quality (e.g., detect artifacts, or monitor the level of the Doppler signal). Subjects and protocol. We investigated 10 male healthy subjects aged 20-54 yr. The subjects reported twice to our laboratory on different days. On the 1st day, the subject received a brief instruction and gave his informed consent. Then the calf ergometer was adjusted, and two or three test series of exercise were performed. The actual experiments were carried out on the 2nd day. The optimal position of the ultrasonic transducer was established while the subject was seated in a chair. After the transducer assembly had been fixed to the leg, the subject moved over to the calf ergometer. The plethysmograph and occlusion cuff were applied, and ECG electrodes for the trigger signal were attached. Doppler calibration was first performed in the supine position. After that the occlusion cuff was removed, and Doppler calibration proceeded in the sitting position and finally in the standing position. Statistics. For each subject, Doppler calibration lines were calculated for the supine, sitting, and standing positions according to the statistical model q = a, + a,D. For each of these positions, the data obtained from two or more exercise series with different work loads were used to estimate the parameters a, and a, by least squares. Pearson’s correlation coefficient and the standard error of estimate (SEE) were calculated as well. The SEE is calculated as the square root of the sum of squared residuals; 95% of the observations will be within the control limits placed at two SEES on either side of the calibration line. For each calibration line the hypothesis that a, = 0 was tested (two-sided t test, P < 0.05). We also tested whether the slope a, changed significantly after a change in position (two-sided t test, P < 0.05).
Evaluation of Doppler calibration in the sitting and standing positions. The validity of the Doppler calibra-
tion in the sitting and standing positions depends on the correct calculation of blood flow from the plethysmogram. A special procedure was applied to 11 exercise series selected at random to evaluate the calculation of the blood flow from the plethysmographic registrations. Results for one typical exercise series are presented in Figs. 4-6. Figure 4 is a recording of the plethysmogram during and after calf exercise in the standing position. At the start of each episode of exercise (E), blood is squeezed out of the venous system by the calf muscle pump, and the calf volume decreases. The large excursions of the plethysmogram during exercise were caused by muscle contractions. When the exercise is interrupted (R), the venous system is refilled with blood, and the calf volume (P) increases. P eventually reaches the final value PF. This value can either be read directly from the registration (R3, R4, and R5, Fig. 4) or estimated by interpolation (Rl and R2, Fig. 4). In our data PF varied slightly after each exercise period, probably due to changes in P not related to blood volume. The difference between PF and P is the volume of blood that is needed to refill the venous system completely PA
=
P F-
P
(1)
For each of the 11 series, PA and q were calculated for each heartbeat during the breaks (R) and after the exercise. To eliminate artifacts in the plethysmographic signal caused by muscle contraction, the heartbeats for which the moment exerted by the foot changed >2 N m have been excluded. By applying this criterion, an interval of ~0.5 s consecutive to the exercise was generally omitted, as well as the interval in which the flow is augmented because of the expansion of the arteries at the moment that the muscle relaxes. In Fig. 5 q/D is plotted against PA (data from Fig. 4). For 1.4 < PA < 3.5, we considered q/D to be constant because it is within 90% of its maximum. Outside this interval, the blood flow q measured by the plethysmograph is underestimated and cannot be used for Doppler RESULTS calibration. In the simplified procedure, the heartbeats Doppler calibration. Typical Doppler calibration lines were used for which the blood flow was maximal. The points were always very near the obtained in the supine, sitting, and standing positions are resulting calibration points selected by the criterion of constant q/D (Fig. 6). shown in Fig. 3. The estimations of a, and a,, Pearson’s correlation coefficient, and SEE of all Doppler calibraFor loads 258 N. m, the length of the interval of PA for tion lines are listed in Table 1. For each estimation 210 which q/D was constant varied between 1.1 and 2.1 ml/ 100 ml in the 11 selected series. For the lowest load (33 data points were available. For each individual subject the Doppler calibrations in N m), the length of the interval was 2.7 ml/100 ml in one extreme case and varied between 0.6 and 0.9 ml/100 ml in the same position but with different loads corresponded well, with a high correlation between q and D. After the the other cases. This means that, during exercise with change from the supine to the sitting position, the slope loads 258 N m, often (but not always) more blood was a, of the calibration line changed significantly in subjects squeezed out of the calf veins than during exercise with blood flow associated 2, 5, and IO, and after the change from the sitting to the 33 No m. Because the maximum standing position, the change in a, was significant in sub- with the lowest load was also much lower than that assojects 2, 3, 6, 8, and 10. In subjects 1, 4, 7, and 9, the slope of ciated with higher loads, there was always a sufficient l
l
l
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1672
DOPPLER ULTRASOUND
CALIBRATED
BY PLETHYSMOGRAPHY
TABLE 1. Results of Doppler calibration of calf blood flow in supine, sitting, and standing postures Supine Subj No.
r
0.98
1 2 3 4 5 6 7 8 9 10
0.91 0.96
0.98 0.95 0.84 0.92 0.94 0.93 0.98
a0
Sitting
SEE
a1
-17.5 1.7* -6.2 -4.1 0.4* 2.1* -4.6 -12.5 -8.8 -5.3
35 14 26 31
r
0.99 0.98 0.97 0.98 0.97 0.95 0.98 0.99 0.93 0.90
1.9 1.4
2.0 1.6 2.4 2.8 2.1 3.3 3.0 2.9
19 21
25 35 33 26
l
a1
-7.9
37 21 21 29 29 23 23 41 27 18
1.4* -3.6 -2.7 -1.7* 2.8 -1.7* -4.6 -2.7 1.2* l
amount of time (1.2-5.6 s) available for the selection of one heartbeat to calibrate the Doppler signal. DISCUSSION
Our experiments demonstrated that during Doppler calibration the relationship between calf blood flow q and Doppler frequency D can be represented by a linear model. For a subject in a fixed position, the Doppler calibration remained valid, because repeating the calibration procedure for various exercise series resulted in a single calibration line. In most subjects, however, the slope of the calibration line changed after a change in posture, indicating that a new calibration was necessary. To explain our experimental results, we compare our model with the well-known Doppler formula q(t) = 0.5 c -
A
ft cos 0
f(t)
(2)
with q(t) the instantaneous blood flow, f(t) the average frequency of the Doppler spectrum, c the velocity of sound in blood, ft the frequency of the transmitted ultrasound, A the cross-sectional area of the vessel within the sample volume, and 4 the angle of insonation. If the cross-sectional area of the vessel is insonated completely, Eq. 2 allows the calculation of blood flow independent of the velocity profile (6). The alteration of the slope of the calibration line after a change in position may have been caused by changes in A or $. A change in @Jmay have been caused by a change in direction of the transducer, the popliteal artery, or both. A change in the direction of the popliteal artery is not very likely, because in the fossa poplitea the popliteal artery is situated on the very bone itself (9). A change in
SEE
a0
Regression: q = a, + a$. Units: a,, ml 100 ml-’ min-‘; a,, ml 100 ml-’ coefficient. * Not significantly different from zero (P > 0.05). l
Standing
l
min-’
l
r
0.99 0.99 0.99
1.7 2.0 3.3 3.6 3.3 3.1 2.2 3.6 6.8 4.4
kHz-‘; SEE, ml
l
-1 100
200
225
4
250
time [s]
Strain gauge plethysmogram during and after calf exercise (80 N m) in standing position. Each sample coincides with an ECG trigger. R, rest; E, exercise. 4.
