Lorilee S. L. Arakaki

Multispectral Analysis for Quantitative Measurements of Myoglobin Oxygen Fractional Saturation in the Presence of Hemoglobin Interference

Quantitative values for myoglobin oxygen fractional saturation were extracted from visible absorption spectra of myoglobin and hemoglobin solutions by analysis with three algorithms: classical least-squares, partial least-squares, and stagewise multiple linear regression. In an effort to mimic in vivo conditions, oxygen tensions and concentrations of myoglobin and hemoglobin solutions in separate cuvettes were varied independently. Transmission measurements were made through both cuvettes so that spectra contained contributions from both myoglobin and hemoglobin. Oxygen tensions in the myoglobin solutions spanned the rapidly varying region of the myoglobin oxygen saturation curve with pO2 ranging from 0 to 4.79 Torr, corresponding to fractional saturation values between 0 and 0.903. A range of hemoglobin oxygenations from fully oxygenated to fully deoxygenated was used. Estimation of myoglobin fractional saturation by the classical least-squares algorithm had a standard error (SEest) of 0.094, while the partial least-squares method resulted in an SEest of 0.070. Partial least-squares estimations resulted in an SEest of 0.041 when a limited wavelength range was used. The stagewise multiple linear regression method had an SEest of 0.052. Results indicate that stagewise regression and partial least-squares yielded estimates of myoglobin fractional saturation that were more accurate than those obtained from classical least-squares.

Myoglobin Oxygen Saturation Measured Independently of Hemoglobin in Scattering Media by Optical Reflectance Spectroscopy

Partial least-squares (PLS) and second-derivative preprocessing were used to obtain estimates of myoglobin oxygen fractional saturation from diffuse reflectance spectra of solutions containing myoglobin, hemoglobin, and a scatterer. A computer model and solutions in vitro were used to simulate several physiological situations. The maximum standard error (SE) was 0.082 for these trials; myoglobin fractional saturation varies between 0 and 1. These results show that a statistical approach can differentiate two highly overlapping absorbance peaks in the presence of diffuse scatter. A robust PLS model was created by using a calibration set with a range of scattering coefficients and concentrations of hemoglobin. Second derivatives of the spectra were less affected by changes in scattering coefficients than were the original spectra. A linear scaling of PLS estimates produced accurate myoglobin saturations from in vitro prediction set spectra that had scattering and absorption coefficients both within and beyond the range represented by the calibration set. Preliminary estimates of myoglobin fractional saturation from spectra acquired from the rat hind limb suggest that this calibration set is appropriate for use in vivo.

Accurate Myoglobin Oxygen Saturation by Optical Spectroscopy Measured in Blood-Perfused Rat Muscle

Optical spectra were acquired from myoglobin and hemoglobin solutions and from the tibialis anterior muscle of Sprague–Dawley rats in the visible region (515 to 660 nm). Validation studies were performed on the in vitro spectra to demonstrate that partial least squares analysis of second-derivative spectra yields accurate measurements of myoglobin saturation in the presence of varying hemoglobin concentrations and saturations. When hemoglobin concentrations were varied between 0.25 and 4 times that of myoglobin, myoglobin saturations were measured with a root mean squared error (RMSE) of 4.9% (n = 56) over the full range from 0 to 1. Myoglobin saturations were also shown to be largely unaffected by hemoglobin saturation. RMSE values of only 1.7% (n = 77) were found when hemoglobin saturations were varied independently from myoglobin saturations. These in vitro validation studies represent the most complete and rigorous done to date using partial least squares analysis on myoglobin and hemoglobin spectra. Analysis of reflectance spectra from the rat hind limb yielded accurate measures of volume-averaged myoglobin fractional saturation in the presence of hemoglobin in vivo. Hemodilution showed that myoglobin fractional saturation measurements in the rat leg are not sensitive to changes in hematocrit, thereby confirming the results from solutions in vitro. Decreases in optical density of 11.3 ± 3.0% (n = 3) were achieved while myoglobin saturation decreased by only 3.1 ± 3.8%. Myoglobin saturation was significantly increased when the fraction of inspired O2 was increased, showing that manipulations of myoglobin saturation are detectable and that myoglobin is not fully saturated in resting muscle. Together, these in vitro and in vivo studies show that cellular oxygenation derived from myoglobin fractional saturation can be measured accurately with little cross-talk from hemoglobin in the visible wavelength region, thereby extending optical spectroscopic studies of cellular and vascular oxygenation beyond the near-infrared regions previously studied.

