Apparatus and method for volumetric capillary cytometry

Claim 3

A method as in claim 1, further comprising diluting the sample after said incubating step and prior to said placing the sample into a capillary tube step.

Claim 4

A method as in claim 1 wherein optically scanning the sample comprises intersecting a horizontally positioned capillary tube with an incident beam positioned directly above the capillary tube.

Claim 5

A method as in claim 1 wherein optically scanning the sample comprises scanning with an incident beam of a wavelength of 600 to 1000 nanometers.

Claim 6

A method as in claim 1 wherein reacting the sample with a fluorescently-labeled binding agent comprises using a binding agent labeled with a fluorophore that is activated at an excitation wavelength in the range of 600 to 1000 nanometers.

Claim 7

A method as in claim 1 wherein optically scanning the sample comprises scanning with an incident beam that is 5 to 15 microns in diameter at an intersection of the incident beam and the capillary tube.

Claim 8

A method as in claim 1 wherein placing the sample in a capillary tube comprises placing the sample in a capillary tube of rectangular cross-section with a shorter dimension of the cross-section defining a depth of the capillary tube and a longer dimension of the cross-section defining a width of the capillary tube.

Claim 9

A method as in claim 1 wherein placing the sample in a capillary tube comprises placing the sample in a capillary tube having a depth dimension of 25 to 225 microns.

Claim 10

A method as in claim 1 wherein optically scanning the sample comprises traversing a path in a first direction transverse to the longitudinal axis of the capillary tube and a second direction along the longitudinal axis of the capillary tube.

Claim 11

A method as in claim 9 wherein optically scanning the sample in a first direction transverse to the longitudinal axis of the capillary tube comprises traversing a scan path that originates and terminates at points beyond respective outer boundaries of the capillary tube.

Claim 12

A method as in claim 1 wherein optically scanning the sample comprises scanning a capillary tube that is in a fixed position.

Claim 13

A method as in claim 1 further comprising determining a concentration of a cellular component of the sample by calculating an amount of enumerated component per specified volume of the capillary tube, the volume being determined by origination and termination points of a scan in a direction along the longitudinal axis of the capillary tube and a cross-sectional area of the capillary tube.

Claim 14

A method as in claim 1 further comprising determining ratios of different cellular components of the sample by calculating an amount of each enumerated component and comparing relative amounts of each enumerated component.

Claim 15

An assay for identifying and enumerating leukocyte subclasses in whole blood via fluorescence, the assay comprising; obtaining a sample of whole uncoagulated blood; incubating the sample with an excess amount of a fluorescently-labeled antibody directed to specific cell surface markers of a leukocyte subclass within the sample to form fluorescent complexes; placing the sample into a capillary tube having specified dimensions; optically scanning the sample with an incident beam of light having a wavelength selected to excite the fluorescent complexes and to minimize interference from red blood cells present in the sample, the incident beam sequentially intersecting the capillary tube in a plurality of beam spots of specified diameter to illuminate a plurality of columnar regions; sequentially detecting emitted fluorescence confined to an interior depth dimension of each columnar region; recording the emitted fluorescence from each columnar region of heightened fluorescence intensity; and enumerating a subclass of leukocytes by determining a presence of the subclass in the sample from the emitted fluorescence.

Claim 16

An assay for identifying and enumerating leukocyte subclasses in whole blood via fluorescence, the assay comprising; obtaining a sample of whole uncoagulated blood; incubating the sample with an excess amount of a fluorescently-labeled antibody directed to specific cell surface markers of a leukocyte subclass within the sample to form fluorescent complexes; placing the sample into a capillary tube having specified dimensions; optically scanning the sample with an incident beam of light having a wavelength selected to excite the fluorescent complexes and to minimize interference from red blood cells present in the sample, the incident beam sequentially intersecting the capillary tube in a plurality of beam spots of specified diameter to illuminate a plurality of columnar regions; sequentially detecting emitted fluorescence confined to an interior depth dimension of each columnar region; recording the emitted fluorescence from each columnar region of heightened fluorescence intensity; and enumerating a subclass of leukocytes by determining a presence of the subclass in the sample from the emitted fluorescence.

Claim 17

An assay as in claim 14, further comprising diluting the sample after said incubating step and prior to said placing the sample into a capillary tube step.

Claim 18

An assay as in claim 14 wherein incubating the sample with a fluorescently-labeled antibody comprises incubating with a plurality of fluorescently-labeled antibodies, each antibody directed to a different specific cell surface marker of a leukocyte subclass, and each antibody labeled with a different fluorophore having different optical characteristics.

