Method and apparatus for automated identification and enumeration of specified blood cell subclasses

• US 4,284,412 A
• Filed: 07/13/1979
• Issued: 08/18/1981
• Est. Priority Date: 07/13/1979
• Status: Expired due to Term

• Current Assignee: Ortho Diagnostics, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Original Assignee: Ortho Diagnostics, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Inventors: Hoffman, Robert A., Hansen, W. Peter
• Primary Examiner(s): Serwin, Ronald
• Application Number:
  US06/057,482
• Time in Patent Office: 767 Days
• Field of Search: 23/230 B, 23/915, 424/8, 424/12, 356/36, 356/39, 250/565, 250/432 R, 250/458, 250/459, 250/461 B, 250/574, 422/81, 422/82, 435/7
• US Class Current: 435/7.24
• CPC Class Codes: G01N 15/042 by centrifuging and investigating centrifugates centrifuges per se B04B
• CPC Class Codes: G01N 15/05 in blood
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/011 with lysing, e.g. of erythrocytes
• CPC Class Codes: G01N 2015/016 White blood cells
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

Specified subclasses of blood cells, such as of lymphocytes, are automatically identified based on utilization of antigenic determinants on the cell surface, their reactivity with antibodies which fluoresce under known circumstances, and utilization of principles of flow cytometry. A blood sample is first incubated with a reagent including antibodies to the lymphocyte subclass to be identified, the antibodies being directly or indirectly made fluorescently responsive to particular light (e. g. argon ion laser). The sample is illuminated, a cell at a time, by such focused coherent light, and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify and enumerate cells of the specified subclass.

Claims

1. An automated method of identifying and enumerating cells of a select subclass of lymphocytes in blood comprising the steps of:

(a) providing an aliquot from the blood to be studied;

(b) selectively tagging cells of said subclass by incubating said aliquot with an antibody which is selectively reactive with distinct antigenic determinants on the surface of cells of said subclass, said antibody having a predetermined fluorescence response to a given optical stimulation;

(c) lysing red cells from said aliquot;

(d) passing said aliquot, substantially a cell at a time, through an area of focused optical stimulation of said given type, while detecting light scattered by and emitted from said cells; and

(e) differentiating cells of said subclass based at least in part on occurrence of said predetermined fluorescence response in said detected light.

2. A method as defined in claim 1 wherein said providing step comprises providing anticoagulated but otherwise substantially unprocessed whole blood.

3. A method as defined in claim 1 wherein said providing step comprises providing a sample of whole blood, centrifuging said sample for a predetermined period of time, isolating the buffy coat layer from said sample, and providing said isolated buffy coat layer as said aliquot.

4. A method as described in claim 1, adapted for identification and enumeration of specified subclasses of T-type lymphocytes, wherein said tagging step comprises incubating said aliquot with anti-said subclass antibody which fluoresces within known intensity and frequency ranges when irradiated by optical stimulation of given wavelengths.

5. A method as described in claim 4, wherein said passing step comprises providing the focus of an argon laser at said area.

6. A method as described in claim 1, wherein said passing step further comprises:

providing respective reference ranges for forward light scatter and right angle scatter;

detecting forward and right angle scatter pulses for a given cell;

comparing respective detected pulses with corresponding reference ranges;

enabling, when both said detected pulses are within their said corresponding ranges, detection of right angle fluorescence of said predetermined response.

7. A method as described in claims 1 or 4 wherein said tagging step comprises incubating said aliquot with a quantity of said antibody which has previously been associated with a fluorescing dye agent.

8. A method as described in claims 1 or 4 wherein said tagging step comprises first incubating said aliquot with a nonfluorescently responsive antibody, then further incubating said aliquot with an antibody reagent which has said predetermined fluorescence response and which is reactive with nonfluorescently responsive antibody.

9. Apparatus for identification and enumeration of specified T-lymphocytes in a sample which has been incubated with a fluorescent responsive antibody to select antigenic determinant on the surface of said specified T-lymphocytes comprising:

(a) a flow cytometry flow cell for passing cells of said sample, rapidly and substantially one at a time, through a given area;

(b) a source of light which stimulates fluorescence in said antibody;

(c) means for coupling and focusing light from said source to cell flow at said given area;

(d) first photosensing means for detecting forward light scatter from said means for coupling and dispersed from said area by a cell passing therethrough;

(e) second photosensing means for detecting light scattered by said cell on an axis substantially normal to the axis of cell flow;

(f) third photosensing means, for sensing fluorescent light pulses;

(g) means for providing respective reference ranges for forward light scatter and right angle light scatter;

(h) means for comparing light pulses detected by said first and second photosensing means with associated ranges from means for providing; and

(i) means, responsive to said means for providing, for selectively enabling said third photosensing means when said detected light pulses occur within associated reference ranges; and

(j) means for displaying fluorescent light pulses as indicia of occurrence of said specified T-lymphocyte cells at said area.

10. Apparatus as described in claim 9 and further including means, responsive to said means for selectively enabling, for compensating signals detected by said third photosensing means to account for pre-selected spurious fluorescence effect, said means for displaying being responsive in turn to said means for compensating.

11. Apparatus as described in claim 9 wherein said means for coupling and focusing is arranged to deliver light from said source substantially orthogonally to cell flow at said given area.

12. Apparatus as described in claim 9 wherein said source of light comprises an argon ion laser, and wherein said third photosensing means comprises means for sensing green fluorescent light pulses.

13. Apparatus as described in claim 12 wherein said third photosensing means is adapted to sense said green light pulses along said normal axis.

14. Apparatus for identification and enumeration of a specified subclass of blood cells in a sample which has been incubated with a fluorescent responsive antibody to a select antigenic determinant on the surface of said specified subclass of blood cells comprising:

(a) a flow cytometry flow cell for passing cells of said sample, rapidly and substantially one at a time, through a given area;

(b) a source of light which stimulates fluorescence in said antibody;

(c) means for coupling and focusing light from said source to cell flow at said given area;

(d) first photosensing means for detecting forward light scatter from said means for coupling and dispersed from said area by a cell passing therethrough;

(e) second photosensing means for detecting light scattered by said cell on an axis substantially normal to the axis of cell flow;

(f) third photosensing means, for sensing fluorescent light pulses;

(g) means for providing respective reference ranges for forward light scatter and right angle light scatter;

(h) means for comparing light pulses detected by said first and second photosensing means with associated ranges from means for providing; and

(i) means, responsive to said means for providing, for selectively enabling said third photosensing means when said detected light pulses occur within associated reference ranges; and

(j) means for displaying fluorescent light pulses as indicia of occurrence of said specified subclass of blood cells at said area.