Consumable tube for use with a flow cytometry-based hematology system

• US 20030030783A1
• Filed: 05/31/2002
• Published: 02/13/2003
• Est. Priority Date: 06/02/2000
• Status: Active Grant

• Current Assignee: Hansen W. Peter, Harold C. Flynn Jr, Harold R. Crews, James W. Russell, John W. Roche, Marcus F. Julian, Michelle L. Coleman
• Original Assignee: Hansen W. Peter, Harold C. Flynn Jr, Harold R. Crews, James W. Russell, John W. Roche, Marcus F. Julian, Michelle L. Coleman
• Inventors: Coleman, Michelle L., Roche, John W., Crews, Harold R., Julian, Marcus F., Russell, James W., Flynn, Harold C. Jr., Hansen, W. Peter
• Granted Patent: US 7,064,823 B2
• Time in Patent Office: Days
• US Class Current: 356/39
• CPC Class Codes: G01N 1/38 Diluting, dispersing or mixing samples
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/0069 with lysing, e.g. of erythrocyts
• CPC Class Codes: G01N 2015/0076 Reticulocytes
• CPC Class Codes: G01N 2015/008 White cells
• CPC Class Codes: G01N 2015/0084 Platelets
• CPC Class Codes: G01N 2015/011 with lysing, e.g. of erythrocytes
• CPC Class Codes: G01N 2015/014 Reticulocytes
• CPC Class Codes: G01N 2015/016 White blood cells
• CPC Class Codes: G01N 2015/018 Platelets
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1493 Particle size
• CPC Class Codes: G01N 35/1095 for supplying the samples to flow-through analysers for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00
• CPC Class Codes: H04L 25/063 Setting decision thresholds using feedback techniques only

Abstract

The present invention is a flow cytometry-based hematology system useful in the analysis of biological samples, particularly whole blood or blood-derived samples. The system is capable of determining at least a complete blood count (CBC), a five-part white blood cell differential, and a reticulocyte count from a whole blood sample. The system preferably uses a laser diode that emits a thin beam to illuminate cells in a flow cell and a lensless optical detection system to measure one or more of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight measurements produced by the cells. The lensless optical detection system contains no optical components, other than photoreactive elements, and does not include any moving parts. Finally, the system uses a unique system of consumable reagent tubes that act as reaction chambers, mixing chambers, and waste chambers for the blood sample analyses. The consumable tubes incorporate reference particles, which act as internal standards to ensure that the dilutions made during processing of the samples have been carried out correctly, and to ensure that the instrument is working properly. The present invention also relates to methods for using the system.

Claims

1. A flow cytometry-based hematology system for classifying and counting biological cells in a blood sample or blood-derived sample, comprising:

a hydraulic system including a flow cell, said hydraulic system being capable of mixing said blood sample or blood-derived sample with the contents of one or more of a disposable vessel, a lyse tube or a sheath tube, and being capable of moving the contents of one or more of said disposable vessel, said lyse tube, said sheath tube, or a mixture of said blood sample or said blood-derived sample with one or more of said contents of said disposable vessel, said lyse tube and said sheath tube through said flow cell;

a diode laser, said diode laser emitting light to irradiate cells present in said flow cell, said cells being derived from said blood sample or said blood-derived sample;

a lensless light detection system, said lensless light detection system having a first portion capable of detecting one or more of axial light loss, low-angle scattered light and high-angle scattered light, and a second portion capable of detecting right angle scattered light scattered at a high numerical aperture, said second portion being physically distinct from said first portion, said first portion and said second portion being capable of converting said axial light loss, said low-angle scattered light, said high-angle scattered light, and said right angle scattered light into electrical signals; and

a signal processor, said signal processor being capable of analyzing said electrical signals generated from said first portion and said second portion of said lensless optical module, and generating data therefrom.

2. The flow cytometry system of claim 1, wherein:

said first portion comprises a photodetector array on a single integrated circuit chip;

said photodetector array being effective to detect at least one of axial light loss, low-angle forward scattered light and high-angle forward scattered light.

3. The flow cytometry system of claim 1, wherein:

said first portion comprises a photodetector array arranged on a plurality of electrically distinct integrated circuit chips;

said photodetector array being effective to detect at least one of axial light loss, low-angle forward scattered light, and high-angle forward scattered light.

