Hemoglobin (Hb) concentration is a metric used for many applications in the medical field, including anemia diagnosis and transfusion guidance. The current strategy for determining Hb concentration is an invasive procedure where blood is drawn from an artery and sent to a laboratory for further analysis. This process is time-consuming, subject to operator error, and carries the risk of infection. Therefore the need exists for a noninvasive technique that can rapidly and accurately predict Hb concentration in human and/or animal tissues. If such a device and method were portable, it would have wide applicability in various areas where rapid Hb measurements are required, such as in the emergency or operating room, in the back of an ambulance, in the battlefield, and in other resource-limited settings.

Hb concentration can also serve as a surrogate marker for neovascularization. In cervical dysplasia, neovascularization has been shown to be associated with poor prognosis and is considered a pathoanatomic feature indicative of a greater risk of recurrence and death [

1,

2]. Early angiogenic changes are also indicative of neoplastic changes in different organ sites, including the prostate [

3], head and neck [

4], and gastrointestinal systems [

5]. Characterization of neovascularization has mostly been performed by invasive and tissue destructive immunohistochemistry on formaldehyde-fixed and paraffin-embedded tissues using various antibodies [

6]. Ratiometric optical spectroscopy could be used to provide noninvasive longitudinal monitoring of neovascularization.

Optical methods show a high degree of accuracy in measuring Hb concentration, but they generally require a sophisticated computational technique such as diffusion approximation [

7] or Monte Carlo modeling [

8–

10] to extract this information from the measured spectra. The focus of the present study is to use simple diffuse reflectance ratios to determine Hb concentration. Optical ratiometric methods involve measurements of reflectance and/or fluorescence at two or more wavelength points to produce a functional form which is subsequently shown to be correlated with a given physical parameter. Oxy- and deoxy-Hb have eight isosbestic points in the ultraviolet-visible (UV-VIS) wavelength range. Isosbestic points indicate wavelengths where two chemical species have the same molar extinction coefficient. Because Hb saturation is calculated from the ratio of oxy-Hb to total Hb, measurements taken at isosbestic points are independent of saturation. Studies from the literature have used ratios involving both isosbestic and non-isosbestic points as a metric to quantify Hb saturation [

11,

12] and Hb concentration [

13].

Several studies in the literature have capitalized on isosbestic points to measure Hb saturation, by using a ratio consisting of one isosbestic point and one where there are maximal differences between oxy- and deoxy-Hb. In one study, a microdensitometer was used to measure oxygen saturation in capillaries of the hamster cheek pouch, through the use of a ratio including the 420 nm isosbestic point and 431 nm non-isosbestic point [

11]. Other studies have used ratios involving two or more isosbestic wavelengths of Hb. One study used 520 and 546 nm to determine the contribution of scattering to optical density measurements of whole blood [

12]. The scattering term could then be used to calculate Hb absorption at the two isosbestic points plus a third wavelength where the oxy- and deoxy-Hb extinction coefficients were different. This, in turn could then be used to calculate Hb saturation. Another study using fluorescence emission measurements from phantoms comprised of flavin adenine dinucleotide, Hb, and polystyrene spheres, found the ratio of fluorescence intensity at two isosbestic points, 500 and 570 nm, could be used to measure total Hb concentration. This ratio was independent of scattering and Hb saturation [

13]. Furthermore, the authors determined that the ratio calculated as the product of 540/560 and 578/560 nm was related to Hb saturation. It is evident from the literature that there is a push towards the development of simple algorithms to measure biologically relevant optical parameters, such as total Hb concentration.

The focus of this present study is to assess the accuracy of total Hb estimation, independent of Hb saturation and scattering, using a simple isosbestic ratiometric analysis of diffuse reflectance intensities developed using Monte Carlo simulations, and whose accuracy was assessed using tissue-mimicking phantoms and *in vivo* human cervical precancer data. Diffuse reflectance spectra were generated using a forward Monte Carlo model, then equations of linear regression between Hb concentration and the ratios were established from the simulations and applied to phantom data. A single reference phantom was used to calibrate the Monte Carlo-generated reflectance to the same scale as the experimentally-measured data. The simulation equations were specific to the probe and instrument used experimentally.

Simulations were conducted for five scattering levels for each of ten absorption levels (Hb concentrations). There were 28 total ratios tested for isosbestic points between 350 and 600 nm. A simple analytical equation was developed to predict Hb concentration. Twenty-five of the 28 ratios had average percent errors within 20% for the simulations when the ratios were averaged over all five scattering levels, four of which were below 5%, nine of which were between 5 and 10%, and seven of which were between 10 and 15%. Linear regression equations from the simulations were then applied to three sets of experimental phantom data. Of the 25 best ratios from the simulations, 12 ratios yielded average percent error within a 20% threshold in extracting Hb concentration from phantoms (Phantom Set 1) with constant Hb concentration and scattering, but variable Hb saturation. Seven of these ratios had errors below 5%, two had errors between 5 and 10%, and three had errors between 15 and 20%. From these 12 ratios, there were a total of six ratios (545/390, 452/390, 570/390, 529/390, 584/390, and 500/390) which could extract Hb concentration from two sets of phantoms (Phantom Sets 2 and 3) with variable Hb concentration and scattering with errors below a 20% threshold. For 452/390 nm, the average percent error was below 15% for both phantom sets. For Phantom Set 2, the average percent error was below 10% for 452/390 nm, between 10 and 15% for 529/390 and 500/390 nm, and between 15 and 20% for 545/390, 570/390, and 584/390 nm. For Phantom Set 3, the average percent error was between 10 and 15% for 545/390, 452/390, 570/390, and 584/390 nm and between 15 and 20% for 529/390 and 500/390 nm.

In order to assess the instrument-independence of this ratiometric method, the six best ratios were tested for two sets of phantoms with variable scattering and Hb concentration measured with a second instrument that used a photomultiplier tube rather than charge-coupled device as the detector. Three of these ratios – 545/390, 452/390, and 529/390 nm – had similarly low errors within 20%, indicating this method can be considered independent of the instrument and probe used. Data from human cervical measurements measured with both instruments was used to test the correlations with Hb concentration extracted with either the ratiometric method or a more complex inverse Monte Carlo model previously developed and validated by our lab [

8,

14,

15]. Both the inverse Monte Carlo model and ratiometric method extracted a wide range of Hb concentration with Pearson linear correlation coefficients of 0.75, 0.76, and 0.88 for the three best ratios, 545/390, 452/390, and 529/390 nm, respectively