INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE POSSIBILITY OF USING LIGHT to measure the oxygen saturation of hemoglobin in vivo has been explored since the 1940s (37). The feasibility of optical blood oximetry stems from the oxygenation dependence of the optical spectrum of hemoglobin. This is illustrated in Fig. 1, which shows the absorption spectra of 100 µM hemoglobin for oxygen saturation values of 0, 20, 40, 60, 80, and 100%. The spectra of Fig. 1 were calculated from published values of the molar extinction coefficients of oxyhemoglobin (HbO₂) and deoxyhemoglobin (Hb) (43, 53).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Near-infrared absorption spectra of 100 µM hemoglobin concentration ([Hb-T], where T stands for total) for different values of the oxygen saturation (SO2) in the range of 0-100%. The curve for SO2 = 0% corresponds to the deoxyhemoglobin (Hb) absorption spectrum, whereas the curve for SO2 = 100% corresponds to the oxyhemoglobin (HbO2) absorption spectrum. These spectra have been computed from published spectra of the molar extinction coefficients of HbO2 and Hb (43, 53).

Oxygen saturation of the pulmonary capillary blood in rabbits has been measured by using dynamic invasive techniques (48). Near-infrared light in the wavelength range from 700 to 900 nm results in a sufficient penetration depth for the noninvasive optical monitoring of skeletal muscle, cerebral gray matter, and breast tissue. As a result, near-infrared techniques allow a noninvasive assessment of hemoglobin saturation for a wide range of applications, such as the study of muscle metabolism (7, 9, 12, 29, 45), the diagnosis of vascular disorders (2, 20, 32, 33, 44, 49), functional brain imaging (3, 10, 24, 30, 35, 50), and breast cancer detection (23, 28, 40, 42, 46).

If near-infrared light is highly sensitive to the oxygen saturation of hemoglobin, then its large penetration depth inside tissues implies that the arterial, venous, and capillary compartments all contribute to the optical signal. The average hemoglobin oxygenation measured with near-infrared spectroscopy (NIRS) (19, 34, 41) is usually referred to as tissue oxygen saturation (St_O2). St_O2 values are assumed to be in between arterial and local venous saturation values (Sa_O2 and Sv_O2, respectively). A number of research studies have investigated the relationship between the near-infrared (noninvasive) measurement of St_O2 and the values of Sa_O2 and local Sv_O2 measured invasively from drawn blood samples (31, 51). The contribution of the arterial compartment to the noninvasive optical signal can be isolated because of its unique temporal dynamics associated with the systolic-diastolic blood pressure variation at the heartbeat frequency (1). The pulsatile component of the optical signals at two or more wavelengths at the heartbeat frequency is used by conventional (1, 36) or self-calibrated (21) pulse oximeters to measure the Sa_O2. Sa_O2 is a parameter that provides information about the ventilation and the oxygen exchange in the lungs. In contrast, Sv_O2 is a parameter that reflects the local balance between blood flow and oxygen consumption. The noninvasive optical measurement of Sv_O2 is complicated by the fact that the isolation of the contribution of the venous compartment to the noninvasive optical signal is not straightforward. There are no clinical devices presently capable of monitoring Sv_O2 noninvasively.

A number of experimental approaches have been proposed to measure Sv_O2 from induced local changes in the venous blood volume. For instance, proposed approaches involve a venous occlusion in a limb (13, 39, 55, 56), tilting the patient's head down by 15 degrees (47), a partial jugular vein occlusion (15, 54), or mechanical ventilation (52). In all these approaches, Sv_O2 is optically measured as the ratio between the increases in the HbO₂ concentration ([HbO₂]) and the total hemoglobin concentration (equal to [HbO₂] + [Hb], where [Hb] is deoxyhemoglobin concentration) induced by the local increase in venous blood volume. To overcome the limitations of these methods, which can either be applied only to the limbs (venous occlusion method) or require an external perturbation (partial jugular vein occlusion, mechanical ventilation, and tilting methods), we propose an alternative approach that is an extension of the method of Wolf et al. (52). This approach involves no external perturbations and is applicable to subjects who are breathing either spontaneously or synchronously with a metronome set at their average respiratory frequency. Furthermore, this method can provide continuous and real-time monitoring of Sv_O2. The basic idea is to measure Sv_O2 from the amplitude of the optically measured [HbO₂] and [Hb] oscillations at the respiratory frequency. The basic hypothesis, originally formulated in this context and tested on the brain of mechanically ventilated infants by Wolf et al., is that the oscillatory components of [HbO₂] and [Hb] at the breathing rate are mostly representative of the venous compartment. Because the venous compliance is ~20 times as large as the arterial compliance (4), a given change in the blood pressure in the veins causes a venous volume change ~20 times as large as the arterial volume change corresponding to the same pressure change in the arteries. During normal breathing, the inspiration phase involves a decrease in the intrathoracic pressure and an increased pressure gradient between the peripheral venous system and the intrathoracic veins. This causes blood to be drawn from the extrathoracic veins into the intrathoracic vessels and heart (26). Because of the vein valves, venous return is increased more by inspiration than it is decreased by expiration (38). The net effect is the so-called respiratory pump that facilitates the venous return from the periphery by the respiration-induced periodic fluctuations in the central venous pressure (38). As a result of the respiratory pump, the peripheral venous blood volume oscillates at the respiratory frequency, decreasing during inspiration and increasing during expiration.

