Method, kit and system for imaging a blood sample

• US 10,093,957 B2
• Filed: 03/29/2016
• Issued: 10/09/2018
• Est. Priority Date: 07/01/2013
• Status: Active Grant

• Current Assignee: S.D. Sight Diagnostics Ltd
• Original Assignee: S.D. Sight Diagnostics Ltd
• Inventors: Pollak, Joseph Joel, Houri Yafin, Arnon, Salpeter, Seth J.
• Primary Examiner(s): Park, Edward
• Application Number:
  US15/083,610
• Publication Number:
  US 20160208306A1
• Time in Patent Office: 924 Days
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• 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 1/38 Diluting, dispersing or mixing samples
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• 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: G02B 21/0012 Surgical microscopes counterbalanced structures for surgical microscopes G02B7/001
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/34 Microscope slides, e.g. mounting specimens on microscope slides
• CPC Class Codes: G02B 21/361 Optical details, e.g. image relay to the camera or image sensor G02B21/364 takes precedence; illumination details G02B21/06 and subgroups
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Provided is a method for imaging a blood sample and a kit and system for executing the method. The method includes introducing a cell suspension including red blood cells onto a base surface of a carrier having a vertical height (H) being greater than or equal to a vertical depth (h) of the cell suspension when on the base carrier, the cell suspension including a cell concentration (C) being determined by a defined function; allowing the cells in the cell suspension to settle on the base surface of the carrier to form a monolayer of cells thereon; and acquiring at least one microscope image of at least a portion of the monolayer of cells; wherein the at least one microscope image is obtained by a microscope set to Depth Of Field that is not more than 20% of the vertical height of the cell suspension settled on the base surface.

Claims

1. A method comprising:

introducing a cell suspension comprising red blood cells, into a carrier that includes a base surface;

allowing the cells in the cell suspension to settle on the base surface of the carrier for a period of time that is at least 1 second per micrometer (μ

m) of a vertical height of the cell suspension when on the base surface, such as to form a monolayer of cells on the base surface of the carrier; and

acquiring at least one microscope image of at least a portion of the monolayer of cells, the at least one microscope image being obtained by a microscope set to Depth Of Field (DOF) that is not more than 20% of the vertical height of the cell suspension when settled on the base surface.

2. The method of claim 1, wherein the vertical height of the cell suspension when settled on the base surface is between 20 μ

m and 1000 μ

m.

3. The method of claim 2, wherein the at least one microscope image is obtained by a microscope set to a DOF that is between 0.5 μ

m and 10 μ

m.

4. The method of claim 1, wherein the at least one microscope image is obtained by a microscope set to a DOF that is not more than 15% of the vertical height of the cell suspension when settled on the base surface.

5. The method of claim 1, further comprising, prior to the introducing, diluting a blood sample comprising the cells by a dilution factor (D) to obtain the cell suspension.

6. The method of claim 5, wherein the dilution factor (D) is between 50 and 300.

7. The method of claim 1, further comprising forming the cell suspension by diluting a blood sample comprising red blood cells to obtain a cell concentration such that, after the cell suspension settles on the base surface, the density of the cells of the cell suspension is between about 10,000 and about 30,000 cells per mm².

8. The method of claim 7, wherein forming the cell suspension comprises forming the cell suspension such that, after the cell suspension settles on the base surface, the cell suspension forms a monolayer having an average base surface coverage of between 40 percent and 90 percent.

9. The method of claim 1, further comprising selecting a microscope objective lens that provides the DOF, wherein the lens permits acquiring an image of at least one object being no more than 3 μ

m long at any dimension thereof.

10. The method of claim 9, wherein selecting the microscope objective lens comprises selecting a lens that permits acquiring an image of at least one object having a height with respect to the base surface of no more than 3 μ

m.

11. The method of claim 1, further comprising analyzing the at least one image to determine based thereon presence or absence of a pathogen in the cell suspension.

12. The method of claim 11, wherein analyzing the at least one image to determine based thereon presence or absence of a pathogen in the cell suspension comprises analyzing the at least one image to determine based thereon presence or absence of a blood infecting protozoa in the cell suspension.

13. The method of claim 12, wherein the blood infecting protozoa is selected from the genus consisting of Trypanosoma, Plasmodium;

Toxoplasma and Babesia, and wherein analyzing the at least one image to determine based thereon presence or absence of the pathogen in the cell suspension comprises analyzing the at least one image to determine based thereon presence or absence of the blood infecting protozoa from the selected genus in the cell suspension.

