阅读说明：本技术 诱导细胞和/或组织片段片状剥脱以增强细胞病理性细胞收集的方法 (Method for inducing exfoliation of cells and/or tissue fragments to enhance pathological cell collection ) 是由 M·鲁滕贝格 D·亚当 E·洛布 于 2019-03-25 设计创作，主要内容包括：使用超声造影剂和超声处理从受试者的器官获得富集的细胞样品的方法和技术。获得了连续的上皮片段,其可用于随后的组织学分析以及用于提供治疗和其他医学考虑的信息。(Methods and techniques for obtaining an enriched cell sample from an organ of a subject using an ultrasound contrast agent and sonication. A continuous epithelial segment is obtained that can be used for subsequent histological analysis and for providing information for treatment and other medical considerations.)

1. A method of obtaining a continuous pancreatic ductal cell sample from a subject, comprising:

administering to the subject a secretin polypeptide in an amount effective to cause pancreatic secretion within one hour before or after the start of sonication with ultrasound energy; sonicating the subject's pancreas with an amount of ultrasound energy from a multi-frequency array having a mechanical index of 0.03 to 1.3 effective to obtain asymmetric ultrasound waves at a predetermined point in the pancreas and cause cell flaking into the subject's pancreatic ducts; then the

Removing a fluid sample containing continuous pancreatic ductal cells from the subject.

2. A method of obtaining a cell sample from an organ of a subject, comprising:

administering to the subject an amount of an ultrasound contrast agent; and

sonicating an organ of the subject with an amount of ultrasound energy effective to cause stable cavitation of an ultrasound contrast agent,

thereby causing the cell or epithelial tissue segment to exfoliate from the organ of the subject.

3. The method of claim 2, wherein the cells comprise epithelial cells.

4. The method of claim 2, wherein the epithelial tissue segment of the epithelium of the organ is exfoliated.

5. The method of claim 2,3 or 4, wherein the organ is a bladder, breast, liver, kidney, pulmonary thyroid, gastrointestinal tract or pancreas.

6. The method of any one of claims 1 to 4, further comprising removing a fluid sample comprising cells from the subject.

7. The method of claim 6, wherein the fluid sample is obtained from an encystment of the organ or a secretion of the organ.

8. A method of obtaining pancreatic cells from a subject, comprising:

administering to the subject an amount of an ultrasound contrast agent; and

sonicating the pancreas of the subject to an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent,

thereby causing cell exfoliation into the pancreatic ducts of the subject.

9. The method of claim 8, wherein the mechanical index of the ultrasonic energy is no more than 0.03, or the mechanical index is no more than 0.05.

10. A method of obtaining pancreatic cells from a subject, comprising:

sonicating the pancreas of the subject with an amount of monochromatic ultrasound energy effective to cause cell exfoliation into the pancreatic ducts of the subject.

11. The method of claim 10, wherein the mechanical index of the ultrasonic energy is from 0.03 to 1.3.

12. A method of obtaining pancreatic cells from a subject, comprising:

sonicating the subject's pancreas with an amount of ultrasound energy from the multi-frequency array effective to obtain asymmetric ultrasound waves at a predetermined point of the pancreas and cause cell flaking into the subject's pancreatic ducts.

13. The method of claim 12, wherein the mechanical index of the ultrasonic energy is from 0.03 to 1.3.

14. The method of any one of claims 8 to 13, further comprising administering to the subject an amount of a secretin polypeptide or other substance that causes pancreatic secretion effective to cause pancreatic secretion within one hour before or after the start of sonication with ultrasonic energy.

15. The method of claim 14, comprising administering the amount of secretin or other substance that causes pancreatic secretion with the amount of ultrasound contrast agent.

16. The method of any one of claims 10 to 15, wherein the secretin is administered and the secretin is human secretin.

17. The method of any one of claims 8 to 16, further comprising placing a catheter or other collection device at the opening of the subject's pancreatic duct.

18. The method of claim 17, wherein the opening of the pancreatic duct is a duodenal papilla.

19. The method of claim 17 or 18, further comprising obtaining a pancreatic secretion sample using the catheter or other collection device.

20. The method of claim 19, further comprising analyzing the obtained pancreatic secretion sample to determine whether the sample comprises pancreatic cancer cells or dysplastic pancreatic cells, or is non-pathological.

21. The method of any one of claims 8 to 20, wherein the method is effective to cause exfoliation of ductal and/or acinar cells of the pancreas.

22. A method according to any one of claims 8 to 21, wherein the method is effective to cause exfoliation of an epithelial tissue segment of the pancreas.

23. The method of any one of claims 2 to 9, wherein the amount of ultrasound energy causes stable cavitation in the pancreas of the subject.

24. The method of any one of claims 1 to 11, wherein the amount of ultrasound energy does not cause inertial cavitation or implosion of microbubbles within the organ of the subject.

25. The method of any one of claims 12 to 24, wherein the amount of ultrasound energy does not cause inertial cavitation or implosion of microbubbles within the pancreas of the subject.

26. The method of any one of claims 1 to 25, further comprising monitoring the subject for implosion of microbubbles or an ultrasound contrast agent during at least a portion of the sonication of the subject.

27. The method of claims 10-26, wherein the amount of ultrasound energy is effective to produce a plurality of microbubbles in an organ of the subject.

28. The method of any one of claims 1 to 7, wherein the ultrasound energy is unfocused in an organ of the subject.

29. The method of any one of claims 8 to 27, wherein the ultrasound energy is unfocused in the pancreas of the subject.

30. The method of claim 28 or 29, wherein the ultrasonic energy is partially unfocused.

31. The method of any one of claims 1 to 7, wherein the ultrasound energy is focused in an organ of the subject.

32. The method of any one of claims 8 to 27, wherein the ultrasound energy is focused in the pancreas of the subject.

33. The method of any one of claims 1 to 9 or 14 to 32, wherein the ultrasound contrast agent comprises microspheres.

34. The method of claim 33, wherein the ultrasound contrast agent comprises sulfur hexafluoride lipid a-type microspheres.

