Method and apparatus for direct spectrophotometric measurements in unaltered whole blood

• US 6,262,798 B1
• Filed: 09/29/1992
• Issued: 07/17/2001
• Est. Priority Date: 09/29/1992
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Shepherd, A. P., Steinke, John M.
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): LAUCHMAN, LAYLA G
• Application Number:
  US07/953,680
• Time in Patent Office: 3,213 Days
• Field of Search: 386/39-41, 356/409, 356/436, 356/442, 250/573, 250/574
• US Class Current: 356/39
• CPC Class Codes: G01N 2021/3148 using three or more wavelengths
• CPC Class Codes: G01N 21/31 Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry G01N21/72 takes precedence
• CPC Class Codes: G01N 33/72 involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood

Abstract

A method and apparatus that allows accurate spectrophotometric determination of the concentrations of various hemoglobin species in whole blood without hemolysis or dilution. To overcome the complex optical properties of whole 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 (including an optical cuvette) all optical parameters, such as sample thickness, detector size and shape, sample-to-detector distance, wavelengths, monochromicity, and maximum angle of light capture by detector, are optimal values so as to minimize the contribution of light scattering to the total optical attenuation of unaltered whole blood and so as to maximize the contribution of true optical absorbance. After making measurements of a blood sample'"'"'s optical density at each of the wavelengths, the invention makes corrections for the effects of light scattering by red blood cells, for light scattering produced by other causes, and (if necessary) for the effects of the finite bandwidth of the substantially monochromatic light.

Claims

1. A method of determining the concentrations of a plurality of constituent components of unaltered whole blood of unknown composition, including:

generating a plurality of substantially monochromatic radiation wavelengths, each wavelength of an absorbance subset of said plurality of wavelengths having been selected by their ability to distinguish the constituent components and having been selected to minimize the effects of radiation scattering and to maximize radiation absorbance by said constituent components, and each wavelength of a scattering subset of said plurality of wavelengths having been selected to maximize the effects of radiation scattering by unaltered whole blood relative to the effects of radiation absorbance by unaltered whole blood;

irradiating a sample of unaltered whole blood of unknown composition with said plurality of radiation wavelengths, through a depth of said sample chosen to minimize radiation scattering by unaltered whole blood;

detecting intensities of said radiation wavelengths, after passing through said depth of said sample, at a distance from said sample, and over a detecting area, both chosen to minimize the effects of radiation scattering by unaltered whole blood on the determination of concentrations of said constituent components; and

calculating concentrations of said plurality of constituent components of said sample of unaltered whole blood corrected for the effects of radiation scattering, based upon detected intensities of each of said plurality of radiation wavelengths, and based upon predetermined molar extinction coefficients for each of said constituent components at each of said plurality of radiation wavelengths.

2. The method of claim 1, wherein said depth of said sample is in the range of 80 to 150 micrometers.

3. The method of claim 2, wherein said depth of said sample is approximately 90 micrometers.

4. The method of claim 1, wherein said detecting area is at least approximately 150 square millimeters.

5. The method of claim 4, wherein said detecting area is at least approximately 600 square millimeters.

6. The method of claim 1, wherein said distance from said sample is within the range of 0 to 10 millimeters.

7. The method of claim 6, wherein said distance from said sample is approximately 1 millimeter.

8. The method of claim 1, wherein said step of detecting is performed over a half-aperture angle of radiation emanating from said sample of at least approximately 30 degrees.

9. The method of claim 8, wherein said step of detecting is performed over a half-aperture angle of radiation emanating from said sample of at least approximately 70 degrees.

10. The method of claim 1, further comprising:

correcting said calculated concentrations of constituent components for the effects of finite spectral bandwidth of the substantially monochromatic wavelengths on the extinction coefficients of each constituent component.

11. The method of claim 1, said plurality of constituent components including HbO₂, HbCO, Hi and Hb, said method further comprising:

before said generating step, selecting four radiation wavelengths by computing an error index for each of HbO₂, HbCO and Hi as the sum of the absolute values of the errors that are induced in the measurement of relative concentrations of HbO₂, HbCO and Hi due to a change in optical density measurements; and

selecting a quadruple of radiation wavelengths having minimum error indices.

12. The method of claim 11, each one of said quadruple of radiation wavelengths being within the range of 510 to 630 nanometers.

13. The method of claim 12, said quadruple of radiation wavelengths comprising 522, 562, 584 and 600 nanometers.

14. The method of claim 12, said quadruple of radiation wavelengths comprising 518, 562, 580 and 590 nanometers.

15. The method of claim 12, said quadruple of radiation wavelengths comprising 520.1, 562.4, 585.2 and 597.5 nanometers.

16. The method of claim 12, said constituent components further including bilirubin, said method further comprising:

before said generating step, selecting a radiation wavelength within the range of 475 to 500 nanometers as the radiation wavelength for the measurement of bilirubin.

17. The method of claim 16, said radiation wavelength for the measurement of bilirubin being 488.4 nanometers.

18. The method of claim 12, said constituent components further including sulfhemoglobin, said method further comprising:

before said generating step, selecting a radiation wavelength within the range of 615 to 625 nanometers as the radiation wavelength for the measurement of sulfhemoglobin.

19. The method of claim 18, said radiation wavelength for the measurement of sulfhemoglobin being 621.7 nanometers.

20. The method of claim 1, further comprising:

correcting said calculated concentrations of constituent components for the effects of light scattering by red blood cells.

21. The method of claim 20, said correcting step comprising, correcting said calculated concentrations of constituent components as a function of the relative concentrations of the constituent components.

