METHOD OF DETERMINING A COMPLETE BLOOD COUNT AND A WHITE BLOOD CELL DIFFERENTIAL COUNT

• US 20090269799A1
• Filed: 04/27/2009
• Published: 10/29/2009
• Est. Priority Date: 04/25/2008
• Status: Active Grant

• Current Assignee: Roche Diagnostic Hematology, Inc.
• Original Assignee: Constitution Medical Investors, Inc. (Roche Holding AG)
• Inventors: Tanasijevic, Milenko, Zahniser, David, Winkelman, James
• Granted Patent: US 9,017,610 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: G01N 1/2813 Producing thin layers of samples on a substrate, e.g. smearing, spinning-on G01N1/30 takes precedence
• CPC Class Codes: G01N 1/312 for samples mounted on planar substrates
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/012 Red blood cells
• CPC Class Codes: G01N 2015/016 White blood cells
• CPC Class Codes: G01N 2015/018 Platelets
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1472 with colour
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: G01N 2035/00138 Slides
• CPC Class Codes: G01N 2035/00465 Separating and mixing arrangements
• CPC Class Codes: G01N 2035/0091 GUI [graphical user interfaces]
• CPC Class Codes: G01N 21/255 Details, e.g. use of specially adapted sources, lighting or optical systems
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G01N 2201/126 Microprocessor processing
• CPC Class Codes: G01N 2333/805 Haemoglobins; Myoglobins
• CPC Class Codes: G01N 33/49 Blood chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80
• CPC Class Codes: G01N 35/00029 provided with flat sample substrates, e.g. slides G01N35/028 takes precedence
• CPC Class Codes: G01N 35/00871 Communications between instruments or with remote terminals
• CPC Class Codes: G01N 35/1016 : Control of the volume dispensed or introduced
• CPC Class Codes: G06F 18/24 : Classification techniques
• CPC Class Codes: G06T 7/0012 : Biomedical image inspection
• CPC Class Codes: H04N 23/56 : provided with illuminating means
• CPC Class Codes: H04N 23/63 : by using electronic viewfinders
• CPC Class Codes: H04N 5/265 : Mixing

Abstract

Systems and methods analyzing body fluids such as blood and bone marrow are disclosed. The systems and methods may utilize an improved technique for applying a monolayer of cells to a slide to generate a substantially uniform distribution of cells on the slide. Additionally aspects of the invention also relate to systems and methods for utilizing multi color microscopy for improving the quality of images captured by a light receiving device.

Claims

1. A system for analyzing cells from blood or bone marrow comprising:

a. an applicator for dispensing a fluid comprising the bone marrow or blood, said applicator comprising an applicator controller and a tip for dispensing the fluid and the cells onto a slide;

b. a light receiving device containing a lens for capturing an image of the cells on the slide;

c. a computer containing a microprocessor and software stored on computer readable media for causing the computer to perform the steps of;

i. instructing the applicator controller to position the tip a certain height above the slide;

said position having an X, Y, and Z directional component;

ii. instructing the applicator controller to move the applicator tip at a certain velocity across the slide;

said velocity having a directional component and a speed component;

iii. instructing the applicator to direct the diluted fluid out of the tip at a certain velocity;

iv. causing the tip to apply a first flow of cells across the slide such that the tip'"'"'s position has a constant Z component;

said flow comprising a monolayer of cells and a flow width;

v. directing the applicator controller to change the position of the tip by a certain distance; and

vi. causing the tip to apply a second flow of cells across the slide such that the tip'"'"'s position has a constant Z component, and the second flow of cells settles into a position near the first flow of cells.

2. The system of claim 1 wherein the first and second flow of cells form a gapless, contiguous monolayer of cells on the slide.

3. The system of claim 1 wherein the certain distance is approximately equal to the flow width.

4. The applicator of claim 1 comprising a first reservoir for storing the blood or bone marrow, a second reservoir for storing a diluent, and a mixer for mixing the blood or bone marrow with the diluent to form a diluted fluid.

5. The system of claim 1 wherein the system comprises the fluid, and the fluid contains blood or bone marrow and a salt-based or protein-based diluent.

6. The system of claim 1 wherein the system comprises the fluid, and the fluid contains blood or bone marrow and bovine albumin, Plasmanate, Ficoll, or Dextran.

