Spectroscopic method and apparatus for total hemoglobin measurement

1. A method of measuring Tot-Hb in a sample, comprising,

i) collecting an absorbance measurement of the sample using one or more than one first or second spectroscopic apparatus comprising a first primary calibration algorithm for one of Oxy-Hb, “Oxy-Hb plus Deoxy-Hb”, or “Total-Hb minus Met-Hb”, and one or more than one second primary calibration algorithm for one or more than one of Met-Hb, Carboxy-Hb, or Sulf-Hb, or comprising a third primary calibration algorithm obtained by adding terms of the first primary calibration algorithm and the terms of the one or more than one second primary calibration algorithm together; and

ii) predicting either:

a) a first value for one of Oxy-Hb, “Oxy-Hb plus Deoxy-Hb,” or “Total-Hb minus MebHb”, in the sample by applying the first primary calibration algorithm to an order derivative of absorbance of the sample at one or more than one wavelength of a standard set of wavelengths, and predicting one or more than one second value for one or more than one of Met-Hb, Carboxy-Hb, or Sulf-Hb in the sample by applying the second primary calibration algorithm to an order derivative of absorbance at one or more than one wavelength of a standard set of wavelengths, and adding the first value and the one or more than one second value together to provide a measurement of Total-Hb; or

b) Total-Hb using the third primary calibration algorithm applied to an order derivative of absorbance of the sample at one or more than one wavelength of a standard set of wavelengths.

2. The method of claim 1 wherein in the step of collecting (step i)), the first primary calibration algorithm and the one or more than one second primary calibration algorithm are generated using an order derivative of absorbance at one or more than one wavelength of a standard set of wavelengths, obtained from one or more than one first apparatus using one or more than one primary calibration set having known reference values for one or more than one of Oxy-Hb, “Oxy-Hb plus Deoxy-Hb”, “Total-Hb minus Met-Hb”, Met-Hb, Carboxy-Hb, or Sulf-Hb, the first primary calibration algorithm is generated for one of Oxy-Hb, “Oxy-Hb plus Deoxy-Hb,” or “Total-Hb minus Met-Hb”, using an order derivative of absorbance at the one or more than one wavelength of a standard set of wavelengths, the known reference values, and a statistical technique and the one or more than one second primary calibration algorithm is generated for one or more than one of Met-Hb, Carboxy-Hb, or Sulf-Hb, using an order derivative of absorbance at the one or more than one wavelength of a standard set of wavelengths, the known reference values, and the statistical technique.

3. The method of claim 2 , wherein the first primary calibration algorithm and the one or more than one second primary calibration algorithm are upgraded, using a small set of unique calibrator materials that are distinct from the one or more than one primary calibration set.

4. The method of claim 2 , wherein:

i) concentration of the Oxy-Hb, the “Oxy-Hb plus Deoxy-Hb”, and the “Total-Hb minus Met-Hb”, are considered to be equivalent if the concentration of Oxy-Hb accounts for about 95% of the concentration of “Oxy-Hb plus Deoxy-Hb”, or about 95% of the concentration of “Total-Hb minus Met-Hb”, in the sample; or

ii) concentration of the Oxy-Hb, the “Oxy-Hb plus Deoxy-Hb”, and the “Total-Hb minus Met-Hb”, are considered to be equivalent if the concentration of “Oxy-Hb plus Deoxy-Hb,” or the concentration of “Total-Hb minus Met-Hb”, in the sample, comprises about 95% Oxy-Hb.

5. The method of claim 2 , wherein the standard set of wavelengths is selected from the range from about 300 nm to about 2500 nm.

6. The method of claim 2 , wherein samples of the one or more than one calibration set comprises from 0% to 100% or any value therebetween, of one or more than one of Oxy-Hb, “Oxy-Hb plus Deoxy-Hb”, “Total-Hb minus Met-Hb”, Carboxy-Hb, Met-Hb, and Sulf-Hb.

7. The method of claim 2 , wherein the statistical technique is selected from the group consisting of simple linear regression, multiple linear regression, and multivariate analysis.

8. The method of claim 7 , wherein the multivariate analysis is selected from the group consisting of partial least squares, principal component analysis, neural network, and genetic algorithm.

9. The method of claim 1 , wherein the first primary calibration algorithm and the one or more than one second primary calibration algorithm, or the third primary calibration algorithm, are installed on, and in operative association with, a second apparatus, and in the step of collecting (step i)), the absorbance of the sample is measured on the second apparatus to produce an absorbance measurement.

10. The method of claim 1 , wherein the one or more than one apparatus is a second apparatus, and wherein a step of data pre-processing follows the step of collecting (step i) and precedes the step of predicting (Step ii).

11. The method of claim 10 , wherein the step of data pre-processing includes a process selected from the group consisting of: calculation of interpolated absorbances; smoothing of absorbances; calculation of a first and higher order derivative of absorbance; multiplicative scatter correction; data transformation;

photometric correction, and a combination thereof.

12. The method of claim 1 , wherein the sample is one of whole blood, serum, plasma, urine, synovial fluid, lymphatic fluid, sputum, feces, or cerebrospinal fluid.

