Quantitative broadband near-infrared spectroscopy of tissue-simulating phantoms containing erythrocytes

We report the use of steady-state diffuse reflectance spectroscopy (SSDRS) to measure the near-infrared absorption spectrum of liquid phantoms containing human erythrocytes in aqueous suspensions of polystyrene spheres which simulate the scattering properties of tissue. The absorption spectra obtained from these SSDRS measurements of intact red cells under oxygenated and deoxygenated conditions are compared with several published spectra of 'stripped' haemoglobin prepared from lysed cells. Two fitting algorithms (nonlinear least squares and singular value decomposition) which exploit the broad spectral range provided by these measurements (170 data points spanning 164 nm in a single acquisition) are used to determine haemoglobin oxygen saturation (SO2) from SSDR spectra collected over a wide range of measured oxygen partial pressures. The validity of these algorithms is assessed by comparing literature values of p50 (the oxygen tension at which haemoglobin is 50% saturated) and the Hill coefficient to values of these parameters determined from the SO2 estimates. The singular value decomposition algorithm can also be used to reconstruct the non-haemoglobin background absorption spectrum without a priori assumptions regarding its constituent chromophores or their concentrations. Using this technique, the absorption spectrum of a small amount of India ink (maximum absorption coefficient (mu(a max)) approximately 0.0006 mm(-1)) added to a phantom containing red cells (mu(a max) approximately 0.026 mm(-1)) was reconstructed over a full range of oxygen saturations. The implications of these measurements for detection of weakly absorbing chromophores (such as cytochrome aa3) in the presence of haemoglobin are discussed.