Determination of white blood cell differential and reticulocyte counts

• US 6,350,613 B1
• Filed: 02/18/1999
• Issued: 02/26/2002
• Est. Priority Date: 03/07/1998
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated, Becton, Dickinson & Co, Wardlaw Partners LP
• Original Assignee: Becton, Dickinson & Co
• Inventors: Wardlaw, Stephen C., Rodriguez, Rodolfo R., Levine, Robert A.
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US09/252,153
• Time in Patent Office: 1,104 Days
• Field of Search: 436/8, 436/10, 436/18, 436/63, 436/164, 436/165, 436/172, 436/174, 435/2, 435/4, 435/29, 435/30, 435/39, 422/55, 422/58, 422/99, 422/102, 356/39, 356/244, 356/246
• US Class Current: 436/10
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/0092 Monitoring flocculation or agglomeration
• CPC Class Codes: G01N 2015/014 Reticulocytes
• CPC Class Codes: G01N 2015/1472 with colour
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]

Abstract

Target nucleated cells, and target cells containing remnant ribosomal material, which are present in a quiescent anticoagulated whole blood sample are optically detected, enumerated, and analyzed in a sample chamber that has a varying through plane thickness due to convergent opposing sample chamber walls. At least one of the convergent walls of the chamber is transparent so that the blood sample can be observed. The chamber'"'"'s varying thickness produces a first lesser thickness region in the chamber wherein individual red cells and quiescent monolayers of red cells in the sample will reside after the sample is introduced into and fills the chamber. Larger formed constituents such as white blood cells and nucleated red blood cells present in the sample will reside in greater thickness regions of the chamber, and non-nucleated red cells which reside in such greater thickness regions will agglomerate to form rouleaux. By admixing fluorescent dyes with the blood sample, target cells in the sample can be enumerated and differentiated by means of a scanning instrument which is able to measure different wave length color signals emitted from the target cells in the sample, and differentiate the target cells one from another by reason of the nature of the emitted color signals.

Claims

1. A method for performing a differential white blood cell count in a sample of anticoagulated whole blood which is contained in a sampling chamber, said method comprising:

a) the step of providing an admixture of at least one fluorescent colorant and anticoagulated whole blood in said sampling chamber, said colorant being operable to differentially highlight different white blood cell types in the whole blood;

b) the step of allowing the admixture to disperse through said chamber so as to form red blood cell aggregates which are associated with quiescent groups of one or more individual white blood cells in optical working fields in the sample;

c) the step of optically performing a multi-field X-Y-Z scan of the dispersed admixture in said chamber under suitable lighting conditions that will cause different white blood cell types to be differentially highlighted by said colorant;

d) the step of photometrically and/or morphometrically separating different white blood cell sub-populations into distinct clusters, which sub-population clusters include lymphocytes, monocytes, granulocytes, eosinophils and basophils, so as to differentiate the sub-populations of white blood cells which are detected in said scanning step;

e) the step of enumerating and categorizing the white blood cells in each sub-population cluster; and

f) the step of deriving a differential count of each of the different sub-populations of white blood cells in each cluster produced by said separating step.

2. The method of claim 1, wherein said chamber is provided with a varying through-plane, or Z axis, thickness which varies from a minimum of about zero to about ten microns to a maximum of about twenty to about fifty microns in the area of said chamber in which said scanning steps are performed.

3. A method for performing a differential nucleated blood cell count in a sample of anticoagulated whole blood which is contained in a sampling chamber having a varying through plane thickness, said method comprising:

a) the step of providing an admixture of at least one fluorescent colorant and anticoagulated whole blood in said sampling chamber, said colorant being operable to differentially highlight different nucleated blood cell types in the whole blood;

b) the step of optically performing a multi-field X-Y-Z scan of the admixture in said chamber under suitable lighting conditions that will cause different nucleated cell types to be differentially highlighted by said colorant;

c) the step of photometrically and/or morphometrically separating at least one nucleated blood cell sub-population into a distinct cluster, wherein said at least one sub-population cluster is one of lymphocytes, monocytes, granulocytes, eosinophils or basophils, so as to differentiate that sub-population of nucleated blood cells from other nucleated cells which are detected in said scanning step;

d) the step of enumerating and categorizing the nucleated blood cells in said nucleated blood cell sub-population cluster; and

e) the step of producing a cell count of at least one of the sub-populations of nucleated blood cells in at least one cluster produced by said separating step.

