Cell analysis method using quantitative fluorescence image analysis

• US 5,733,721 A
• Filed: 12/20/1992
• Issued: 03/31/1998
• Est. Priority Date: 11/20/1992
• Status: Expired due to Term

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Rao, Jian Yu, Hurst, Robert E., Hemstreet, George P. III, Bonner, Rebecca B.
• Primary Examiner(s): Scheiner, Toni R.
• Application Number:
  US07/984,191
• Time in Patent Office: 1,927 Days
• Field of Search: 435/7.23, 435/6, 435/968, 435/973, 364/413.08, 382/10, 382/28, 382/36, 436/800, 436/805
• US Class Current: 435/6.14
• CPC Class Codes: G01N 2333/4712 Muscle proteins, e.g. myosin, actin, protein
• CPC Class Codes: G01N 2333/485 Epidermal growth factor [EGF] (urogastrone)
• CPC Class Codes: G01N 2333/82 Translation products from oncogenes
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/57407 Specifically defined cancers
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/813 Cancer

Abstract

A system for evaluating one or more biochemical markers for evaluating individual cancer risk, cancer diagnosis and for monitoring therapeutic effectiveness and cancer recurrence, particularly of bladder cancer. The system uses automated quantitative fluorescence image analysis of a cell sample collected from a body organ. Cells are treated with a fixative solution which inhibits crystal formation. Cell images are selected and stored as grey level images for further analysis. Cell images may be corrected for autofluorescence using a novel autofluorescence correction method. A neural net computer may be used to distinguish true-positive images from false-positive images to improve accuracy of cancer risk assessment. Cells having images positive for a marker amy be compared to threshold quantities related to predetermined cancer risk.

Claims

1. A method of analyzing a cell sample derived from urine or from a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide, the portion of the cell sample treated with a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting formation of substantially all of the crystals in the cell sample prior to application of the portion of the cell sample to the slide leaving the prepared slide substantially free of crystals for improving microscopic analysis of the cell on the prepared slide, then treating the slide with a fluorescent label for labeling the cytological marker to form a labeled cytological marker;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in a cell to emit fluorescent light having an emission wavelength for forming a field image;

using a microscope means to select cell images on the field image;

obtaining a number related to the selected cell images;

and

outputting the number for use in classifying the cell sample.

2. The method of claim 1 wherein in the step of providing a prepared slide the cytological marker is selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

3. The method of claim 1 wherein in the step of providing a prepared slide the fluorescent label is comprised of a fluorochrome bound to an affinity probe.

4. The method of claim 3 wherein in the step of providing a prepared slide the fluorochrome is selected from the group consisting of Texas Red, bodipy and fluorescein.

5. The method of claim 1 wherein in the step of providing a prepared slide the fluorescent label is comprised of a dye specific for DNA.

6. The method of claim 5 wherein in the step of providing a prepared slide the fluorescent label is Hoechst 33258.

7. The method of claim 1 wherein the step of using a microscope means is preceded by the step of correcting the field image for autofluorescence.

8. The method of claim 1 wherein the step of obtaining a number is preceded by the step of reviewing the selected cell images with a confirmation means confirming that the selected cell images represent cells of a desired type.

9. The method of claim 8 wherein in the step of reviewing the selected cell images the confirmation means is a neural net computing means.

10. The method of claim 1 wherein the step of outputting the number is followed by the step of classifying the cell sample.

11. The method of claim 1 wherein in the step of providing a prepared slide, the prepared slide is further defined as having been prepared by treating the slide with a second fluorescent label for labeling a second cytological marker to form a labeled second cytological marker.

12. The method of claim 11 further comprising the additional step of analyzing the prepared slide for the second cytological marker comprising:

irradiating a second portion of the prepared slide with an amount of a second excitation wavelength of light effective in causing the cells containing the second fluorescent label to emit fluorescent light having a second emission wavelength for forming a second field image wherein the second portion may be the same as the first portion,

using the microscope means to select second cell images on the second field image,

obtaining a second number related to the selected second cell images, and

outputting the second number for use in classifying the cell sample.

13. The method of claim 12 wherein the step of using the microscope means to select second cell images is preceded by the step of correcting the second field image for autofluorescence.

14. The method of claim 12 wherein the step of obtaining a second number is preceded by the step of reviewing the selected second cell images with a confirmation means for confirming that the selected second cell images represent cells of a desired type.

15. The method of claim 14 wherein in the step of reviewing the selected second cell images the confirmation means is a neural net computing means.