Claim 19

A scanning imaging cytometer for non-flowing fluids in a capillary tube, the cytometer comprising: a transparent capillary tube containing a non-flowing fluid having complexes of cells and fluorescently-labeled binding agent in suspension with free fluorescently-labeled binding agent, said capillary tube having outer and inner walls; a beam of light impinging upon the outer wall of said capillary tube transverse to a longitudinal axis of said capillary tube in a beam spot having a first diameter illuminating a columnar region of the fluid, said beam of light having an excitation wavelength for stimulating fluorescent emission from the complexes and the free binding agent; a light detector spaced apart from said capillary tube and responsive to the fluorescent emission; a wide angle light collector positioned proximate said capillary tube, said light collector configured to gather fluorescent emission from the illuminated columnar region and to transmit the fluorescent emission towards said light detector in a retrobeam; a spatial filter having a pinhole aperture, said spatial filter positioned between said light collector and said detector, the pinhole aperture disposed to intercept the retrobeam and having a second diameter admitting only a portion of the retrobeam to said detector, the second diameter of the pinhole aperture substantially exceeding the first diameter of the beam spot, so as to confine a depth of detection to an interior depth dimension of said capillary tube; and means for providing motion of said beam of light relative to said capillary tube to cause said beam of light to sequentially impinge upon said capillary tube in a plurality of beam spots, so as to illuminate within the capillary tube a plurality of columnar regions whose total volume can be determined.

Claim 20

A scanning imaging cytometer for non-flowing fluids in a capillary tube, the cytometer comprising: a transparent capillary tube containing a non-flowing fluid having complexes of cells and fluorescently-labeled binding agent in suspension with free fluorescently-labeled binding agent, said capillary tube having outer and inner walls; a beam of light impinging upon the outer wall of said capillary tube transverse to a longitudinal axis of said capillary tube in a beam spot having a first diameter illuminating a columnar region of the fluid, said beam of light having an excitation wavelength for stimulating fluorescent emission from the complexes and the free binding agent; a light detector spaced apart from said capillary tube and responsive to the fluorescent emission; a wide angle light collector positioned proximate said capillary tube, said light collector configured to gather fluorescent emission from the illuminated columnar region and to transmit the fluorescent emission towards said light detector in a retrobeam; a spatial filter having a pinhole aperture, said spatial filter positioned between said light collector and said detector, the pinhole aperture disposed to intercept the retrobeam and having a second diameter admitting only a portion of the retrobeam to said detector, the second diameter of the pinhole aperture substantially exceeding the first diameter of the beam spot, so as to confine a depth of detection to an interior depth dimension of said capillary tube; and means for providing motion of said beam of light relative to said capillary tube to cause said beam of light to sequentially impinge upon said capillary tube in a plurality of beam spots, so as to illuminate within the capillary tube a plurality of columnar regions whose total volume can be determined.

Claim 21

A scanning imaging cytometer as in claim 17 wherein the fluorescently-labeled binding agent contains a fluorophore that is activated at an excitation wavelength in the range of 600 to 1000 nanometers.

Claim 22

A scanning imaging cytometer as in claim 17 wherein the binding agent is an antibody.

Claim 23

A scanning imaging cytometer as in claim 17 wherein the binding agent is specific to cell surface markers.

Claim 24

A scanning imaging cytometer as in claim 17 wherein the binding agent is an intercalating dye.

Claim 25

A scanning imaging cytometer as in claim 17 wherein the binding agent is a nucleic acid probe.

Claim 26

A scanning imaging cytometer as in claim 17 wherein the binding agent reacts with specific receptors.

Claim 27

A scanning imaging cytometer as in claim 17 wherein the beam of light impinging upon the outer wall of the capillary tube passes through a central portion of the wide angle light collector and then impinges on the outer wall of the capillary tube and is focused into an interior depth of the capillary tube.

Claim 28

A scanning imaging cytometer as in claim 17 further comprising particle counting means associated with the detector for distinguishing background fluorescence associated with free binding agent from fluorescence associated with complexes.

Claim 29

A scanning imaging cytometer as in claim 17 wherein the means for providing motion causes the plurality of spots to partially overlap each other.

Claim 30

A scanning imaging cytometer as in claim 17 wherein the means for providing motion causes the said beam of light to overscan lateral edges of the capillary tube.

Claim 31

A scanning imaging cytometer as in claim 17 wherein the means for providing motion causes the said beam of light to sequentially impinge upon the capillary tube in a two-dimensional array of spots, illuminating a two-dimensional array of columnar regions whose total volume can be determined.

Claim 32

A scanning image cytometer as in claim 17, wherein said light detector comprises a spectral dispersion device positioned between said spatial filter and first and second light sensitive members, the spectral dispersion device configured for separating a plurality of fluorescent emission wavelengths and directing the separated wavelengths to the first and second light sensitive members.

Claim 33

A scanning imaging cytometer as in claim 17 wherein the volume probed is determined by a known cross-sectional area of the capillary tube and a distance that has been scanned along the capillary tube.

Claim 34

A scanning imaging cytometer as in claim 17 wherein the volume is determined by a known cross-sectional area of the capillary tube and by providing specific identification marks on the capillary tube to define distance.