35. The method of any one of claims 1 to 34, wherein the ultrasound energy is emitted from a transducer in contact with or adjacent to the subject's skin, and wherein a closest point of the transducer is within 2-4 inches of the subject's pancreatic surface.

36. The method of claim 35, wherein the mechanical index does not exceed 0.03.

37. The method of claim 35, wherein the mechanical index does not exceed 0.05.

38. The method of claim 35, 36 or 37, wherein the frequency of the ultrasonic energy sonicated comprises 1.5MHz to 3.0 MHz.

39. The method of any one of claims 1 to 34, wherein the ultrasonic energy is emitted from a transducer in contact with or adjacent to the skin of the subject, and wherein a closest point of the transducer is located at a distance of greater than 4 inches and up to 9 inches from the surface of the pancreas of the subject.

40. The method of claim 39, wherein the mechanical index does not exceed 0.03.

41. The method of claim 39, wherein the mechanical index does not exceed 0.05.

42. The method of claim 39, 40 or 41, wherein the frequency of the ultrasonic energy sonicated comprises 1.0MHz to 1.5 MHz.

43. The method of any of claims 1 to 42, wherein the ultrasound energy is emitted from a transducer array having a length of 3 to 5 inches by a width of 1 to 3 inches.

44. The method of any one of claims 1 to 43, wherein the ultrasound energy is emitted from a transducer array that is substantially triangular in shape.

45. The method of any one of claims 1 to 43, wherein the ultrasound energy is emitted from a transducer array that is substantially rectangular in shape.

46. The method of any one of claims 1 to 44, wherein the ultrasound is emitted from a transducer array connected to the subject by a strap.

47. The method of any one of claims 1 to 45, wherein the subject is sonicated with the amount of ultrasound energy for more than 5 minutes.

48. The method of any one of claims 1 to 46, wherein the subject is sonicated with the amount of ultrasound energy for less than 15 minutes.

49. The method of any one of claims 1 to 9 or 14 to 47, wherein the amount of ultrasound contrast agent is administered to the subject within 1 hour before the subject begins sonication with the amount of ultrasound energy.

50. The method of claim 48, wherein the amount of ultrasound contrast agent is administered to the subject within 30 minutes before the subject begins sonication with the amount of ultrasound energy.

51. The method of any one of claims 8, 9, or 14-49, wherein the amount of secretin is administered to the subject within 1 hour of beginning sonication of the subject with the amount of ultrasonic energy.

52. The method of any one of claims 8, 9, or 14-50, wherein the amount of secretin is administered to the subject within 30 minutes of initiating sonication of the subject with the amount of ultrasonic energy.

53. The method of claim 50 or 51, wherein the amount of secretin is administered to the subject before sonication with the amount of ultrasound energy is initiated.

54. The method of claim 50 or 51, wherein the amount of secretin is administered to the subject after sonication is initiated with the amount of ultrasound energy.

55. The method of claim 53, wherein the amount of secretin is administered to the subject within less than 10 minutes after terminating sonication with the amount of ultrasonic energy.

56. The method of claim 50 or 51, wherein the amount of secretin is administered to the subject during sonication with the amount of ultrasonic energy.

57. The method of any one of claims 50-53 or 55, wherein the amount of secretin and ultrasound contrast agent are administered to the subject simultaneously.

58. The method of any one of claims 1 to 56, wherein ultrasound imaging is used to assist in the placement of a transducer emitting ultrasound energy over an organ or over a pancreas.

59. A method according to any one of claims 8 to 57, wherein ultrasound imaging is used to assist in the placement of a catheter or other collection device at the opening of the subject's pancreatic duct.

60. The method of any one of claims 1 to 7, wherein ultrasound imaging is used to assist in placement of a catheter or other fluid collection device in or near an organ of the subject.

61. The method of claim 10, 11, 12, or 13, wherein no ultrasound contrast agent is administered to the subject.

62. The method of any one of claims 1 to 9 or 14 to 59, wherein the ultrasound contrast agent is administered intravenously.

63. The method of claim 61, wherein the ultrasound contrast agent is administered intravenously only.

64. The method of any one of claims 1 to 9 or 12 to 62, wherein the ultrasonic energy is multi-frequency and/or multi-phase.

65. The method of any one of claims 1 to 63, wherein the subject is dynamically sonicated with ultrasound energy.

66. The method of any one of claims 1 to 63, wherein the amount of ultrasonic energy is sonicated from more than one location on the first side.

67. The method of any one of claims 1-63, wherein the amount of ultrasonic energy is sonicated from more than one location on a second side perpendicular to the first side.

68. The method of any one of claims 8 to 66, wherein the pancreas of the subject is exposed to an amount of ultrasound radiation at least two different locations on the coronal, sagittal, or transverse planes of the pancreas.

69. The method of any one of claims 1 to 7, wherein the organ of the subject is exposed to an amount of ultrasound radiation at least two different locations on a coronal, sagittal, or transverse plane of the organ.

70. The method of any one of claims 1-68, wherein the subject is supine.

71. The method of any one of claims 1-69, wherein the subject is a human.

72. A method of treating a pancreatic disorder in a subject, comprising:

a) determining whether the subject has dysplastic or pancreatic cancer cells in the pancreas thereof by the method of any one of claims 20-58, 60-67, 69, or 70, and

b) administering chemotherapy, radiation therapy, immunotherapy or pancreatectomy to the subject found in a) to have dysplastic pancreatic or pancreatic cancer cells in their pancreas.

73. The method of claim 71, wherein determining whether the subject has dysplastic pancreatic or pancreatic cancer cells in its pancreas is performed by one or more of a cytomorphological analysis, a histomorphological analysis, or a biomolecular marker analysis.

