Technique for determining whether a cell is malignant as opposed to non-malignant using extrinsic fluorescence spectroscopy

• US 5,635,402 A
• Filed: 04/29/1994
• Issued: 06/03/1997
• Est. Priority Date: 03/05/1992
• Status: Expired due to Fees

• Current Assignee: Cheng H. Liu, Robert R. Alfano, Wei L. Sha, Yury Budansky
• Original Assignee: Cheng H. Liu, Robert R. Alfano, Wei L. Sha, Yury Budansky
• Inventors: Budansky, Yury, Sha, Wei L., Liu, Cheng H., Alfano, Robert R.
• Primary Examiner(s): SNAY, JEFFREY R
• Application Number:
  US08/236,861
• Time in Patent Office: 1,131 Days
• Field of Search: 436/63-64, 436/172, 436/800, 436/813, 436/149, 436/811, 250/461.2, 250/459.1
• US Class Current: 436/63
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6423 Spectral mapping, video display
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72

Abstract

A technique for determining whether a cell is malignant as opposed to non-malignant using extrinsic fluorescence spectroscopy. The technique is premised on the principle that certain fluorescent dyes preferentially stain malignant cells as opposed to non-malignant cells. Accordingly, by exposing a cell to the fluorescent dye, irradiating the cell with light of such a wavelength as to cause the dye to fluoresce, measuring the intensity of fluorescence at a wavelength indicative of fluorescence of the dye, and comparing the fluorescence intensity to standards obtained from malignant cells and non-malignant cells, it is possible for one to accurately classify the cell as being either malignant or non-malignant. The present invention also relates to an automated system which applies the principles of the aforementioned technique to depict the spatial distribution of cells within an area of a Pap smear-type sample and to characterize each of the cells as being malignant or non-malignant.

Claims

1. A method of examining the cells present within an area of a Pap smear for purposes of detecting cancer, the method comprising the steps of:

a) exposing the cells present within the area of the Pap smear to a fluorescent dye which preferentially stains malignant cells as opposed to non-malignant cells;

b) illuminating the area with light of a wavelength sufficient to cause the fluorescent dye to fluoresce whereby fluorescent light is emitted from the area;

c) measuring the intensity of fluorescent light emitted from the area as a function of location within the area at a wavelength indicative of fluorescence of the fluorescent dye, said measuring step comprising passing the fluorescent light through a filter selective for said wavelength indicative of fluorescence of said fluorescent dye and then converting the filtered fluorescent light from optical signals into electrical signals corresponding in intensity thereto;

d) comparing the electrical signals obtained in step (c) to appropriate standards obtained from cells known to be malignant and non-malignant; and

e) using the results of said comparisons to generate a map of the area, said map conveying the spatial distribution of cells within the area and a characterization of each cell within the area as being either malignant or non-malignant.

2. The method as claimed in claim 1 wherein said fluorescent dye is selected from the group consisting of hematoporphyrin, hematoporphyrin derivative, dihematoporphyrin ether/ester, tetramicin, rhodamine, and cyanine.

3. The method as claimed in claim 2 wherein said fluorescent dye is hematoporphyrin derivative.

4. The method as claimed in claim 2 wherein said fluorescent dye is selected from the group consisting of hematoporphyrin, hematoporphyrin derivative, dihematoporphyrin ether/ester.

5. The method as claimed in claim 4 wherein the fluorescent dye used has an absorption spectrum and wherein said illuminating step comprises illuminating the area with light at a wavelength within the absorption spectrum for the fluorescent dye used.

6. The method as claimed in claim 5 wherein said fluorescent dye is hematoporphyrin and wherein said measuring step comprises measuring the intensity of fluorescence at a wavelength of about 820 nm or about 860 nm.

7. The method as claimed in claim 5 wherein said illuminating step comprises illuminating the area with light having a wavelength between about 300 nm and about 400 nm.

8. The method as claimed in claim 7 wherein said illuminating step comprises illuminating the area with light having a wavelength between about 350 nm and about 400 nm.

9. The method as claimed in claim 5 wherein said fluorescent dye is hematoporphyrin derivative or dihematoporphyrin ether/ester and wherein said measuring step comprises measuring the intensity of fluorescence at a wavelength of about 630 nm or about 690 nm.

10. The method as claimed in claim 9 wherein said fluorescent dye is hematoporphyrin derivative and wherein said measuring step comprises measuring the intensity of fluorescence at a wavelength of about 630 nm.

11. The method as claimed in claim 10 wherein said exposing step comprises adding an appropriate amount of hematoporphyrin derivative to the area and removing any excess hematoporphyrin derivative from the area.

12. The method as claimed in claim 11 wherein the hematoporphyrin derivative added to the cell is in solution at a concentration of approximately 1.0×

10^-3 Mol/L.

13. A method of examining the cells present within an area of a Pap smear for purposes of detecting cancer, the method comprising the steps of:

a) exposing the cells within the area of the Pap smear to a fluorescent dye which preferentially stains malignant cells as opposed to non-malignant cells;

b) illuminating the area with light of a wavelength sufficient to cause the fluorescent dye to fluoresce whereby fluorescent light is emitted from the area;

c) measuring the intensity of fluorescent light emitted from the area as a function of location within the area at a reference wavelength and at a wavelength indicative of fluorescence of the fluorescent dye, said measuring step comprising passing the fluorescent light alternately through a filter selective for said wavelength indicative of fluorescence of the fluorescent dye and through a filter selective for said reference wavelength and then converting the filtered fluorescent light from optical signals into electrical signals corresponding in intensity thereto;

d) determining the ratio of intensities of said electrical signals corresponding to the fluorescent light at said reference wavelength and at said wavelength indicative of fluorescence of the fluorescent dye;

e) comparing said ratio to standard ratios obtained from malignant and non-malignant cells; and

f) using the results of said comparisons to generate a map of the area, said map conveying the spatial distribution of cells within the area and a characterization of each cell within the area as being either malignant or non-malignant using said comparisons.