16. The method of claim 12 wherein the step of outputting the second number is followed by the step of using the first number and the second number to classify the cell sample.

17. The method of claim 11 wherein in the step of providing a prepared slide, the prepared slide is further defined as having been prepared by treating the slide with a third fluorescent label for labeling a third cytological marker to form a labeled third cytological marker.

18. The method of claim 17 further comprising the additional step of analyzing the prepared slide for the third cytological marker comprising:

irradiating a third portion of the prepared slide with an amount of a third excitation wavelength of light effective in causing the cells containing the third fluorescent label to emit fluorescent light having a third emission wavelength for forming a third field image, wherein the third portion may be the same as the second portion or the first portion,

using the microscope means to select third cell images on the field third image,

obtaining a third number related to the selected third cell images, and

outputting the third number for use in classifying the cell sample.

19. The method of claim 18 wherein the step of using the microscope means to select third cell images is preceded by the step of correcting the third field image for autofluorescence.

20. The method of claim 18 wherein the step of obtaining a third number is preceded by the step of reviewing the selected third cell images with a confirmation means for confirming that the selected third cell images represent cells of a desired type.

21. The method of claim 20 wherein in the step of reviewing the selected third cell images the confirmation means is a neural net computing means.

22. The method of claim 18 wherein the step of outputting the third number is followed by the step of using the first number, the second number and the third number to classify the cell sample.

23. A method of analyzing a cell sample derived from urine or from a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide, the portion of the cell sample treated with a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting formation of substantially all of the crystals in the cell sample prior to application of the portion of the cell sample to the slide leaving the prepared slide substantially free of crystals for improving microscopic analysis of the cell on the prepared slide, then treating the slide with a first fluorescent label for labeling the first cytological marker to form a labeled first cytological marker and the second fluorescent label for labeling the second cytological marker to form a labeled second cytological marker;

irradiating a first portion of the prepared slide with an amount of a first excitation wavelength of light effective in causing the first fluorescent label in the cell to emit fluorescent light having a first emission wavelength for forming a first field image;

using a microscope means to select first cell images on the first field image;

obtaining a first number related to the selected first cell images;

irradiating the second portion of the prepared slide with a second excitation wavelength of light effective in causing the second fluorescent label to emit fluorescent light having a second emission wavelength for forming a second field image wherein the second portion may be the same as the first portion;

using the microscope means to select second cell images on the second field image;

obtaining a second number related to the selected second cell images; and

outputting the first number and the second number for use in classifying the cell sample.

24. The method of claim 23 wherein the step of using a microscope means to select first cell images is preceded by the step of correcting the first field image for autofluorescence.

25. The method of claim 23 wherein the step of obtaining a first number is preceded by the step of reviewing the selected first cell images with a confirmation means for confirming that the selected cell images represent cells of a desired type.

26. The method of claim 25 wherein in the step of reviewing the selected first cell images the confirmation means is a neural net computing means.

27. The method of claim 23 wherein the step of obtaining a second number is preceded by the step of reviewing the selected second cell images with a confirmation means for confirming that the selected second cell images represent cells of a desired type.

28. The method of claim 27 wherein in the step of reviewing the selected second cell images the confirmation means is a neural net computing means.

29. The method of claim 23 wherein in the step of providing a prepared slide the first cytological marker and the second cytological marker are selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

30. The method of claims 23 wherein in the step of providing a prepared slide the first fluorescent label and the second fluorescent label are selected from the group consisting of Hoechst 33258, M344 plus a fluorescent conjugate, an anti-HER-2/neu protein probe plus a fluorescent conjugate, an anti-EGFR probe plus a fluorescent conjugate, and DNase I plus a fluorescent conjugate.

31. The method of claim 30 wherein in the step of providing a prepared slide the fluorescent conjugate is selected from the group consisting of Texas Red, bodipy and fluorescein.

32. The method of claim 23 wherein the first cytological marker is DNA and the second cytological marker is the p300 protein antigen.

33. The method of claim 23 wherein in the step of providing a prepared slide the first cytological marker is DNA and the second cytological markers is actin.

34. The method of claim 23 wherein the step of outputting the first number and the second number is followed by the step of classifying the cell sample.

35. The method of claim 23 wherein in the step of providing a prepared slide, the prepared slide is further defined as having been prepared by treating the slide with a third fluorescent label for labeling a third cytological marker to form a labeled third cytological marker.