It is on this oscillatory component at the respiratory frequency that we base our near-infrared measurement of the Sv_O2. We coin the term spiroximeter to indicate an instrument for measuring the Sv_O2 from respiration-induced oscillations in the venous blood pressure and in the venous volume fraction in tissues. It must be observed that respiration may also induce perturbations to the heart rate (respiratory sinus arrhythmia) and consequently to the cardiac output and arterial blood pressure. As a result, the arterial compartment volume may, in general, also oscillate at the respiratory frequency; thus near-infrared spiroximetry data must be carefully examined to guarantee a reliable reading of Sv_O2.

We report a validation study conducted on the hind leg of three piglets, in which we compared the near-infrared measurements of Sv_O2 (Sv_O2-NIRS) with the Sv_O2 values obtained by the gas analysis of venous blood samples (Sv_O2-blood). To show the applicability of spiroximetry to human subjects, we also conducted a preliminary test on the vastus medialis and vastus lateralis muscles of healthy volunteers at rest and postexercise.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tissue spectrometer. The near-infrared measurements were performed with a frequency-domain tissue spectrometer (model 96208, ISS, Champaign, IL) (18, 25). This instrument uses two parallel photomultiplier tube detectors that are time shared by eight multiplexed laser diodes emitting at 636, 675, 691, 752, 780, 788, 830, and 840 nm, respectively. The frequency of intensity modulation is 110 MHz, and heterodyne detection is performed with a cross-correlation frequency of 5 kHz. The multiplexing rate, i.e., the frequency of sequential laser switching, is 100 Hz. As a result, 50 cross-correlation periods are acquired during the on time of each laser diode, and a complete acquisition cycle over the eight wavelengths is completed every 80 ms. The laser diodes and the photomultiplier tubes are all coupled to fiber optics. The eight individual illumination fibers, each 400 µm in internal diameter, are arranged into a fiber bundle having a rectangular cross-section of 3.5 × 2.0 mm². The collecting circular fiber bundles are 3.0 mm in internal diameter. The optical fibers are placed in contact with the skin by means of a flexible plastic probe. The optical probe arranges the tips of the illuminating and collecting fiber bundles along a line, with the two collecting fiber bundles at distances of 1.0 and 2.0 cm from the single illuminating bundle. In some cases, we have used a second tissue spectrometer to perform simultaneous measurements on both legs (piglets 2 and 3) or at different locations on the same leg (human subjects). In the second tissue spectrometer (which used the optical probes PL and HVL defined below), the 840-nm laser diode was replaced by a laser diode emitting at 814 nm.