74. A method of increasing the efficacy of collecting a cell sample from a tissue in an assay procedure on a subject, comprising sonicating the tissue of the subject with an amount of ultrasonic energy effective to cause stable cavitation of an ultrasonic contrast agent, thereby causing exfoliation of cells or tissue fragments of the subject, prior to collecting the cell sample from the tissue, and then collecting the cell sample from the tissue of the subject.

75. The method of claim 73, wherein the tissue comprises epithelial cells.

76. A method of assaying a cell or tissue sample of a subject to determine whether the cell or tissue comprises a cancerous or precancerous cell or tissue, comprising:

a) receiving a cell or tissue sample, wherein the sample has been previously obtained by: administering to the subject an amount of an ultrasound contrast agent; and sonicating the organ of the subject with an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent, thereby causing flaking of the cells or epithelial tissue segments from the organ of the subject, and collecting the flaked cells or tissue sample;

b) performing one of a cellular morphology analysis, a tissue morphology analysis, or a biomolecular marker analysis to determine whether the cell or tissue comprises a cancerous or precancerous cell or tissue.

77. The method of claim 75, further comprising administering the amount of an ultrasound contrast agent to the subject and sonicating an organ of the subject with an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent, thereby causing exfoliation of cells or epithelial tissue segments from the organ of the subject, and collecting the exfoliated cell or tissue sample.

78. A method for early detection of dysplastic and carcinomic cells in the pancreas comprising applying ultrasound energy directed at the pancreas to induce cell exfoliation and then endoscopically collecting pancreatic fluid containing exfoliated cells and cell clusters for molecular and microscopic morphological examination.

79. The method of claim 77, wherein ultrasound energy directed at the pancreas to increase cell flaking into pancreatic juice is applied to the patient's body in vitro.

80. The method of claim 77, wherein the ultrasonic energy directed at the pancreas to increase cell flaking into pancreatic juice is applied endoscopically.

81. The method of claim 77, wherein the administration of ultrasonic energy to increase exfoliation is combined with the administration of secretin (a hormone that increases pancreatic fluid production).

82. The method of claim 77, wherein the pancreatic juice is examined by means of a neural network-based computer-assisted microscopy system.

83. An isolated sample of bodily fluid, wherein the sample is directly obtained from a subject whose tissue or organ has been sonicated, wherein the sample comprises epithelial or other cells from the tissue or organ at an enrichment level greater than 2-fold compared to the level of epithelial or other cells in an otherwise identical sample obtained from a subject that has not been sonicated.

84. The isolated bodily fluid sample of claim 82, wherein the subject is also administered an ultrasound contrast agent present during sonication of the tissue or organ.

85. The isolated bodily fluid sample of claim 82 or 83, wherein the pancreas of the subject has been sonicated and secretin has been administered to the subject prior to obtaining a sample directly from the subject.

86. The isolated bodily fluid sample of claim 82 or 83, wherein the organ is a bladder, breast, liver, kidney, lung, thyroid, gastrointestinal tract, or cyst.

Background

More than 95% of cancers (cancer) in adults are epithelial malignancies (carcinomas), which means that they originate from the epithelial lining of the organ. In many cases, these epithelia are in direct contact with surrounding body fluids, allowing the epithelial cells to continually slough off into the surrounding body fluids as part of the natural process of epithelial regeneration. Examples are pancreatic ductal cells that are continually shed into the pancreatic juice, lung cells that are continually shed into the sputum, and bladder cells that are continually shed into the urine. Dysplastic and cancerous cells, which may be present in the epithelium of a subject, are also naturally shed into the surrounding fluid along with normal epithelial cells. In many cases, these fluids can be obtained by non-invasive or minimally invasive sampling methods. The exfoliated cells found in these sampled fluids can then be concentrated using standard laboratory techniques and subsequently used for microscopic evaluation by cytopathologists, theoretically enabling early detection of dysplasia and cancer. Although cytopathological examination of cells exfoliated into surrounding bodily fluids is often highly specific, its sensitivity to dysplasia and cancer detection is often limited by the fact that: the slow rate of natural exfoliation of epithelial cells into the surrounding fluid results in very few samples with few cells available for examination by a pathologist. Currently, this limits the cytopathological detection of dysplasia and cancer in the following sites: mediastinum, pleura, pericardium, peritoneum, lung, breast, salivary gland, meninges, pancreatic duct, pancreatic capsule, kidney, liver, bladder, ovary, and the like.

For example, the pancreas is composed of multiple cell types, each of which may cause a different type of cancer. Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the united states. This is due to its late diagnosis, usually at the metastatic stage, its aggressive biology and only partial response to known chemotherapy. Cytological evaluation of pancreatic juice has been shown to have high sensitivity, above 79% in diagnosing pancreatic cancer.

Pancreatic cancer is often already advanced when found because it usually has little or no early symptoms. For this reason, pancreatic cancer is expected to be the second leading cause of cancer death in the united states by 2030 (1).

Pancreatic screening and monitoring of patients at elevated risk for developing pancreatic cancer due to their family history of pancreatic cancer, mutation in pancreatic cancer susceptibility genes, or accidental detection of pancreatic cysts.

Current pancreatic cancer examinations include Endoscopic Ultrasound (EUS) and endoscopic magnetic resonance imaging/magnetic resonance cholangiopancreatography (ERCP). Although these tests are accurate for detecting pancreatic cysts (2,3,4), they are less suitable for detecting small solid pancreatic cancers, as evidenced by the number of patients who, despite regular monitoring, still develop pancreatic cancer. This reflects a missed opportunity for early detection (5).

Cytopathology is an important clinical standard for diagnosing disease and has been examined as a diagnostic tool for pancreatic cancer. For example, in 1974, yoshiko Endo and colleagues first reported a cytological diagnosis of pancreatic cancer after collection of pancreatic juice with a duodenoscope (6). They reported a sensitivity of 79%. In the next decades, many researchers have attempted to determine pancreatic malignancy using cytological examination of pancreatic juice, but the range of sensitivity has remained persistent at around 70-80% (7, 8). The lack of sensitivity observed by many endoscopists over the years may reflect the deficiencies of typical cell collection and analysis methods. In addition, obtaining low levels of cells or tissue fragments in a fluid sample for performing histological analysis in bioassays is a problem with many types of bioassays.