7. The system of claim 1 wherein the system comprises the fluid, and the fluid contains blood or bone marrow and a diluent containing four parts Plasmalyte for every one part of Plasmanate.

8. The system of claim 1 wherein the system comprises the fluid, and the fluid contains two parts blood for every one part of a diluent containing 5% albumin by volume.

9. The system of claim 1 wherein the software causes the computer to perform the steps of:

a. instructing a light receiving device to capture an image of the first flow of the cells;

b. analyzing the image to determine the width of the first flow of cells; and

c. directing the controller to move the tip a distance equal to the width of the first flow of cells.

10. The system of claim 1 wherein the software causes the computer to perform the steps of:

a. directing the applicator controller to apply a third flow of cells across the slide along a line having a fixed Y and Z component and an increasing X component;

b. directing the applicator controller to apply a fourth flow of cells across the slide along a line having a fixed Y and Z component and a decreasing X component; and

c. wherein the first, second, third, and fourth flows of cells form a gapless, contiguous, monolayer flow of cells on the slide.

11. The system of claim 1 comprising:

a discharge unit for cleaning the slide;

a dispenser for dispensing stain onto the slide;

a slide tilter for tipping the slide to remove excess stain;

a light receiving device for taking an image of the slide;

a slide mover for positioning the slide under the light receiving device; and

a light emission device for directing light onto or through the slide.

12. The system of claim 1 wherein the software causes the computer to instruct:

a. the controller to position the tip about 70 microns above the slide;

b. the applicator to direct fluid out of the tip at a rate of about 0.1 microliters per second; and

c. the controller to move the tip along the slide at speed of about 30 mm per second.

13. The system of claim 1 comprising:

a platform having;

a. a feeder for storing new slides;

b. a first station for applying a monolayer of cells onto the slide;

c. a second station for staining, fixating, and drying the slides,

d. a third station for illuminating and imaging the slides;

e. a collector for receiving processed slides; and

f. an advancer for moving the slides through the system starting from the feeder, moving the slides to a first station, the second station, the third station; and

then the collector.

14. The system of claim 1 wherein the software causes the computer to instruct a light emission source to:

a. emit white light for illuminating the slide;

b. apply a first light filter to the light to restrict the emission of the light to a first wavelength; and

c. apply a second light filter to the light to restrict the emission of the light to a second wavelength.

15. The system of claim 14 wherein the software causes the computer to instruct a light emission source to apply a third light filter to the light to restrict the emission of the light to a third wavelength.

16. The system of claim 14 wherein the first wavelength is 430 nm and the second wavelength is 570 nm.

17. The system of claim 15 wherein the first, second, and third wavelengths are 430 nm, 500 nm, 525 nm or 600 nm.

18. The system of claim 14 wherein the software causes the computer to:

a. refine the images by compensating for spatial shifts or other distortions; and

b. combine two or more images captured by the light receiving device when at least two separate wavelengths of lights were directed at the slide to generate multi color images for a display.

19. The system of claim 14 wherein the software causes the computer to determine spatial, densitometric, calorimetric and texture features of the cells for classification of cell type.

20. The system of claim 14 wherein the software of the computer causes:

a. computer to instruct the light receiving source to capture a black and white image of the slide;

b. the computer to correct focus and image quality by adjusting a focal distance of the lens; and

c. the computer to shift focal position of the lens while a number of filters are interposed between the slide and the light source.

21. The light emission source of claim 14 comprising a halogen bulb for generating the white light, and a rotational motor for changing the positions of the filter.

22. The system of claim 1 wherein the software causes the computer to instruct a light source to:

a. cause a first LED to emit light at a first wavelength; and

b. cause a second LED to emit light at a secondwavelength.

23. A process for analyzing cells from blood or bone marrow comprising:

a. dispensing fluid containing blood or bone marrow onto a slide using a tip;

b. capturing an image of the slide with a light receiving device;

c. instructing an applicator controller to position the tip a certain height above the slide;

said position having an X, Y, and Z directional component;

d. instructing the applicator controller to move the applicator tip at a certain velocity across the slide;

said velocity having a directional component and a speed component;

e. instructing an applicator to direct the diluted fluid out of the tip at a certain velocity;

f. causing the tip to apply a first flow of cells across the slide such that the tip'"'"'s position has a constant Z component;

said flow comprising a monolayer of cells and a flow width;

g. directing the applicator controller to change the position of the tip by a certain distance; and

h. causing the tip to apply a second flow of cells across the slide such that the tip'"'"'s position has a constant Z component, and the second of flow cells settles into a position near the first flow of cells.