36. The method of claim 35 further comprising the additional step of analyzing the prepared slide for the third cytological marker comprising:

irradiating a third portion of the prepared slide with an amount of a third excitation wavelength of light effective in causing the cells containing the third fluorescent label to emit fluorescent light having a third emission wavelength for forming a third field image wherein the third portion may be the same as the second portion or the first portion,

using the microscope means to select third cell images on the third field image,

obtaining a third number related to the selected third cell images, and

outputting the third number for use in classifying the cell sample.

37. The method of claim 36 wherein the step of using the microscope means to select third cell images is preceded by the step of correcting the third field image for autofluorescence.

38. The method of claim 36 wherein the step of obtaining a third number is preceded by the step of reviewing the selected third cell images with a confirmation means for confirming that the selected third cell images represent cells of a desired type.

39. The method of claim 38 wherein in the step of reviewing the selected third cell images the confirmation means is a neural net computing means.

40. The method of claim 36 wherein the step of outputting the third number is followed by the step of classifying the cell sample.

41. A method of analyzing a cell sample derived from urine or from a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide, the portion of the cell sample treated with a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting formation of substantially all of the crystals in the cell sample prior to application of the portion of the cell sample to the slide leaving the prepared slide substantially free of crystals for improving microscopic analysis of the cell on the prepared slide, then treating the slide with a first fluorescent label for labeling the first cytological marker to form a labeled first cytological marker, a second fluorescent label for labeling the second cytological marker to form a labeled second cytological marker, and a third fluorescent label for labeling the third cytological marker to form a labeled third cytological marker;

irradiating a first portion of the prepared slide with an amount of a first excitation wavelength of light effective in causing the first fluorescent label in the cell to emit fluorescent light having a first emission wavelength for forming a first field image;

using a microscope means to select first cell images on the first field image;

obtaining a first number related to the selected first cell images;

irradiating a second portion of the prepared slide with a second excitation wavelength of light effective in causing the second fluorescent label to emit fluorescent light having a second emission wavelength for forming a second field image visible wherein the second portion may be the same as the first portion;

using the microscope means to select second cell images on the second field image;

obtaining a second number related to the selected second cell images;

irradiating a third portion of the prepared slide with a third excitation wavelength of light effective in causing the third fluorescent label to emit fluorescent light having a third emission wavelength for forming a third field image wherein the third portion may be the same as the second portion or the first portion;

using the microscope means to select third cell images on the third field image;

obtaining a third number related to the selected third cell images; and

outputting the first number, the second number, and the third number for use in classifying the cell sample.

42. The method of claim 41 wherein the step of using a microscope means to select first cell images is preceded by the step of correcting the first field image for autofluorescence.

43. The method of claim 41 wherein the step of obtaining a first number is preceded by the step of reviewing the selected first cell images with a confirmation means for confirming that the selected cell images represent cells of a desired type.

44. The method of claim 43 wherein in the step of reviewing the selected first cell images the confirmation means is a neural net computing means.

45. The method of claim 41 wherein the step of using the microscope means to select second cell images is preceded by the step of correcting the second field image for autofluorescence.

46. The method of claim 41 wherein the step of obtaining a second number is preceded by the step of reviewing the selected second cell images with a confirmation means for confirming that the selected second cell images represent cells of a desired type.

47. The method of claim 46 wherein in the step of reviewing the selected second cell images the confirmation means is a neural net computing means.

48. The method of claim 41 wherein the step of using the microscope means to select third cell images is preceded by the step of correcting the third field image for autofluorescence.

49. The method of claim 41 wherein the step of obtaining a third number is preceded by the step of reviewing the selected third cell images with a confirmation means for confirming that the selected third cell images represent cells of a desired type.

50. The method of claim 49 wherein in the step of reviewing the selected third cell images the confirmation means is a neural net computing means.

51. The method of claim 41 wherein in the step of providing a prepared slide the first cytological marker, the second cytological marker and the third cytological marker are selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

52. The method of claim 41 wherein in the step of providing a prepared slide the first fluorescent label, the second fluorescent label and the third fluorescent label are selected from the group consisting of Hoechst 33258, M344 plus a fluorescent conjugate, an anti-HER-2/neu protein probe plus a fluorescent conjugate, an anti-EGFR probe plus a fluorescent conjugate, and DNase I plus a fluorescent conjugate.

53. The method of claim 52 wherein in the step of providing a prepared slide the fluorescent conjugate is selected from the group Texas Red, bodipy and fluorescein.