Thus, there is a need to increase the yield of cells exfoliated from various organ and tissue types for collection and subsequent analysis in order to enhance the specificity of these diagnostic tests. It is also desirable to induce exfoliation of intact tissue, which can be used to provide an anteriorly located view of a tissue segment. Particularly with respect to pancreatic cancer, there is a need for improved cell collection and tissue fragment collection techniques for detecting rare cells from the normal cellular background of the pancreas.

Disclosure of Invention

The embodiments of the invention set forth herein provide methods and systems for obtaining cells and/or tissue fragments and other molecules at much greater ratios than previously observed by different means. In one embodiment, a method includes administering an ultrasound contrast agent that forms microbubbles in the circulatory system of a patient and sonicating (sonicating) the subject. Following the introduction of the microbubbles, the organ or tissue (e.g., pancreas) is subjected to wide-area ultrasound energy. The ultrasound application of embodiments of the present invention may be described as low intensity non-focused ultrasound (LINFU). In embodiments of the invention, the combination of ultrasonic energy and the energy applied by the microbubbles results in the breakdown and/or exfoliation of pancreatic cells and optionally tissue fragments. In the case of obtaining a pancreatic sample, the patient is then injected with secretin, a drug that induces pancreatic secretion. In this regard, some exfoliated and mobilized cells and tissue fragments may be deposited into pancreatic juice and then collected endoscopically. The cells and/or tissue fragments in the enriched sample obtained are then subjected to morphological analysis and/or analysis using molecular biomarkers to detect the presence or absence of cellular abnormalities.

These procedures can significantly increase the total number of cells expressed in pancreatic juice. In addition, the procedures set forth herein can induce the isolation of intact tissue fragments from the pancreas.

These procedures, when applied to other organs or body parts (e.g., mediastinum, pleura, pericardium, peritoneum, lung, breast, salivary gland, meninges, pancreatic ducts, pancreatic cysts, kidney, liver, bladder, or ovary) can significantly increase the total number of cells expressed in the surrounding fluid of these organs. In addition, the procedures set forth herein may additionally induce the separation of intact tissue fragments from the exemplary organs described above. For example, the procedures set forth herein are used to induce the exfoliation of lung cells into the surrounding sputum or to induce the exfoliation of bladder cells and bladder tissue into the surrounding urine.

There is provided a method of obtaining a cell sample from an organ of a subject, the method comprising: administering to a subject an amount of an ultrasound contrast agent; and sonicating the organ of the subject with an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent, thereby causing the cells or epithelial tissue segments to exfoliate from the organ of the subject.

Also provided is a method of obtaining pancreatic cells from a subject, the method comprising:

administering to a subject an amount of an ultrasound contrast agent; and

sonicating the pancreas of the subject to an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent,

thereby causing cell exfoliation into the pancreatic ducts of the subject.

Also provided is a method of obtaining pancreatic cells from a subject, the method comprising: sonicating a pancreas of a subject with an amount of monochromatic ultrasound energy effective to cause cell exfoliation into pancreatic ducts of the subject.

Also provided is a method of obtaining pancreatic cells from a subject, the method comprising: sonicating the pancreas of the subject with an amount of ultrasound energy from the multi-frequency array effective to achieve asymmetric ultrasound waves at a predetermined point of the pancreas of the subject and to cause cell flaking into the pancreatic ducts of the subject.

Also provided is a method of treating a pancreatic disorder in a subject, the method comprising:

a) determining whether the subject has dysplastic pancreatic or pancreatic cancer cells in its pancreas by the methods described herein, and

b) administering chemotherapy, radiation therapy, immunotherapy or pancreatectomy to the subject found in a) to have dysplastic pancreatic or pancreatic cancer cells in their pancreas.

In some embodiments, the subject is determined to have dysplastic pancreatic or pancreatic cancer cells in its pancreas by one or more of a cellular morphology analysis, a histomorphology analysis, or a biomolecular marker analysis.

Also provided is a method of increasing the efficacy of collecting a cell sample from a tissue in an assay procedure on a subject, the method comprising sonicating a tissue of the subject with an amount of ultrasonic energy effective to cause stable cavitation of an ultrasonic contrast agent prior to collecting the cell sample from the tissue, thereby causing exfoliation of cells or tissue fragments of the subject, and then collecting the cell sample from the tissue of the subject.

Also provided is a method of assaying a cell or tissue sample of a subject to determine whether the cell or tissue comprises a cancerous or precancerous cell or tissue, the method comprising:

a) receiving a cell or tissue sample, wherein the sample has been previously obtained by: administering to a subject an amount of an ultrasound contrast agent; and sonicating the organ of the subject with an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent, thereby causing flaking of the cells or epithelial tissue segments from the organ of the subject, and collecting the flaked cells or tissue sample;

b) performing one of a cellular morphology analysis, a tissue morphology analysis, or a biomolecular marker analysis to determine whether the cell or tissue comprises a cancerous or precancerous cell or tissue.

Also provided is a method for early detection of dysplastic and cancerous cells in the pancreas comprising applying ultrasound energy directed at the pancreas to induce cell exfoliation and then endoscopically collecting pancreatic fluid containing exfoliated cells and cell clusters for molecular examination and microscopic morphological examination.

Also provided is an isolated sample of bodily fluid, wherein the sample is obtained directly from a subject whose tissue or organ has been sonicated, wherein the sample comprises epithelial or other cells from the tissue or organ enriched at a level greater than 2-fold compared to the level of epithelial or other cells in an otherwise identical sample obtained from a subject that has not been sonicated.