Muscle oxygenation measurement in humans by noninvasive optical spectroscopy and Locally Weighted Regression

We have developed a method to make real-time, continuous, noninvasive measurements of muscle oxygenation (Mox) from the surface of the skin. A key development was measurement in both the visible and near infrared (NIR) regions. Measurement of both oxygenated and deoxygenated myoglobin and hemoglobin resulted in a more accurate measurement of Mox than could be achieved with measurement of only the deoxygenated components, as in traditional near-infrared spectroscopy (NIRS). Using the second derivative with respect to wavelength reduced the effects of scattering on the spectra and also made oxygenated and deoxygenated forms more distinguishable from each other. Selecting spectral bands where oxygenated and deoxygenated forms absorb filtered out noise and spectral features unrelated to Mox. NIR and visible bands were scaled relative to each other in order to correct for errors introduced by normalization. Multivariate Curve Resolution (MCR) was used to estimate Mox from spectra within each data set collected from healthy subjects. A Locally Weighted Regression (LWR) model was built from calibration set spectra and associated Mox values from 20 subjects using 2562 spectra. LWR and Partial Least Squares (PLS) allow accurate measurement of Mox despite variations in skin pigment or fat layer thickness in different subjects. The method estimated Mox in five healthy subjects with an RMSE of 5.4%.

Simultaneous Optical Spectroscopic Measurement of Hemoglobin and Myoglobin Saturations and Cytochrome aa3 Oxidation In Vivo

A method to simultaneously measure oxygenation in vascular, intracellular, and mitochondrial spaces from optical spectra acquired from muscle has been developed. In order to validate the method, optical spectra in the visible and near-infrared regions (600–850 nm) were acquired from solutions of myoglobin, hemoglobin, and cytochrome oxidase that included Intralipid as a light scatterer. Spectra were also acquired from the rabbit forelimb. Three partial least squares (PLS) analyses were performed on second-derivative spectra, each separately calibrated to myoglobin oxygen saturation, hemoglobin oxygen saturation, or cytochrome aa3 oxidation. The three variables were measured from in vitro and in vivo spectra that contained all three chromophores. In the in vitro studies, measured values of myoglobin saturation, hemoglobin saturation, and cytochrome aa3 oxidation had standard errors of 5.9%, 7.4%, and 12.2%, respectively, with little cross-talk between the in vitro measurements. In the progression from normal oxygenation to ischemia in the rabbit forelimb, hemoglobin desaturated first, followed by myoglobin, while cytochrome aa3 reduction occurred last. The ability to simultaneously measure oxygenations in the vascular, intracellular, and mitochondrial compartments will be valuable in physiological studies of muscle metabolism and in clinical studies when oxygen supply or utilization are compromised.

Optical spectroscopy demonstrates elevated intracellular oxygenation in an endotoxic model of sepsis in the perfused heart.