FIG.
l
2.5 1.8 2.9 7.0 4.9 5.8 4.3 3.1 4.7 5.8
ml-‘. min-‘. r, Pearson’s correlation
0 R2 x R3
I
OI
32 30 29 33 26 16 26 27 26 34
the direction of the transducer is more likely, because the transducer was mounted to the upper leg and it was observed that the form of the upper leg changed after a change in position from sagging of the relaxed muscles. The same explanation holds for the different slopes of the calibration lines for different subjects in the same position: the variation in the form of the upper leg resulted in a variation of the direction of the transducer. Although the radius of an artery and hence the crosssectional area A is a function of the transmural pressure, it is very unlikely that the increased transmural pressure in the sitting or standing position altered A significantly. The normal pulse pressure of -5.3 kPa (40 mmHg) results in a 1.2% variation in the radius of the femoral artery (13). In the popliteal artery this variation will be the same or less, because arteries generally become stiffer when the distance to the heart increases. In the standing position, the increase in pressure in the popliteal artery is -6 kPa (45 mmHg), which is slightly higher than the pulse pressure. If the active vascular tone does not change, we would therefore expect that the radius increases with - 1.4% in standing position and the crosssectional area with 2.8%. The changes in the slope of the calibration lines were much greater. Equation 2 yields a calibration line that passes the origin, whereas in our model a significant negative intercept
lo-
I
100
SEE
a1
-1.6 -1.6 -1.2* -7.2 -6.5 5.5 -5.9 1.5* -11.9 -17.8
0.91 0.97 0.92 0.97 0.97 0.96 0.95
#
,a
a0
0
+
R4
A
R5
I
1 2 PA (ml/100
1
I
I
3
4
5
ml]
FIG. 5. Ratio of q measured by plethysmography to D as a function of available volume (PA) during refilling. Curved line represents average result (curve fitted by eye). Symbols represent consecutive periods of rest in Fig. 4.
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DOPPLER
n-
CALIBRATED
100 0
c 80 -d E
2
ULTRASOUND
0
1.4
pA( 1.4 ml/100 ml ( PA{ 3.5 ml/100 ml
60
0 0-
40 I & 20 CT 0
0
0.5
1
I.5 D (kHz)
2
2.5
FIG. 6. Relationship between q measured by plethysmography D for 3 ranges of P,. Filled symbols were selected in standard dure, in which heartbeats were used at maximal blood flow.
3 and proce-
a, was found in most cases. We ascribe this to the influence of the velocity profile. At high blood flows, the velocity profile is flattened and f,,,(t) approaches f(t). As the blood flow decreases, the velocity profile gradually becomes parabolic, with fm,,( t) approaching 2f( t). This will result in a curved calibration diagram, which could be fitted ve ry well with a straight line with (negative) intercept a0 9 but only wi th in the limited range of blood flows that occurred during the calibration procedure. Extrapolation of the fitted line is therefore not allowed. Hiatt et al. (10) compared blood flow measurements with VOP with a 23-cm-wide cuff around the thigh and Doppler ultrasound in the superficial femoral artery at rest and after exercise of the calf muscles. According to Eq. 2 they measured A and estimated 4. They used the mean of the instanta neous flow velocity distribution in the sample volume to calcu late the blood flow. Because we used a 1.2-cm-wide cuff for venous occlusion and D, the results are not directly comparable. Despite these differences, their diagram of plethysmograph flow vs. Doppler flow is a straight line with a negative 1 ntercept just like most of our calibration diagrams. Hiatt et al. ascribed this negative intercept to an underestimation of blood flow by VOP. Our results demonstrate that such an intercept also occurs without venous occlusion. In the sitting and standing positions it was shown that, for some time after exercise (1.2-5.6 s), the blood flow calculated from plethysmography was proportional to D. In this interval no outflow occurred, and P, (Fig. 5) decreased from 3.5 to 1.4 ml/100 ml. For PA < 1.4, blood flow was u.nderestimated by the ple thysmograph as blood started to flow out of the venous system. For PA > 3.5 di rectly after the exercise, blood flow was also underestim ated, probably because the increase in calf volume in the segment enclosed by the plethysmograph is not yet representative of the increase in calf volume in the rest of the calf. None of the subjects had an abnormally low P, or an ab norma JlY fast increase in calf volume after exercise or had any clinical indications of venous incompetence. An important application of the calibration model will be the prediction of the calf blood flow from the Doppler frequency during rhythmic calf exercise. Such a predic-
BY
PLETHYSMOGRAPHY
1673
tion will be valid if the Doppler frequency is within the range that occurred during the calibration, so long as the calibration was not affected by exercise and displacements of the ultrasonic transducer did not occur or affect the Doppler frequency. In the sitting and standing positions the calibration was performed between the muscle contractions during exercise, i.e., when the calf muscles were relaxed. It was demonstrated by repeated measurements that the calibration remained valid during this procedure. A small increase in mean arterial pressure may have occurred (lo), but it would be smaller than the pulse pressure of 5.3 kPa, so that the increase in A will be negligible. It also means that $ did not change and proves that the transducer assembly succeeded in retaining 4 between the contractions. The same arguments hold for the supine position, so that the calibration performed after exercise remains valid during exercise between the contractions. The use of f,,,(t) greatly reduced the susceptibility to displacements of the ultrasonic transducer. A frequency correction as applied by Lubbers et al. (12) was therefore not necessary. Furthermore, measurement of the sound level demonstrated that the transducer assembly