Isolated blood-tissue phantom: tissue hemoglobin index sensitivity to total hemoglobin
The experiments using the tissue phantom model provide evidence that the THI metric is specific to (Figure
1) and sensitive to (Figure
2) the total amount of hemoglobin for a given optical path length or volume that the detected light signal interrogates. The dual-layer phantom results of Figure
1 show that, at a fixed blood layer thickness, the THI signal doubled - changing from 5.8 to 11.4 - when Hbt was doubled from 6 to 12 g/dl. At constant Hbt (12 g/dl), an increase in the THI from 11.4 to 18.0 was similarly proportional to the increase in blood layer thickness (from 1.0 to 1.5 mm). These results suggest that both vascular Hbt and vascular density or thickness (diameter) would influence THI readings
in vivo.
The results of Figure
2 show that the optical scattering properties of tissue can also influence a THI reading. As the optical scattering coefficient increases, the mean distance between scattering events (1/Optical scattering coefficient) increases and therefore the increased traversed distance of detected light (optical path length) results in more total hemoglobin absorption events and thus a greater THI value. The four-factor range in background optical scattering was chosen to estimate the sensitivity of THI to a change in tissue optical scattering. The resultant <10% THI reading change per 1/cm scattering coefficient change provides a basis for studying how THI might change
in vivo using tissue optical scattering properties reported in the published literature.
The reported absolute value for tissue optical scattering measured
in vivo on human limbs varies widely, from about 4 to 10/cm, and appears specific to a measured tissue bed, to the reporting research institution, or to the measurement equipment used [
22‐
24]. Information regarding optical scattering changes for forearm tissue within a fixed NIRS measurement device suggests that optical scattering changes, observed in intersubject variability studies [
22,
23,
25] or ischemia studies [
26], does not exceed a factor of two. Although the authors have found no report of optical scattering variation in the thenar eminence tissue bed, the variability in this bed may be less than that in forearm since subcutaneous tissue thickness either from fat or edema is reported to be less variable at the thenar site [
27]. The
in vivo THI error from an extreme change in optical scattering (twofold change) therefore appears to be less than 20% of the THI reading.
Human study volunteers: normal tissue hemoglobin index range
The present study is the first investigation and report of the THI range in a nonhospitalized large group of human volunteers. The THI mean (14.1) and standard deviation (1.6) values are similar to the reference range for blood hemoglobin concentration (13.5 to 15.1 g/dl) [
28]. This closeness of normal THI to normal Hbt reflects how the THI was calibrated against Hbt in a phantom tissue model mimicking the optical attenuation of tissue with normal levels of hemoglobin when measured with a 15 mm optical probe spacing. Different optical probe spacings and tissue locations can yield significantly different results. For instance, the author's stomach, which has about 1 inch of adipose thickness, measured a THI of 5 units compared with near 14 units on the thenar when using a 15 mm optical probe spacing distance. Adipose tissue has less dense vasculature than muscle and is estimated to have one-third of the THC of muscle (0.05 mM vs. 0.15 mM, respectively) [
29]. A 25 mm optical spacing on the thenar can produce a normal THI value near 22 units [
6] since the 25 mm probe, compared with a 15 mm probe, results in a significantly larger optical path length. The PSF of Equation (
1) would need to be utilized to allow different optical probe spacings to have a common THI scale.
Our previous research identified that the mean THI from 10 healthy volunteer subjects and 10 sepsis patients closely resembled the mean Hbt measurements in both groups [
5]. The correlation of THI to Hbt for individually paired measurements within the sepsis patient cohort, however, had only weak linear correlation to Hbt (
r2 = 0.14) [
30], similar to the results of the porcine hind limb hemodilution study.
Variation in optical scattering properties of the thenar eminence tissue between the 434 measurement subjects is unknown. The observed coefficient of variation for the THI, equal to 11%, is less than the calculated coefficients of variation for other hemodynamic variables of this study (15% for systolic blood pressure and 16% for heart rate) (Table
1). The relatively low coefficient of variation for normal THI indicates that optical scattering variation does not significantly confound THI measurements. We have seen THI values near 4 units in hospitalized sepsis patients [
30], which indicates that the THI in patients can be well outside the normal reference range in nonhospitalized study subjects (14.1 ± 1.6 units). More investigation is needed to determine whether an abnormally low THI reading is diagnostically useful or relevant to a patient's health status or treatment.
Human study volunteers: induced upper-extremity ischemia and exsanguination
A total of 30 human subjects underwent acute arm ischemia conditions evoked by arterial occlusion, venous occlusion, and blood volume exsanguination. Head-of-bed elevation, used clinically to mitigate ventilator-associated pneumonia and elevated intracranial pressure [
31,
32], was evaluated for its effect on THI and StO
2 variability. The main findings of the present study are that the THI trend during cuff-induced ischemia differentiated arterial and venous blood flow occlusions, and that the residual THI signal when extrapolated to 100% blood volume exsanguination was 3.7 ± 2.0 THI units. Since the blood hemoglobin concentration would be fairly constant during the study measurements, the results indicate that regional ischemia and posture could confound any correlation between the THI and Hbt.