22. The method of claim 21, said correcting step further comprising:

iteratively determining a red blood cell scattering vector for the particular composition of the whole blood sample being analyzed; and

using said red blood cell scattering vector to correct said calculated constituent component concentrations.

23. The method of claim 1, further comprising:

correcting said calculated constituent component concentrations for the effects of non-specific light scattering.

24. The method of claim 23, said correcting step comprising, correcting said calculated concentrations of constituent components as a function of the relative concentrations of the constituent components under consideration.

25. The method of claim 24, said correcting step further comprising:

iteratively determining a non-specific scattering vector for the particular composition of the whole blood sample being analyzed; and

using said non-specific scattering vector to correct said calculated constituent component concentrations.

26. The method of claim 1, further comprising, correcting said calculated concentrations of constituent components for the effects of light scattering by red blood cells and for the effects of non-specific light scattering.

27. The method of claim 26, said correcting step comprising, correcting said calculated concentrations of constituent components as a function of the relative concentrations of the constituent components under consideration.

28. The method of claim 27, said correcting step further comprising:

iteratively determining a red blood cell scattering vector for the particular composition of the whole blood sample being analyzed;

iteratively determining a non-specific scattering vector for the particular composition of the whole blood sample being analyzed; and

using said non-specific scattering vector and said red blood cell scattering vector to correct said calculated constituent component concentrations.

29. The method of claim 28, said plurality of constituent components including HbO₂, HbCO, Hi and Hb, said method further comprising:

before said generating step, selecting four radiation wavelengths by computing an error index for each of HbO₂, HbCO and Hi as the sum of the absolute values of the errors that are induced in the measurement of relative concentrations of HbO₂, HbCO and Hi due to a change in optical density measurements; and

selecting a quadruple of radiation wavelengths having minimum error indices.

30. The method of claim 29, each one of said quadruple of radiation wavelengths being within the range of 510 to 630 nanometers.

31. The method of claim 30, said constituent components further including bilirubin, said method further comprising:

before said generating step, selecting a radiation wavelength within the range of 475 to 500 nanometers as the radiation wavelength for the measurement of bilirubin.

32. The method of claim 31, said constituent components further including sulfhemoglobin, said method further comprising:

before said generating step, selecting a radiation wavelength within the range of 615 to 625 nanometers as the radiation wavelength for the measurement of sulfhemoglobin.

33. The method of claim 32, further comprising, before said generating step, selecting a radiation wavelength within the range of 635 to 645 nanometers as an additional radiation wavelength for the measurement of sulfhemoglobin.

34. The method of claim 20, said correcting step comprising,

correcting said calculated concentrations of constituent components as a function of wavelength.

35. The method of claim 23, said correcting step comprising,

correcting said calculated concentrations of constituent components as a function of wavelength.

36. The method of claim 26, said correcting step comprising,

correcting said calculated concentrations of constituent components as a function of wavelength.

37. A method of determining the concentrations of a plurality of k constituent components of unaltered whole blood, k being an integer, comprising:

generating a plurality of n different substantially monochromatic radiation wavelengths, where n is an integer and n>

k, k of said n wavelengths having been selected to measure radiation absorption by said k constituent components, and n-k of said n wavelengths having been selected to compensate for errors due to n-k scattering factors in unaltered whole blood;

irradiating a sample of unaltered whole blood with said n radiation wavelengths;

detecting intensities of said n radiation wavelengths after passing through said sample of unaltered whole blood; and

calculating concentrations of said k constituent components of said sample of unaltered whole blood, corrected for the effects of radiation scattering, as a function of said detected intensities of said n radiation wavelengths.

38. The method of claim 37, said calculating step comprising:

calculating a vector of n optical densities of said sample of unaltered whole blood, each optical density being a function of a respective one of said n detected intensities; and

calculating said concentrations of said k constituent components using a set of n linear equations that equate said vector of n optical densities with a linear combination of k light absorbance vectors and n-k light scattering vectors, real coefficients of said k absorbance vectors in said linear combination being equal to said concentrations of said k constituent components.

39. The method of claim 38, wherein each of said k light absorbance vectors corresponds to a specific one of said k constituent components, the entries of each light absorbance vector being extinction coefficients of a corresponding constituent component at each of said n wavelengths.

40. The method of claim 38, wherein each of said n-k light scattering vectors corresponds to an identifiable scattering factor, said method further comprising, iteratively determining each of said n-k scattering vectors as functions of said concentrations of said k constituent components present in said sample of unaltered whole blood.

41. The method of claim 37, said calculating step comprising, correcting said calculated concentrations of said k constituent components for the effects of finite spectral bandwidth of the n substantially monochromatic wavelengths on the extinction coefficients corresponding to each constituent component.

42. The method of claim 37, said generating step comprising:

selecting four radiation wavelengths by computing an error index for each of HbO₂, HbCO and Hi as the sum of the absolute values of the errors that are induced in the measurement of relative concentrations of HbO₂, HbCO and Hi due to a change in optical density measurements; and

selecting a quadruple of radiation wavelengths having minimum error indices.

43. The method of claim 37, said calculating step comprising, correcting said calculated concentrations of said k constituent components as a function of the relative concentrations of the k constituent components.

44. The method of claim 37, said calculating step comprising:

iteratively determining a red blood cell scattering vector for the particular composition of the unaltered whole blood sample being analyzed;

iteratively determining a non-specific scattering vector for the particular composition of the unaltered whole blood sample being analyzed; and

using said red blood cell scattering vector and said nonspecific scattering vector to correct said calculated concentrations of said k constituent components.