effectively prevented displacements of the ultrasonic transducer during exercise. The calibration procedure that we applied in the supine position was basically the same as the procedure that was used by Bernink et al. (2) and Lubbers et al. (12). However, their calibration diagrams were curved, even when a frequency correction was applied to compensate for the effects of small displacements of the transducer. As we argued before, this cannot be caused by a change in A. Subsequent application of spectral analysis to the signals measured by Lubbers et al. revealed that, during high blood velocities the upper frequency limit (7 kHz) of their equipment was surpassed. It was this deficiency that caused the curvature. Wallare and Wesche (15) measured the blood flow in the femoral artery with pulsed Doppler equipment during exercise of the quadriceps muscle group. To prevent displacements of the transducer and changes in 4, they allowed only small movements of the leg. The cross section of the femoral artery was assumed to be circular, and the area was estimated by measuring the vessel diameter with an ultrasonic method. The 4 was estimated to be 45” by virtue of ultrasonic echo scans. Thus they calculated the quantitative blood flow for two subjects. Compared with the method applied by Wallse and Wesche, our method of blood flow measurement by ultrasound has the advantage that no separate measurements are necessary to obtain A and +. Conclusions. 1) Two methods have been developed for in vivo calibration of ultrasonic Doppler equipment. One method can be applied in the supine position and the other in sitting and standing positions. With these methods we demonstrated that the Doppler calibration remains valid in a fixed position but that after a change in position a new calibration is required. 2) The maximum Doppler frequency is superior to the zero-crossing frequency that was used previously (12) because it is less susceptible to displacements of the ultrasonic transducer. 3) The calibrated Doppler equipment enables the
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1674
DOPPLER
ULTRASOUND
CALIBRATED
BY
measurement of calf blood flow in the supine, sitting, and standing positions between rhythmic muscle contractions during calf exercise. These measurements can be used to study the local effects of both posture and exercise on calf blood flow in individuals. Blood flow also can be measured after exercise, provided the Doppler frequency is within the range that occurred during the calibration .. Calibrated Doppler measuremen ts of the blood flow at rest are therefore not possible. We thank the volunteers who participated in this study. This study was supported by Dutch Foundation for Medical and Health Research MEDIGON Project 900-517-169. Address for reprint requests and construction details of the adjustable transducer holder: G. J. Barendsen, Laboratorium voor Medische Fysica, Bloemsingel 10, 9712 KZ Groningen, The Netherlands. Received
7 August
1990; accepted
in final
form
31 October
8.
9. 10.
1991. 11.
REFERENCES 1. BARENDSEN, G. J., H. VENEMA, AND Jw. VAN DEN BERG. Semicontinuous blood flow measurement by triggered venous occlusion plethysmography. J. Appl. Physiol. 31: 288-291, 1971. 2. BERNINK, P. J. L. M., J. LUBBERS, G. J. BARENDSEN, AND J. VAN DEN BERG. Blood flow in the calf during and after exercise: measurements with Doppler ultrasound and venous occlusion plethysmography in healthy subjects and patients with arterial occlusive disease. Angiology 33: 146-160, 1982. 3. BLOMQVIST, C. G., AND H. L. STONE. Cardiovascular adjustments to gravitational stress. In: Handbook of Physiology. The Cardiovascular System. Peripheral Circulation and Organ Blood Flow. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. III, pt. 2, chapt. 28, p. 1025-1063. 4. BRAKKEE, A. J. M. Plethysmographic measurement of peripheral
12.
13. 14.
15.
16.
PLETHYSMOGRAPHY
circulatory parameters in man. Adv. Cardiovasc. Phys. 5, Pt. II: 53-66, 1983. BRAKKEE, A. J. M., AND A. J. H. VENDRIK. Strain-gauge plethysmography; theoretical and practical notes on a new design. J. Appl. Physiol. 21: 701-704, 1966. BRODY, W. R., AND J. D. MEINDL. Theoretical analysis of the CW Doppler ultrasonic flowmeter. IEEE Trans. Biomed. Eng. 21: 183192, 1974. CALEYA, D. DE, K. P. BETHGE, AND K. BARBEY. Methodische Aspekte zur pneumatischen Segmentplethysmographie. I. Das Problem der Lagerung und der distalen venosen Okklusion. 2. Kardiol. 64: 625-635, 1975. FOLKOW, B., U. HAGLUND, M. JODAL, AND 0. LUNDGREN. Blood flow in the calf muscle of man during heavy rhythmic exercise. Acta Physiol. Stand. 81: 157-163, 1971. HAFFERL, A. Lehrbuch der Topografischen Anatomie (3rd ed.). Berlin: Springer-Verlag, 1969, p. 858-859. HIATT, W. R., S. Y. HUANG, J. G. REGENSTEINER, A. J. MICCO, G. ISHIMOTO, M. MANCO-JOHNSON, J. DROSE, AND J. T. REEVES. Venous occlusion plethysmography reduces arterial diameter and flow velocity. J. Appl. Physiol. 66: 2239-2244, 1989. HOKANSON, D. E., D. S. SUMNER, AND D. E. STRANDNESS. An electrically calibrated plethysmograph for direct measurement of limb blood flow. IEEE Trans. Biomed. Eng. 22: 25-29, 1975. LUBBERS, J., P. J. L. M. BERNINK, G. J. BARENDSEN, AND J. VAN DEN BERG. A continuous wave Doppler velocimeter for monitoring blood flow in the popliteal artery, compared with venous occlusion plethysmography of the calf. Pfluegers Arch. 382: 241-248, 1979. MILNOR, W. R. Hemodynamics. Baltimore, MD: Williams & Wilkins, 1989, p. 58-101. TBNNESEN, K. H., AND P. SEJRSEN. Washout of ““Xenon after intramuscular injection and direct measurement of blood flow in skeletal muscle. J. Clin. Lab. Invest. 25: 71-81, 1970. WALLBE, L., AND J. WESCHE. Time course and magnitude of blood flow changes in the human quadriceps muscles during and following rhythmic exercise. J. Physiol. Lond. 405: 257-273, 1988. WHITNEY, R. J. The measurement of volume changes in human limbs. J. Physiol. Lond. 121: l-27, 1953.