4. The flow cytometry system of claim 1, further comprising a servo circuit capable of correcting for the influence of dark current noise and non-scattered light incident on said lensless light detection system.

5. The flow cytometry system of claim 2, wherein said photodetector array has at least three separate and electrically distinct light detection elements.

6. The flow cytometry system of claim 5, wherein said elements are made from a single piece of photoreactive material.

7. The flow cytometry system of claim 5, further comprising a Mie mask placed over said photodetector array.

8. The flow cytometry system of claim 5, wherein:

a first one of said light detection elements is capable of detecting axial light loss;

a second one of said light detection elements is capable of detecting forward scattered light scattered at an angle between about 1 and about 3 degrees; and

a third one of said light detection elements is capable of detecting forward scattered light scattered at an angle between about 4 and about 9 degrees.

9. The flow cytometry system of claim 1, wherein said second portion comprises a high numerical aperture light detector capable of collecting light scattered at an angle between about 50 and about 130 degrees.

10. The flow cytometry system of claim 5, wherein:

said first, second and third light detection elements are aligned on a first axis that is substantially orthogonal to a second axis defined by a line of travel of cells through a flow cell of said flow cytometer and further is substantially orthogonal to a third axis defined by a line of travel of laser light incident on said cells in said flow cell; and

said second axis and said third axis are substantially orthogonal to each other.

11. The flow cytometry system of claim 1, wherein said diode laser is capable of emitting a laser light beam having a height between about 0.1 and about 10 μ

m, and a width between about 0.1 and about 200 μ

m.

12. The flow cytometry system of claim 1, further comprising a fluorescent light detector capable of detecting light emitted from a fluorescent compound present in said flow cell upon illumination of said fluorescent compound by said diode laser.

13. The flow cytometry system of claim 1, further comprising a bar code reader, said bar code reader being capable of reading information encoded on a bar code label present on a surface of said disposable vessel;

said information including one or more of type of test, lot number of reference particles present in said disposable vessel, expiration date of said disposable vessel, a serial number of said disposable vessel, a reference particle concentration, a mean value of axial light loss and coefficient of variation of axial light loss for said reference particles, a mean value of low-angle forward light scatter and coefficient of variation of low-angle forward light scatter for said reference particles, a mean value of high-angle forward light scatter and coefficient of variation of high-angle forward light scatter for said reference particles, a mean value of right angle light scatter and coefficient of variation of right angle light scatter for said reference particles, a mean value of time-of-flight and coefficient of variation of time-of-flight for said reference particles, absorbance of a sample mixture, transmission of a sample mixture, reflectance of a sample mixture, or fluorescence of a sample mixture.

14. The flow cytometry system of claim 1, wherein said hydraulic system comprises not more than one flow cell.

15. The flow cytometry system of claim 1, wherein said hydraulic system further comprises:

a reservoir for a sheath solution;

at least one syringe connected to said reservoir, said at least one syringe being driven by a motor;

a single HGB module, said HGB module being connected on an inlet side to a sample inlet;

said sample inlet being connected to said flow cell by a single flow path passing through said HGB module;

a single flow cell, said single flow cell being connected to said HGB module and said at least one syringe on an inlet side of said flow cell, and to a waste container on an outlet side of said flow cell.

16. The flow cytometry system of claim 1, wherein said hydraulic system further comprises a plurality of valves to control flow through said hydraulic system, and wherein the number of said valves does not exceed five.

17. A lensless light detection system for a flow cytometer.

18. The lensless light detection system of claim 17, said lensless light detection system having a first portion capable of detecting one or more of axial light loss, low-angle scattered light, and high-angle scattered light, and a second portion capable of detecting right angle scattered light scattered at a high numerical aperture, said second portion being physically distinct from said first portion, and said first portion and said second portion being capable of converting said axial light loss, said low-angle scattered light, said high-angle scattered light, and said right angle scattered light into electrical signals.

19. The lensless light detection system of claim 17, comprising:

said first portion comprises a photodetector array on a single integrated circuit chip;

said photodetector array being effective to detect at least one of axial light loss, low-angle forward scattered light, and high-angle forward scattered light.

