METHODS AND SYSTEMS FOR ANALYZING IMAGES OF SPECIMENS PROCESSED BY A PROGRAMMABLE QUANTITATIVE ASSAY

• US 20120163681A1
• Filed: 11/29/2011
• Published: 06/28/2012
• Est. Priority Date: 11/29/2010
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Dako Denmark A/S (Agilent Technologies, Inc.)
• Inventors: Lohse, Jesper, Jain, Rohit, Pedersen, Hans Christian, Schmid, Joachim, Caron, Jeff, Briscoe, Thomas
• Granted Patent: US 9,970,874 B2
• Time in Patent Office: Days
• US Class Current: 382/128
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• 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 21/6456 Spatial resolved fluorescence measurements; Imaging

Abstract

Disclosed are methods and systems for analyzing images of specimens processed by a programmable quantitative assay or more specifically a robust programmable quantitative dot assay, PDQA, that enable specimens to be imaged and assessed across a wide variety of conditions and applications. Specific embodiments directed to immunohistochemical applications provide more quantitative methods of imaging and assessing biological samples including tissue samples.

Claims

1. A method of optically quantifying expression of at least one target molecule in at least one region of interest of a specimen comprising:

producing optically recognizable dots at sites of the specimen wherein at least one dot corresponds to a single target molecule,

wherein the dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the number of dots produced at the sites within a given region are representative of a fractional sub-population of a total population of target molecules within that region;

imaging the specimen;

selecting at least one region of interest within the image;

recognizing at least one dot within the at least one region of interest and;

quantifying the dots within the at least one region of interest.

2. The method of claim 1, further comprising assessing the expression of target molecules within the at least one region of interest.

3. The method of claim 1, wherein the at least one region of interest includes the entire specimen.

4. The method of claim 1, wherein the number of dots produced at the sites within a given region is representative of a localized expression of the target molecule present within the region

5. The method of claim 1, wherein the recognizing and quantifying steps are performed across the entire region of interest without performing a background separation step.

6. The method of claim 1, wherein the at least one additional programmable optical feature includes at least one of fluorescence, diffraction level, sharpness, hue, intensity, and saturation.

7. The method of claim 1, wherein quantifying the dots includes at least one of counting the dots, measuring a total area of the dots, calculating a density of the dots in a unit area.

8. The method of claim 1, wherein quantifying the dots includes calculating statistical measures of the predetermined optical features of recognized candidate dots;

comparing the optical features of a candidate object with the calculated statistical measures; and

adjusting a dot count based at least in part on the results of the comparing step.

9. The method of claim 1, wherein the programmable feature of shape includes at least one of sphericity, eccentricity, compactness, elongation, min to max feret ratio, roundness, and concentric-ringedness.

10. The method of claim 1, wherein the programmable feature of size includes at least one of length, width, diameter, and area.

11. The method of claim 1, wherein the region of interest is selected prior to the quantifying step.

12. The method of claim 1, wherein the step of producing optically recognizable dots is performed using an immunohistochemical detection system.

13. The method of claim 1, wherein the step of producing optically recognizable dots is performed using a non-antibody-based detection system.

14. The method of claim 1, wherein the step of producing optically recognizable dots is performed using an enzyme-labeled molecular probe detection system.

15. The method of claim 1, wherein the step of producing optically recognizable dots comprises chromogenic staining.

16. The method of claim 1, wherein the step of producing optically recognizable dots comprises fluorescence staining.

17. The method of claim 1, wherein at least a percentage of the dots chosen from 70%, 80%, or 90% are capable of being optically recognized at a specified magnification when imaged at two different focus plane depths within the specimen, and wherein the two focus plane depths are separated by a distance that is at least a distance chosen from 10%, 20%, or 50% of the thickness of the specimen.

18. The method of claim 1, wherein imaging the specimen comprises producing an image of the specimen at a resolution within a range in microns per pixel chosen from 0.6 to 0.9, 0.9 to 1.2, 1.2 to 2.5, or 2.5 to 5 and at least 90% of the produced dots are capable of being optically recognized at the chosen resolution.

19. The method of claim 1, wherein imaging the specimen comprises producing an image of the specimen at a resolution of less than 1 micron per pixel, and at least a percentage of the produced dots chosen from 70%, 80%, or 90% are capable of being optically recognized at the resolution.