24. The process of claim 23 comprising the steps of storing the blood or bone marrow in a first reservoir;

storing diluent in a second reservoir;

mixing the blood or bone marrow and the diluent in a mixer to form a diluted fluid; and

dispensing the fluid and the cells onto a slide using a tip.

25. The process of claim 23 wherein the first and second flow of cells form a gapless, contiguous monolayer of cells on the slide.

26. The process of claim 23 wherein the certain distance is equal to the flow width.

27. The process of claim 23 wherein the fluid comprises a salt based or protein based diluent.

28. The process of claim 23 wherein the fluid comprises bovine albumin, Plasmanate, Ficoll, or Dextran.

29. The process of claim 23 wherein the fluid comprises a diluent containing four parts Plasmalyte for every one part of Plasmanate.

30. The process of claim 23 wherein the fluid contains two parts blood for every one part of a diluent containing 5% albumin by volume.

31. The process of claim 23 comprising the steps of:

a. instructing a light receiving device to capture an image of the first flow of the cells;

b. analyzing the image to determine the width of the first flow of cells; and

c. moving the tip a distance equal to the width of the first flow of cells.

32. The process of claim 23 comprising the steps of:

a. applying a third flow of cells across the slide along a line having a fixed Y and Z component and an increasing X component;

b. applying a fourth flow of cells across the slide along a line having a fixed Y and Z component and a decreasing X component; and

c. wherein the first, second, third, and fourth flows of cells form a gapless, contiguous, monolayer flow of cells on the slide.

33. The process of claim 23 comprising the steps of:

providing a discharge unit for cleaning the slide;

providing a dispenser for dispensing stain onto the slide;

providing a slide tilter for tipping the slide to remove excess stain;

providing a light receiving device for taking an image of the slide;

providing a slide mover for positioning the slide under the light receiving device;

providing a light emission device for directing light onto or through the slide.

34. The process of claim 23 comprising the steps of:

a. positioning the tip about 70 microns above the slide;

b. directing fluid out of the tip at a rate of about 0.1 microliters per second; and

c. moving the tip along the slide at speed of about 30 mm per second.

35. The process of claim 23 comprising the steps of:

a. providing a platform comprising;

a feeder for storing new slides;

b. providing a first station for applying a monolayer of cells onto the slide;

c. providing a second station for staining, fixating, and drying the slides;

d. providing a third station for illuminating and imagining the slides;

e. providing a collector for receiving processed slides; and

f. moving the slides through the system starting from the feeder, moving the slide to the first station, the second station, the third station; and

then the collector.

36. The process of claim 23 comprising the steps of:

a. emitting white light for illuminating the slide;

b. applying a first light filter to the light to restrict the emission of the light to a first wavelength; and

c. applying a second light filter to the light to restrict the emission of the light to a second wavelength.

37. The process of claim 23 comprising the steps of applying a third light filter to the light to restrict the emission of the light to a third wavelength.

38. The process of claim 36 wherein the first wavelength is 430 nm and the second wavelength is 570 nm.

39. The process of claim 37 wherein the first, second, and third wavelengths are 430 nm, 500 nm, 525 nm or 600 nm.

40. The process of claim 36 comprising the steps of:

a. refining the images by compensating for spatial shifts; and

b. combining two or more images captured by the light receiving device when at least two separate wavelengths of lights were directed at the slide to generate multi color images for a display.

41. The process of claim 36 comprising the steps of determining spatial, densitometric, colorimetric and texture features of the cells for classification of cell type.

42. The process of claim 36 comprising the steps of:

a. instructing the light receiving source to capture a black and white image of the slide;

b. correcting focus and image quality by adjusting a focal distance of the lens; and

c. shifting focal position of the lens while a number of filters are interposed between the slide and the light source.

43. The process of claim 36 comprising the steps of providing a halogen bulb for generating the white light, and a rotational motor for changing the positions of the filter.

44. The process of claim 23 comprising the steps of:

a. causing a first LED to emit light at a first wavelength; and

b. causing a second LED to emit light at a second wavelength.