4. A method for performing a reticulocyte count in a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with one or more fluorescent colorants that are operable to differentially highlight remnants of nucleated material in the reticulocytes in the blood sample, said blood sample and colorants being contained in a viewing chamber having varying thickness regions therein, said method comprising:

a) the step of scanning the blood sample with an optical instrument having a field of view;

b) the step of locating fields of view in said quiescent blood sample which fields of view contain individual red cells or monolayers of red cells;

c) the step of illuminating said located fields of view in the quiescent blood sample with selected wavelengths of light so as to differentially highlight any remnants of nucleated material in red cells in said located fields of view; and

d) the step of enumerating any differentially highlighted red cells in said fields of view.

5. A method for performing a differential white blood cell count on a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with one or more fluorescent colorants that are operable to differentially highlight nucleated material in the white cells in the blood sample, said blood sample and colorants being contained in a viewing chamber having varying thickness regions therein, said method comprising:

a) the step of scanning the blood sample with an optical instrument having a field of view;

b) the step of locating and scanning fields of view in said quiescent blood sample which fields of view contain individual white blood cells and aggregates of red blood cells, and which fields of view have a viewing chamber through plane thickness of about fifty microns or less;

c) the step of illuminating said fields of view in the quiescent blood sample with selected wavelengths of light so as to differentially highlight any nucleated material in white blood cells in said located fields of view; and

d) the step of categorizing and enumerating all differentially highlighted white blood cells in said located and scanned fields of view so as to obtain differential white blood cell counts, said categorizing step being based on differences in light emissions emanating from said differentially highlighted white blood cells.

6. The method of claim 5 wherein said colorant is operable to differentiate types of white cells one from another by reason of different signal characteristics emanating from various types of white cells, whereby a differential white cell count can be derived from said blood sample.

7. The method of claim 5 wherein said categorizing step involves the use of both photometric and morphometric cell information obtained from said differences in light emissions.

8. A method for performing a differential white cell count in a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with one or more fluorescent colorants that are operable to differentially highlight intracellular dye-binding material in the white cells in the blood sample, said blood sample and colorants being contained in a viewing chamber having varying through plane thickness regions therein, said method comprising:

a) the step of locating usable fields of view having a through plane thickness of less than about fifty microns in said quiescent blood sample which contain at least a predetermined number of individual white blood cells which are separated from aggregates of red blood cells;

b) the step of illuminating said usable fields of view with at least one predetermined wavelength of light that will differentially highlight intracellular dye-binding material contained in white blood cells and will cause illuminated white blood cells to emit light;

c) the step of measuring emitted light from said illuminated white blood cells in two or more different wavelengths; and

d) the step of analyzing said different wavelengths of emitted light in a manner which will differentiate types of white blood cells from each other.

9. The method of claim 8 wherein said differentiating step is performed by using photometric information and/or morphometric information derived from said emitted light, and further including the step of counting such differentiated types of white blood cells.

10. A method for identifying at least one sub-population of target cells containing nucleated material or containing intracellular remnants of nucleated material, which target cells are contained in a substantially undiluted quiescent sample of anticoagulated whole blood that is admixed with a fluorescent colorant, and which is dispersed in a viewing chamber-that includes a region that contains individually separated cells, said region having a through plane thickness that is in the range of about zero microns to about forty microns, said method comprising:

a) the step of optically scanning selected fields of view in said region which fields contain individual red blood cells, and/or monolayers of red blood cells, and/or red blood cell aggregates;

b) the step of illuminating said selected fields of view with a light source of one or more preselected wavelengths operable to cause said fluorescent colorant to produce nucleated material or remnant nucleated material fluorescent emissions of a plurality of known wavelengths, which emissions are characteristic of sub-populations of cells; and

c) the step of analyzing said emissions at said plurality of known wavelengths so as to identify the sub-populations of cells in the fields of view.

11. The method of claim 10 wherein the viewing chamber includes additional regions which have a through plane thickness which is greater than about forty microns.

12. The method of claim 10 wherein the colorant is a supravital stain.

13. The method of claim 10 wherein the sub-population of target cells is a sub-population of white blood cells.

14. The method of claim 10 wherein the sub-population of target cells is a sub-population of red blood cells.

15. The method of claim 10 wherein the colorant is part of a binding particle that is directed against an epitope on the sub-population of target cells.

16. The method of claim 10 wherein said emissions are characterized by photometric and/or morphometric characteristics which can be used to identify the sub-population in question.

