Spectral characterization of liquid hemoglobin phantoms with varying oxygenation states

Abstract

Significance: For optical methods to accurately assess hemoglobin oxygen saturation in vivo, an independently verifiable tissue-like standard is required for validation. For this purpose, we propose three hemoglobin preparations and evaluate methods to characterize them.

Aim: To spectrally characterize three different hemoglobin preparations using multiple spectroscopic methods and to compare their absorption spectra to commonly used reference spectra.

Approach: Absorption spectra of three hemoglobin preparations in solution were characterized using spectroscopic collimated transmission: whole blood, lysed blood, and ferrous-stabilized hemoglobin. Tissue-mimicking phantoms composed of Intralipid, and the hemoglobin solutions were characterized using spatial frequency-domain spectroscopy (SFDS) and enhanced perfusion and oxygen saturation (EPOS) techniques while using yeast to deplete oxygen.

Results: All hemoglobin preparations exhibited similar absorption spectra when accounting for methemoglobin and scattering in their oxyhemoglobin and deoxyhemoglobin forms, respectively. However, systematic differences were observed in the fitting depending on the reference spectra used. For the tissue-mimicking phantoms, SFDS measurements at the surface of the phantom were affected by oxygen diffusion at the interface with air, associated with higher values than for the EPOS system.

Conclusions: We show the validity of different blood phantoms and what considerations need to be addressed in each case to utilize them equivalently.

Keywords: hemoglobin; oxygen saturation; tissue simulating phantom.

Fig. 1

(a) Schematic drawing of setup used for spectral collimated transmission (SCT) measurements and (b) schematic drawing of setup used for SFDS. DMD, digital micromirror device.

Fig. 2

Normalized total attenuation coefficient, ${\mu }_t(\lambda )$, of WB, LB, and ferrous-stabilized hemoglobin ($A_0$) in (a) oxyhemoglobin samples and (b) deoxyhemoglobin samples measured using the SCT setup. For comparison, normalized reference absorption spectra, ${\mu }_{a,\mathrm{HbO}2}(\lambda )$ and ${\mu }_{a,\mathrm{Hb}}(\lambda )$, from Prahl and Zijlstra et al. are given.

Fig. 3

Normalized hemoglobin absorption spectra, ${\mu }_{a,\mathrm{Hb}+\mathrm{HbO}2,N}(\lambda )$ when compensating for MetHb absorption and light scattering in the SCT measurements, according to Eqs. (4) and (7), (a) for oxyhemoglobin preparations using Prahl’s reference spectra and (b) for deoxyhemoglobin preparations using Zijlstra et al.’s reference spectra. Corresponding normalized residual spectra (${\epsilon }_{{\mu }_{t,N}}(\lambda )$) when fitting using reference spectra from [(c), (d)] Prahl and [(e), (f)] Zijlstra et al. and the optimal reference spectra, (g) Prahl’s for oxyhemoglobin and (h) Zijlstra et al.’s for deoxyhemoglobin.

Fig. 4

Normalized absorption spectra, ${\mu }_{a,N}$, calculated by SFDS on tissue-mimicking phantoms with Intralipid and hemoglobin for WB, LB, and ferrous-stabilized hemoglobin ($A_0$). Data are given for oxyhemoglobin, deoxyhemoglobin in bulk media, and deoxyhemoglobin at time point 3 where a thin plastic film was used to prevent surface oxygenation (End). Normalized residual spectra (${\epsilon }_{{\mu }_{a,N}}(\lambda )$) calculated based on Prahl’s, Zijlstra et al.’s, and the optimal set of reference spectra, respectively.

Fig. 5

Comparison of simultaneous ${\mathrm{SO}}_2$ estimations (oxygen saturation), for WB, LB, and ferrous-stabilized hemoglobin ($A_0$) measured by SFDS at the surface of the phantom relative to EPOS (submerged $\sim 2\mathrm{cm}$). Filled markers indicate ${\mathrm{SO}}_2$ values measured $\sim 5$ to 10 min after plastic film covered the surface of the phantom. The discrepancy in rate of oxygenation change (24%) is represented by the difference in slope between the dashed line and the dotted line (unity).