Also provided is a method of obtaining a continuous pancreatic ductal cell sample from a subject, the method comprising:

administering to the subject, within one hour before or after sonication with ultrasonic energy, an amount of a secretin polypeptide effective to cause pancreatic secretion;

sonicating a subject's pancreas with an amount of ultrasound energy at a predetermined point of the pancreas and causing cell sheets to exfoliate into the subject's pancreatic ducts; then the

Removing a fluid sample containing continuous pancreatic ductal cells from the subject.

Brief description of the drawings

FIG. 1: as described in example 1 below, large sections of pancreatic epithelial tissue were obtained from pancreatic fluid collected after sonication of the animals.

Detailed Description

There is provided a method of obtaining a cell sample from an organ of a subject, the method comprising:

administering to a subject an amount of an ultrasound contrast agent; and

sonicating an organ of a subject with an amount of ultrasound energy effective to cause stable cavitation of an ultrasound contrast agent,

thereby causing the cell or epithelial tissue segment to exfoliate from the organ of the subject.

In some embodiments, the cell comprises an epithelial cell.

In some embodiments, the epithelial tissue segment of the organ epithelium is exfoliated.

In some embodiments, the organ is the bladder, breast, liver, kidney, pulmonary thyroid, gastrointestinal tract, or pancreas. The organ from which the cell sample is collected may be referred to as the target organ.

In some embodiments, the sonication is applied so as not to sonicate any other organs than the target organ. In some embodiments, the sonication is applied so as not to induce stable cavitation in any organ other than the target organ. In some embodiments, an ultrasound contrast agent is applied so as to selectively accumulate in the target organ. In some embodiments, the sonication and/or ultrasound contrast agent is applied over time (temporally) so as to selectively not induce stable cavitation in any organ other than the target organ. In some embodiments, the sonication and/or ultrasound contrast agent is applied spatially so as to selectively not induce stable cavitation in any organ other than the target organ. In some embodiments, the ultrasound treatment and/or ultrasound contrast agent is applied temporally and spatially so as to selectively not induce stable cavitation in any organ other than the target organ. In some embodiments, the sonication is applied temporally so as to only selectively sonicate the target organ. In some embodiments, the sonication is applied spatially so as to only selectively sonicate the target organ. In some embodiments, the sonication is applied temporally and spatially so that only the target organ is selectively sonicated.

In some embodiments, the method further comprises removing a fluid sample containing cells of the subject.

In some embodiments, the fluid sample is obtained from an encapsulation of the organ.

In some embodiments, the fluid sample is obtained from secretions of the organ.

Also provided is a method of obtaining pancreatic cells from a subject, the method comprising:

administering to a subject an amount of an ultrasound contrast agent; and

sonicating the pancreas of the subject to an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent,

thereby causing cell exfoliation into the pancreatic ducts of the subject.

In some embodiments, the mechanical index of the ultrasonic energy is no more than 0.03, or the mechanical index is no more than 0.05.

Also provided is a method of obtaining pancreatic cells from a subject, the method comprising:

sonicating a pancreas of a subject with an amount of monochromatic ultrasound energy effective to cause cell exfoliation into pancreatic ducts of the subject.

In some embodiments, the mechanical index of the ultrasonic energy is from 0.03 to 1.3.

Also provided is a method of obtaining pancreatic cells from a subject, the method comprising:

sonicating a pancreas of a subject with an amount of ultrasound energy from the multi-frequency array effective to achieve asymmetric ultrasound at a predetermined point of the pancreas and cause cell flaking into pancreatic ducts of the subject.

In some embodiments, the mechanical index of the ultrasonic energy is from 0.03 to 1.3.

Also provided is a method of obtaining a continuous pancreatic ductal cell sample from a subject, the method comprising:

administering to the subject a secretin polypeptide in an amount effective to cause pancreatic secretion within one hour before or after the start of sonication with ultrasound energy; sonicating a subject's pancreas with an amount of ultrasound energy at a predetermined point of the pancreas and causing cell sheets to exfoliate into the subject's pancreatic ducts; a fluid sample containing continuous pancreatic ductal cells of the subject is then removed. A continuous cell is two or more cells, wherein each cell is linked to at least one other cell. For example, a segment of epithelial tissue or a cell sheet.

In some embodiments, the mechanical index of the ultrasonic energy is from 0.03 to 1.3.

In some embodiments, the method further comprises administering to the subject an amount of a secretin polypeptide or other substance that causes secretion by the pancreas effective to cause secretion by the pancreas within one hour before or after the sonication with ultrasonic energy is initiated.

In some embodiments, the method comprises administering an amount of secretin or other substance that causes pancreatic secretion with an amount of ultrasound contrast agent.

In some embodiments, secretin is administered, and secretin is human secretin.

In some embodiments, the method further comprises positioning a catheter or other collection device at the opening of the pancreatic duct of the subject.

In some embodiments, the opening of the pancreatic duct is the duodenal papilla.

In some embodiments, the method further comprises obtaining a pancreatic secretion sample using a catheter or other collection device.

In some embodiments, the method further comprises analyzing the obtained pancreatic secretion sample to determine whether the sample contains pancreatic cancer cells or dysplastic pancreatic cells, or is non-pathological.

In some embodiments, computer-assisted analysis is used to identify whether a cell is cancerous or dysplastic. In some embodiments, cell morphology analysis, histomorphology analysis, and/or molecular biology analysis is used to identify whether a cell is cancerous or dysplastic.

In some embodiments, the method is effective to cause exfoliation of ductal and/or acinar cells of the pancreas.

In some embodiments, the method is effective to cause exfoliation of an epithelial tissue segment of the pancreas.

In some embodiments, the amount of ultrasound energy causes stable cavitation in the pancreas of the subject.

In some embodiments, the amount of ultrasound energy does not cause inertial cavitation or implosion of the microbubbles within the organ of the subject.

In some embodiments, the amount of ultrasound energy does not cause inertial cavitation or implosion of microbubbles within the pancreas of the subject.

In some embodiments, the method further comprises monitoring the subject for microbubble implosion or an ultrasound contrast agent during at least a portion of the sonication of the subject.

In some embodiments, the amount of ultrasound energy is effective to produce a plurality of microbubbles in the organ of the subject.