20. The method of claim 1, wherein recognizing at least one dot includes identifying dot origins of at least two dots;

and

quantifying the at least one dot includes calculating at least one factor based on at least one distance measured between two dot origins.

21. The method of claim 1, wherein a total time to produce the optically recognizable dots is less than 100 minutes.

22. The method of claim 1, wherein producing the dots is carried out at a substantially fixed temperature.

23. The method of claim 22, wherein the substantially fixed temperature is less than 30 degrees C.

24. A system for optically quantifying expression of at least one target molecule in at least one region of interest in a specimen comprising:

a first kit for detecting a fractional sub-population of the at least one target molecule in the specimen;

a second kit for producing optically recognizable dots at sites of the specimen wherein at least one dot corresponds to a single target molecule,

wherein the dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the number of dots produced at the sites within a given region are representative of a fractional sub-population of a total population of target molecules within that region.

25. The system according to claim 24, further comprising

a stainer adapted to execute a staining protocol using the first and second kits;

an imager adapted to image the specimen; and

a processor configured to;

recognize at least one dot within the at least one region of interest and;

quantify the dots within the at least one region of interest.

26. The system according to claim 24, wherein the first kit comprises a first binding agent and a predetermined portion of the first binding agent comprises a label;

and

the second kit comprises a substrate including a staining precipitate.

27. The system according to claim 25, wherein the processor is further configured to assess the expression of target molecules within the at least one region of interest.

28. The system according to claim 25, wherein the at least one region of interest includes the entire specimen.

29. The system according to claim 24, wherein the number of dots produced at the sites within a given region is representative of a localized expression of the target molecule present within the region.

30. The system according to claim 25, wherein the processor is further configured to quantify the dots across the entire region of interest without performing a background separation step.

31. The system according to claim 24, wherein the at least one additional programmable optical feature includes at least one of fluorescence, diffraction level, sharpness, hue, intensity, and saturation.

32. The system according to claim 25, wherein the processor configured to quantify the dots is further configured to perform at least one of counting the dots, measuring a total area of the dots, calculating a density of the dots in a unit area,

33. The system according to claim 25, wherein the processor configured to quantify the dots is further configured to calculate statistical measures of the predetermined optical features of recognized candidate dots;

compare the optical features of a candidate object with the calculated statistical measures; and

adjust a dot count based at least in part on the results of the comparison.

34. The system according to claim 24, wherein the programmable feature of shape includes at least one of sphericity, eccentricity, compactness, elongation, min to max feret ratio, roundness, and concentric-ringedness.

35. The system according to claim 24, wherein the programmable feature of size includes at least one of length, width, diameter, and area.

36. The system according to claim 24, wherein the first kit uses an immunohistochemical detection system.

37. The system according to claim 24, wherein the first kit uses a non-antibody-based detection system.

38. The system according to claim 26, wherein the label of the first kit is an enzyme.

39. The system according to claim 25, wherein at least a percentage of the dots chosen from 70%, 80%, or 90% are capable of being optically recognized at a specified magnification when imaged at two different focus plane depths within the specimen, and wherein the two focus plane depths are separated by a distance that is at least a distance chosen from 10%, 20%, or 50% of the thickness of the specimen.

40. The system according to claim 25, wherein the imager is adapted to produce an image of the specimen at a resolution within a range in microns per pixel chosen from 0.6 to 0.9, 0.9 to 1.2, 1.2 to 2.5, or 2.5 to 5 and at least 90% of the produced dots are capable of being optically recognized at the chosen resolution.

41. The system according to claim 25, wherein the imager is adapted to produce an image of the specimen at a resolution of less than 1 micron per pixel, and at least a percentage of the produced dots chosen from 70%, 80%, or 90% are capable of being optically recognized at the resolution.

42. The system according to claim 25, wherein the processor is further configured to

identify dot origins of at least two dots;

and

quantify at least one dot based on at least one distance measured between two dot origins.

43. The system according to claim 24, wherein the first kit and the second kit are adapted to detect the target molecules and produce the optically recognizable dots in less than 100 minutes.

44. The system according to claim 24, wherein the first kit and the second kit are adapted to detect the target molecules and produce the optically recognizable dots at a substantially fixed temperature.