54. The method of claim 41 wherein in the step of providing a prepared slide the first cytological marker is DNA and the second cytological marker is selected from the group consisting of p300, actin, EGFR and HER-2/neu protein.

55. The method of claim 41 wherein in the step of providing a prepared slide the first cytological marker is DNA, the second cytological marker is the p300 protein antigen and the third cytological marker is selected from the group consisting of EGFR, actin and HER-2/neu protein.

56. The method of claim 41 wherein in the step of providing a prepared slide the first cytological marker is DNA, the second cytological marker is actin and the third cytological marker is selected from the group consisting of p300, EGFR and HER-2/neu protein.

57. The method of claim 41 wherein the step of outputting the first number, the second number and the third number is followed by the step of classifying the cell sample.

58. A method of analyzing a cell sample, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a fluorescent label for labeling a cytological marker to form a labeled cytological marker;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in the cell to emit fluorescent light having an emission wavelength for forming a field image;

digitizing the field image;

converting the field image into a plurality of digitized pixels;

obtaining for each pixel of the field image an intensity measurement of the peak emission wavelength;

correcting the field image for autofluorescence by selecting an off peak emission wavelength, obtaining an intensity measurement of the off peak emission wavelength for each pixel of the field image, and subtracting the intensity measurement of the off peak emission wavelength from the intensity measurement of the peak emission wavelength for each pixel of the field image wherein is obtained a corrected field image;

using a microscope means to select cell images on the corrected field image;

obtaining a number related to the selected cell images; and

outputting the number for use in classifying the cell sample.

59. The method of claim 58 wherein in the step of providing a prepared slide the cytological marker is selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

60. The method of claim 58 wherein in the step of providing a prepared slide the fluorescent label is comprised of a fluorochrome bound to an affinity probe.

61. The method of claim 60 wherein in the step of providing a prepared slide the fluorochrome is selected from the group consisting of Texas Red, bodipy and fluorescein.

62. The method of claim 58 wherein in the step of providing a prepared slide the fluorescent label is comprised of a dye specific for DNA.

63. The method of claim 62 wherein in the step of providing a prepared slide the fluorescent label in Hoechst 33258.

64. The method of claim 58 wherein the step of obtaining a number is preceded by the step of reviewing the selected cell images with a confirmation means confirming that the selected cell images represent cells of a desired type.

65. The method of claim 64 wherein in the step of reviewing the selected cell images the confirmation means is a neural net computing means.

66. The method of claim 58 wherein in the step of providing a prepared slide, the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting crystal formation.

67. The method of claim 58 wherein the step of providing a prepared slide, the cell sample is collected by washing a body organ, collected from a body fluid, or collected from a needle aspiration of a body gland or organ.

68. A method of analyzing a cell sample, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a first fluorescent label for labeling a first cytological marker to form a labeled first cytological marker and a second fluorescent label for labeling a second cytological marker to form a labeled second cytological marker;

irradiating a first portion of the prepared slide with an amount of a first excitation wavelength of light effective in causing the first fluorescent label in the cell to emit fluorescent light having a first teak emission wavelength for forming a first field image;

converting the first field image into a plurality of digitized pixels;

obtaining for each pixel of the first field image an intensity measurement of the first peak emission wavelength;

correcting the first field image for autofluorescence by selecting a first off peak emission wavelength, obtaining an intensity measurement of the first off peak emission wavelength for each pixel of the first field image, and subtracting the intensity measurement of the first off peak emission wavelength from the intensity measurement of the first peak emission wavelength for each pixel of the first field image wherein is obtained a corrected first field image;

using a microscope means to select first cell images on the corrected first field image;

obtaining a first number related to the selected first cell images;

irradiating a second portion of the prepared slide with a second excitation wavelength of light effective in causing the second fluorescent label to emit fluorescent light having a second emission wavelength for forming a second field image wherein the second portion may be the same as the first portion;

converting the second field image into a plurality of digitized pixels;

obtaining for each pixel an intensity measurement of the second peak emission wavelength;

correcting the second field image for autofluorescence by selecting a second off peak emission wavelength, obtaining an intensity measurement of the second off peak emission wavelength for each pixel of the second field image, and subtracting the intensity measurement of the second off peak emission wavelength from the intensity measurement of the second peak emission wavelength for each pixel of the second field image wherein is obtained a corrected second field image;

using the microscope means to select second cell images on the corrected second field image;

obtaining a second number related to the selected second cell images; and

outputting the first number and the second number for use in classifying the cell sample.