Venous occlusion with a pneumatic cuff initially stops venous blood flow until the venous pressure increases above the occlusion pressure. A reduced venous flow resumes once the venous pressure rises above the cuff pressure [
33]. This could explain why StO
2 during venous occlusion had limited change (an approximately 14 StO
2 unit decrease) compared with arterial occlusion (an approximately 54 StO
2 unit decrease). While StO
2 decreased during both venous and arterial occlusion, the THI increased 1.5 ± 1.0 units with venous occlusion and decreased 4.0 ± 2.0 units with arterial occlusion. These results suggest that the THI trend during ischemia might help to identify whether a flow resistance or blockage is emanating from the venous or arterial vascular compartment, similar to other studies measuring NIRS-derived relative THC changes in muscle free flaps [
34]. The porcine hind limb THI readings always increased during distal vena cava cross-clamp occlusion (Figures
3 and
4b), while aorta cross-clamping caused the THI to always drop as indicated in Figure
3. While a rise in the THI during venous occlusion is expected because of venous pooling and blood volume congestion, decreases in the THI during arterial occlusion may have been caused by blood volume reduction. Other NIRS researchers have noted a decrease in total hemoglobin NIRS signals during arterial occlusion [
35‐
37]. In compliant blood vessels, a decrease in arterial vascular pressure would reduce the vascular volume and hence cause the THC and THI to decrease.
The posture of the limb and the influence of gravity might also affect blood drainage and movement out of the measured vascular space. The head-of-bed elevation results of Table
3 show that StO
2 decreased on average 6 units with 60° of elevation while the average THI slightly increased 0.6 units. The sitting upright and reclined results in Table
2 also show that the average StO
2 was 5 units lower while the average THI was 0.5 units higher with an upright posture. These results confirm that limb posture can have a measurable influence on StO
2 and THI measurements. With the arm positioned below heart level, the hand's venous pressure and venous blood volume would increase. The lower oxygen saturated venous blood could then become a larger fraction of the total blood volume that StO
2 is measuring, and thus lower the StO
2.
Other researchers have used scintillation X-ray measures to demonstrate that Esmarch bandage exsanguination of the lower forearm causes a 69% reduction in tissue blood volume [
38]. We assume that the similar exsanguination technique of our study produces a similar 69% reduction in tissue blood volume. The nadir THI during the exsanguination procedure was 7.0 ± 1.6 units. Extrapolation to 100% blood volume reduction from a baseline THI value of 14.6 (Equation (
2)) results in an estimated residual THI value of 3.7 ± 2.0 units. This residual value may indicate that the maximum potential contribution of myoglobin to the THI signals is approximately ≤25% of the baseline THI values in healthy volunteers. The 3.7 ± 2.0 nadir THI value in the present study compared with the 2.8 ± 0.6 residual THI value determined in the porcine hind limb study can possibly be explained by the higher concentration of myoglobin reported in human muscle compared with porcine muscle (4.7 mg/g and <1 mg/g wet weight, respectively) [
39,
40]. The nonzero venous Hbt level (0.5 g/dl) would elevate the residual THI in the porcine study and cause an overestimation of the myoglobin contribution. Deviations from the 69% blood volume exsanguination assumption for the human limb exsanguination experiments would change the estimated myoglobin contribution to human thenar THI measurements.
Since the average THI value in 434 human thenar tissue sites (14.1 ± 1.6) was greater than the average THI value in five porcine hind limbs (8.3 ± 1.1), the potential contribution of myoglobin to the human thenar THI is lower. However, it is evident that the contribution of myoglobin to a THI signal potentially changes for any given level of THI. For instance, in human thenar tissue with a normal THI value of 14.1, the myoglobin contribution may be limited to 25%; but at a THI level of 5, the myoglobin contribution might approach 75%. A THI measurement can therefore help to identify the tissue compartment, cellular versus vascular, where StO
2 is pre-dominantly measured. It is not clear whether myoglobin and change concomitantly or independently in resting StO
2 muscle [
41]. Our previous work involving measurement of StO
2 in oxygen consumption-inhibited porcine organs having myoglobin (heart and hind limb) and not having myoglobin (kidney) suggested that myoglobin did not significantly influence StO
2 specificity to hemoglobin oxygen saturation measured from arterial and venous blood samples [
11]. Regardless of myoglobin's contribution to NIRS signals, a low StO
2 resulting from either myoglobin or hemoglobin desaturation would indicate a lower oxygen reserve available to the tissue.