45. The system according to claim 44, wherein the substantially fixed temperature is less than 30 degrees C.

46. A method of optically quantifying expression of at least one target molecule in at least one region of interest in a specimen comprising:

producing optically recognizable dots at sites of the specimen wherein at least one dot corresponds to a single target molecule,

wherein the dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the number of dots produced at the sites within a given region are representative of a fractional sub-population of a total population of target molecules within that region;

wherein each dot is representative of a predetermined number of target molecules to within predetermined statistical limits;

imaging the specimen;

selecting the at least one region of interest within the image;

recognizing at least one dot within the at least one region of interest; and

quantifying the dots within the at least one region of interest.

47. The method according to claim 46, further comprising assessing the expression of target molecules within the at least one region of interest.

48. The method according to claim 46, wherein the number of dots produced at the sites within a given region is representative of a localized expression of the target molecule present within the region.

49. The method according to claim 46, wherein producing the optically recognizable dots comprises

detecting substantially all target molecules within the specimen with a first binding agent;

wherein the number of target molecules represented by each dot is determined by a predetermined portion of the first binding agent that comprises a label.

50. The method according to claim 49, wherein the label is an enzyme.

51. The method according to claim 49, wherein the portion of first binding agent that comprises a label is proportional to the number of target molecules represented by each dot.

52. The method according to claim 47, wherein quantifying the dots comprises calculating a dot density.

53. A system for optically quantifying expression of at least one target molecule in at least one region of interest in a specimen comprising:

a first kit for detecting a fractional sub-population of the at least one target molecule in the specimen; and

a second kit for producing optically recognizable dots at sites of the specimen wherein at least one dot corresponds to a single target molecule,

wherein the dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the number of dots produced at the sites within a given region are representative of a fractional sub-population of a total population of the at least one target molecule within that region; and

wherein each dot is representative of a predetermined number of target molecules to within predetermined statistical limits.

54. The system according to claim 53, further comprising

a stainer adapted to execute a staining protocol using the first and second kits;

an imager adapted to image the specimen; and

a processor configured to;

recognize at least one dot within the at least one region of interest; and

quantify the dots within the at least one region of interest.

55. The system according to claim 54, wherein the processor is further configured to assess the expression of target molecules within the at least one region of interest.

56. The system according to claim 53, wherein the number of dots produced at the sites within a given region is representative of a localized expression of the target molecule present within the region.

57. The system according to claim 53, wherein the first kit comprises a first binding agent and a predetermined portion of the first binding agent comprises a label;

wherein the predetermined portion of the first binding agent comprising a label determines the number of target molecules that each dot is representative of;

wherein the second kit comprises a substrate including a staining precipitate.

58. The system according to claim 57, wherein the label is an enzyme.

59. The system according to claim 57, wherein the portion of the first binding agent that comprises a label is proportional to the number of target molecules represented by each dot.

60. The system according to claim 54, wherein the processor configured to quantify optically recognizable dots is further configured to calculate a dot density.

61. A method of optically quantifying expression of at least one target molecule in at least one region of interest in a specimen comprising:

imaging the specimen with an imager;

selecting at least one region of interest within the image;

recognizing at least one optically recognizable dot at sites of the specimen within the at least one region of interest wherein the at least one dot corresponds to a single target molecule;

wherein the dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the number of dots recognized at the sites within a given region are representative of a fractional sub-population of a total population of target molecules within that region;

wherein each dot is representative of a predetermined number of target molecules to within predetermined statistical limits; and

performing at least one image analysis step based on the at least one recognized dot.

62. The method according to claim 61, wherein the at least one image analysis step includes

quantifying the dots within the at least one region of interest;

and

assessing the expression of target molecules within the at least one region of interest.

63. The method according to claim 61, wherein the number of dots at the sites within a given region is representative of a localized expression of the target molecule present within the region.

64. The method according to claim 61, wherein the at least one image analysis step is performed by a processor without user supervision.

65. The method according to claim 61, wherein the at least one image analysis step is performed by a processor under user supervision.

66. The method according to claim 62, wherein quantifying the dots includes counting the dots.

67. The method according to claim 62, wherein quantifying the dots includes counting clusters of dots.

68. The method according to claim 61, wherein the at least one image analysis step includes a feature extraction on the at least one region of interest based on the at least one recognized dot.

69. The method according to claim 61, wherein the at least one image analysis step includes recognizing geometric objects in the image based on the at least one recognized dot.