20. The lensless light detection system of claim 17, comprising:

said first portion comprises a photodetector array arranged on a plurality of electrically distinct integrated circuit chips;

said photodetector array being effective to detect at least one of axial light loss, low-angle forward scattered light, and high-angle forward scattered light.

21. The lensless light detection system of claim 17, further comprising a servo circuit capable of correcting for the influence of dark current noise and non-scattered light incident on said lensless light detection system.

22. The lensless light detection system of claim 17, wherein said second portion comprises a high numerical aperture light detector capable of detecting right angle scattered light scattered at an angle between 50 and 130 degrees.

23. The lensless light detection system of claim 19, wherein said photodetector array has at least three separate and electrically distinct light detection elements.

24. The lensless light detection system of claim 23, wherein said elements are made from a single piece of photoreactive material.

25. The lensless light detection system of claim 19, further comprising a Mie mask placed over said photodetector array.

26. The lensless light detection system of claim 23, wherein:

a first one of said light detection elements is capable of detecting axial light loss;

a second one of said light detection elements is capable of detecting forward scattered light scattered at an angle between 1 and 3 degrees; and

a third one of said separate light detection elements is capable of detecting forward scattered light scattered at an angle between 4 and 9 degrees.

27. The lensless light detection system of claim 17, wherein said second portion comprises a high numerical aperture light detector capable of detecting right angle scattered light scattered at an angle between 50 and 130 degrees.

28. The lensless light detection system of claim 23, wherein:

said first, second and third light detection elements are aligned on a first axis that is substantially orthogonal to a second axis defined by a line of travel of cells through a flow cell of said flow cytometer and further is substantially orthogonal to a third axis defined by a line of travel of laser light incident on said cells in said flow cell; and

said second axis and said third axis are substantially orthogonal to each other.

29. A disposable vessel for use in a flow cytometry-based hematology system, comprising:

a tube;

a known number of reference particles in said tube, each of said reference particles having a predetermined diameter such that, when said reference particles are illuminated by light, said light is scattered by said reference particles such that said scattered light falls into at least one of a plurality of predetermined light scatter channels; and

at least one reagent in said tube.

31. The disposable vessel of either of claims 29 or 30, wherein said diameter of each of said reference particles is between 1 micron and 10 microns.

32. The disposable vessel of either of claims 29 or 30, wherein said diameter of each of said reference particles is 4.0±

0.5 microns.

33. The disposable vessel of either of claims 29 or 30, wherein said known number of reference particles is between about 10³ and about 10⁵ per μ

l of a sample solution.

34. The disposable vessel of either of claims 29 or 30, wherein said known number of reference particles is present at a concentration of 10,000±

1,000 per μ

l of a sample solution.

35. The disposable vessel of either of claims 29 or 30, wherein said reference particles are selected from the group consisting of polystyrene latex particles, fixed cells, pollen, glass, and large colloids.

36. The disposable vessel of either of claims 29 or 30, wherein said reference particles are polystyrene latex particles.

37. The disposable vessel of either of claims 29 or 30, wherein said reference particles are styrene divinylbenzene latex particles.

38. The disposable vessel of claim 32, wherein said known number of reference particles is present at a concentration of 10,000±

1,000 per μ

l of a sample solution.

39. The disposable vessel of either of claims 29 or 30, wherein said reagent is effective to enable said flow cytometry-based hematology system to count at least one of red blood cells and reticulocytes in a blood sample or blood-derived sample.

40. The disposable vessel of either of claims 29 or 30, wherein said reagent is effective to stain one or more cells in a blood sample or blood-derived sample.

41. The disposable vessel of either of claims 29 or 30, wherein said reagent includes a reactant capable of staining ribonucleic acid in reticulocytes.

42. The disposable vessel of claim 41, wherein said reactant capable of staining ribonucleic acid in reticulocytes is new methylene blue.

43. The disposable vessel of claim 42, wherein said new methylene blue is present at a concentration between about 0.1 and about 0.5 grams per liter of a sample solution.

44. The disposable vessel of either of claims 29 or 30, wherein said reagent includes a reactant capable of modifying the shape of red blood cells but otherwise leaving said red blood cells substantially intact for a period of at least one minute.

45. The disposable vessel of claim 44, wherein said reactant capable of modifying the shape of red blood cells but otherwise leaving said red blood cells substantially intact is a nonzwitterionic surfactant.