In some embodiments, the ultrasound energy is unfocused in an organ of the subject.

In some embodiments, the ultrasound energy is unfocused in the pancreas of the subject.

In some embodiments, the ultrasound energy is partially unfocused.

In some embodiments, the ultrasound energy is focused in an organ of the subject.

In some embodiments, the ultrasound energy is focused in the pancreas of the subject.

In some embodiments, the ultrasound contrast agent comprises microspheres.

In some embodiments, the contrast agent is not administered directly to the pancreas, but is administered intravenously.

In some embodiments, the ultrasound contrast agent comprises sulfur hexafluoride lipid a-type microspheres.

In some embodiments, the ultrasound energy is emitted from a transducer that contacts or is adjacent to the skin of the subject, and wherein a closest point of the transducer is within 2-4 inches of the surface of the pancreas of the subject.

In some embodiments, the mechanical index does not exceed 0.03.

In some embodiments, the mechanical index does not exceed 0.05.

In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 1.5MHz to 3.0 MHz.

In some embodiments, the ultrasound energy is emitted from a transducer that contacts or is adjacent to the skin of the subject, and wherein a closest point of the transducer is at a location that is greater than 4 inches and up to 9 inches from the surface of the pancreas of the subject.

In some embodiments, the mechanical index does not exceed 0.03.

In some embodiments, the mechanical index does not exceed 0.05.

In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 1.0MHz to 1.5 MHz.

In some embodiments, the ultrasound energy is emitted from a transducer array that is 3 to 5 inches in length by 1 to 3 inches in width.

In some embodiments, ultrasound energy is emitted from the imaging probe and the ultrasound imaging system.

In some embodiments, the ultrasound energy is transmitted from a volume probe and an ultrasound imaging system.

In some embodiments, the ultrasound energy is emitted from a transducer array that is substantially triangular in shape.

In some embodiments, the ultrasound energy is emitted from a transducer array that is substantially rectangular in shape.

In some embodiments, the ultrasound energy is emitted from a flexible transducer array.

In some embodiments, ultrasound energy is emitted from a transducer array that includes a plurality of cavitation detectors for monitoring and locating cavitation during operation.

In some embodiments, ultrasound is emitted from a transducer array that is connected to the subject by a strap.

In some embodiments, the subject is sonicated with the amount of ultrasound energy for greater than 5 minutes.

In some embodiments, the subject is sonicated with the amount of ultrasound energy for less than 15 minutes.

In some embodiments, the amount of ultrasound contrast agent is administered to the subject within 1 hour before the subject begins sonication with the amount of ultrasound energy.

In some embodiments, the amount of ultrasound contrast agent is administered to the subject within 30 minutes before the subject begins sonication with the amount of ultrasound energy.

In some embodiments, the amount of secretin is administered to the subject within 1 hour after the subject begins sonication with the amount of ultrasonic energy.

In some embodiments, the amount of secretin is administered to the subject within 30 minutes after the subject is initially sonicated with the amount of ultrasound energy.

In some embodiments, the amount of secretin is administered to the subject prior to beginning sonication with the amount of ultrasound energy.

In some embodiments, the amount of secretin is administered to the subject after the sonication with the amount of ultrasound energy is initiated.

In some embodiments, the amount of secretin is administered to the subject less than 10 minutes after terminating sonication with the amount of ultrasonic energy.

In some embodiments, the amount of secretin is administered to the subject during sonication with the amount of ultrasonic energy.

In some embodiments, the amount of secretin and ultrasound contrast agent are administered to the subject simultaneously.

In some embodiments, ultrasound imaging is used to assist in the placement of a transducer that emits ultrasound energy over an organ or over the pancreas.

In some embodiments, ultrasound imaging is used to assist in placement of a catheter or other collection device at the opening of the subject's pancreatic duct.

In some embodiments, ultrasound imaging is used to assist in the placement of a catheter or other fluid collection device in or near an organ of a subject.

In some embodiments, no ultrasound contrast agent is administered to the subject.

In some embodiments, the ultrasound contrast agent is administered intravenously.

In some embodiments, the ultrasound contrast agent is administered intravenously only.

In some embodiments, the ultrasonic energy is multi-frequency and/or multi-phase.

In some embodiments, the subject is dynamically sonicated with ultrasound energy.

In some embodiments, the amount of ultrasonic energy is sonicated from more than one location on the first side.

In some embodiments, the amount of ultrasonic energy is sonicated from more than one location on a second side perpendicular to the first side.

In some embodiments, the pancreas of the subject is exposed to an amount of ultrasound radiation at least two different locations on the coronal, sagittal, or transverse planes of the pancreas.

In some embodiments, the organ of the subject is exposed to an amount of ultrasound radiation at least two different locations on a coronal, sagittal, or transverse plane of the organ.

In some embodiments, the subject is supine.

In some embodiments, the subject is a human.

Also provided is a method of treating a pancreatic disorder in a subject, the method comprising:

a) determining whether the subject has dysplastic pancreatic or pancreatic cancer cells in its pancreas by the methods described herein, and

b) administering chemotherapy, radiation therapy, immunotherapy or pancreatectomy to the subject found in a) to have dysplastic pancreatic or pancreatic cancer cells in their pancreas.

In some embodiments, determining whether the subject has dysplastic pancreatic or pancreatic cancer cells in its pancreas is performed by one or more of a cellular morphology analysis, a histomorphology analysis, or a biomolecular marker analysis.

Also provided is a method of increasing the efficacy of collecting a cell sample from a tissue in an assay procedure on a subject, the method comprising sonicating the tissue of the subject with an amount of ultrasonic energy effective to cause stable cavitation of an ultrasonic contrast agent, thereby causing exfoliation of cells or tissue fragments of the subject, prior to collecting the cell sample from the tissue, and then collecting the cell sample from the tissue of the subject.

In some embodiments, the tissue comprises epithelial cells.