14. The method of claim 1, wherein allowing the cells in the cell suspension to settle on the base surface of the carrier to form the monolayer of cells on the base surface of the carrier comprises allowing a period of time of less than 5 minutes for the cells to settle as a monolayer on the base surface.

15. The method of claim 1, wherein a vertical height of the carrier is between 20 μ

m and 300 μ

m.

16. The method of claim 1, wherein acquiring the at least one microscope image comprises acquiring a plurality of microscope images, at least two of which are provided under different conditions, the different conditions being selected from the group consisting of:

different portions of the base surface being imaged, and different illumination conditions being used.

17. The method according to claim 1, wherein introducing the cell suspension into the carrier comprises introducing the cell suspension into the carrier, the cell suspension having a concentration that is such that when substantially all of the cells in the cell suspension are allowed to settle on the base surface of the carrier to form the monolayer of cells, a monolayer is formed on the surface with substantially no overlap between the cells.

18. A method for imaging a blood sample, the method comprising:

introducing a cell suspension, onto a base surface of a carrier, wherein the cells are human cells comprising at least 75% red blood cells and at least a portion of the cells are suspected of being infected with a plasmodium parasite selected from the group consisting of Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium ovale (P. ovale), Plasmodium malariae (P. malariae), and Plasmodium knowlesi (P.knowlesi);

mixing the cells with one or more stains, the one or more stains including at least one fluorescent stain;

allowing the cells in the cell suspension to settle on the base surface of the carrier, such as to form a monolayer of cells on the base surface of the carrier; and

acquiring at least one microscope image of at least a portion of the monolayer of cells, the at least one microscope image being obtained by a microscope set to Depth Of Field (DOF) that is not more than 20% of a vertical height of the cell suspension when settled on the base surface, the acquiring of the at least one microscope image comprising acquiring a fluorescent image that is configured to facilitate detecting staining with the fluorescent stain.

19. A system for imaging a blood sample, the blood sample including human cells that include at least 75% red blood cells, at least a portion of the cells being suspected of being infected with a plasmodium parasite selected from the group consisting of Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium ovale (P. ovale), Plasmodium malariae (P. malariae), and Plasmodium knowlesi (P.knowlesi), the blood sample including a blood cell diluting agent, the system comprising:

one or more stains configured to be mixed with the blood sample, the one or more stains comprising at least one fluorescent stain;

a blood sample preparing unit configured;

to receive the blood sample, and

to facilitate forming therefrom a blood cell suspension, that is such that the cell suspension forms a monolayer of the cells when allowed to settle on a base surface of a carrier;

a microscope image acquisition unit configured to acquire at least one image of the blood cell suspension when on the base surface of the carrier, the at least one image including at least one fluorescent image that is configured to facilitate detecting of staining the fluorescent stain;

a controller configured to drive the microscope image acquisition unit to acquire the at least one microscope image of the cell suspension using a microscope set to a Depth Of Field (DOF) that is not more than 20% of a vertical height of the cell suspension when settled on the base surface.

20. The system of claim 19, wherein the controller is configured to drive the microscope image acquisition unit to acquire a plurality of microscope images of the monolayer, at least two of which are provided under different conditions, the different conditions being selected from the group consisting of:

different portions of the monolayer being imaged, and different illumination conditions being used.

21. The system according to claim 19, wherein the blood sample preparing unit is configured to facilitate forming a blood cell suspension, that is such that that when substantially all of the cells in the cell suspension are allowed to settle on the base surface of the carrier to form the monolayer of cells, a monolayer is formed on the base surface with substantially no overlap between the cells.

22. A system for imaging a blood sample comprising red blood cells and at least one blood cell diluting agent, the system comprising:

a blood sample preparing unit comprising a carrier having a base surface, the blood sample preparing unit being configured;

to receive the blood sample, and

to facilitate forming therefrom a blood cell suspension, that is such that the cell suspension forms a monolayer of the cells when allowed to settle on the base surface of the carrier, by allowing the cells in the cell suspension to settle on the base surface of the carrier for a period of time that is at least 1 second per micrometer (μ

m) of a vertical height of the cell suspension when on the base surface;

a microscope image acquisition unit configured to acquire at least one image of the blood cell suspension when on the base surface of the carrier;

a controller being configured to drive the microscope image acquisition unit to acquire at least one microscope image of the cell suspension by a microscope set to a Depth Of Field (DOF) that is not more than 20% of a vertical height of the cell suspension when settled on the base surface.