Measurements on piglets. We performed measurements on three piglets that were 15 ± 1 days old and weighed 5 ± 1 kg. The experimental arrangement for the piglet measurements is schematically illustrated in Fig. 2. The piglets were anesthetized by inhalation of 3-4% isoflurane administered by means of a breathing mask applied to the piglet's snout. The animals were not mechanically ventilated, and they breathed freely throughout the experiment. A strain-gauge belt (Sleepmate/Newlife Technologies, Resp-EZ) was placed around the piglet's thorax to continuously monitor the respiratory excursion. A pulse oximeter (Nellcor, N-200) continuously recorded the heart rate at the foot of the right hind leg. The analog outputs from the strain gauge and the pulse oximeter were fed to the auxiliary input ports of the tissue spectrometer for continuous coregistration of optical and physiological data. A femoral cutdown was performed into the left inferior femoral vein to insert a catheter for periodic blood sampling. The femoral venous blood samples were run through a commercial blood-gas analyzer (Instrumentation Laboratory, model 1304 pH/blood-gas analyzer) to obtain invasive readings of Sv_O2-blood. One optical probe (identified as probe PR) was always located on the right (noncatheterized) hind leg. In piglets 2 and 3, a second probe (probe PL) was placed on the catheterized (left) leg. The protocol consisted of varying the femoral Sv_O2 over the approximate range of 20-95% by modulating the volume fraction of oxygen inspired by the piglet (FI_O2) over the range of 10-100%. The oxygenation cycles performed on the three piglets are illustrated in Fig. 3. Each cycle consisted of varying the FI_O2 approximately every 4-6 min through the values of ~40, 15, 10, and 100% (piglets 1 and 2) or ~40, 20, 17.5, 15, 12.5, 10, and 100% (piglet 3). We performed two FI_O2 cycles on piglet 1, four on piglet 2, and three on piglet 3. For each specific value of FI_O2, we acquired about 3,000 optical data points [4 min × (60 s/min)/(80 ms/data point)] or more. During cycles C and D on piglet 2, the optical probe PR was slightly moved with respect to the location examined during cycles A and B, to collect data on two different muscle volumes during the two cycle pairs A-B and C-D. Optical probe PR always collected data on the right hind leg, whereas probe PL was placed on the left hind leg during cycles A and B of piglet 2 and cycles A and B of piglet 3 (we did not collect data with the optical probe PL on piglet 1, during cycles C-D on piglet 2, and during cycle C on piglet 3). In all three piglets, the invasive measurement of Sv_O2 from a femoral vein blood sample was performed at the end of each FI_O2 interval, as shown in Fig. 3. Motion artifacts were minimized in the optical data by securing the piglet's legs to the operating table. The protocol was approved by the Institutional Review Board of the Massachusetts General Hospital, where the piglet experiments were performed.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Experimental arrangement for the piglet study. A breathing mask applied to the piglet's snout provided the 3-4% isoflurane anesthetic and was connected to the oxygen line for variations in the fraction of inspired oxygen (FIO2). A strain-gauge belt and a pulse oximeter monitored the respiratory excursion and the heart rate, respectively, and their analog outputs were directed to the auxiliary inputs of the frequency-domain tissue spectrometer (ISS, Champaign, IL, model 96208). One or two optical probes (PR on the right hind leg and PL on the left hind leg) of the tissue spectrometer were used to measure the near-infrared tissue absorption with a time resolution of 80 ms. The absorption oscillations at the respiratory frequency were processed to provide measurement of the venous O2 saturation (SvO2-NIRSresp) (NIRS is near-infrared spectroscopy). Invasive measurements of the venous O2 saturation (designated SvO2-blood) were obtained by gas analysis of venous blood samples collected by a femoral vein catheter. aux, Auxiliary; optical I/O, optical input/output.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Schematic representation of FIO2 cycles for piglet 1 (A), piglet 2 (B), and piglet 3 (C). , Time at which venous blood samples were run through the blood-gas analyzer for SvO2-blood measurements.

Measurements on human subjects. We performed measurements on eight healthy human subjects (6 men and 2 women; mean age of 24.5 yr, age range of 20-35 yr). The subjects sat on a comfortable chair and rested for 10-15 min before the experimental protocol was started. A pneumatic cuff was placed around the right thigh of the subject to later induce a venous occlusion by inflating the cuff to a pressure of 70 mmHg. A pulse oximeter probe (Nellcor, N-200) was placed on the index finger of the left hand. A strain-gauge belt (Sleepmate/Newlife Technologies, Resp-EZ) was placed around the subject's upper abdomen to monitor the respiratory excursion. As in the piglet experiment, we used the analog outputs of the pulse oximeter and strain gauge for continuous coregistration of the physiological and near-infrared data. Two optical probes were placed on the right thigh; the first probe (probe HVM) was positioned on top of a visible superficial vein of the vastus medialis muscle, and the second probe (probe HVL) was placed on the vastus lateralis muscle, far from visible superficial veins. During the measurements, we asked the subject to breathe regularly, following a metronome whose frequency was set to the average breathing rate of the subject at rest (typically 14-15 breaths/min). During the whole experiment, the subject was asked to breathe at the same frequency as the metronome pace. No subjects experienced any discomfort or difficulties with this procedure. The measurement protocol consisted of 2 min of baseline (we acquired 1,535 optical data points at 80 ms/point), followed by 40 s of venous occlusion, and a final recovery period of a few minutes. A few subjects performed an additional exercise routine to test the effect of exercise on the measured value of Sv_O2-NIRS_resp on the muscle. The exercise consisted of raising the right foot, voluntarily contracting the leg muscles (isometric contraction), until the subject felt tired. The human study was approved by the Institutional Review Board of Tufts University, where the human experiments were performed; all subjects gave their written, informed consent.