46. The disposable vessel of claim 45, wherein said nonzwitterionic surfactant is selected from the group consisting of an alkylphenol ethoxylate and an alcohol ethoxylate.

47. The disposable vessel of claim 44, wherein said reactant capable of modifying the shape of red blood cells but otherwise leaving said red blood cells substantially intact is Plurafac-A-39-Prill.

48. The disposable vessel of either of claims 29 or 30, wherein said reagent includes new methylene blue and a nonzwitterionic surfactant.

49. The disposable vessel of either of claims 29 or 30, wherein said reagent includes:

a) new methylene blue, at a concentration between about 0.1 and about 0.5 grams per liter of a sample solution;

b) Plurafac-A-39-Prill, at a concentration between about 0.1 and about 0.6 grams per liter of a sample solution;

c) sodium bicarbonate, at a concentration between about 6.0 and about 10.0 grams per liter of a sample solution;

d) sodium chloride, at a concentration between about 1.0 and about 5.0 grams per liter of a sample solution;

e) Tricine, at a concentration between about 1.0 and about 5.0 grams per liter of a sample solution;

f) disodium EDTA, at a concentration between about 0.5 and about 3.0 grams per liter of a sample solution;

g) ethyl paraben at a concentration between about 0.1 and about 0.5 grams per liter of a sample solution; and

h) methyl paraben at a concentration between about 0.1 and about 0.3 grams per liter of a sample solution.

50. The disposable vessel of either of claims 29 or 30, wherein said reagent is effective to enable said flow cytometry-based hematology system to measure at least one of a red blood cell count, a reticulocyte count, a platelet count, a two-part white blood cell differential, a five-part white blood cell differential, and hemoglobin concentration.

51. The disposable vessel of either of claims 29 or 30, wherein said reagent is effective to enable said flow cytometry-based hematology system to measure a complete blood count, a five-part white blood cell differential, and a reticulocyte count.

52. The disposable vessel of either of claims 29 or 30, wherein said reagent is effective to enable said flow cytometry-based hematology system to measure platelet aggregation.

53. The disposable vessel of either of claims 29 or 30, wherein said reagent is effective to enable said flow cytometry-based hematology system to utilize one of a clottable assay and an aggregation assay.

54. The disposable vessel of either of claims 29 or 30, wherein said reagent is effective to enable said flow cytometry-based hematology system to utilize a member selected from the group consisting of a prothrombin time assay, an activated partial thromboplastin time assay, and a thrombin time assay.

55. The disposable vessel of either of claims 29 or 30, wherein said reagent comprises a fluorescent compound.

56. The disposable vessel of either of claims 29 or 30, wherein said reagent comprises an antibody or antibody derivative.

57. The disposable vessel of either of claims 29 or 30, wherein said reagent comprises a member selected from the group consisting of an enzyme, an enzyme substrate, and an enzyme inhibitor.

58. The disposable vessel of either of claims 29 or 30, wherein said reagent comprises a member selected from the group consisting of a receptor, a receptor body, a receptor agonist, and a receptor antagonist.

59. The disposable vessel of either of claims 29 or 30, wherein said disposable vessel is an open tube.

60. The disposable vessel of either of claims 29 or 30, wherein said disposable vessel is a closed tube.

30. A disposable vessel for use in a flow cytometry-based hematology system, comprising:

a tube;

a known number of reference particles in said tube, each of said reference particles having one of a plurality of predetermined diameters such that, when said reference particles are illuminated by light from a laser, said light is scattered by said reference particles such that said scattered light falls into one of a plurality of predetermined light scatter channels; and

at least one reagent in said tube.

61. A hydraulic system for a flow cytometer, comprising:

a reservoir for a sheath solution;

at least one syringe connected to said reservoir, said at least one syringe being driven by a motor;

a single HGB module, said HGB module being connected on an inlet side to a sample inlet;

said sample inlet being connected to said flow cell by a single flow path passing through said HGB module;

a single flow cell, said single flow cell being connected to said HGB module and said at least one syringe on an inlet side of said flow cell, and to a waste container on an outlet side of said flow cell.

62. A hydraulic system according to claim 61, wherein said sample inlet includes a needle effective to pierce a stopper on a blood collection tube and aspirate a sample in said tube.