Also provided is a method of assaying a cell or tissue sample of a subject to determine whether the cell or tissue comprises a cancerous or precancerous cell or tissue, the method comprising:

a) receiving a cell or tissue sample, wherein the sample has been previously obtained by: administering to a subject an amount of an ultrasound contrast agent; and sonicating the organ of the subject with an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent, thereby causing flaking of the cells or epithelial tissue segments from the organ of the subject, and collecting the flaked cells or tissue sample;

b) performing one of a cellular morphology analysis, a tissue morphology analysis, or a biomolecular marker analysis to determine whether the cell or tissue comprises a cancerous or precancerous cell or tissue.

In some embodiments, the method further comprises administering the amount of an ultrasound contrast agent to the subject and sonicating an organ of the subject with an amount of ultrasound energy effective to cause stable cavitation of the ultrasound contrast agent, thereby causing the cells or epithelial tissue segments to exfoliate from the organ of the subject, and collecting the exfoliated cells or tissue sample.

Also provided is a method for early detection of dysplastic and carcinomic cells in the pancreas comprising applying ultrasound energy directed at the pancreas to induce cell exfoliation and then endoscopically collecting pancreatic fluid containing exfoliated cells and cell clusters for molecular and microscopic morphological examination.

In some embodiments, ultrasound energy directed at the pancreas to increase cell flaking into pancreatic juice is applied to the patient's body outside the patient's body.

In some embodiments, the ultrasound energy directed at the pancreas to increase cell flaking into pancreatic juice is applied endoscopically.

In some embodiments, the administration of ultrasonic energy to increase exfoliation is combined with the administration of secretin, a hormone that increases pancreatic fluid production.

In some embodiments, pancreatic juice is examined by means of a neural network-based computer-assisted microscopy system.

Also provided is an isolated sample of bodily fluid, wherein the sample is obtained directly from a subject whose tissue or organ has been sonicated, wherein the sample comprises epithelial or other cells from the tissue or organ enriched at a level greater than 2-fold compared to the level of epithelial or other cells in an otherwise identical sample obtained from a subject that has not been sonicated.

In some embodiments, the ultrasound contrast agent present during the sonication of the tissue or organ is also administered to the subject.

In some embodiments, the pancreas of the subject has been sonicated and secretin has been administered to the subject prior to obtaining the sample directly from the subject.

In some embodiments, the organ is bladder, breast, liver, kidney, lung, thyroid, gastrointestinal tract, or cysts (cist). To increase the yield of cells of interest (e.g., abnormal cells in a bodily fluid sample), the present invention utilizes the application of ultrasonic energy, preferably at a level safe for diagnostic purposes (e.g., a mechanical index ("MI") of 1.9 or less). In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.01. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.02. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.03. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.04. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.05. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.06. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.07. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.08. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.09. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.1. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.11. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.12. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.13. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.14. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.15. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.16. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.17. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.18. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.19. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.2. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.3. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.4. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.5. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.6. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.7. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.8. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 0.9. In some embodiments, the subject is sonicated with ultrasound energy having a MI of 1.0. In some embodiments, the subject is sonicated with ultrasound energy having a MI of 1.1. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 1.2. In some embodiments, the subject is sonicated with ultrasound energy having a MI of 1.3. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 1.4. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 1.5. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 1.6. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 1.7. In some embodiments, the subject is sonicated with ultrasound energy having a MI of 1.8. In some embodiments, the subject is sonicated with ultrasound energy having an MI of 1.9. In some embodiments, the subject is sonicated with ultrasound energy having a MI of 2.0.

The patient is subjected to ultrasound energy for a period of time before the cells are collected. In some embodiments, the subject is sonicated with ultrasound energy for 1 minute. In some embodiments, the subject is sonicated with ultrasound energy for 2 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 3 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 4 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 5 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 6 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 7 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 8 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 9 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 10 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 10-15 minutes. In some embodiments, the subject is sonicated with ultrasound energy for 15-20 minutes.

In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 1.0MHz to 1.5 MHz. In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 1.0MHz to 3.0 MHz. In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 1.5MHz to 3.0 MHz. In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 1.5MHz to 2.0 MHz. In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 2.0MHz to 3.5 MHz. In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises 2.5MHz to 3.0 MHz. In some embodiments, the frequency of the ultrasonic energy that is sonicated comprises less than 1.0 MHz.

In some embodiments, the waveform of ultrasonic energy sonicated includes a negative peak having an amplitude greater than a positive peak. In some embodiments, the waveform of ultrasonic energy sonicated includes a negative peak having an amplitude less than the positive peak. In some embodiments, the waveform of ultrasonic energy sonicated includes a negative peak having an amplitude equal to a positive peak. Such enhanced ultrasound can be achieved by means known in the art, see, for example, U.S. patent No. 7,905,836 to Dan Adam issued 3, 15, 2011, which is incorporated herein by reference.

In some embodiments, the ultrasound energy sonicated does not cause heating of the skin or tissue of the subject. In some embodiments, the ultrasonic energy for sonication is applied to the subject in vitro. In some embodiments, the ultrasound energy for sonication is applied from within the subject, e.g., via an endoscopic ultrasound transducer.

In embodiments of the invention, sonication of the ultrasound energy is performed prior to or simultaneously with administration of the microbubble ultrasound contrast agent to the subject. In some embodiments, the contrast agents are commercially availableIn some embodiments, the ultrasound contrast agent comprises sulfur hexafluoride lipid type a microspheres (sulfurr hexafluoride lipid type a microspheree).

In embodiments of the invention, the ultrasound energy is combined with microbubbles that circulate in the patient's circulatory system, providing sufficient energy to induce exfoliation of cells and optionally whole tissue fragments from the epithelium of the organ, preferably the organ.

In embodiments relating to the pancreas, pancreatic juice is obtained during upper gastrointestinal endoscopy procedures. For example, after intravenous administration of secretin, a disposable aspiration catheter is passed into the duodenum to collect pancreatic juice. Alternatively, pancreatic juice can be collected by direct suction of duodenal fluid through the suction channel of an endoscope. After obtaining pancreatic juice, its cellular contents may be fixed, for example, on one or more sample slides. The sample slides may then be processed and prepared for diagnostic analysis.

