Identification of blood elements using inverted microscopy

• US 20080138852A1
• Filed: 02/11/2005
• Published: 06/12/2008
• Est. Priority Date: 02/13/2004
• Status: Active Grant

• Current Assignee: Roche Diagnostic Hematology, Inc.
• Original Assignee: Roche Diagnostic Hematology, Inc.
• Inventors: Winkelman, James W., Tanasijevic, Milenko J., Zahniser, David
• Granted Patent: US 9,176,121 B2
• Time in Patent Office: Days
• US Class Current: 435/40.5
• 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 2015/144 Imaging characterised by its optical setup
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80

Abstract

A method of identifying the elements of a blood sample including placing an aliquot of blood on a transparent substrate such as a coverslip. The blood is allowed to stand and the cells to settle to form a layer or matrix. Inverted microscopy is used to identify the elements in the sample. Various forms of illumination may be used alone or in multiple combinations. The method improves the accuracy due to homogenous distribution of formed elements in the wet drop or aliquot, simplifies the method, lowers the cost of the test and results in a shortened analytical cycle time.

Claims

1. A method of examining the elements in an aliquot of blood, said method comprising:

placing an aliquot of blood containing element on a transparent surface;

allowing the elements to settle to the surface of the transparent surface; and

examining the aliquot from below the transparent surface.

2. The method of claim 1, wherein the aliquot is stained, diluted or a combination thereof.

3. The method of claim 1, wherein the transparent surface is a glass or plastic

4. The method of claim 3, wherein the transparent surface is a coverslip.

5. The method of claim 3, wherein the aliquot is a stable droplet formed by natural or external forces.

6. The method of claim 1, wherein the aliquot is examined with an inverted microscope or other similar optics.

7. The method of claim 6, wherein the aliquot is examined under illumination using at least one light source selected from the group consisting of reflected light, transmitted light or a combination thereof.

8. The method of claim 7, wherein the light source may include more than one source, is perpendicular or at an angle to the plane of the glass or a combination thereof.

9. The method of claim 7, wherein the examination is visual or completed using digital image analysis.

10. The method of claim 7, wherein the at least one light source may be selected from white light, monochromatic light of one or multiple wavelengths, from the ultraviolet through the infrared spectrum and polarized light.

11. The method of claim 7, wherein the transmitted light course is a column, an annulus or a single source from one angle.

12. The method of claim 7, wherein the light source is parallel, divergent or convergent.

13. The method of claim 7, wherein the combination of light sources is simultaneous or sequential.

14. The method of claim 7, wherein the angle of illumination may be continuously altered.

15. The method of claim 6, wherein the microscope having changeable lenses having smaller and larger magnification or smaller or larger depths of field.

16. The method of claim 1, wherein the blood sample has a hemoglobin content which may be determined by measuring an absorption or reflection of wavelengths of individual cells.

17. The method of claim 1, wherein the number of red blood cells, white blood cells and platelets may be counted.

18. The method of claim 1, wherein an hematocrit is determined.

19. The method of claim 1, wherein a white blood cell differential count and/or red blood cell morphology assessment is performed.

20. The method of claim 1, wherein abnormal white blood cell assessment is performed.

21. The method of claim 17, wherein red blood cell indices are obtained including a hemoglobin assessment.

22. The method of claim 17, wherein red blood cell indices are obtained including a red blood cell count.

23. The method of claim 17, wherein red blood cell indices are obtained including a hematocrit.

24. The method of claim 17, wherein the characteristic of the formed cellular elements of the blood may be determined visually directly through the optics or using digital images, or by automatically by analyzing digital images.

25. The method of claim 7, wherein the reflected light is darkfield illumination.