69. The method of claim 68 wherein in the step of providing a prepared slide, the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting crystal formation.

70. The method of claim 68 comprising the additional step of using the first number and the second number to classify the cell sample.

71. The method of claim 68 wherein the step of obtaining a first number is preceded by the step of reviewing the selected first cell images with a confirmation means for confirming that the selected cell images represent cells of a desired type.

72. The method of claim 71 wherein in the step of reviewing the selected first cell images the confirmation means is a neural net computing means.

73. The method of claim 68 wherein the step of obtaining a second number is preceded by the step of reviewing the selected second cell images with a confirmation means for confirming that the selected second cell images represent cells of a desired type.

74. The method of claim 73 wherein in the step of reviewing the selected second cell images the confirmation means is a neural net computing means.

75. The method of claim 68 wherein in the step of providing a prepared slide the first cytological marker and the second cytological marker are selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

76. The method of claim 68 wherein in the step of providing a prepared slide the first fluorescent label and the second fluorescent label are selected from the group consisting of Hoechst 33258, M344 plus a fluorescent conjugate, an anti-HER-2/neu protein probe plus a fluorescent conjugate, an anti-EGFR probe plus a fluorescent conjugate, and DNase I plus a fluorescent conjugate.

77. The method of claim 76 wherein in the step of providing a prepared slide the fluorescent conjugate is selected from the group consisting of Texas Red, bodipy and fluorescein.

78. The method of claim 68 wherein the first cytological marker is DNA and the second cytological marker is the p300 protein antigen.

79. The method of claim 68 wherein in the step of providing a prepared slide the first cytological marker is DNA and the second cytological marker is actin.

80. The method of claim 68 wherein the step of providing a prepared slide, the cell sample is collected by washing a body organ, collected from a body fluid, or collected from a needle aspiration of a body gland or organ.

81. A method of analyzing a cell sample, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a first fluorescent label for labeling a first cytological marker to form a labeled first cytological marker, a second fluorescent label for labeling a second cytological marker to form a labeled second cytological marker, and a third fluorescent label for labeling a third cytological marker to form a labeled third cytological marker;

irradiating a first portion of the prepared slide with an amount of a first excitation wavelength of light effective in causing the first fluorescent label in the cell to emit fluorescent light having a first peak emission wavelength for forming a first field image;

converting the first field image into a plurality of digitized pixels;

obtaining for each pixel of the first field image an intensity measurement of the first peak emission wavelength;

correcting the first field image for autofluorescence by selecting a first off peak emission wavelength, obtaining an intensity measurement of the first off peak emission wavelength for each pixel of the first field image, and subtracting the intensity measurement of the first off peak emission wavelength from the intensity measurement of the first peak emission wavelength for each pixel of the first field image wherein is obtained a corrected first field image;

using a microscope means to select first cell images on the corrected first field image;

obtaining a first number related to the selected first cell images;

irradiating a second portion of the prepared slide with a second excitation wavelength of light effective in causing the second fluorescent label to emit fluorescent light having a second emission wavelength for forming a second field image wherein the second portion may be the same as the first portion;

converting the second field image into a plurality of digitized pixels;

obtaining for each pixel an intensity measurement of the second peak emission wavelength;

correcting the second field image for autofluorescence by selecting a second off peak emission wavelength, obtaining an intensity measurement of the second off peak emission wavelength for each pixel of the second field image, and subtracting the intensity measurement of the second off peak emission wavelength from the intensity measurement of the second peak emission wavelength for each pixel of the second field image wherein is obtained a corrected second field image;

using the microscope means to select second cell images on the corrected second field image;

obtaining a second number related to the selected second cell images;

irradiating a third portion of the prepared slide with a third excitation wavelength of light effective in causing the third fluorescent label to emit fluorescent light having a third peak emission wavelength for forming a third field image wherein the third portion may be the same as the second portion or the first portion;

correcting the third field image for autofluorescence by selecting a third off peak emission wavelength, obtaining an intensity measurement of the third off peak emission wavelength for each pixel of the third field image, and subtracting the intensity measurement of the third off peak emission wavelength from the intensity measurement of the third peak emission wavelength for each pixel of the third field image wherein is obtained a corrected third field image;

using the microscope means to select third cell images on the corrected third field image;

obtaining a third number related to the selected third cell images; and

outputting the first number, the second number, and the third number for use in classifying the cell sample.

82. The method of claim 81 wherein the step of obtaining a first number is preceded by the step of reviewing the selected first cell images with a confirmation means for confirming that the selected first cell images represent cells of a desired type.

