The non-invasive quantification of cutaneous haemoglobin and its oxygenation by reflectance spectrophotometry

Abstract

The work detailed in this study describes the design of a rapid scanning multiwavelength reflectance spectrophotometer and its application to the measurement of cutaneous pigments. In this instrument the radiation from a 6V, 20 W quartz halogen lamp is transmitted to the reflectance probe by fibre optics and illuminates the skin surface perpendicularly. The diffuse reflected light is then collected and transmitted to a rotating circular variable filter, CVF. The CVF is driven by a BBC Microcomputer and the spectrum from 356 to 721 nm is measured at 200 equally spaced wavelengths in 2.8 s. Based upon the spectral differences between the absorbances of oxy and reduced haemoglobin, two indices were defined for quantifying the amount of haemoglobin, the "haemoglobin index", and oxygen saturation, the "oxygenation index", in a blood containing sample. The haemoglobin index is derived from the spectrum of haemoglobin as the difference between two gradients calculated between three isobestic points (527.5,544, and 573 nm). To measure the oxygenation index, two isobestic points (544 and 573 nm) and a point at 558.5 nm, where the specific absorption coefficient of oxy and reduced haemoglobin differ, are used. Linear calibration curves of the haemoglobin index against haemoglobin content in-vitro using collimated and diffused light were obtained. The oxygenation index was converted to percentage oxygen saturation by comparing the oxygenation index of a sample with that of reduced and fully oxygenated blood. The validity of the method was examined by measuring the oxygen dissociation curve of human blood at pCO2 of 40 mmHg, pH of 7.33, and 37 °C and comparing the results with between measured and published curves confirms that the oxygen saturation of blood can be accurately measured by the present method. The scattering properties of the epidermis were studied by measuring the angular distribution of light scattered by a sheet of epidermis. It was found that most of the radiation transmitted through the epidermis is forwardly orientated; 67% of the incident radiation at 630 nm remains within 22.5 degrees of the incident beam. To facilitate the study of the absorption properties of epidermis a new index called the "melanin index" was introduced and quantified using a synthetic melanin compound for calibration purposes. The melanin index is based on the slope of the LIR spectrum of skin surface between 650 -700 nm, and was used to investigate the melanin pigmentation levels of different ethnic groups. Reflectance spectrometry was used to study the amount of haemoglobin and oxygen saturation of the superficial blood in skin. Variations in the haemoglobin index of an arm and a finger tip were studied under different conditions of temperature and height of the limb relative to the heart. The effects of applying a topical vasodilating chemical and of the thermal regulatory function of blood at three different sites on a hand 'were also studied. In all these measurements the results were in good agreement with the biological prediction and confirmed the satisfactory operation of the instrument, the validity of the indices and their usefulness in quantifying "skin colour" changes. To find the correlation between in-vivo haemoglobin index and the true amount of haemoglobin in blood in g/dl, skin reflectance measurements and blood samples were taken from patients who attended a blood clinic. The correlation coefficient between haemoglobin content (g/dl) and the haemoglobin index of the patients were 0.4 and 0.2 respectively. The weak correlation coefficient is attributable to the difficulty of defining the volume of blood observed when measuring the haemoglobin index. The instrument was used to measure changes in the blood content of the skin of animal and human subjects following treatment by photochemotherapy