13. The method of claim 12 , wherein the Total-Hb, is used as an indicator of hemolysis.

14. The method of claim 1 , wherein the sample is exposed to atmospheric oxygen prior to the absorbance measurement.

15. The method of claim 1 , wherein the Total-Hb is a combination of endogenous Hb and exogenous Hb, and wherein the exogenous Hb is one or more than one Hb-based blood substitute, and wherein the Met-Hb is a combination of endogenous Met-Hb and the Met-Hb form of the one or more than one Hb-based blood substitute.

16. The method of claim 1 , wherein by measuring the proportion of Total-Hb that is in the form of Met-Hb, provides a method of monitoring degradation or reversal of degradation of one or more than one Hb-based blood substitute in the sample.

17. A reagentless spectroscopic method for measuring Total-Hb in a sample comprising:

i) collecting an absorbance measurement of the sample using one or more than one spectroscopic apparatus comprising a primary calibration algorithm for Total-Hb, the sample having been exposed to atmospheric oxygen; and

ii) predicting a value for Total-Hb in the sample by applying the primary calibration algorithm to an order derivative of absorbance of the sample at one or more than one wavelength of a standard set of wavelengths, to provide a measurement of Total-Hb,

wherein in the step of collecting (step i)), the primary calibration algorithm for Total-Hb is generated using an order derivative of absorbance at one or more than one wavelength of a standard set of wavelengths obtained from one or more than one first apparatus, known reference values obtained from a primary calibration set, and a statistical technique, wherein each sample of the calibration set is exposed to atmospheric oxygen before spectroscopic measurement, and wherein the calibration set comprises known reference values from about 0 to about 100% or any value therebetween, of one or more than one of Oxy-Hb, Deoxy-Hb. Met-Hb, Carboxy-Hb, and Sulf-Hb, and

wherein the first primary calibration algorithm is upgraded using a small set of unique calibrator materials that are distinct from the primary calibration set prior to obtaining the absorbance measurement.

18. The method of claim 17 , wherein the primary calibration algorithm is installed on, and is in operative association with, a second apparatus, and in the step of collecting (step i)), the absorbance of the sample is measured on the second apparatus to produce an absorbance measurement.

19. The method of claim 17 , wherein a step of data pre-processing follows the step of collecting (step i)), and before the step of predicting (step (ii)).

20. The method of claim 19 , wherein the step of data pre-processing includes a process selected from the group consisting of: calculation of interpolated absorbances; smoothing of absorbances; calculation of a first and higher order derivative of absorbance; multiplicative scatter correction; data transformation; photometric correction, and a combination thereof.

21. The method of claim 17 , wherein the standard set of wavelengths is selected from the range from about 300 nm to about 2500 nm, or any amount therebetween.

22. The method of claim 17 , wherein the statistical technique is selected from the group consisting of simple linear regression, multiple linear regression, and multivariate analysis.

23. The method of claim 22 , wherein the multivariate analysis is selected from the group consisting of partial least squares, principal component analysis, neural network, and genetic algorithm.

24. The method of claim 17 , wherein the sample is one of whole blood, serum, plasma, urine, synovial fluid, lymphatic fluid, sputum, feces, or cerebrospinal fluid.

25. The method of claim 24 , wherein the Total-Hb, is used as an indicator of hemolysis.

26. The method of claim 17 , wherein the Total-Hb is a combination of endogenous Hb and exogenous Hb, and wherein the exogenous Hb is one or more than one Hb-based blood substitute.

27. A spectroscopic apparatus, comprising:

a) a source of electromagnetic radiation (EMR);

b) a first aperture located between the source of EMR and a sample slot to produce a light path therebetween;

c) the sample slot in the apparatus for receiving a sample vessel to be placed within the light path;

d) a second aperture located in the light path, between the sample slot and one or more than one photodetector, the one or more than one photodetector in operative association with the spectroscopic apparatus; and either

e) a first primary calibration algorithm in operative association with the spectroscopic apparatus, the first primary calibration algorithm generated for one of Oxy-Hb, “Oxy-Hb plus Deoxy-Hb,” or “Total-Hb minus Met-Hb”, using an order derivative of absorbance at one or more than one wavelength of a standard set of wavelengths obtained from one or more than one first apparatus using one or more than one calibration set having known reference values for one or more than one of Oxy-Hb, “Oxy-Hb plus Deoxy-Hb”, “Total-Hb minus Met-Hb”, Met-Hb, Carboxy-Hb, or Sulf-Hb, and a statistical technique, and one or more than one second primary calibration algorithm in operative association with the spectroscopic apparatus, the one or more than one second primary calibration algorithm generated for one or more than one of MetHb, Carboxy-Hb, or Sulf-Hb, using an order derivative of absorbance at the one or more than one wavelength of a standard set of wavelengths, the known reference values, and the statistical technique; or

f) a third primary calibration algorithm in operative association with the spectroscopic apparatus, the third primary calibration algorithm obtained by adding the terms of the first primary calibration algorithm and the terms of the one or more than one second primary calibration algorithm together.