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LEEUWEN,
J. LUBBERS,
Department of Medical Physics, Faculty 9712 KZ Groningen, The Netherlands
G. J. BARENDSEN,
of Medicine,
VANLEEUWEN,B.E.,J. LUBBERS,G.J. BARENDSEN,AND L. DE PATER.Calf blood flow and posture: Doppler ultrasound calibrated by plethysmography. J. Appl. Physiol. 72(5): 1668-1674, 1992.-A procedure was developed that enables measurement of rapid variations in calf blood flow during voluntary rhythmic contraction of the calf muscles in supine, sitting, and standing positions. During the exercise, maximum blood velocity is measured by Doppler ultrasound equipment in the popliteal artery. The Doppler signals are calibrated by plethysmography to enable calculation of blood flow during exercise in ml. 100 Knowledge of the cross-sectional area of the vesml-l min. sel and the angle of insonation is not required in this procedure. Evaluation of the calibration method with 10 healthy volunteers showed that for each subject a new calibration was necessary after a change in posture; the relationship between the blood flow and the maximum Doppler frequency averaged over one heart cycle was linear for each calibration. l
rhythmic emia
muscle contractions;
calf ergometry;
exercise hyper-
DURINGRHYTHMICEXERCISE oftheleginthesittingand
standing positions, the muscle pumps of the upper and lower leg contribute significantly to the systemic circulation (3). There are also effects on local hemodynamics, because these muscle pumps increase the perfusion pressure (arterial-to-venous pressure difference) in the leg by lowering the local venous pressure. As a result, leg blood flow may be higher during exercise in the sitting and standing positions than in the supine position. To investigate whether this is true, leg blood flow must be measured during exercise in the supine position as well as in the sitting and standing positions. The only method that has been used to compare calf blood flow during exercise in supine, sitting, and standing positions is the 133Xe clearance method (8). With this method, small depots of 133Xe are injected in the muscle and the clearance rate is used as a measure for muscle blood flow. Major limitations of this method, however, are that 1) 133Xe clearance rate is influenced by the trauma of injection and by inhomogeneous composition and perfusion of the muscle (14) and 2) the method has insufficient resolution in time to distinguish the blood flow during muscle contraction from that during relaxation. The Doppler effect of ultrasound has been used during leg exercise in the supine position to monitor changes in blood flow in the upper leg (15) and the calf (2). These measurements demonstrated that the method has suffi1668
0161-7567/92
$2.00
Groningen
AND
L. DE PATER
University,
cient resolution in time to distinguish the variations in blood flow that occur during muscle contraction and relaxation. For quantitative measurement of blood flow with ultrasound, one must either measure the cross-sectional area of the vessel and the angle of insonation (15) or apply a calibration procedure. In the procedure that was used by Lubbers et al. (12), continuous-wave (CW) ultrasonic Doppler equipment with a zero-crossing detector was calibrated immediately after calf exercise in the supine position by comparing the zero-crossing frequency with the blood flow measured by venous occlusion plethysmography. A major limitation of this procedure was the susceptibility of the zero-crossing frequency to displacements of the ultrasonic transducer that are likely to occur during exercise. Furthermore the procedure cannot be applied in the sitting or standing positions because venous occlusion plethysmography can be used only in the supine position. The objectives of the present study therefore were I) to develop and evaluate a new calibration procedure that can be used in sitting and standing positions and that enables measurement of blood flow during exercise in these positions, 2) to apply a signal-processing technique that minimizes the susceptibility of the Doppler frequency to displacements of the ultrasonic transducer, 3) to investigate whether a calibration performed during or after a certain amount of exercise remains valid during and after another amount of exercise, and 4) to investigate whether a calibration performed in the supine position remains valid in the sitting and standing positions. METHODS Principle of Doppler calibration. In normal subjects the calf blood flow can be considered equal to the blood flow in the popliteal artery. This is so because in normal subjects there are no significant functional collaterals at the knee (9) and, consequently, almost all the blood to the lower leg passes the popliteal artery. Furthermore, during (exercise) hyperemia, the blood flow through the foot is negligible. This is evident from measurements with venous occlusion plethysmography (7): during hyperemia there was no significant difference between calf blood flow obtained with and without a distal occlusion cuff around the ankle to exclude the foot blood flow. The purpose of the calibration procedure is to quantify the relationship between Doppler frequency and calf blood flow. The ultrasonic transducer was positioned
Copyright 0 1992 the American Physiological Society
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DOPPLER
ULTRASOUND
CALIBRATED
BY
1669
PLETHYSMOGRAPHY
rn
above the popliteal artery. From the ultrasonic signal an average Doppler frequency was derived in a way that \ B minimized the susceptibility to displacements of the I$ \ transducer. This Doppler frequency was compared with C the calf blood flow measured by strain gauge plethysmoFPhy. A linear statistical model was used to fit the re~~ sults. Calf ergometry. A series of different values of calf blood flow is needed for the Doppler calibration. In the sitting and standing positions these were obtained during calf exercise. In the supine position the Doppler calibration was performed immediately after exercise as the blood - \ flow decreased and returned to the value at rest. Exercise was performed on a calf ergometer. Details of this ergomete r have been published previously (12). The exercise consisted of 90 treads (rhythmic exten sions and flexions) of the right foot on a pedal, with a frequency of 0.5 Hz indicated by a metronome. The angle- through which the pedal rotated was 15". Work was performed only during the extension of the foot. The moment imposed to the pedal by a spring determined the work load of each tread. During flexion of the foot, the calf muscles were completely relaxed, and the pedal returned slowly 1. Experimental setup of supine position and details of transto the initial position by the combined action of the ducerFIG. assembly: calf ergometer (A); strain gauge plethysmograph (B); spring and a damper. occlusion cuff (C); metal strip (D) for fixation of transducer assembly The work that the calf muscles performed was varied (E). Details of transducer assembly: block (K) is fixed to leg with metal by application of various moments and variation of the strip (D); block (L) can rotate with respect to block (K) around axis (x); (M) can slide with respect to block (L) by means of screw (y); number of treads. The duty cycle (the duration of the block holder (N) can rotate with respect to block (M) around axis (2); holder muscle contraction divided by the total du ration of con- (N) contains ultrasonic transducer (T), which is pushed to skin (S) and traction and relaxation during on.e tread) was S0.5 and directed to blood vessel (V). such that it could be easily reproduced by the subject. This was checked during exercise by recording two elec- ment of 92 N m (work 2,170 J) was applied if the subject trical signals, one representing the angle of rotation of could manage it. the pledal and the other representi .ng th .e exerted mODoppler equipment. Commercially available bidirecment. tional pulsed Doppler equipment with a built-in spectral In the supine position, the subject lay on his back with analyzer (TC2-64B, Eden Medizinische Technik) and a his shoulders supported by stops. The position of the flat ~-MHZ transducer was used. This transducer was right leg was adapted to the application of venous occlu- manufactured from our specifications