Recent clinical studies of patients with sepsis have shown that the delivery of adequate oxygen alone does not necessarily result in improved organ function or survival. This study was undertaken to determine if optical spectroscopy could detect higher intracellular oxygenations in isolated, perfused guinea pig hearts that have been treated with endotoxin (lipopolysaccharide [LPS]) than in controls. Four hours after intraperitoneal injection with LPS, adult guinea pigs were anesthetized, and hearts were excised and perfused in the Langendorff manner. Six control and eight LPS-exposed guinea pigs were studied. Myoglobin oxygen saturation was determined from analysis of optical reflectance spectra acquired from the left ventricular free wall. Myoglobin saturation was significantly higher at baseline with LPS than in controls (96.0% ± 0.8% vs. 89.4% ± 1.7%, P < 0.001). At the end of 30 s of ischemia, myoglobin saturation decreased to 15% ± 1% in controls, but to only 60% ± 7% in the LPS group. Myocardial performance was determined by measured left ventricular developed pressure, which was significantly depressed in the LPS-exposed hearts relative to controls (30 ± 4 mmHg vs. 67 ± 9 mmHg, P < 0.001). Myocardial oxygen consumption, calculated from measurements of arterial and venous PO2 and coronary flow, was lower in LPS hearts relative to controls (0.199 ± 0.021 mL oxygen·min−1·g−1 vs. 0.157 ± 0.006 mL oxygen·min−1·g−1). In this model of sepsis in the perfused guinea pig heart, intracellular oxygenation was higher and oxygen consumption was lower than in controls. Cellular dysfunction seen in sepsis may be caused by compromised oxygen use rather than insufficient oxygen delivery. Optical spectroscopy has the potential to noninvasively monitor patients and their responses to therapy.

Muscle Oxygenation as an Early Predictor of Shock Severity in Trauma Patients

Introduction: We evaluated the potential utility of a new prototype noninvasive muscle oxygenation (MOx) measurement for the identification of shock severity in a population of patients admitted to the trauma resuscitation rooms of a Level I regional trauma center. The goal of this project was to correlate MOx with shock severity as defined by standard measures of shock: systolic blood pressure, heart rate, and lactate. Methods: Optical spectra were collected from subjects by placement of a custom-designed optical probe over the first dorsal interosseous muscles on the back of the hand. Spectra were acquired from trauma patients as soon as possible upon admission to the trauma resuscitation room. Patients with any injury were eligible for study. MOx was determined from the collected optical spectra with a multiwavelength analysis that used both visible and near-infrared regions of light. Shock severity was determined in each patient by a scoring system based on combined degrees of hypotension, tachycardia, and lactate. MOx values of patients in each shock severity group (mild, moderate, and severe) were compared using two-sample t tests. Results: In 17 healthy control patients, the mean MOx value was 91.0 ± 5.5%. A total of 69 trauma patients were studied. Patients classified as having mild shock had a mean MOx of 62.5 ± 26.2% (n = 33), those classified as in moderate shock had a mean MOx of 56.9 ± 26.9% (n = 25) and those classified as in severe shock had a MOx of 31.0 ± 17.1% (n = 11). Mean MOx for each of these groups was statistically different from the healthy control group (P < 0.05). Receiver operating characteristic analyses show that MOx and shock index (heart rate/systolic blood pressure) identified shock similarly well (area under the curves [AUC] = 0.857 and 0.828, respectively). However, MOx identified mild shock better than shock index in the same group of patients (AUC = 0.782 and 0.671, respectively). Conclusions: The results obtained from this pilot study indicate that MOx correlates with shock severity in a population of trauma patients. Noninvasive and continuous MOx holds promise to aid in patient triage and to evaluate patient condition throughout the course of resuscitation.

