Cell analysis method using quantitative fluorescence image analysis

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.