and contained the sion plethysmography: the right knee was slightly bent, same active element as the commercially available 4with the lower leg -15 cm above heart level. For each MHz pencil probe. To obtain a calibration that could be subject, calf exercise in this position consisted of two se- reproduced, an arrangement was applied that minimized ries of 90 treads and moments of 33 and 58 N. m (work the displacements of the ultrasonic transducer and the 780 and 1,370 J, respectively). Each series was followed changes in the angle of insonation in each position and by a 3-min period during which Doppler calibration was allowed us to (re)direct the ultrasonic transducer without performed. altering the angle of insonation. The arrangement conIn the sitting position, the subject was seated in a sisted of the flat transducer fitted in a small holder faschair. The trunk was vertical and the upper right leg tened to the upper leg with a strip of spring steel. The horizontal. The angle between upper and lower right leg position of the transducer was such that the popliteal was the same as in the supine position. The calf exercise artery was insonated at the back of the knee in the region in this position consisted of two or three series of 90 of the fossa poplitea. (Fig. 1). treads each with short breaks (duration -8 s) after the To minimize the susceptibility of the Doppler freZnd, 5th, 15th, and 30th tread. Moments of 33,58, and (if quency to displacements of the ultrasonic transducer, we the subject could manage) 80 No m (work 1,880 J) were used the maximum Doppler frequency [ f,,,(t)] of the applied. During each break and immediately after com- Doppler spectrum. This quantity is proportional to the pletion of the 90 treads, one blood flow value was availinstantaneous maximum blood velocity [umax(t)]. Disable for Doppler calibration. placements of the ultrasonic transducer can be allowed In the standing position, the subject was supported by so long as the part of the cross section that contains a bicycle saddle. The right leg was not stretched com- V max (t) is insonated. The f,,,(t) was averaged over each pletely, inasmuch as the angle between upper and lower heart cycle, and this quantity (D) was compared with the leg was the same as in the supine and sitting positions. calf blood flow (q) measured by plethysmography. Calf exercise and Doppler calibration were performed in Displacements of the transducer were monitored by the same way as in the sitting position, but an extra mo- the sound level of the (forward) Doppler signal. At the d
l
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1670
DOPPLER
ULTRASOUND
CALIBRATED
BY PLETHYSMOGRAPHY
ECG trigger
start of each series of exercise, the sound level was maximized by directing
the ultrasound
beam to the center of
the vessel and by adjusting the depth range of the sample volume. A minimum signal level of 38 dB for reliable measurements was established from test bench measure-
ments. This minimum level should be considered typical for our equipment. When in the course of an exercise series the sound level fell below 38 dB, all signals were rejected and the transducer was redirected.
The quantity f,,,(t) was available only for blood flow in the forward direction. This is not a limitation, because there is no blood flow in the backward direction during exercise, at least not between contractions (2) and for some time after exercise. To detect the reoccurrence of blood flow in the backward direction after exercise, we used the zero-crossing frequency (the number of times the Doppler signal crosses its baseline per unit of time) of the bidirectional Doppler signal. Plethysmography. Strain gauge plethysmography has become an established method for noninvasive physiological or clinical studies of blood circulation in extremities (4) since its introduction by Whitney (16) in 1953. Our strain gauge plethysmograph
consisted of two mercury-
filled tubes of silicon rubber mounted mechanically parallel and electrically in series. This assembly was stretched around the thickest part of the calf. With relaxed muscles, relative changes in calf volume dV/V were equal to
I I I I I I i I I I I I I I 4r
fd W-M
321IP'I
measurement of calf blood flow. We therefore developed an alternative method of strain gauge plethysmography that could be used in the sitting
and standing positions.
With this method the blood flow was calculated from the increase in calf volume that occurred after one or more
relative changes in resistance dRlR of the mercury con-
treads of the foot.
ductor. These changes in resistance were measured with the circuitry proposed by Hokanson et al. (11): a con-
Essential for this method of strain gauge plethysmography is a properly functioning calf muscle pump, so
gauge, and
that the venous system of the calf is emptied during the
changes in resistance were measured as changes in voltage. Voltage and current terminals were separated (by
treads of the foot; i.e., the venous valves must be competent. Moreover, there had to be no outflow of blood from
the d-lead method) to rule out errors caused by the resis-
the venous system for some interval
tance of lead wires. For calibration of the plethysmograph, the electrical method described by Brakkee and
treads were completed. A special procedure was applied to 11 exercise series selected at random to confirm that
Vendrik
these conditions
stant current
was sent through
the strain
(5) was used.
were fulfilled
of time after the
(see RESULTS).
Because
In the supine position, the calf blood flow was determined by venous occlusion plethysmography (VOP)
all the blood that flowed into the venous system during that interval was used for refilling, the rate of volume
after exercise. Venous occlusion was achieved by inflat-
increase was equal to that of the calf blood flow.
ing a C&cm-wide occlusion cuff around the upper leg, just above the transducer assembly, to 6.6 kPa (50 mmHg).
The procedure of Doppler calibration in the sitting and standing positions was as follows. During the breaks after the Znd, 5th, 15th, and 30th tread and directly after completion of the 90 treads, the blood flow was measured for each heartbeat. As the amount of work increased,
The lower leg was positioned above heart level, and the cuff was alternately inflated and deflated for periods of three heartbeats to keep the venous pressure relatively low. This ensured that the increase in calf volume during
the occlusions was proportional to arterial blood flow. To enable short occlusion periods, and beat-to-beat calculation of blood flow, the occlusions were triggered by an
blood flow also increased. During each of the breaks and
after each series of exercise the venous system is filled rapidly, so we selected the heartbeats for which the blood flow was maximal.
The blood flow during these selected
the blood flow through the foot is negligible during exercise hyperemia (7).
heartbeats was then compared with the corresponding averaged Doppler frequencies, D. Data acquisition and processing. A personal computer system (Olivette M280 with 40-Mbyte hard disk and Labmaster analog-to-digital board) was used for data ac-
When the cuff was inflated, the position of the ultrasonic transducer relative to the vessel sometimes differed from that when the cuff was empty. The value of D was therefore calculated between occlusions. The corresponding blood flow value was obtained by linear interpolation (Fig. 2). The calibration was terminated when blood flow in the backward direction started to occur. VOP can only be applied in the supine position for
quisition and semiautomatic data processing. The following signals were sampled: plethysmogram and calibration pulse, ECG trigger, exerted moment and angle of rotation, forward f,,,(t), and sound level of forward and reverse Doppler signals. These signals and the forward and reverse zero-crossing frequencies of the Doppler signal were recorded on a strip chart recorder (SiemensElema Mingograf 800). This registration was used during
electrocardiogram (ECG) (1). The first occlusion began within two heartbeats after the exercise period. A distal occlusion cuff around the ankle was not applied, because
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DOPPLER
ULTRASOUND
100
100
supr
100
ne
r
r
80.