17. A method for identifying at least one sub-population of nucleated blood cells which are contained in a substantially undiluted quiescent sample of anticoagulated whole blood that is admixed with a fluorescent colorant and is dispersed in a viewing chamber that includes a region that contains aggregates of red blood cells, and individually separated nucleated cells, said region having a through plane thickness which is no greater than about fifty microns, said method comprising:

a) the step of scanning selected fields of view in said region with an optical scanning instrument;

b) the step of illuminating said selected fields of view with a light source of a preselected wavelength that is operable to cause said fluorescent colorant to produce fluorescent emissions at a plurality of known wavelengths, which emissions are characteristic of sub-populations of nucleated cells; and

c) the step of analyzing said emissions at said plurality of known wavelengths so as to identify any sub-populations of nucleated cells in the fields of view.

18. A method of identifying sub-populations of nucleated cells contained in a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with a fluorescent colorant and which is dispersed in a viewing chamber that includes a region that contains individually separated nucleated cells, said region having a through plane thickness that is no greater than about fifty microns, said method comprising:

a) the step of scanning selected fields of view in said region with an optical scanning instrument;

b) the step of illuminating said selected fields of view with a light source of a preselected wavelength that is operable to cause said fluorescent colorant to produce fluorescent emissions at a plurality of known wavelengths, which emissions are characteristic of a sub-population of nucleated cells; and

c) the step of analyzing said emissions at said plurality of known wavelengths so as to identify the sub-population of nucleated cells in the fields of view.

19. A method of identifying target sub-populations of nucleated cells contained in a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with a fluorescent colorant, and which is dispersed in a viewing chamber that includes a region that contains individual red blood cells, and/or monolayers of red blood cells, and/or aggregates of red blood cells, and which region has a through plane thickness that is no greater than about fifty microns, said method comprising:

a) the step of scanning selected fields of view in said region with an optical scanning instrument;

b) the step of illuminating said selected fields of view with a light source of one or more preselected wavelengths that are operable to cause said fluorescent colorant to produce fluorescent emissions at one or more known wavelengths, which emissions are characteristic of said target sub-populations of nucleated cells; and

c) the step of analyzing said emissions at said known wavelengths so as to identify and differentiate said target sub-populations of nucleated cells.

20. The method of claim 19 wherein said viewing chamber is substantially wedge-shaped and includes additional regions which have through plane thicknesses that are greater than about forty microns.

21. A method of identifying reticulocyte cells contained in a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with a fluorescent colorant, and which is dispersed in a viewing chamber that includes a region that contains individual red blood cells, and/or monolayers of red blood cells, and which region has a through plane thickness that is no greater than about twenty microns, said method comprising:

a) the step of scanning selected fields of view in said region with an optical scanning instrument;

b) the step of illuminating said selected fields of view with a light source of one or more preselected wavelengths that are operable to cause said fluorescent colorant to produce fluorescent emissions at one or more known wavelengths, which emissions are characteristic of remnants of nucleated material contained in said reticulocyte cells; and

c) the step of analyzing said emissions at said known wavelengths so as to identify and differentiate said reticulocyte cells from other cells in said selected fields of view in the sample.

22. A method for performing a reticulocyte count in a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with one or more fluorescent colorants that are operable to differentially highlight remnants of nucleated material in the reticulocytes in the blood sample, said blood sample and colorants being contained in a viewing chamber, said method comprising:

a) the step of scanning the blood sample with an optical instrument having a field of view;

b) the step of locating fields of view in said quiescent blood sample which fields of view contain individual red cells or monolayers of red cells;

c) the step of illuminating said located fields of view in the quiescent blood sample with selected wavelengths of light so as to differentially highlight any remnants of nucleated material in red cells in said located fields of view; and

d) the step of enumerating any differentially highlighted red cells in said fields of view.

23. A method of identifying target sub-populations of cells or other formed bodies contained in a substantially undiluted quiescent sample of anticoagulated whole blood which is admixed with a fluorescent colorant, and which is dispersed in a viewing chamber that includes a region that contains individual red blood cells, and/or monolayers of red blood cells, and/or aggregates of red blood cells, said method comprising:

a) the step of scanning selected fields of view in said region with an optical scanning instrument, which selected fields of view have a through plane thickness of about fifty microns or less;

b) the step of illuminating said selected fields of view with a light source of one or more preselected wavelengths that are operable to cause said fluorescent colorant to produce fluorescent emissions at one or more known wavelengths, which emissions produce photometric and morphometric information which are operable to differentiate said target sub-populations of cells and other formed bodies; and

c) the step of analyzing said emissions so as to identify and differentiate said target sub-populations of cells and formed bodies both photometrically and morphometrically.