Near-infrared data processing for the measurement of Sv_O2. We used a modified Beer-Lambert law approach (14) to translate the temporal intensity ratio collected at each wavelength [I(, t)/I(, 0), where I is intensity,  is wavelength, and t is time] at a distance of 1.0 cm from the illumination point, into a time variation in the tissue absorption [µ_a(, t), where µ_a is the tissue absorption coefficient]. This approach was implemented by applying the following equation (14)

(1)

where L_eff is the effective optical pathlength from the illuminating point to the light collection point. We measured L_eff by quantifying µ_a and the reduced scattering coefficient (µ'_s) using the frequency-domain multidistance method (17). The diffusion-theory relationship that gives L_eff in terms of µ_a, µ_s', and the source-detector separation (r) in a semi-infinite turbid medium (where the illumination and collection points are at the boundary of the turbid medium) is the following (17)

(2)

More details on this hybrid frequency-domain [to measure L_eff()] and continuous wave (modified Beer-Lambert law) approach are given in Refs. 14, 17, 21, and 22. Equation 2 shows that for typical values of the near-infrared µ_a and µ'_s, say µ_a = 0.1 cm¹ and µ'_s = 10 cm¹, the value of L_eff is ~5.5 cm for r = 1 cm. The multi-distance scheme was implemented by considering the data collected by the two fiber bundles located at two different distances (1.0 and 2.0 cm) from the source fiber bundle. At these source detector distances, the diffusion regime of light propagation in tissues is already established (18). As an alternative to the diffusion equation model to describe the spatial dependence of the optical signal, empirical approaches have been proposed (6). The different sensitivity of the two detector channels was accounted for by a preliminary calibration measurement on a synthetic tissue-like sample. The applicability of the initial calibration to the whole data set was verified at the end of each measurement session by repositioning the optical probe on the calibration sample. We typically reproduced the calibration values of the block optical coefficients to within 10%. In the piglet experiments, we updated the measured values of L_eff at each wavelength every time the FI_O2 was changed. Specifically, L_eff was computed, according to Eq. 2, from average measurements of µ_a and µ'_s over the last 80 s of each period corresponding to a specific FI_O2 value. For the measurements on human subjects, we computed an initial value of L_eff (at each wavelength) over the first 2 min of baseline, and we used this value for the analysis of the data over the whole measurement session. The long integration time for the mean pathlength measurements (80 s in the piglet experiment, 120 s in the human subjects measurements) realized a low-pass filter that minimized the time-varying contributions from the Hb oscillations caused by the arterial pulsation and breathing. Furthermore, the two-distance measurement scheme for the mean pathlength measurement also provided some level of spatial averaging. In contrast, the optical data for the measurement of Sv_O2 were acquired with an 80-ms temporal resolution and with the use of a single source-detector distance (1 cm).

(3)

(4)

(5)

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Figure 4 reports average spectra of L_eff measured for a source-detector separation of 1 cm. Figure 4A refers to piglet measurements conducted at two different values of FI_O2, whereas Fig. 4B refers to human measurements with probes HVM and HVL. The error bars in Fig. 4 represent the standard deviations over multiple measurements (multiple FI_O2 cycles and piglets for Fig. 4A and multiple subjects for Fig. 4B).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Near-infrared spectra of the effective optical pathlength (Leff) measured on the piglet's leg (A) and a human thigh muscle (B) for a source-detector separation (r) of 1 cm. In A, different symbols refer to 2 different values of FIO2. In B, different symbols refer to 2 different thigh muscles (vastus medialis for probe HVM and vastus lateralis for probe HVL). The lines join the points as an aid to the eye.

In the piglet experiment, we discarded 11 (from a total of 67) Sv_O2-NIRS_resp(FFT) measurements because the standard deviation over 800 FFTs exceeded 15%. These discarded Sv_O2-NIRS_resp(FFT) readings occurred as follows: one (of 8) in piglet 1, two (of 26) in piglet 2, and eight (of 33) in piglet 3. One discarded reading was assigned to motion artifacts, whereas the other ten discarded measurements all occurred at low-FI_O2 values (10-17.5%) corresponding to Sv_O2-blood values of 20-50%. We were not able to apply the BP method to piglet 2 and to the FI_O2 cycles A and B of piglet 3 because of irregular absorption oscillation waveforms that were not reliably processed by the BP + MA approach.