In embodiments relating to the pancreas, the secretin administered is human. In some embodiments, the secretin administered is human, bovine, or porcine. One useful form of human secretin is produced by ChiRhoClin, Inc (Burtonsville, Md), under the trade name "CHIRHOSTIM". In one embodiment, Hum (a secretin) has the sequence His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val; 1 in SEQ ID NO. One useful form of porcine secretin is produced by ChiRhoClin, Inc (Burtonsville, Md.) and sold under the trade name "secerfo" by Repligen Corporation (Waltham, Mass.). Another useful form of porcine secretin is produced by ChiRhoClin, Inc (Burtonsville, Md.) under the trade name "secemax". One useful form of HUMAN SECRETIN is manufactured and sold by ChiRhoClin, inc. Secretin can be combined in a composition with a pharmaceutically acceptable carrier and administered in the form of such a composition. Secretin can be administered by any means known in the art. In one embodiment, secretin or a composition containing secretin is administered intravenously.

As used herein, "and/or", e.g., option a and/or option B, encompasses the following individual embodiments: (i) option a, (ii) option B, and (iii) option a plus option B.

All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

When introducing elements of the present invention or the preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

Where a range of values is provided herein, it is understood that all subsets of the range of values, and all individual integers encompassed therein, to the second decimal place, are provided as part of the invention. Thus, frequencies having a length of 1.5 to 2.0MHz include a frequency subset of 1.51 to 1.61MHz, a frequency subset of 1.7 to 2.0MHz, and a frequency subset of 1.65 to 1.85MHz, and so on, unless explicitly stated otherwise.

While the invention has been described in conjunction with the embodiments outlined above, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the exemplary embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention.

Experimental examples

Example 1

Pigs previously fasted for 24 hours were anesthetized for comfort and placed in a lateral supine position. A collection catheter was placed into the duodenum at the exit of the porcine pancreas using fluoroscopy. Intravenous administration of ultrasound contrast agent (of Bracco Imaging) to pigsSulfur hexafluoride lipid type a microspheres). Prior to sonication of the animals with ultrasound, the area adjacent to the pancreatic exit (near the Vater ampulla/Oddi sphincter) was cleaned using ductal suction. The animals were sonicated using a General Electric visual E9 ultrasound system for 10 minutes using a GE 4VD probe with ultrasound having a mechanical index of 0.03 (frequency of 1.5Mhz to 3.0Mz) through a transducer placed on the animal's skin approximately 3-4 inches directly above the animal's pancreas. Ultrasound transducer by observing the appearance of opacity in the pancreas when ultrasound is properly placed to achieve stable cavitation of the ultrasound agentAnd (4) placing the components correctly. At the end of the sonication, porcine secretin (0.2 to 0.4mg/kg body weight) was administered intravenously to the animals. The animal's pancreas then produces pancreatic fluid that is collected to a volume of 50mL (which takes about 5 minutes) using a catheter placed in the duodenum near the Vater ampulla/Oddi sphincter. Pulsating flow was observed.

Subsequent histological analysis of the samples obtained showed that individual cells as well as large fragments of pancreatic epithelial tissue (see fig. 1) had been induced to exfoliate into the pancreatic fluid by ultrasound techniques.

Example 2

Pigs previously fasted for 24 hours were anesthetized for comfort and placed in a lateral supine position. A collection catheter was placed into the duodenum at the exit of the porcine pancreas using fluoroscopy. Prior to sonication of the animals with ultrasound, the area adjacent to the pancreatic exit (near the Vater ampulla/Oddi sphincter) was cleaned using ductal suction. The animals are sonicated for 10 minutes with a single frequency ultrasound (e.g., at 2.0 Mz) with a mechanical index of 1.3 to 1.4 by a transducer placed on the animal's skin approximately 3-4 inches directly above the animal's pancreas. When the ultrasound is properly placed, proper placement of the ultrasound transducer is achieved by observing the opaque appearance in the pancreas. At the end of the sonication, the animals were administered secretin intravenously (0.2 to 0.4mg/kg body weight). The animal's pancreas then produces pancreatic fluid that is collected to a volume of 50mL (which takes about 5 minutes) using a catheter placed in the duodenum near the Vater ampulla/Oddi sphincter. Pulsating flow was observed.

Subsequent histological analysis of the samples obtained showed that individual cells of the pancreatic epithelial tissue had been induced to exfoliate into the pancreatic fluid by ultrasound techniques. However, it was also observed that animals exhibited clear signs of pancreatitis, which is sometimes an undesirable side effect of pancreatic fluid collection. It was determined that a lower mechanical index of 1.3 to 1.4 is preferred to reduce possible pancreatitis.

Example 3

Pigs previously fasted for 24 hours were anesthetized for comfort and placed in a lateral supine position. A collection catheter was placed into the duodenum at the exit of the porcine pancreas using fluoroscopy. Prior to sonication of the animals with ultrasound, the area adjacent to the pancreatic exit (near the Vater ampulla/Oddi sphincter) was cleaned using ductal suction. The animal is sonicated for 10 minutes with multi-frequency (and optionally multi-phase, asymmetric-wave) ultrasound (at frequencies of 1.5Mhz to 3.0Mz) with a mechanical index greater than 0.03 but not more than 1.3 by a transducer placed on the animal's skin approximately 3-4 inches directly above the animal's pancreas. When the ultrasound is properly placed, proper placement of the ultrasound transducer is achieved by observing the opaque appearance in the pancreas. At the end of the sonication, the animals were administered secretin intravenously (0.2 to 0.4mg/kg body weight). Pancreatic fluid was collected to a volume of 50mL using a catheter placed in the duodenum near the Vater ampulla/Oddi sphincter and samples were histologically analyzed.

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