80.
60.
60.
40.
40.
20.
20.
o 33Nm D SBNm o 80Nm x
0’ 0
. I
’ 2 D
(kHz)
’ 3
’ 4
0’ 0
’ I
’ 2 D (kHz)
3
’ 4
oL 0
*
*
I
2 D
.
3
92Nm
’
4
(kHz1
3. Calibration diagrams of flow (q) against Doppler frequency for subj I, in supine, sitting, and standing positions.
FIG.
(D)
1671
BY PLETHYSMOGRAPHY
the calibration line did not change significantly after a change in position. The intercept a, for zero Doppler frequency differed significantly from zero in most calibrations (Table 1).
uprlghl
slllnO
c
CALIBRATED
the experiments to check the signal quality (e.g., detect artifacts, or monitor the level of the Doppler signal). Subjects and protocol. We investigated 10 male healthy subjects aged 20-54 yr. The subjects reported twice to our laboratory on different days. On the 1st day, the subject received a brief instruction and gave his informed consent. Then the calf ergometer was adjusted, and two or three test series of exercise were performed. The actual experiments were carried out on the 2nd day. The optimal position of the ultrasonic transducer was established while the subject was seated in a chair. After the transducer assembly had been fixed to the leg, the subject moved over to the calf ergometer. The plethysmograph and occlusion cuff were applied, and ECG electrodes for the trigger signal were attached. Doppler calibration was first performed in the supine position. After that the occlusion cuff was removed, and Doppler calibration proceeded in the sitting position and finally in the standing position. Statistics. For each subject, Doppler calibration lines were calculated for the supine, sitting, and standing positions according to the statistical model q = a, + a,D. For each of these positions, the data obtained from two or more exercise series with different work loads were used to estimate the parameters a, and a, by least squares. Pearson’s correlation coefficient and the standard error of estimate (SEE) were calculated as well. The SEE is calculated as the square root of the sum of squared residuals; 95% of the observations will be within the control limits placed at two SEES on either side of the calibration line. For each calibration line the hypothesis that a, = 0 was tested (two-sided t test, P < 0.05). We also tested whether the slope a, changed significantly after a change in position (two-sided t test, P < 0.05).
Evaluation of Doppler calibration in the sitting and standing positions. The validity of the Doppler calibra-
tion in the sitting and standing positions depends on the correct calculation of blood flow from the plethysmogram. A special procedure was applied to 11 exercise series selected at random to evaluate the calculation of the blood flow from the plethysmographic registrations. Results for one typical exercise series are presented in Figs. 4-6. Figure 4 is a recording of the plethysmogram during and after calf exercise in the standing position. At the start of each episode of exercise (E), blood is squeezed out of the venous system by the calf muscle pump, and the calf volume decreases. The large excursions of the plethysmogram during exercise were caused by muscle contractions. When the exercise is interrupted (R), the venous system is refilled with blood, and the calf volume (P) increases. P eventually reaches the final value PF. This value can either be read directly from the registration (R3, R4, and R5, Fig. 4) or estimated by interpolation (Rl and R2, Fig. 4). In our data PF varied slightly after each exercise period, probably due to changes in P not related to blood volume. The difference between PF and P is the volume of blood that is needed to refill the venous system completely PA
=
P F-
P
(1)
For each of the 11 series, PA and q were calculated for each heartbeat during the breaks (R) and after the exercise. To eliminate artifacts in the plethysmographic signal caused by muscle contraction, the heartbeats for which the moment exerted by the foot changed >2 N m have been excluded. By applying this criterion, an interval of ~0.5 s consecutive to the exercise was generally omitted, as well as the interval in which the flow is augmented because of the expansion of the arteries at the moment that the muscle relaxes. In Fig. 5 q/D is plotted against PA (data from Fig. 4). For 1.4 < PA < 3.5, we considered q/D to be constant because it is within 90% of its maximum. Outside this interval, the blood flow q measured by the plethysmograph is underestimated and cannot be used for Doppler RESULTS calibration. In the simplified procedure, the heartbeats Doppler calibration. Typical Doppler calibration lines were used for which the blood flow was maximal. The points were always very near the obtained in the supine, sitting, and standing positions are resulting calibration points selected by the criterion of constant q/D (Fig. 6). shown in Fig. 3. The estimations of a, and a,, Pearson’s correlation coefficient, and SEE of all Doppler calibraFor loads 258 N. m, the length of the interval of PA for tion lines are listed in Table 1. For each estimation 210 which q/D was constant varied between 1.1 and 2.1 ml/ 100 ml in the 11 selected series. For the lowest load (33 data points were available. For each individual subject the Doppler calibrations in N m), the length of the interval was 2.7 ml/100 ml in one extreme case and varied between 0.6 and 0.9 ml/100 ml in the same position but with different loads corresponded well, with a high correlation between q and D. After the the other cases. This means that, during exercise with change from the supine to the sitting position, the slope loads 258 N m, often (but not always) more blood was a, of the calibration line changed significantly in subjects squeezed out of the calf veins than during exercise with blood flow associated 2, 5, and IO, and after the change from the sitting to the 33 No m. Because the maximum standing position, the change in a, was significant in sub- with the lowest load was also much lower than that assojects 2, 3, 6, 8, and 10. In subjects 1, 4, 7, and 9, the slope of ciated with higher loads, there was always a sufficient l
l
l
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1672
DOPPLER ULTRASOUND
CALIBRATED
BY PLETHYSMOGRAPHY
TABLE 1. Results of Doppler calibration of calf blood flow in supine, sitting, and standing postures Supine Subj No.