63. A closed hydraulic system according to claim 61, comprising at least two syringes connected to said reservoir, each of said at least two syringes being driven by a motor.

64. A closed hydraulic system according to claim 61, further comprising a plurality of valves to control flow through said hydraulic system, wherein the number of said valves is not greater than five.

65. A method for analyzing a blood sample or a blood-derived sample using a flow cytometry-based hematology system, comprising the steps of:

a) mixing a portion of said blood sample or blood-derived sample with a reagent in a disposable vessel containing a known number of reference particles, said reference particles having one of a plurality of predetermined diameters such that when said reference particles are illuminated by light, said light is scattered such that said scattered light falls into one of a plurality of predetermined light scatter channels;

b) standardizing said sample using said reference particles by measuring at least one of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each of said reference particles, and comparing said measurements to predetermined values obtained for said reference particles;

c) counting at least a portion of cells present in said sample;

d) measuring at least one parameter from the group measured in step b) for each of one or more cells in said sample, to produce a measurement value for each parameter; and

e) analyzing said measurement values to classify each cell.

67. The method of either of claims 65 or 66, wherein said low-angle forward scattered light is light scattered at an angle between about 1 and about 3 degrees.

68. The method of either of claims 65 or 66, wherein said high-angle forward scattered light is light scattered at an angle between about 4 and about 9 degrees.

69. The method of either of claims 65 or 66, where said right angle scattered light is scattered at an angle between about 50 and about 130 degrees.

70. The method of either of claims 65 or 66, wherein said low-angle scattered light is light scattered at an angle between about 1 and about 3 degrees, said high-angle scattered light is light scattered at an angle between about 4 and about 9 degrees, and said right angle scattered light is scattered at an angle between about 50 and about 130 degrees.

71. The method of either of claims 65 or 66, further comprising measuring a hemoglobin content of said blood sample or blood-derived sample.

72. The method of either of claims 65 or 66, further comprising measuring an amount of light emitted from a fluorescent compound present in said blood sample or blood-derived sample after said reagent is mixed with said blood sample or blood-derived sample.

66. A method for analyzing a blood sample or a blood-derived sample using a flow cytometry-based hematology system, comprising the steps of:

a) mixing a first portion of said blood sample or blood-derived sample with a reagent in a disposable vessel containing a known number of reference particles to form a red cell solution, said reference particles having one of a plurality of predetermined diameters such that when said reference particles are illuminated by light, said light is scattered such that said scattered light falls into one of a plurality of predetermined light scatter channels;

b) standardizing said red cell solution and said flow cytometry-based hematology system using said reference particles by measuring at least one of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each of said reference particles, and comparing said measurements to predetermined values obtained for said reference particles;

c) counting at least a portion of red blood cells, reticulocytes, and platelets present in said red cell solution;

d) measuring at least one parameter from the group measured in step b) for each of one or more cells in said red cell solution, to produce a red cell solution measurement value for each parameter for one or more of said red blood cells, reticulocytes, and platelets from said sample;

e) mixing a second portion of said blood sample or blood-derived sample with a lyse solution in said disposable vessel containing a known number of reference particles to form a white cell solution;

f) counting at least a portion of white blood cells present in said white cell solution;

g) measuring at least one parameter from the group measured in step b) for each of one or more cells in said white cell solution, to produce a white cell solution measurement value for each parameter for said white blood cells; and

h) analyzing said red cell solution measurement values and said white cell solution measurement values to classify each cell.

73. A method for analyzing a blood sample or a blood-derived sample using a flow cytometry-based hematology system, comprising the steps of:

a) mixing said blood sample or blood-derived sample with a reagent in a disposable vessel containing a known number of reference particles, said reference particles having one of a plurality of predetermined diameters such that when said reference particles are illuminated by light, said light is scattered such that said scattered light falls into one of a plurality of predetermined light scatter channels;

b) standardizing said sample using said reference particles by measuring at least one of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each of said reference particles, and comparing said measurements to predetermined values obtained for said reference particles;

c) counting one or more cells present in said sample;

d) measuring at least one parameter for said sample, said parameter having been measured in step b), by using one of a platelet aggregation assay and a clottable assay, to produce a measurement value for each parameter; and

e) analyzing said measurement values.