Method and apparatus for direct spectrophotometric measurements in unaltered whole blood

Abstract

A method and apparatus that allows accurate spectrophotometric determination of the concentrations of various hemoglobin species in whole undiluted blood. The invention employs 1) an optical apparatus designed to maximize the true optical absorbance of whole blood and to minimize the effects of light scattering on the spectrophotometric measurements of the concentrations of various constituent components, and 2) methods to correct the hemoglobin concentration measurements for light scattering and for the effects of the finite bandwidth of the substantially monochromatic light. In the optical apparatus optical parameters, such as sample thickness, detector size, sample-to-detector distance, wavelengths, monochromicity, and maximum angle of light capture by detector, are selected so as to minimize the contribution of light scattering and to maximize the contribution of true optical absorbance. After making measurements of a blood sample's optical density at each of the wavelengths, corrections are made for the effects of light scattering.

Description

[0001] A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. FIELD OF THE INVENTION [0002] This invention relates to a method and apparatus to assess the optical transmittance of a sample of unaltered whole blood at multiple wavelengths to attain an accurate measurement of its total hemoglobin concentration, the concentration of bilirubin, and the concentrations of oxy-, deoxy-, carboxy-, met-, and sulfhemoglobin. BACKGROUND [0003] To the best of the Applicants' knowledge, no prior art whether patented or not has ever successfully exploited the optical transmittance of unaltered whole blood to achieve an accurate measurement of the total hemoglobin concentration (THb) in a bl...

AI Technical Summary

Benefits of technology

The present invention provides a method for measuring the concentrations of hemoglobin species in unaltered whole blood without the need for hemolysis or chemical conversion. This is achieved by using a spectrophotometric apparatus and a set of optimal measuring wavelengths that minimize the effects of light scattering and maximize the contribution of true optical absorbance. The method also takes into account the errors caused by light scattering and other factors, and provides corrections for these errors based on the wavelength of the measuring radiation and the concentrations of the various hemoglobin species present in each analyzed sample. Overall, the present invention offers a non-destructive and accurate optical test for blood samples that preserves them for further analysis.

Problems solved by technology

Because the individual hemoglobin species differ significantly from each other in their optical absorbance spectra, no method that employs a single wavelength can measure the total hemoglobin concentration spectrophotometrically.

The disadvantages of the above-mentioned chemical conversion methods are that 1) hemolysis and accurate dilutions are needed, 2) the reagents are often toxic, 3) chemical conversion is slow, 4) the red blood cells and the chemical nature of the whole blood sample are so drastically altered that the same sample cannot be used for further subsequent analyses of other blood constituents, and 5) the chemical conversion of the various hemoglobin derivatives into a single species makes it impossible to measure the original concentrations of the several individual species.

The latter disadvantage is a serious limitation because the concentrations of oxy-, carboxy-, met-, deoxy-, and sulfhemoglobin provide valuable diagnostic information in many different medical applications.

These prior methods eliminate the need for chemically converting the various hemoglobin derivatives into a single species, but they suffer from the disadvantage of requiring a complex, bulky, and expensive apparatus comprised of pumps, plumbing, associated control circuitry, and in some cases, ultrasonic hemolyzers to dilute and hemolyze the blood sample.

Finally, the techniques that employ hemolysis have two additional disadvantages: 1) their complicated plumbing systems are prone to clogging by blood residue, and 2) they aspirate and destroy the blood sample so that it cannot be retrieved and subjected to further analysis of other constituents.

In whole blood, the major obstacle to making optical measurements is the intense light scattering caused by the highly concentrated red blood cells, e.g. 5.4×106 RBCs / μl for human males.

Prior art that relies on hemolysis to eliminate the intense light scattering by the red blood cells has the further disadvantage that even after thorough hemolysis the sample can be relatively turbid (L. R. Sehgal et al., Critical Care Medicine, 12:907-909, 1984).

Nevertheless, the residual turbidity in hemolyzed blood causes troublesome errors in the hemoglobin measurements.

Thus, an additional disadvantage of prior apparatus that rely on hemolysis is the residual turbidity that results from a small number of unlysed red blood cells, lipid particles such as chylomicrons (a normal constituent of plasma that persists after hemolysis), light-scattering cell fragments produced by the hemolysis process, and other causes that are unknown.

First, as mentioned previously, the turbidity of hemolyzed blood is insignificant in magnitude in comparison with the light scattering of unhemolyzed whole blood.

Prior art was not designed to accommodate the greater magnitude of the scattering effects of unaltered whole blood.

The Applicants have discovered that, even though most of the light is scattered at small angles, the magnitude of large-angle light scattering is sufficient to cause serious errors in spectrophotometric measurements on whole blood.

Prior art does not address the problem of designing practical instruments by capturing the large-angle light scattering of unhemolyzed blood.

Prior art does not address the complex wavelength dependence of the scattering effects of unaltered whole blood.

Prior art does not address the complex dependence of light scattering on the concentrations of the individual hemoglobin species in unaltered whole blood.

Fifth, many poorly understood, uncontrolled processes occur in unaltered, whole blood that change its optical properties.

As shown in the comparative example below, the apparatus of Lundsgaard, U.S. Pat. No. 4,997,769, fails to yield valid results when measuring a sample of unaltered (unhemolyzed) whole blood.

Similarly, other prior art that relies on hemolysis such as Brown et al., U.S. Pat. No. 4,134,678; Raffaele, U.S. Pat. No. 4,013,417; and Johansen et al., U.S. Pat. No. 3,972,614 would also fail to yield valid results on unhemolyzed whole blood because these methods also fail to capture the light scattered at large angles by the sample, and because their measurements do not take into account the magnitude, the wavelength dependence, or the hemoglobin-composition-dependence of the light-scattering effects of unaltered whole blood.

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