Method and apparatus for direct spectrophotometric measurements in unaltered whole blood

• US 20030202170A1
• Filed: 12/06/2002
• Published: 10/30/2003
• Est. Priority Date: 02/23/1989
• Status: Active Grant

• 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: Steinke, John M., Shepherd, A. P.
• Granted Patent: US 7,075,628 B2
• Time in Patent Office: Days
• 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 which allow accurate spectrophotometric determinations 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. Four of the seven wavelengths of light are chosen by a criterion that minimizes the spectrophotometric error in the measurements of the concentrations of oxy-, carboxy-, and methemoglobin. Additional wavelengths enable the invention to assess light scattering quantitatively and to measure the concentrations of bilirubin and sulfhemoglobin. 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. By eliminating the need for hemolysis or dilution, the present invention overcomes disadvantages of prior art that requires pumps, plumbing, ultrasonic hemolyzers, and associated control circuitry, yet it makes accurate spectrophotometric measurements of the bilirubin concentration, the total hemoglobin concentration, and the relative concentrations of oxy-, deoxy-, carboxy-, met-, and sulfhemoglobin. Because the sample is neither hemolyzed nor diluted, it can be subjected to further chemical or hematological analysis.

Claims

1. A method of determining the concentrations of a plurality of constituent components of unaltered whole blood, 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 with said plurality of radiation wavelengths, through a depth of said sample chosen to minimize radiation scattering by unaltered whole undiluted 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 concentration of said constituent components; and

calculating concentrations of said plurality of constituent components of said sample of unaltered whole blood, 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 radiation wavelengths of said absorbance subset.

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, said quadruple of radiation wavelengths each 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, said quadruple of radiation wavelengths each 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.