Figure 5 shows typical temporal traces of the relative [HbO₂] and [Hb] measured on the piglet's leg (with optical probe PR) (Fig. 5A) and on the human vastus medialis muscle at rest (Fig. 5B) and during venous occlusion on the upper thigh (optical probe HVM) (Fig. 5C). The temporal traces of [Hb] and [HbO₂] are obtained by fitting the measured spectrum of µ_a(,t) (whose value at each wavelength was obtained from Eq. 2) with a linear combination of the HbO₂ and Hb extinction spectra. This procedure results in the application of Eqs. 3 and 4 without the superscript "resp" on µ_a, [HbO₂], and [Hb]. Two oscillatory components are clearly visible in the relative [HbO₂] and [Hb] traces of Fig. 5A: the first one, associated with the heartbeat (as shown by the pulse oximeter data; top trace in Fig. 5) is at a frequency of ~2.5 Hz, whereas the second one, associated with respiration (as shown by the strain gauge signal; second trace from the top in Fig. 5), is at a frequency of ~0.65 Hz. Only the latter oscillatory component (at a frequency of ~0.23 Hz in human subjects) is clearly visible in Fig. 5B, whereas neither is present in Fig. 5C. Figure 5, B and C, shows additional low-frequency oscillations associated with changes in blood pressure and heart rate. We observe that the strain-gauge signal (second trace from the top in Fig. 5) increases during inspiration and decreases during expiration. The BP filter described in the previous section aims at isolating the oscillatory component at the respiratory frequency by filtering out higher and lower frequency components. The relative [HbO₂] and [Hb] traces after BP filtering are shown in Fig. 5, bottom. In the case reported in Fig. 5, which is representative of the results reported in this article for Sv_O2-NIRS_resp, the oscillatory components of [HbO₂] and [Hb] at the respiratory frequency are in phase with each other and disappear during venous occlusion.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Representative traces of the relative HbO2 concentrations ([HbO2]) and Hb concentrations ([Hb]) measured on the piglet's leg (with optical probe PR) (A) and on the human vastus medialis muscle at rest (B) and during venous occlusion on the upper thigh (optical probe HVM) (C). Bottom panels report the [Hb] traces after processing with the digital band-pass filter [band pass (BP) + modeling algorithm (MA)] designed to isolate the oscillations at the respiratory frequency. Top trace represents the piglet's heartbeat monitored by the pulse oximeter. Second trace from the top is the strain gauge signal that monitors the respiratory excursion. The strain gauge signal increases during inspiration and decreases during expiration. a.u., Arbitrary units.

Figure 6 illustrates representative µ spectra measured on the piglet's leg (probe PR) (Fig. 6A) and on the human vastus medialis muscle at rest (Fig. 6B) and during venous occlusion on the upper thigh (probe HVM) (Fig. 6C). The y-axis of each panel of Fig. 6 refers to the values of µ obtained with the BP filter method. The values of µ computed with the FFT method are normalized by a wavelength-independent factor to match the BP value of µ at 636 nm. The relatively high value of ^fit during venous occlusion (Fig. 6C) is an indication of the poor fit, which in turn results from the lack of hemoglobin oscillations at the respiratory frequency (see Fig. 5C, bottom, and the y-axis values of Fig. 5C compared with those of Fig. 5B). Figure 6 also shows the best fit of the hemoglobin absorption spectrum to the BP µ and to the FFT µ. The best-fit hemoglobin spectra represent the oxygen saturation of hemoglobin, as illustrated in Fig. 1. The value of Sv_O2-NIRS_resp is given by Eq. 5.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Representative change in respiratory tissue absorption coefficient (µ) spectra measured with the BP and fast Fourier transform (FFT) methods on the piglet's leg (probe PR) (A) and on the human vastus medialis muscle at rest (B) and during venous occlusion on the upper thigh (probe HVM) (C). The experimental 8-point µ spectra were fitted with the hemoglobin absorption spectrum (with the oxy- and deoxyhemoglobin concentrations as fitting parameters). The values of fit (defined in the text) for the BP and FFT spectra give a measure of the quality of the fit.

