Attempt to Detect Carboxy-Hemoglobin in Human Blood Using Optical Methods

M. Carnohan

Department of Physics, College of Charleston, Charleston, SC

Reflectance spectra are taken for human blood both before and after a patient smokes. Observed changes in the reflectance spectra–for wavelengths which are absorbed by the hemoglobin–indicate that the patient's smoking invokes a change in the mixture of hemoglobin in the blood. The shift of spectral intensities in the blood indicating a change from Dieoxyhemoglobin to Carboxyhemoglobin–is an widely understood phenomenon in biology. The focus presented here then becomes the method for which the hemoglobin is detected. Spectrophotometer detection, in this case, allows for the observation of a potentially dangerous toxin in a non-invasive manner.

INTRODUCTION

Currently, the detection of almost all chemicals in the blood comes from taking a sample of blood from the patient–a process that is not always a safe one and is sometimes objectionable. The long range goals of this researcher are to develop alternative methods of detection which are quick and non-invasive. The method explored by this particular research is spectrophotometry–exploiting a property which many chemicals exhibit: to absorb light.

Biological chromophores absorb light because the conjugated bonds which hold them together are excited by wavelengths of light which carry the same energy. This absorption can be observed by a spectrophotometer–meaning optical means are all that are necessary to detect the presence of many prescribed drugs, harmful toxins, and photosensitizers. Most chemicals which enter the body, like those sought by this research, are bonded in organic chains by highly conjugated bonds and therefore can be detected because of thier chromophore nature.

Applications of research in the optical detection field are far reaching and are crucial to the advancement of techniques such as Photodynamic Therapy (PDT). Non-invasive measurements of diffuse reflectance have been used by various researchers for detection of photosensitizing dyes in malignant tissues–methodologies have included fiber optic light sources in combination with fiber optic probes, CCD cameras or integrating sphere detectors [Star Phys med Biol 1997]. Another such application is the monitoring of toxicity due to environmental conditions in factories where potentially harmful exposure to chemicals is a risk. Where blood sampling may be intrusive or time consuming, spectrophotometry would serve as a good alternative.

A Spectrophotometer has a polychromatic source of light which can split into narrow wavelength bands through the use of diffraction grating and a prism. In this instrument, the resulting light is sent through a fiber optic light bundles to an integrating sphere which collects the light which is reflected from the sample. The spectrophotometer yields a plot of the intensity of the reflected light as a function of wavelength. The resulting graph is inversely related to the absorption spectrum for that material. In this way, one can compare a reflectance graph of a known substance to its absorption spectrum–allowing for detection of a specific chromophore in a complex medium like human blood.

METHODS

The patient is to abstain from smoking for a time exceeding four hours to insure that Carboxyhemoglobin levels are suppressed. Spectra is gathered by placing the integrating sphere of the spectrophotometer on the patients abdomen and collecting reflectance values for wavelengths in the visible spectrum. The patient then smokes–upon returning from doing so spectra is collected.

 

FIGURE 1 Integrating Sphere. Nearly monochromatic is generated and sent through fiber bundles into the sphere. The light is directional and is shown on the sample which is placed in the path on the light, at the sensor. The reflected light from the sample is collected inside the sphere and sent through a bundle back to the spectrophotometer ‘s detector which outputs the percentage of the light collected to that of the intensity of the light sent to the sphere.

Spectral data was also observed at a single wavelength–thought to be most affected by the hemoglobin shift–550nm, for a period of 15 minutes. In one instance, this was performed after an abstinence of smoking and in another the patient smoked while being monitored by the spectrophotometer. The non-smoking period yielded no net change in the transmittance percentage over the time span observed, but the observation while the patient was smoking yielded a transmittance increase from 28.9% total reflectance to 31.3%: a larger gain than the peak oscillations observed in the non-smoking test.

FIGURE 2 Calculated Absorption (k) Spectra of Hemoglobin Derivatives; (a) Oxyhemoglobin; (b) Deoxyhemoglobin; (c) Carboxyhemoglobin; (d) Hemoglobin Nitrite. [Joshua PHIRI PhD Diss. IIT 19993]

DISCUSSION

In addition to error evaluation, there are several immediate limitations which should be discussed. Patterson makes the case that the field needs to improve clinical instrumentation so that it is capable of measuring reflectance changes of a few percent. He also estimates that it will take a calibration of the reflectance technique before the wide range benefits possible will be realized [Patterson et al P&P 1987].

CONCLUSION

While simple detection of Carboxyhemoglobin–and not analysis of that found–is the scope of this research, it stands that the reach of this research could easily be extended to include such an analysis. One huge gain in attempting detection is the building of a calibration table for the molecule which one seeks. In most cases calibrations need to be preformed for every molecule which interacts or combines in the mixture one is observing. In this way, optical analysis of human blood requires prior understanding of light interactions with melanin in the dermis, water in the blood, and with the different hemoglobins.

The detection of shifts in the hemoglobin composition by this research represents the first awareness in the field of photobiology–that light interactions can tell us things about our bodies. The interests of this researcher are served both in this realization, and ones which come from seeing the physicals effects of smoking. The (biological) optical advancements taking place today present so many new challenges–and yet with those come possibilities unfathomable, so that in just a few years photo-medical practices are sure to be as common as physical medicine's practice is today.

Acknowledgements

I thank Dr. Jeff Wragg for helpful discussion; Dr. Linda Jones for providing resources, laboratory space, and helpful guidance