r
0.98
1 2 3 4 5 6 7 8 9 10
0.91 0.96
0.98 0.95 0.84 0.92 0.94 0.93 0.98
a0
Sitting
SEE
a1
-17.5 1.7* -6.2 -4.1 0.4* 2.1* -4.6 -12.5 -8.8 -5.3
35 14 26 31
r
0.99 0.98 0.97 0.98 0.97 0.95 0.98 0.99 0.93 0.90
1.9 1.4
2.0 1.6 2.4 2.8 2.1 3.3 3.0 2.9
19 21
25 35 33 26
l
a1
-7.9
37 21 21 29 29 23 23 41 27 18
1.4* -3.6 -2.7 -1.7* 2.8 -1.7* -4.6 -2.7 1.2* l
amount of time (1.2-5.6 s) available for the selection of one heartbeat to calibrate the Doppler signal. DISCUSSION
Our experiments demonstrated that during Doppler calibration the relationship between calf blood flow q and Doppler frequency D can be represented by a linear model. For a subject in a fixed position, the Doppler calibration remained valid, because repeating the calibration procedure for various exercise series resulted in a single calibration line. In most subjects, however, the slope of the calibration line changed after a change in posture, indicating that a new calibration was necessary. To explain our experimental results, we compare our model with the well-known Doppler formula q(t) = 0.5 c -
A
ft cos 0
f(t)
(2)
with q(t) the instantaneous blood flow, f(t) the average frequency of the Doppler spectrum, c the velocity of sound in blood, ft the frequency of the transmitted ultrasound, A the cross-sectional area of the vessel within the sample volume, and 4 the angle of insonation. If the cross-sectional area of the vessel is insonated completely, Eq. 2 allows the calculation of blood flow independent of the velocity profile (6). The alteration of the slope of the calibration line after a change in position may have been caused by changes in A or $. A change in @Jmay have been caused by a change in direction of the transducer, the popliteal artery, or both. A change in the direction of the popliteal artery is not very likely, because in the fossa poplitea the popliteal artery is situated on the very bone itself (9). A change in
SEE
a0
Regression: q = a, + a$. Units: a,, ml 100 ml-’ min-‘; a,, ml 100 ml-’ coefficient. * Not significantly different from zero (P > 0.05). l
Standing
l
min-’
l
r
0.99 0.99 0.99
1.7 2.0 3.3 3.6 3.3 3.1 2.2 3.6 6.8 4.4
kHz-‘; SEE, ml
l
-1 100
200
225
4
250
time [s]
Strain gauge plethysmogram during and after calf exercise (80 N m) in standing position. Each sample coincides with an ECG trigger. R, rest; E, exercise. 4.
FIG.
l
2.5 1.8 2.9 7.0 4.9 5.8 4.3 3.1 4.7 5.8
ml-‘. min-‘. r, Pearson’s correlation
0 R2 x R3
I
OI
32 30 29 33 26 16 26 27 26 34
the direction of the transducer is more likely, because the transducer was mounted to the upper leg and it was observed that the form of the upper leg changed after a change in position from sagging of the relaxed muscles. The same explanation holds for the different slopes of the calibration lines for different subjects in the same position: the variation in the form of the upper leg resulted in a variation of the direction of the transducer. Although the radius of an artery and hence the crosssectional area A is a function of the transmural pressure, it is very unlikely that the increased transmural pressure in the sitting or standing position altered A significantly. The normal pulse pressure of -5.3 kPa (40 mmHg) results in a 1.2% variation in the radius of the femoral artery (13). In the popliteal artery this variation will be the same or less, because arteries generally become stiffer when the distance to the heart increases. In the standing position, the increase in pressure in the popliteal artery is -6 kPa (45 mmHg), which is slightly higher than the pulse pressure. If the active vascular tone does not change, we would therefore expect that the radius increases with - 1.4% in standing position and the crosssectional area with 2.8%. The changes in the slope of the calibration lines were much greater. Equation 2 yields a calibration line that passes the origin, whereas in our model a significant negative intercept
lo-
I
100
SEE
a1
-1.6 -1.6 -1.2* -7.2 -6.5 5.5 -5.9 1.5* -11.9 -17.8
0.91 0.97 0.92 0.97 0.97 0.96 0.95
#
,a
a0
0
+
R4
A
R5
I
1 2 PA (ml/100
1
I
I
3
4
5
ml]
FIG. 5. Ratio of q measured by plethysmography to D as a function of available volume (PA) during refilling. Curved line represents average result (curve fitted by eye). Symbols represent consecutive periods of rest in Fig. 4.
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DOPPLER
n-
CALIBRATED
100 0
c 80 -d E
2
ULTRASOUND
0
1.4
pA( 1.4 ml/100 ml ( PA{ 3.5 ml/100 ml
60
0 0-
40 I & 20 CT 0
0
0.5
1
I.5 D (kHz)
2
2.5
FIG. 6. Relationship between q measured by plethysmography D for 3 ranges of P,. Filled symbols were selected in standard dure, in which heartbeats were used at maximal blood flow.
3 and proce-
a, was found in most cases. We ascribe this to the influence of the velocity profile. At high blood flows, the velocity profile is flattened and f,,,(t) approaches f(t). As the blood flow decreases, the velocity profile gradually becomes parabolic, with fm,,( t) approaching 2f( t). This will result in a curved calibration diagram, which could be fitted ve ry well with a straight line with (negative) intercept a0 9 but only wi th in the limited range of blood flows that occurred during the calibration procedure. Extrapolation of the fitted line is therefore not allowed. Hiatt et al. (10) compared blood flow measurements with VOP with a 23-cm-wide cuff around the thigh and Doppler ultrasound in the superficial femoral artery at rest and after exercise of the calf muscles. According to Eq. 2 they measured A and estimated 4. They used the mean of the instanta neous flow velocity distribution in the sample volume to calcu late the blood flow. Because we used a 1.2-cm-wide cuff for venous occlusion and D, the results are not directly comparable. Despite these differences, their diagram of plethysmograph flow vs. Doppler flow is a straight line with a negative 1 ntercept just like most of our calibration diagrams. Hiatt et al. ascribed this negative intercept to an underestimation of blood flow by VOP. Our results demonstrate that such an intercept also occurs without venous occlusion. In the sitting and standing positions it was shown that, for some time after exercise (1.2-5.6 s), the blood flow calculated from plethysmography was proportional to D. In this interval no outflow occurred, and P, (Fig. 5) decreased from 3.5 to 1.4 ml/100 ml. For PA < 1.4, blood flow was u.nderestimated by the ple thysmograph as blood started to flow out of the venous system. For PA > 3.5 di rectly after the exercise, blood flow was also underestim ated, probably because the increase in calf volume in the segment enclosed by the plethysmograph is not yet representative of the increase in calf volume in the rest of the calf. None of the subjects had an abnormally low P, or an ab norma JlY fast increase in calf volume after exercise or had any clinical indications of venous incompetence. An important application of the calibration model will be the prediction of the calf blood flow from the Doppler frequency during rhythmic calf exercise. Such a predic-
BY
PLETHYSMOGRAPHY
1673