70. The method according to claim 61, wherein the at least one image analysis step includes classifying the recognized dots into at least two classes of objects;

and quantifying the classified objects.

71. A system for optically quantifying the expression of at least one target molecule in at least one region of interest in a specimen comprising:

an imager adapted to image the specimen;

at least one processor configured to;

select at least one region of interest within the image;

recognize at least one optically recognizable dot at sites of the specimen within the at least one region of interest wherein at least one dot corresponds to a single target molecule;

wherein the dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the number of dots recognized at the sites within a given region are representative of a fractional sub-population of a total population of target molecules within that region;

wherein each dot is representative of a predetermined number of target molecules to within predetermined statistical limits; and

perform at least one image analysis step based on the at least one recognized dot.

72. The system according to claim 71, wherein the at least one image analysis step includes

quantifying the at least one dot within the at least one region of interest;

and

assessing the expression of target molecules within the at least one region of interest.

73. The method according to claim 71, wherein the number of dots at the sites within a given region is representative of a localized expression of the target molecule present within the region.

74. The system according to claim 71, wherein the at least one image analysis step is performed by the processor without user supervision.

75. The system according to claim 71, wherein the at least one image analysis step is performed by the processor under user supervision.

76. The system according to claim 72, wherein quantifying the dots includes counting the dots.

77. The system according to claim 72, wherein quantifying the dots includes counting clusters of dots.

78. The system according to claim 71, wherein the at least one image analysis step includes a feature extraction on the at least one region of interest based on the at least one recognized dot.

79. The system according to claim 71, wherein the at least one image analysis step includes recognizing geometric objects in the image based on the at least one recognized dot.

80. The system according to claim 71, wherein the at least one image analysis step includes classifying the recognized dots into at least two classes of objects;

and

quantifying the classified objects.

81. A method of optically quantifying multiplexed diagnostic indicators in a tissue specimen comprising:

producing a first multiplexed diagnostic indicator within at least one region of interest in a tissue specimen;

wherein the first multiplexed diagnostic indicator comprises a first set of optically recognizable dots at sites of the specimen wherein at least one dot corresponds to a single first target molecule,

wherein the first set of dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the first set of dots produced at the sites within a given region are representative of a fractional sub-population of a total population of first target molecules within that region;

producing a second multiplexed diagnostic indicator within the at least one region of interest;

imaging the specimen;

assessing the first multiplexed diagnostic indicator and the second multiplexed diagnostic indicator;

wherein assessing the first multiplexed diagnostic indicator comprises

recognizing at least one of the first set of dots within the at least one region of interest; and

quantifying the first set of dots within the at least one region of interest; and

determining an overall diagnostic assessment of the tissue specimen based at least in part on first and second multiplexed diagnostic indicator assessments.

82. The method according to claim 81,

wherein the second multiplexed diagnostic indicator comprises a second set of optically recognizable dots at sites of the specimen wherein at least one dot corresponds to a single second target molecule,

wherein the second set of dots are characterized by in-situ programmable features of size and shape and at least one additional programmable optical feature,

wherein the second set of dots produced at the sites within a given region are representative of a fractional sub-population of a total population of second target molecules within that region;

and

assessing the second multiplexed diagnostic indicator comprises

recognizing at least one of the second set of dots within the at least one region of interest; and

quantifying the second set of dots within the at least one region of interest.

83. The method according to claim 82,

wherein the first set of dots is a set of target dots and the second set of dots is a set of reference dots;

wherein the target dots and the reference dots may be differentiated by at least one programmable optical feature; and

the method further comprises assessing the expression of target molecules in the at least one region of interest based at least in part on the ratio of the quantified first set of target dots to the quantified second set of reference dots.

84. The method according to claim 81, wherein producing the second multiplexed diagnostic indicator comprises processing the specimen with at least one of immunohistochemical staining, in situ hybridization;

hematoxylin and eosin staining; and

assessing the second multiplexed diagnostic indicator comprises quantifying at least one feature in the at least one region of interest based on the processing.

85. The method according to claim 81, wherein producing the second multiplexed diagnostic indicator assessment comprises processing the specimen with at least one of immunohistochemical staining, in situ hybridization;

hematoxylin and eosin staining; and

assessing the second multiplexed diagnostic indicator comprises recognizing morphological features in the specimen based on the processing.