Photon path depth in tissue phantoms: A comparison of visible and Near-Infrared (NIR) wavelengths

Optical spectroscopy is being used increasingly in medical applications to noninvasively investigate tissues below the skin. In order to assure adequate sampling of tissues underlying the skin, photon penetration depth must be known. Photon penetration in tissues has been studied with near-infrared (NIR) light, but experimental study of visible light propagation in tissue has been limited. In this study, a micro-motion system coupled with a reflectance spectroscopy system was used to determine the penetration depth of visible-range and NIR photons (535-800 nm) in phantoms composed of Intralipid and hemoglobin. An absorbing target was placed at intervals of 0.1mm along a 15mm line perpendicular to and bisecting the line between the ends of the source and detector optical fiber bundles. Comparisons between detected light intensities at different target positions were used to determine the most probable photon path depths at 576 nm and at 760 nm. Scattering coefficients, hemoglobin concentrations, and source-detector separations were varied to evaluate their effects on the penetration depth of photons. Results from phantoms containing Intralipid only showed that the most-probable penetration depth at 576 nm was comparable to that at 760 nm. Larger sourcedetector separations resulted in deeper photon penetration depths for both spectral regions. Changes in scattering over a 4-fold range did not affect the photon path depth appreciably. In the presence of hemoglobin with a source-detector separation of 13 mm, the most probable depth of photon penetration in the visible range was greater than 2.5 mm, and was within 1 mm of the most probable depth of photon penetration in the NIR. This study demonstrates the feasibility of using the visible and NIR regions in transcutaneous reflectance spectroscopy.

Direct optical measurement of intraoperative myocardial oxygenation during congenital heart surgery

This study demonstrates use of novel technology to measure cellular oxygenation during corrective congenital heart surgery. Cellular oxygenation was measured using a custom-designed optical probe placed on the free wall of the right ventricle. Cellular oxygenation, determined from myoglobin saturation, was calculated using multiwavelength analysis. Timing of bypass, aortic cross-clamp, infusion of cardioplegic solution, and length of intensive care unit (ICU) stay were recorded. Baseline cellular oxygenation was approximately 50% just before aortic cross-clamp and decreased to approximately 20% during cardioplegia. Cellular oxygenation remained low throughout cardioplegia and returned toward baseline after bypass. In four cases, cellular oxygenation did not return as quickly to baseline as in the other three cases. Among the four patients demonstrating slow recovery, the average ICU length of stay was 2.25 days compared with an average stay of 1.33 days for those patients exhibiting rapid cellular oxygenation recovery (p = 0.06). The slow recovery group had an average cross-clamp time of 40.1 ± 28.4 minutes, compared with 26.0 ± 8.5 minutes for the fast recovery group (p = 0.34). This study demonstrates for the first time that myocyte cellular oxygenation can be measured intraoperatively during cardiac surgery. Measurement of cellular oxygenation may be useful for improving myocardial preservation techniques.

Muscle oxygenation as an indicator of shock severity in patients with suspected severe sepsis or septic shock.

Purpose The aim of this pilot study was to evaluate the potential of a new noninvasive optical measurement of muscle oxygenation (MOx) to identify shock severity in patients with suspected sepsis. Methods We enrolled 51 adult patients in the emergency department (ED) who presented with possible sepsis using traditional Systematic Inflammatory Response Syndrome criteria or who triggered a “Code Sepsis.” Noninvasive MOx measurements were made from the first dorsal interosseous muscles of the hand once potential sepsis/septic shock was identified, as soon as possible after admission to the ED. Shock severity was defined by concurrent systolic blood pressure, heart rate, and serum lactate levels. MOx was also measured in a control group of 17 healthy adults. Results Mean (± SD) MOx in the healthy control group was 91.0 ± 5.5% (n = 17). Patients with mild, moderate, and severe shock had mean MOx values of 79.4 ± 21.2%, 48.6 ± 28.6%, and 42.2 ± 4.7%, respectively. Mean MOx for the mild and moderate shock severity categories were statistically different from healthy controls and from each other based on two-sample t-tests (p < 0.05). Conclusions We demonstrate that noninvasive measurement of MOx was associated with clinical assessment of shock severity in suspected severe sepsis or septic shock. The ability of MOx to detect even mild septic shock has meaningful implications for emergency care, where decisions about triage and therapy must be made quickly and accurately. Future longitudinal studies may validate these findings and the value of MOx in monitoring patient status as treatment is administered.