tion will be valid if the Doppler frequency is within the range that occurred during the calibration, so long as the calibration was not affected by exercise and displacements of the ultrasonic transducer did not occur or affect the Doppler frequency. In the sitting and standing positions the calibration was performed between the muscle contractions during exercise, i.e., when the calf muscles were relaxed. It was demonstrated by repeated measurements that the calibration remained valid during this procedure. A small increase in mean arterial pressure may have occurred (lo), but it would be smaller than the pulse pressure of 5.3 kPa, so that the increase in A will be negligible. It also means that $ did not change and proves that the transducer assembly succeeded in retaining 4 between the contractions. The same arguments hold for the supine position, so that the calibration performed after exercise remains valid during exercise between the contractions. The use of f,,,(t) greatly reduced the susceptibility to displacements of the ultrasonic transducer. A frequency correction as applied by Lubbers et al. (12) was therefore not necessary. Furthermore, measurement of the sound level demonstrated that the transducer assembly effectively prevented displacements of the ultrasonic transducer during exercise. The calibration procedure that we applied in the supine position was basically the same as the procedure that was used by Bernink et al. (2) and Lubbers et al. (12). However, their calibration diagrams were curved, even when a frequency correction was applied to compensate for the effects of small displacements of the transducer. As we argued before, this cannot be caused by a change in A. Subsequent application of spectral analysis to the signals measured by Lubbers et al. revealed that, during high blood velocities the upper frequency limit (7 kHz) of their equipment was surpassed. It was this deficiency that caused the curvature. Wallare and Wesche (15) measured the blood flow in the femoral artery with pulsed Doppler equipment during exercise of the quadriceps muscle group. To prevent displacements of the transducer and changes in 4, they allowed only small movements of the leg. The cross section of the femoral artery was assumed to be circular, and the area was estimated by measuring the vessel diameter with an ultrasonic method. The 4 was estimated to be 45” by virtue of ultrasonic echo scans. Thus they calculated the quantitative blood flow for two subjects. Compared with the method applied by Wallse and Wesche, our method of blood flow measurement by ultrasound has the advantage that no separate measurements are necessary to obtain A and +. Conclusions. 1) Two methods have been developed for in vivo calibration of ultrasonic Doppler equipment. One method can be applied in the supine position and the other in sitting and standing positions. With these methods we demonstrated that the Doppler calibration remains valid in a fixed position but that after a change in position a new calibration is required. 2) The maximum Doppler frequency is superior to the zero-crossing frequency that was used previously (12) because it is less susceptible to displacements of the ultrasonic transducer. 3) The calibrated Doppler equipment enables the
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1674
DOPPLER
ULTRASOUND
CALIBRATED
BY
measurement of calf blood flow in the supine, sitting, and standing positions between rhythmic muscle contractions during calf exercise. These measurements can be used to study the local effects of both posture and exercise on calf blood flow in individuals. Blood flow also can be measured after exercise, provided the Doppler frequency is within the range that occurred during the calibration .. Calibrated Doppler measuremen ts of the blood flow at rest are therefore not possible. We thank the volunteers who participated in this study. This study was supported by Dutch Foundation for Medical and Health Research MEDIGON Project 900-517-169. Address for reprint requests and construction details of the adjustable transducer holder: G. J. Barendsen, Laboratorium voor Medische Fysica, Bloemsingel 10, 9712 KZ Groningen, The Netherlands. Received
7 August
1990; accepted
in final
form
31 October
8.
9. 10.
1991. 11.
REFERENCES 1. BARENDSEN, G. J., H. VENEMA, AND Jw. VAN DEN BERG. Semicontinuous blood flow measurement by triggered venous occlusion plethysmography. J. Appl. Physiol. 31: 288-291, 1971. 2. BERNINK, P. J. L. M., J. LUBBERS, G. J. BARENDSEN, AND J. VAN DEN BERG. Blood flow in the calf during and after exercise: measurements with Doppler ultrasound and venous occlusion plethysmography in healthy subjects and patients with arterial occlusive disease. Angiology 33: 146-160, 1982. 3. BLOMQVIST, C. G., AND H. L. STONE. Cardiovascular adjustments to gravitational stress. In: Handbook of Physiology. The Cardiovascular System. Peripheral Circulation and Organ Blood Flow. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. III, pt. 2, chapt. 28, p. 1025-1063. 4. BRAKKEE, A. J. M. Plethysmographic measurement of peripheral
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circulatory parameters in man. Adv. Cardiovasc. Phys. 5, Pt. II: 53-66, 1983. BRAKKEE, A. J. M., AND A. J. H. VENDRIK. Strain-gauge plethysmography; theoretical and practical notes on a new design. J. Appl. Physiol. 21: 701-704, 1966. BRODY, W. R., AND J. D. MEINDL. Theoretical analysis of the CW Doppler ultrasonic flowmeter. IEEE Trans. Biomed. Eng. 21: 183192, 1974. CALEYA, D. DE, K. P. BETHGE, AND K. BARBEY. Methodische Aspekte zur pneumatischen Segmentplethysmographie. I. Das Problem der Lagerung und der distalen venosen Okklusion. 2. Kardiol. 64: 625-635, 1975. FOLKOW, B., U. HAGLUND, M. JODAL, AND 0. LUNDGREN. Blood flow in the calf muscle of man during heavy rhythmic exercise. Acta Physiol. Stand. 81: 157-163, 1971. HAFFERL, A. Lehrbuch der Topografischen Anatomie (3rd ed.). Berlin: Springer-Verlag, 1969, p. 858-859. HIATT, W. R., S. Y. HUANG, J. G. REGENSTEINER, A. J. MICCO, G. ISHIMOTO, M. MANCO-JOHNSON, J. DROSE, AND J. T. REEVES. Venous occlusion plethysmography reduces arterial diameter and flow velocity. J. Appl. Physiol. 66: 2239-2244, 1989. HOKANSON, D. E., D. S. SUMNER, AND D. E. STRANDNESS. An electrically calibrated plethysmograph for direct measurement of limb blood flow. IEEE Trans. Biomed. Eng. 22: 25-29, 1975. LUBBERS, J., P. J. L. M. BERNINK, G. J. BARENDSEN, AND J. VAN DEN BERG. A continuous wave Doppler velocimeter for monitoring blood flow in the popliteal artery, compared with venous occlusion plethysmography of the calf. Pfluegers Arch. 382: 241-248, 1979. MILNOR, W. R. Hemodynamics. Baltimore, MD: Williams & Wilkins, 1989, p. 58-101. TBNNESEN, K. H., AND P. SEJRSEN. Washout of ““Xenon after intramuscular injection and direct measurement of blood flow in skeletal muscle. J. Clin. Lab. Invest. 25: 71-81, 1970. WALLBE, L., AND J. WESCHE. Time course and magnitude of blood flow changes in the human quadriceps muscles during and following rhythmic exercise. J. Physiol. Lond. 405: 257-273, 1988. WHITNEY, R. J. The measurement of volume changes in human limbs. J. Physiol. Lond. 121: l-27, 1953.
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