14. The method as claimed in claim 13 wherein said fluorescent dye is selected from the group consisting of hematoporphyrin, hematoporphyrin derivative, dihematoporphyrin ether/ester, tetramicin, rhodamine, and cyanine.

15. The method as claimed in claim 13 wherein said fluorescent dye is selected from the group consisting of hematoporphyrin, hematoporphyrin derivative, and dihematoporphyrin ether/ester.

16. The method as claimed in claim 15 wherein the fluorescent dye used has an absorption spectrum and wherein said illuminating step comprises illuminating the area with light at a wavelength within the absorption spectrum for the fluorescent dye used.

17. The method as claimed in claim 16 wherein said illuminating stem comprises illuminating the area with light at a wavelength between about 300 nm and about 400 nm.

18. The method as claimed in claim 17 wherein said illuminating step comprises illuminating the area with light having a wavelength between about 350 nm and about 400 nm.

19. The method as claimed in claim 18 wherein said fluorescent dye is hematoporphyrin derivative, or dihematoporphyrin ether/ester and wherein said measuring step comprises measuring the intensity of fluorescence at a reference wavelength of about 440 nm, about 560 nm, or about 580 nm and at a wavelength indicative of fluorescence of said fluorescent dye of about 630 nm or about 690 nm.

20. The method as claimed in claim 18 wherein said fluorescent dye is hematoporphyrin and wherein said measuring step comprises measuring the intensity of fluorescence from the cell at a reference wavelength of about 440 nm about 560 nm or about 580 nm. at a wavelength at which the fluorescent dye fluoresces of about 620 nm or about 660 nm.

21. A method of making a map of the spatial distribution of cells within an area of a Pap smear and for characterizing each of the cells within the area as being either malignant or non-malignant, the method comprising the steps of:

a) exposing the cells within the area of the Pap smear to a fluorescent dye which preferentially stains malignant cells as opposed to non-malignant cells;

b) illuminating the area with light of a wavelength sufficient to cause the fluorescent dye to fluoresce whereby fluorescent light is emitted from the area;

c) measuring at a wavelength indicative of fluorescence of the fluorescent dye the fluorescence intensity of the entire illuminated area as a function of location therewithin, said measuring step comprising passing the fluorescent light through a filter selective for said wavelength indicative of fluorescence of the fluorescent dye and then converting the filtered fluorescent light from optical signals into electrical signals corresponding in intensity thereto;

d) comparing the electrical signals emitted from each location of the entire illuminated area to standards which permit each location to be characterized as corresponding to a malignant cell, a non-malignant cell or background;

e) characterizing each location on the basis of said comparisons; and

f) displaying the results of said characterizations in the form of a map.

22. A method of examining the cells present within an area of a Pap smear for purposes of detecting cancer, the method comprising the steps of:

a) exposing the cells within the area of the Pap smear to a fluorescent dye which preferentially stains malignant cells as opposed to non-malignant cells;

b) illuminating the area with light of a wavelength sufficient to cause the fluorescent dye to fluoresce whereby fluorescent light is emitted from the area;

c) measuring the intensity of fluorescent light emitted from the area as a function of location within the area at a reference wavelength and at a wavelength indicative of fluorescence of the fluorescent dye, said measuring step comprising passing the fluorescent light alternately through a filter selective for said wavelength indicative of fluorescence of the fluorescent dye and through a filter selective for said reference wavelength and then converting the filtered fluorescent light from optical signals into electrical signals corresponding in intensity thereto;

d) determining the difference in intensities of the electrical signals corresponding to the optical signals at said reference wavelength and at said wavelength indicative of fluorescence of the fluorescent dye;

e) comparing said difference to standard differences obtained from malignant and non-malignant cells; and

f) using said comparisons to generate a map conveying the spatial distribution of cells within the area and a characterization of each cell within the area as being either malignant or non-malignant.

23. A method of cancer-screening a cell sample containing cells whose cancer condition are unknown, the method comprising the steps of:

a) exposing the cell sample to a fluorescent dye which preferentially stains malignant cells as opposed to non-malignant cells;

b) preparing a monolayer spread with the stained cell sample;

c) illuminating an area of the monolayer spread with light of a wavelength sufficient to cause the fluorescent dye to fluoresce whereby fluorescent light is emitted from the area;

d) measuring the intensity of fluorescent light emitted from the area as a function of location within the area at a wavelength indicative of fluorescence of the fluorescent dye, said measuring step comprising passing the fluorescent light through a filter selective for said wavelength indicative of fluorescent light and then converting the filtered fluorescent light from optical signals into electrical signals corresponding in intensity thereto;

e) comparing the electrical signals obtained in step (d) to appropriate standards obtained from cells known to be malignant and non-malignant; and

f) using said comparisons to generate a map conveying the spatial distribution of cells within the area and a characterization of each cell within the area as being either malignant or non-malignant.

24. The method as claimed in claim 23 wherein said illuminating step comprises illuminating the entirety of the area at once.

25. The method as claimed in claim 23 wherein said illuminating step comprises illuminating successive portions of the area.