Claim 35

A scanning imaging cytometer as in claim 17 wherein the beam is focused to a location within the capillary tube.

Claim 36

A scanning imaging cytometer as in claim 17 wherein the beam of light has a wavelength in the range of 600 to 1000 nanometers.

Claim 37

A scanning imaging cytometer for non-flowing fluids in a capillary tube, the cytometer comprising: a transparent capillary tube containing a non-flowing fluid having complexes of cells and fluorescently-labeled binding agent, the capillary having outer and inner walls; a beam of light impinging upon the outer wall of said capillary tube transverse to a longitudinal axis of said capillary tube in a beam spot having a first diameter illuminating a columnar region of the fluid, said beam of light having an excitation wavelength for stimulating fluorescent emission from the complexes; a light detector spaced apart from said capillary tube and responsive to the fluorescent emission; a wide angle light collector positioned proximate said capillary tube, said light collector configured to gather fluorescent emission from the illuminated columnar region and to transmit the fluorescent emission towards said light detector in a retrobeam; a spatial filter having a pinhole aperture, said spatial filter positioned between said light collector and said detector, the pinhole aperture disposed to intercept the retrobeam and having a second diameter admitting most of the retrobeam to said detector, the second diameter of the pinhole aperture substantially exceeding the first diameter of the beam spot, so as to confine a depth of detection to an interior depth dimension of said capillary tube; and means for providing motion of said beam of light relative to said capillary tube to cause said beam of light to sequentially impinge upon said capillary tube in a plurality of beam spots, so as to illuminate within the capillary tube a plurality of columnar regions whose total volume can be determined.

Claim 38

A scanning imaging cytometer for non-flowing fluids in a capillary tube, the cytometer comprising: a transparent capillary tube containing a non-flowing fluid having complexes of cells and fluorescently-labeled binding agent, the capillary having outer and inner walls; a beam of light impinging upon the outer wall of said capillary tube transverse to a longitudinal axis of said capillary tube in a beam spot having a first diameter illuminating a columnar region of the fluid, said beam of light having an excitation wavelength for stimulating fluorescent emission from the complexes; a light detector spaced apart from said capillary tube and responsive to the fluorescent emission; a wide angle light collector positioned proximate said capillary tube, said light collector configured to gather fluorescent emission from the illuminated columnar region and to transmit the fluorescent emission towards said light detector in a retrobeam; a spatial filter having a pinhole aperture, said spatial filter positioned between said light collector and said detector, the pinhole aperture disposed to intercept the retrobeam and having a second diameter admitting most of the retrobeam to said detector, the second diameter of the pinhole aperture substantially exceeding the first diameter of the beam spot, so as to confine a depth of detection to an interior depth dimension of said capillary tube; and means for providing motion of said beam of light relative to said capillary tube to cause said beam of light to sequentially impinge upon said capillary tube in a plurality of beam spots, so as to illuminate within the capillary tube a plurality of columnar regions whose total volume can be determined.

Claim 41

An apparatus as in claim 35 wherein the capillary tube is positioned horizontally and the incident beam of the optical scanning means intersects the capillary tube from a position directly above the capillary tube.

Claim 42

An apparatus as in claim 35 wherein the wavelength of the incident beam is 600 to 1000 nanometers.

Claim 43

An apparatus as in claim 35 wherein the diameter of the beam spot is in the range of 5 to 15 microns.

Claim 44

An apparatus as in claim 35, wherein the capillary tube has a depth in the range of 25 to 225 microns.

Claim 45

An apparatus as in claim 35 wherein the incident beam of the optical scanning means traverses a path in a first direction along the width of the capillary tube and a second direction along a longitudinal axis of the capillary tube.

Claim 46

An apparatus as in claim 35 wherein the optical scanning means traverses a path in a direction along the width of the capillary tube and the path originates and terminates at points beyond the respective outer boundaries of the capillary tube.

Claim 47

An apparatus as in claim 35, wherein said detection means comprises a spectral dispersion device positioned between said spatial filter and first and second light sensitive members, the spectral dispersion device configured for separating a plurality of fluorescent emission wavelengths and directing the separated wavelengths to the first and second light sensitive members.

Claim 48

An apparatus as in claim 35 further comprising a lens, a central portion of the lens being used by the optical scanning means for passage of the incident beam before intersection of the incident beam with the capillary tube, and the entire lens being used by the collection means for gathering of fluorescent emission from the capillary tube.

Claim 51

The apparatus of claim 44, wherein the depth of said capillary is in the range of 25 to 225 microns.

Claim 52

The apparatus of claim 45, wherein the excitation wavelength of the beam of light from said means for generating is in the range of 600 to 1000 nanometers.

Claim 53

The apparatus of claim 46, wherein the beam spot has a diameter in the range of five to fifteen microns.