83. The method of claim 82 wherein in the step of reviewing the selected first cell images the confirmation means is a neural net computing means.

84. The method of claim 81 wherein the step of obtaining a second number is preceded by the step of reviewing the selected second cell images with a confirmation means for confirming that the selected second cell images represent cells of a desired type.

85. The method of claim 84 wherein in the step of reviewing the selected second cell images the confirmation means is a neural net computing means.

86. The method of claim 81 wherein the step of obtaining a third number is preceded by the step of reviewing the selected third cell images with a confirmation means for confirming that the selected third cell images represent cells of a desired type.

87. The method of claim 81 wherein in the step of reviewing the selected third cell images the confirmation means is a neural net computing means.

88. The method of claim 81 wherein in the step of providing a prepared slide, the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting crystal formation.

89. The method of claim 81 wherein in the step of providing a prepared slide the first cytological marker, the second cytological marker and the third cytological marker are selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

90. The method of claim 81 wherein in the step of providing a prepared slide the first fluorescent label, the second fluorescent and the third fluorescent label are selected from the group consisting of Hoechst 33258, M344 plus a fluorescent conjugate, an anti-HER-2/neu protein probe plus a fluorescent conjugate, an anti-EGFR probe plus a fluorescent conjugate, and DNase I plus a fluorescent conjugate.

91. The method of claim 90 wherein in the step of providing a prepared slide, the fluorescent conjugate is selected from the group Texas Red, bodipy and fluorescein.

92. The method of claim 81 wherein in the step of providing a prepared slide the first cytological marker is DNA and the second cytological marker is selected from the group consisting of p300, actin, EGFR and HER-2/neu protein.

93. The method of claim 81 wherein in the step of providing a prepared slide the first cytological marker is DNA, the second cytological is the p300 protein antigen and the third cytological marker is selected from the group consisting of EGFR, actin and HER-2/neu protein.

94. The method of claim 81 wherein in the step of providing a prepared slide the first cytological marker is DNA, the second cytological marker is actin and the third cytological marker is selected from the group consisting of p300, EGFR and HER-2/neu protein.

95. The method of claim 81 wherein the step of providing a prepared slide, the cell sample is collected by washing a body organ, collected from a body fluid, or collected from a needle aspiration of a body gland or organ.

96. The method of claim 81 comprising the additional step of using the first number, the second number, and the third number to classify the cell sample.

97. A method of analyzing a cell sample derived from urine or from a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a fluorescent label for labeling a cytological marker to form a labeled cytological marker and wherein the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting formation of substantially all of the crystals in the sample;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in the cell to emit fluorescent light having an emission wavelength for forming a field image;

using a microscope means to select cell images on the field image;

reviewing the selected cell images with a neural net computing means for confirming that the selected cell images represent cells of a desired type;

obtaining a number related to the selected cell images; and

outputting the number for use in classifying the cell sample.

98. The method of claim 97 wherein in the step of providing a prepared slide the cytological marker is selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

99. The method of claim 97 wherein in the step of providing a prepared slide the fluorescent label is comprised of a fluorochrome bound to an affinity probe.

100. The method of claim 99 wherein in the step of providing a prepared slide the fluorochrome is selected from the group consisting of Texas Red, bodipy and fluorescein.

101. The method of claim 97 wherein in the step of providing a prepared slide the fluorescent label is comprised of a dye specific for DNA.

102. The method of claim 101 wherein in the step of providing a prepared slide the fluorescent label is Hoechst 33258.

103. The method of claim 97 wherein the step of using a microscope means is preceded by the step of correcting the field image for autofluorescence.

104. The method of claim 97 wherein the step of providing a prepared slide, the cell sample is collected by washing a body organ, collected from a body fluid, or collected from a needle aspiration of a body gland or organ.

105. A method of analyzing a cell sample, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a fluorescent label for labeling a cytological marker to form a labeled cytological marker;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in the cell to emit fluorescent light having a peak emission wavelength for forming a field image;

converting the field image into a plurality of digitized pixels;

obtaining for each pixel of the field image an intensity measurement of the peak emission wavelength;

correcting the field image for autofluorescence by selecting an off peak emission wavelength, obtaining an intensity measurement of the off peak emission wavelength for each pixel of the field image, and subtracting the intensity measurement of the off peak emission wavelength from the intensity measurement of the peak emission wavelength for each pixel of the field image wherein is obtained a corrected field image;

using a microscope means to select cell images on the corrected field image;

classifying a cell image as positive or negative for a predetermined guantity of the fluorescent label;

using a neural net computing means to further classify a positive cell image as a true-positive cell image or as a false-positive cell image;

obtaining a parameter related to the number of true-positive cell images; and

outputting the parameter for use in classifying the cell sample.