Porcine hind limb: blood hemoglobin dilution
The isovolumetric hemodilution experiments indicate that the THI is not a direct measure of Hbt, although THI trends might indicate a changing Hbt. Whereas tissue phantom experiments indicated a strong linear correlation (
r2 > 0.99; Figure
2) of the THI to total hemoglobin changes, the
in vivo correlation of the THI to Hbt was nonlinear from 14 to near 0 g/dl and was only weakly linear (
r2 = 0.26; Figure
4a) within the physiologic relevant range, from 4 to 16 g/dl.
NIRS optical signals originate primarily from the arteriolar, capillary, and venule microvascular compartments [
42]. The relative amount of blood in each of these compartments determines where an attenuated or absorbed optical signal is being measured. In resting muscle tissue, one-third to one-half of the capillaries are open and perfused with blood [
43]. Vascular tone reduction (vasodilation) and a resultant capillary recruitment from higher capillary pressure could explain why THC, and hence the THI, might be preserved even though dramatic changes in blood hemoglobin concentration occur. The general increase in cardiac output and femoral artery blood flow (Table
4) during Hbt dilution would result from microvascular arteriole vasodilation and additional blood flow to newly opened capillaries. In human volunteer subjects in whom the blood Hbt was reduced to 5.0 g/dl, researchers have observed decreased systemic vascular resistance and increased cardiac index [
44]. A reduction in blood viscosity from hemodilution would decrease flow resistance and would also contribute to the observed increase in the systemic and local blood flows [
45].
Myoglobin could be a significant factor contributing to the lack of correlation between the THI and Hbt as well as between StO
2 and mixed venous hemoglobin oxygen saturation (Table
4) since hemoglobin and myoglobin have similar absorption characteristics in the near-infrared wavelength region (650 to 900 nm) [
46]. The estimated contribution of myoglobin to NIRS-derived hemoglobin signals is unclear. Reports have ranged from suggesting that nearly all of the NIRS-derived signal is from myoglobin [
47] to suggesting 90% of the signal is coming from hemoglobin [
42,
48]. Other studies have suggested that the myoglobin contribution is linked to the blood hemoglobin concentration since the constant concentration of tissue myoglobin becomes a larger fraction of the NIRS signal as the hemoglobin signal is diluted [
49]. The results of our porcine study suggest that the THI may be useful in interpreting the potential contribution of myoglobin to NIRS-derived signals such as StO
2. The residual THI of 2.8 ± 0.6 units, observed when the femoral vein hemoglobin concentration was 0.5 g/dl, suggests that the average myoglobin signal was no more than 40% of the average THI value (equal to 6.8 ± 1.0) when blood Hbt was near 4 g/dl, and was no more than 34% of the average THI value (equal to 8.3 ± 1.0) with blood Hbt near 13 g/dl. Other researchers have found in isolated porcine hearts that the myoglobin contribution may range from 63 to 46%, with one-half (5.1 ± 0.4 g/dl Hbt) and whole blood perfusate mixtures, respectively [
49].
Venous cuff occlusion techniques have been used to isolate NIRS signals to the nonpulsating venous blood compartment [
50,
51]. The increased venous pressure causes venous pooling and a subsequent increase in THI. In Figure
4 the right limbs have slightly higher THI, and ΔTHI could be from venous obstruction from the flow transducer applied to the right hind limb femoral vein. The magnitude of the THI increase following a 3-minute period of 50 mmHg venous occlusion pressure produced a differential THI signal (ΔTHI) that had a significantly better correlation to Hbt compared with steady-state THI measurements. The
y intercept of 0.07 in Figure
4b indicates that any offset from myoglobin absorption is potentially removed and a stronger linear relationship to Hbt is obtained (
r2 = 0.62 vs.
r2 = 0.26; Figure
4). Further studies may be warranted to examine whether the THI combined with venous occlusion techniques can be optimized to produce a reliable continuous non-invasive measurement indicative of Hbt status.
After surgical preparation and before beginning the porcine hemodilution experiments, it was observed that the starting hind limb THI was approximately one-half of the average THI value (14.1 ± 1.6 units) measured on the thenar eminence of human subjects. Postoperative porcine Hbt levels were low, about 10 g/dl, possibly stemming from the age of the pigs and from the fluids given during the surgical phase of the experiment. A 20 mg bolus of furosemide diuretic was used to hemoconcentrate three of the five animals to boost the starting hemoglobin level. At 13.6 ± 0.9 g/dl Hbt, the average hind limb THI value of 8.3 ± 1.1 units was still much lower than that observed in humans. Differences in tissue blood volume, resulting from differences in vascular density or myoglobin concentration between the human thenar and porcine hind limb, could account for the significantly different observed baseline THI values. The results of the human blood exsanguination experiments (see the exsanguination discussion section), however, indicate that myoglobin differences between human and porcine muscle may be less relevant to this THI difference. Differences in the optical scattering properties of porcine hind limb and human thenar eminence could also be a contributing factor for the lower observed THI baseline in the porcine hind limb. The subcutaneous tissue thickness of the hind limbs, more similar to that in the thenar eminence (about ≤1.5 mm), would make optical scattering differences less likely to have caused the THI differences.