Figure 7 compares the measurements of Sv_O2-NIRS_resp(BP), Sv_O2-NIRS_resp(FFT), and Sv_O2-blood during cycle A of piglet 1 and during cycle C of piglet 3. The Sv_O2-NIRS_resp(BP) traces reported in Fig. 7 were obtained by performing a running average of the breath-to-breath values obtained with the BP method. In Fig. 7, the averaging procedure consists of a 5-point (in Fig. 7A) or 15-point (in Fig. 7B) running average. The assessment of the agreement between the measurements of Sv_O2-NIRS_resp(FFT) and Sv_O2-blood in the full piglet study is carried out according to the procedure described by Bland and Altman (5). Figure 8A plots the results of the NIRS method based on the respiratory oscillations of the tissue absorption against the invasive measurement of Sv_O2-blood. The shape of the symbols in Fig. 8A indicates the piglet number, whereas the type of fill indicates the location of the NIRS measurement. The range of Sv_O2-blood values considered in this study is ~20-95%. The error bars in Fig. 8A are the standard deviations (SD) computed from the results of ~800 successive FFTs (as described in MATERIALS AND METHODS). Figure 8B displays the difference between the two readings vs. their average, and it quantifies the discrepancy between the two methods and the possible dependence of such a difference on the level of Sv_O2. The mean difference between Sv_O2-NIRS_resp and Sv_O2-blood over the full oxygenation range considered in this study is 1.0% (a measurement of the bias of the Sv_O2-NIRS_resp measurement), and the SD of the difference is 5.8%. Figure 8B does not show any striking dependence of the difference on the mean. If we take the values of mean difference ± 2 SD as the limits of agreement of the two methods (5), we get an estimate of the maximal discrepancies between Sv_O2-NIRS_resp(FFT) and Sv_O2-blood of 10.6% and +12.6%.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Comparison between the continuous measurement of SvO2-NIRSresp (BP) and the discontinuous measurements of SvO2-NIRSresp(FFT) and SvO2-blood. A refers to cycle A of piglet 1, whereas B refers to cycle C of piglet 3. The values of FIO2 (%, left y-axes) during the experiment are indicated by the shaded profiles.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Comparison of SvO2-NIRSresp (FFT) and SvO2-blood in the piglet study. The shape of the symbols refer to the piglet (circles, piglet 1; squares, piglet 2; triangles, piglet 3), whereas the filling indicates the measurement side (filled symbols, right leg, i.e., probe PR; open symbols, left leg, i.e., probe PL). A: SvO2-NIRSresp(FFT) is plotted vs. SvO2-blood. B: difference is plotted vs. the average of the 2 measurements. Two horizontal lines indicate the range given by mean difference ± 2 SD (SD is the standard deviation of the difference between the 2 measurements).

In the human experiment, we found that the NIRS values of Sv_O2 measured with probe HVM (placed on top of a visible vein) were typically smaller than those measured with probe HVL (placed far from any visible vein). Furthermore, the amplitude of the oscillatory absorption at the respiration (heartbeat) frequency was typically greater (smaller) for the data collected with probe HVM than with probe HVL. Of the 16 Sv_O2-NIRS_resp measurements (8 subjects, 2 locations), we discarded only 2 measurements (because of a value of ^fit greater than twice the error in µ), both collected with probe HVL. Figure 9 compares the Sv_O2-NIRS_resp values measured in the human subjects at rest in which the FFT method and the BP filtering approach were used. Figure 9A shows the good agreement of the two measurements, and Fig. 9B quantifies the average difference (0.9%) and the maximum discrepancies of 5.1 and +6.9%, as given by the mean ± 2 SD of the differences. Figure 10 reports a similar comparison between Sv_O2-NIRS_resp(FFT) and Sv_O2-NIRS_vo. As described by Yoxall and Weindling (56), under the assumption that a venous occlusion induces an initial increase in the venous blood volume, Sv_O2-NIRS_vo is given by [HO₂]₀/[H T]₀, where the dots indicate a time derivative and the subscript 0 indicates the initial time that immediately follows the onset of venous occlusion. The agreement between Sv_O2-NIRS_resp(FFT) and Sv_O2-NIRS_vo is good, with an average deviation of 0.8% and maximum discrepancies of 4.2 and +5.8%. Two horizontal lines in Figs. 8B and 9B indicate the range given by the mean difference ± 2 SD. The maximum discrepancy among Sv_O2-NIRS_resp(FFT), Sv_O2-NIRS_resp(BP), and Sv_O2-NIRS_vo is less than the maximum deviation between Sv_O2-NIRS_resp(FFT) and Sv_O2-blood found in piglets (see Fig. 8B).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Comparison of SvO2-NIRSresp (FFT) and SvO2-NIRSresp(BP) in the human study. , Vastus medialis muscle; i.e., probe HVM. , Vastus lateralis muscle; i.e., probe HVL. Probe HVM was place on top of a visible superficial vein, whereas probe HVL was far from visible veins. A: SvO2-NIRSresp(FFT) is plotted vs. SvO2-NIRSresp(BP). B: difference is plotted vs. the average of the 2 measurements. Two horizontal lines in B indicate the range given by mean difference ± 2 SD.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Comparison of SvO2-NIRSresp (FFT) and SvO2-NIRSvo (venous occlusion) in the human study. , Vastus medialis muscle; i.e., probe HVM. , Vastus lateralis muscle; i.e., probe HVL. Probe HVM was place on top of a visible superficial vein, whereas probe HVL was far from visible veins. A: SvO2-NIRSresp(FFT) is plotted vs. SvO2-NIRSvo. B: difference is plotted vs. the average of the 2 measurements. Two horizontal lines in B indicate the range given by mean difference ± 2 SD.