106. The method of claim 105 wherein in the step of providing a prepared slide the cytological marker is selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

107. The method of claim 105 wherein in the step of providing a prepared slide the fluorescent label is comprised of a fluorochrome bound to an affinity probe.

108. The method of claim 107 wherein in the step of providing a prepared slide the fluorochrome is selected from the group consisting of Texas Red, bodipy and fluorescein.

109. The method of claim 107 wherein in the step of providing a prepared slide the fluorescent label is Hoechst 33258.

110. The method of claim 105 wherein in the step of providing a prepared slide the fluorescent label is comprised of a dye specific for DNA.

111. The method of claim 110 wherein in the step of providing a prepared slide the fluorescent label is Hoechst 33258.

112. The method of claim 105 wherein in the step of providing a prepared slide the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting crystal formation.

113. The method of claim 105 wherein the step of providing a prepared slide, the cell sample is collected by washing a body organ, collected from a body fluid, or collected from a needle aspiration of a body gland or organ.

114. A method of analyzing a cell sample derived from urine or from a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a fluorescent label for labeling a cytological marker to form a labeled cytological marker and wherein the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting the formation of substantially all of the crystals in the sample;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in the cell to emit fluorescent light having an emission wavelength for forming a field image;

correcting the field image for autofluorescence;

using a microscope means to select cell images on the field image;

obtaining a number related to the selected cell images; and

outputting the number for use in classifying the cell sample.

115. The method of claim 114 wherein in the step of providing a prepared slide the cytological marker is selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

116. The method of claim 114 wherein in the step of providing a prepared slide the fluorescent label is comprised of a fluorochrome bound to an affinity probe.

117. The method of claim 116 wherein in the step of providing a prepared slide the fluorochrome is selected from the group consisting of Texas Red, bodipy and fluorescein.

118. The method of claim 114 wherein in the step of providing a prepared slide the fluorescent label is comprised of a dye specific for DNA.

119. The method of claim 114 wherein the step of obtaining a number is preceded by the step of reviewing the selected cell images with a confirmation means confirming that the selected cell images represent cells of a desired type.

120. The method of claim 119 wherein in the step of reviewing the selected cell images the confirmation means is a neural net computing means.

121. A method of analyzing a cell sample derived from urine or from a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a fluorescent label for labeling a cytological marker to form a labeled cytological marker and wherein the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting the formation of substantially all of the crystals in the sample;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in the cell to emit fluorescent light having an emission wavelength for forming a field image;

correcting the field image for autofluorescence;

using a microscope means to select cell images on the field image;

reviewing the selected cell images with a neural net computing means for confirming that the selected cell images represent cells of a desired type;

obtaining a number related to the selected cell images; and

outputting the number for use in classifying the cell sample.

122. The method of claim 121 wherein in the step of providing a prepared slide the cytological marker is selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

123. The method of claim 121 wherein in the step of providing a prepared slide the fluorescent label is comprised of a fluorochrome bound to an affinity probe.

124. The method of claim 123 wherein in the step of providing a prepared slide the fluorochrome is selected from the group consisting of Texas Red, bodipy and fluorescein.

125. The method of claim 121 wherein in the step of providing a prepared slide the fluorescent label is comprised of a dye specific for DNA.

126. The method of claim 125 wherein in the step of providing a prepared slide the fluorescent label is Hoechst 33258.

127. A method of assessing an individual'"'"'s risk for bladder cancer, comprising:

providing a prepared slide having a population of cells affixed thereto, the prepared slide having been prepared by applying a portion of a cell sample provided by the individual to a slide then treating the slide with at least a first fluorescent label for labeling cellular DNA and at least a second fluorescent label for labeling cellular p300 protein;

irradiating the prepared slide with an amount of a first excitation wavelength of light effective in causing the first fluorescent label in the cells on the prepared slide to emit fluorescent light having a first emission wavelength;

obtaining a first population parameter related to the number of cells in the population of cells having quantities of cellular DNA which exceed a predetermined threshold quantity of cellular DNA;

irradiating the prepared slide with a second excitation wavelength of light effective in causing the second fluorescent label in the cells on the prepared slide to emit fluorescent light having a second emission wavelength;

obtaining a second population parameter related to the number of cells in the population of cells having quantities of p300 protein which exceed a predetermined threshold quantity of p300 protein;

comparing the first population parameter to a set of predetermined first parameter thresholds;

comparing the second population parameter to a set of predetermined second parameter thresholds; and

assigning a predetermined risk for bladder cancer to the individual based on which first parameter thresholds and second parameter thresholds are exceeded.