45. The process of claim 23 wherein the process does not include attaching a coverslip or oil objective to the slide.

46. A system for analyzing cells from blood or bone marrow comprising:

a. an applicator for dispensing a fluid comprising the bone marrow or blood, said applicator comprising an applicator controller and a tip for dispensing the fluid and the cells onto a slide;

b. a computer containing a microprocessor and software stored on computer readable media for causing the computer to perform the steps of;

i. instructing the applicator controller to position the tip a certain height above the slide;

said position having an X, Y, and Z directional component;

ii. instructing the applicator controller to move the applicator tip at a certain velocity across the slide;

said velocity having a directional component and a speed component;

iii. instructing the applicator to direct the diluted fluid out of the tip at a certain velocity;

iv. causing the tip to apply a first flow of cells across the slide such that the tip'"'"'s position has a constant Z component;

said flow comprising a monolayer of cells and a flow width;

v. directing the applicator controller to change the position of the tip by a certain distance; and

vi. causing the tip to apply a second flow of cells across the slide such that the tip'"'"'s position has a constant Z component, and the second flow of cells settles into a position near the first flow of cells.

47. The system of claim 46 comprising two light receiving devices, wherein the computer causes the first light receiving device to capture an image of the unstained and nonfixed slide, and the computer causes the second light receiving device to capture an image of the slide after it has been stained and fixed.

48. A process for analyzing cells from blood or bone marrow comprising:

a. dispensing fluid containing blood or bone marrow onto a slide using a tip;

b. instructing an applicator controller to position the tip a certain height above the slide;

said position having an X, Y, and Z directional component;

c. instructing the applicator controller to move the applicator tip at a certain velocity across the slide;

said velocity having a directional component and a speed component;

d. instructing an applicator to direct the fluid out of the tip at a certain velocity;

e. causing the tip to apply a first flow of cells across the slide such that the tip'"'"'s position has a constant Z component;

said flow comprising a monolayer of cells and a flow width;

f. directing the applicator controller to change the position of the tip by a distance equal to the flow width; and

g. causing the tip to apply a second flow of cells across the slide such that the tip'"'"'s position has a constant Z component, and the second of flow cells settles into a position near the first flow of cells.

49. The process of claim 48 comprising the steps of instructing a first light receiving device to capture an image of a unstained and nonfixed slide, and instructing a second light receiving device to capture an image of the slide after it has been stained and fixed.

50. A system for analyzing cells from blood or bone marrow comprising:

a. an applicator for dispensing a fluid comprising the bone marrow or blood, said applicator comprising a tip for dispensing the fluid and the cells onto a slide;

b. a slide controller for moving the slide;

c. a computer containing a microprocessor and software stored on computer readable media for causing the computer to perform the steps of;

i. instructing the slide controller to position the slide a certain height below the tip;

said position having an X, Y, and Z directional component;

ii. instructing the slide controller to move the slide at a certain velocity across the tip;

said velocity having a directional component and a speed component;

iii. instructing the applicator to direct the diluted fluid out of the tip at a certain velocity;

iv. causing the tip to apply a first flow of cells across the slide such that the slide'"'"'s position has a constant Z component;

said flow comprising a monolayer of cells and a flow width;

v. directing the slide controller to change the position of the tip by a certain distance; and

vi. causing the tip to apply a second flow of cells across the slide such that the slide'"'"'s position has a constant Z component, and the second flow of cells settles into a position near the first flow of cells.

51. A process for analyzing cells from blood or bone marrow comprising:

a. dispensing fluid containing blood or bone marrow onto a slide using a tip;

b. instructing a slide controller to position the slide a certain height below the tip;

said position having an X, Y, and Z directional component;

c. instructing the slide controller to move the slide at a certain velocity across the slide;

said velocity having a directional component and a speed component;

d. directing the fluid out of the tip at a certain velocity;

e. causing the tip to apply a first flow of cells across the slide such that the tip'"'"'s position has a constant Z component;

said flow comprising a monolayer of cells and a flow width;

f. instructing the slide controller to change the position of the slide by a certain distance; and

g. causing the tip to apply a second flow of cells across the slide such that the slide'"'"'s position has a constant Z component, and the second of flow cells settles into a position near the first flow of cells.