The effect of muscle exercise on the measurement of Sv_O2-NIRS_resp(BP) on top of a visible superficial vein (probe HVM) is illustrated in Fig. 11. Although Sa_O2 (measured with a pulse oximeter) is unaffected by the exercise, Sv_O2-NIRS_resp(BP) shows a significant postexercise decrease from a baseline value of 75-78% down to a minimum value of ~54%. The recovery to the baseline value of Sv_O2-NIRS_resp(BP) occurs after ~30 s. By using the BP approach, we could monitor Sv_O2-NIRS_resp at every breathing period, i.e., every ~5 s, thus achieving a real-time monitoring of Sv_O2. We observe that we could not obtain meaningful measurements of Sv_O2-NIRS_resp during exercise because of motion artifacts.

View larger version (15K): [in this window] [in a new window]   Fig. 11.   Continuous measurement of SvO2-NIRSresp(BP) with optical probe HVM (vastus medialis muscle, on top of a visible superficial vein) on a healthy human subject during baseline and after isometric muscle exercise (recovery).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Various methods for measuring Sv_O2. The method presented in this article to measure Sv_O2 from the near-infrared absorption oscillations at the respiratory frequency (spiroximetry) can be implemented by using a FFT or a digital BP filter in conjunction with a MA. We have indicated the measurements of Sv_O2 obtained with these two approaches with the notations Sv_O2-NIRS_resp(FFT) and Sv_O2-NIRS_resp(BP), respectively. An alternative method for measuring Sv_O2 with NIRS is based on a previously described venous occlusion protocol (13, 39, 55, 56). We have identified the results of this measurement procedure with the notation Sv_O2-NIRS_vo. In the human study, the NIRS measurements were conducted at two locations on the thigh. One location was on top of a visible superficial vein of the vastus medialis muscle (probe HVM), and the second location was far from visible superficial veins on the vastus lateralis muscle (probe HVL). Finally, the invasive measurement of Sv_O2 performed by the gas analysis of venous blood samples is indicated with Sv_O2-blood. In this section, we discuss the different features of these measurements of Sv_O2, and the comparison of their results, as reported in Figs. 7-9.

The FFT and BP filter approaches to near-infrared spiroximetry. The major advantage of the BP approach is that it allows for a real-time measurement of Sv_O2 by providing a reading of Sv_O2-NIRS_resp(BP) at every respiration cycle. Consequently, this method is particularly effective during transients, as illustrated by the recovery of the Sv_O2-NIRS_resp(BP) traces corresponding to the sudden increase of FI_O2 to 100% in piglets (see Fig. 6, A and B), or to the end of the exercise period in human subjects (see Fig. 11). On the other hand, the BP filter + MA method is susceptible to fluctuations in the respiratory frequency and to irregular respiration patterns. This accounts for the fact that we did not get reliable readings of Sv_O2-NIRS_resp(BP) in piglet 2 and in FI_O2 cycles A and B of piglet 3. The FFT method was more robust, producing reliable readings in 56 of 67 cases (84%) in the piglet study and in 14 of 16 cases (87%) in the human study. It is important to observe that 10 of the 11 discarded readings in piglets occurred at low-Sv_O2-NIRS-blood values (20-50%), and one was assigned to motion artifacts. Both discarded readings in the human study were collected with probe HVL, which was placed far from visible veins. Therefore, we have found indications that the measurement of Sv_O2-NIRS_resp(FFT) is particularly robust at Sv_O2 values >50% (in piglets) and when the optical probe is placed on top of a visible superficial vein (in human subjects). Although the FFT method, which is based on a measurement of the integrated peak at the respiratory frequency, is less sensitive than the BP method to irregular respiration patterns, it is not applicable during transients. In fact, we did not obtain reliable readings of Sv_O2 when the time frame used to compute Sv_O2-NIRS_resp(FFT) (80 s in piglets, 80-120 s in human subjects) included significant changes in the Sv_O2. When both the FFT and the BP methods can be applied, they provide Sv_O2-NIRS_resp measurements that are in excellent agreement, as shown in Figs. 6 and 9. The differences between the two measurements (SD of 3.0%) are comparable with measurement errors and significantly less than the maximum deviation between Sv_O2-NIRS_resp(FFT) and Sv_O2-blood (approximately ±10%) observed in the piglet study (see Fig. 8B).