128. The method of claim 127 wherein in the step of providing a prepared slide, the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting crystal formation.

129. The method of claim 127 wherein the step of obtaining a first population parameter is preceded by the step of using confirmation means for confirming that the cells labeled with the first fluorescent label represent cells of a desired type.

130. The method of claim 127 wherein in the step of providing a prepared slide, the portion of the cell sample used in preparing the slide was a urine or bladder wash sample fixated using a fixative composition comprising a salt of ethylene-diaminetetraacetic acid effective in inhibiting crystal formation.

131. The method of claim 127 wherein the step of obtaining a second population parameter is preceded by the step of using confirmation means for confirming that the cells labeled with the second fluorescent label represent cells of a desired type.

132. A method of analyzing a cell sample derived from urine or from a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a fluorescent label for labeling a cytological marker to form a labeled cytological marker and wherein the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting formation of substantially all of the crystals in the sample;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in the cell to emit fluorescent light having an emission wavelength for forming a field image;

using a microscope means to select cell images on the field image;

classifying the cell image as positive or negative for the fluorescent label;

using a neural net computing means to further classify a positive cell image as a true-positive cell image or as a false-positive cell image;

obtaining a parameter related to the number of true-positive cell images; and

outputting the parameter for use in classifying the cell sample.

133. The method of claim 132 wherein the step of using a microscope means to select cell images is preceded by the step of correcting the field image for autofluorescence.

134. The method of claim 132 wherein the step of classifying the cell image is preceded by the step of reviewing the selected cell images with a confirmation means for confirming that the selected cell images represent cells of a desired type.

135. The method of claim 132 wherein in the step of providing a prepared slide the cytological marker is selected from the group consisting of DNA, p300, actin, EGFR and HER-2/neu protein.

136. The method of claim 135 wherein the cytological marker is the p300 protein antigen.

137. The method of claim 135 wherein in the step of providing a prepared slide the cytological marker is actin.

138. The method of claim 132 wherein the step of providing a prepared slide the fluorescent label is selected from the group consisting of Hoechst 33258, M344 plus a fluorescent conjugate, an anti-HER2/neu protein probe plus a fluorescent conjugate, an anti-EGFR probe plus a fluorescent conjugate, and DNase I plus a fluorescent conjugate.

139. The method of claim 138 wherein in the step of providing a prepared slide the fluorescent conjugate is selected from the group consisting of Texas Red, bodipy and fluorescein.

140. The method of claim 132 wherein the step of providing a prepared slide, the cell sample is collected by washing a body organ, collected from a body fluid, or collected from a needle aspiration of a body gland or organ.

141. The method of claim 132 wherein the step of outputting the number is followed by the step of classifying the cell sample as to cancer risk.

142. The method of claim 132 wherein the step of classifying the cell sample further comprises using at least one additional variable.

143. A method of analyzing a cell sample derived from urine or a bladder wash, comprising:

providing a prepared slide, the prepared slide having been prepared by applying a portion of a cell sample to a slide then treating the slide with a fluorescent label for labeling a cytological marker to form a labeled cytological marker and wherein the portion of the cell sample used in preparing the slide was fixated using a fixative composition comprising a salt of ethylenediaminetetraacetic acid effective in inhibiting formation of substantially all of the crystals in the sample;

irradiating a portion of the prepared slide with an amount of an excitation wavelength of light effective in causing the fluorescent label in the cell to emit fluorescent light having an emission wavelength for forming a field image;

using a microscope means to select cell images on the field image;

classifying the cell image as positive or negative for the fluorescent label;

using a neural net computing means to further classify a positive cell image as a true-positive cell image or as a false-positive cell image, wherein the neural net computing means has been previously trained with a training set comprising;

a true-positive image set comprising positive cell images derived from one or more subjects diagnosed as having cancer, and

a false-positive image set comprising positive cell images derived from one or more subjects diagnosed as free of cancer;

obtaining a parameter related to the number of true-positive cell images; and

outputting the parameter for use in classifying the cell sample.