Method and device for identification of nucleated red blood cells from a maternal blood sample

• US 8,774,488 B2
• Filed: 03/11/2011
• Issued: 07/08/2014
• Est. Priority Date: 03/11/2010
• Status: Expired due to Fees

• Current Assignee: Cellscape Corporation
• Original Assignee: Cellscape Corporation
• Inventors: Parikh, Bhairavi, Brody, Michael D., Stone, James, Halderman, Jonathan D.
• Primary Examiner(s): CHEN, SHIN LIN
• Application Number:
  US13/046,543
• Publication Number:
  US 20110311960A1
• Time in Patent Office: 1,215 Days
• Field of Search: 382/129, 382/130, 382/133, 382/134, 424/130.1, 435/372, 435/968
• US Class Current: 382/134
• CPC Class Codes: C12N 5/0087 Purging against subsets of blood cells, e.g. purging alloreactive T cells
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

A method for concentrating and isolating nucleated cells, such as a maternal and fetal nucleated red blood cells (NRBC'"'"'s), in a maternal whole blood sample. The invention also provides methods and apparatus for preparing to analyze and analyzing the sample for identification of fetal genetic material as part of prenatal genetic testing. The invention also pertains to methods and apparatus for discriminating fetal nucleated red blood cells from maternal nucleated red blood cells obtained from a blood sample taken from a pregnant woman.

Claims

1. A method of identifying a nucleated fetal cell, the method comprising the steps of:

obtaining a maternal blood sample;

removing non-nucleated cells from the maternal blood sample to thereby create an enriched maternal blood sample;

adhering cells from the enriched maternal blood sample to a surface;

staining the cells with a nuclear stain to thereby produce a plurality of cells having stained nuclei;

generating a pair of images corresponding to the same cell wherein one image of the pair of images shows the stained nucleus of the cell;

applying an algorithm to the pair of images to determine that the shown cell is a nucleated red blood cell;

treating the nucleated red blood cell with a fetal identifier after the applying an algorithm step to thereby generate a treated cell; and

performing an analysis on the treated cell using the fetal identifier to identify the treated cell as a nucleated fetal cell.

2. The method of claim 1 wherein the analysis is selected from the group consisting of in situ hybridization and immunohistochemistry, the method further comprising scanning the surface with an automated microscope after the adhering step.

3. The method of claim 2 wherein performing comprises treating with an antibody selected from the group consisting of anti-zeta hemoglobin and anti-epsilon hemoglobin if the analysis is immunohistochemistry.

4. The method of claim 1 wherein generating a pair of images comprises generating a first image with transmitted illumination and a second image with coaxial illumination.

5. The method of claim 4 wherein generating a first image with transmitted illumination comprises generating a first image with transmitted illumination with a wavelength between about 380 nm and 800 nm.

6. The method of claim 4 wherein generating a first image with transmitted illumination and a second image with coaxial illumination comprises generating the second image with coaxial illumination with a wavelength between 350 nm and 364 nm.

7. The method of claim 1 further comprising selectively placing fetal identifiers on a plurality of candidate fetal cells.

8. The method of claim 1 wherein applying an algorithm comprises the steps of:

flattening at least one image;

segmenting at least one image and thereby defining foreground and background pixels;

removing background pixels from at least one image to generate a transformed image;

enumerating nuclei in the transformed image to generate enumerated nuclei; and

calculating at least one of complexity and brightness for at least one enumerated nuclei, wherein low complexity or high brightness indicates a fetal cell character.

9. The method of claim 1 wherein applying an algorithm comprises calculating a brightness of a background of the surface and a brightness of a foreground of the surface, and comparing a measurement of the pair of images to one or both of the background brightness and the foreground brightness.

10. The method of claim 1 further comprising fixing the sample with a non-crosslinking fixative before the performing an analysis step.

11. The method of claim 1 further comprising physically segregating an identified nucleated fetal cell from other cells adhered to the surface.

12. The method of claim 11, wherein physically segregating comprises microdissecting the nucleated fetal cell.

13. The method of claim 11, wherein physically segregating comprises picking up the nucleated fetal cell from surface.

14. The method of claim 1 wherein performing an analysis comprises using an anti-embryonic hemoglobin antibody to thereby identify the treated cell as a nucleated fetal cell.

15. The method of claim 1, wherein staining the cells comprises treating the cells with 4′

,6-diamidino-2-phenylindole (DAPI).

16. A method of identifying a nucleated fetal cell, the method comprising the steps of:

obtaining a maternal blood sample;

removing non-nucleated cells from the maternal blood sample to thereby create an enriched maternal blood sample;

creating a monolayer of cells from the enriched maternal blood sample on a surface;

staining the nuclei of the cells in the monolayer with a nuclear stain;

transmitting a hemoglobin absorbable light through the monolayer;

generating a pair of images showing a cell in a portion of the monolayer, wherein the first image shows the stained nucleus of said cell and a second image shows the absorbance of the hemoglobin absorbable light through said portion;

applying an algorithm to the pair of images to categorize said cell as a white cell, a non-nucleated red cell, or a nucleated red cell;

performing an analysis using a fetal identifier on said cell if the cell is categorized as a nucleated red blood cell by the algorithm; and

identifying said cell as a nucleated fetal red cell after the fetal identifier indicates the presence of fetal cellular material in said cell.

17. The method of claim 16 wherein generating a pair of images comprises generating a first image with transmitted illumination and a second image with coaxial illumination.

18. The method of claim 17, wherein generating a first image with transmitted illumination comprises generating a first image with transmitted illumination with a wavelength between about 380 nm and 800 nm.

19. The method of claim 17, wherein generating a first image with transmitted illumination comprises generating a first image with transmitted illumination with a wavelength around 420 nm.

20. The method of claim 16, wherein performing an analysis comprises selectively placing fetal identifiers on a plurality of portions of the surface, wherein each portion contains a candidate fetal cell.

21. The method of claim 16, wherein applying an algorithm comprises calculating a brightness of a background of the surface and a brightness of a foreground of the surface, and comparing a measurement of the pair of images to one or both of the background brightness and the foreground brightness.

22. The method of claim 16 further comprising storing a location of the pair of images.

23. The method of claim 22 further comprising locating a nucleated cell based on the stored location.

24. The method of claim 16, wherein the performing step comprises treating with a stabilizer.

25. The method of claim 16, wherein the applying an algorithm step comprises categorizing said cell as a nucleated red cell after the second image shows the absorbance of hemoglobin absorbable light coincident with the location of the stained nucleus shown in the first image.

26. The method of claim 16 further comprising physically segregating a cell categorized by the algorithm as a nucleated red blood cell from other cells in the monolayer.

27. The method of claim 26, wherein physically segregating comprises microdissecting the cell.

28. The method of claim 26, wherein physically segregating comprises picking up said cell from the monolayer.

29. The method of claim 16, wherein staining the nuclei comprises treating the cells with 4′

,6-diamidino-2-phenylindole (DAPI).

30. The method of claim 16 further comprising adjusting density of cells in the maternal blood sample before the creating a monolayer step.