Measurements of Sv_O2-NIRS_resp and Sv_O2-NIRS_vo. Both of these NIRS methods to measure the Sv_O2 (resp and vo) rely on a change in the volume fraction of venous blood in the tissue. The two major differences between the two methods are as follows. 1) The vo method requires an external perturbation consisting of a pneumatic-cuff-induced venous occlusion, whereas the resp method is only based on the intrinsic blood pressure oscillations induced by normal respiration and can be applied continuously. 2) The vo method can be applied only to limbs, whereas the resp method can, in principle, be applied to any tissue and in particular to the brain, as already shown by Wolf et al. (52). However, we stress that it is always important to verify that the [HbO₂] and [Hb] oscillate in phase at the respiratory frequency for the resp method to provide reliable measurements of Sv_O2. For instance, Elwell et al. (16) reported out-of-phase oscillations of [Hb] and [HbO₂] in the human brain, which would indicate a blood flow rather than volume oscillations, thus rendering the resp method inapplicable. In our human study, we found an excellent agreement between Sv_O2-NIRS_resp(FFT) and Sv_O2-NIRS_vo, with a maximum deviation on the order of ±4-5% (see Fig. 10).

Optical probes PR, PL, HVM, and HVL. In the piglet study, we have found no significant difference between the Sv_O2-NIRS_resp(FFT) data collected with probes PR (on the right leg) and PL (on the left leg, where the venous catheter was inserted) (see Fig. 8A). This result indicates that noninvasive measurements of Sv_O2 on one leg can be meaningfully compared with invasive measurements of Sv_O2 on the other leg. In the human study, we found some differences between the Sv_O2-NIRS measurements with probe HVM (placed on top of a visible superficial vein in the vastus medialis muscle) and with probe HVL (placed far from visible veins on the vastus lateralis muscle). As shown in Figs. 8 and 9, the Sv_O2-NIRS readings (with both the NIRS_resp and NIRS_vo method) of probe HVM (see Figs. 8 and 9) were typically smaller than the readings of probe HVL (see Figs. 8 and 9). We assign this result to a partial contribution from the capillary and/or arterial compartments picked up by probe HVL. In fact, although the optical data from probe HVM shown in Fig. 4, B and C, do not show any visible contribution from the arterial pulsation, data from probe HVL (not shown) do contain pulsatile components at the heartbeat frequency. As a result, we believe that the optical probe should be placed on top of visible superficial veins for more accurate readings of Sv_O2-NIRS_resp on human subjects. We believe that the reason that Sv_O2-NIRS_resp readings in the piglet study were in close agreement with the invasive measurement of Sv_O2, despite the evident arterial pulsation in Fig. 5A, is related to the smaller extent of respiratory sinus arrhythmia in piglets with respect to humans. In fact, respiratory sinus arrhythmia is the main origin of the arterial oscillations at the respiratory frequency (38). The larger role played by respiratory sinus arrhythmia in human subjects with respect to piglets will probably require a more careful interpretation of the optical data for spiroximetry. However, the results of Fig. 11 show the practical applicability of spiroximetry to human subjects, so that we do not expect respiratory sinus arrhythmia to introduce an intrinsic limitation of the method.

Noninvasive vs. invasive measurements of Sv_O2. The comparison between Sv_O2-NIRS_resp(FFT) and Sv_O2-blood in the piglet study shows a maximum deviation range of 10.6% to +12.6%. The local character of the Sv_O2 (as opposed to the systemic nature of the Sa_O2) requires some caution in the comparison of invasive (Sv_O2-blood) and noninvasive (Sv_O2-NIRS) measurements of Sv_O2. In fact, in our piglet study, Sv_O2-blood was measured from blood samples drawn from the femoral vein, whereas Sv_O2-NIRS_resp was measured with an optical probe placed on the leg muscle. It is likely that the NIRS oscillatory signal (at the respiratory frequency) is not just representative of the femoral vein and may therefore be indicative of the oxygen consumption at different tissue areas than those affecting the femoral vein saturation. This fact may not lead to significant differences under rest conditions, but it may be important under stress. Although we found a good agreement between Sv_O2-NIRS_resp(FFT) and Sv_O2-blood over the whole range of FI_O2 values considered (see Fig. 8), we observed a meaningfully greater SD of the differences over the 20-55% Sv_O2-blood range (SD = 7.8%) than in the 55-95% range (SD = 3.6%).

Effect of muscle exercise on Sv_O2-NIRS_resp(BP). The result reported in Fig. 11 serves the purpose of further illustrating the potential of the Sv_O2-NIRS_resp(BP) measurement approach. In fact, Fig. 11 shows the feasibility of monitoring the Sv_O2 in real time on a breath-to-breath basis (one data point every 4-5 s). Furthermore, the baseline Sv_O2-NIRS_resp(BP) value of 75-78% and the exercise-induced drop indicate the venous origin of the saturation measurement, since the Sa_O2 measurement provided by the pulse oximeter stayed constant at 98 ± 1% for the whole measurement period. On the other hand, Fig. 11 reports only one representative case, and more studies are required to quantify the effect of muscle exercise on the measurement of Sv_O2-NIRS_resp.