Apparatus and method for volumetric capillary cytometry

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.

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.

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.

An apparatus for making volumetric fluorescence measurements of a cell suspension, the apparatus comprising:

• a capillary tube of rectangular cross-section with a shorter dimension of the cross-section defining a depth of said capillary tube and a longer dimension of the cross-section defining a width of said capillary tube, said capillary tube containing a cell suspension therein;
• optical scanning means for generating an incident beam of a wavelength selected to excite the cell suspension of the capillary tube, the incident beam sequentially intersecting the capillary tube in a plurality of beam spots of specified diameter to illuminate a plurality of columnar regions;
• detection means having a light sensitive member for detection of fluorescent emission;
• collection means for gathering of fluorescent emission from the columnar region and for directing the fluorescent emission to said detection means;
• a spatial filter with a specified pinhole aperture for allowing detection of fluorescent light emitted from the entire columnar region, said spatial filter disposed between said collection means and said detection means, so as to confine a depth of detection to an interior depth dimension of said capillary tube;
• and
• a data reader in communication with said detection means for recordation of fluorescent emission signals.

An apparatus for the detection of subclasses of blood cells, the apparatus comprising:

• a transparent capillary having a known rectangular cross-sectional area and having a known depth, thereby providing a known volume, wherein the depth substantially exceeds ten microns;
• means for generating a beam of light having an excitation wavelength of at least 600 nanometers and configured to impinge upon the upper surface of said capillary so as to provide a beam spot having a diameter of at least five microns;
• means for providing motion of the beam of light relative to said capillary for causing the beam of light to impinge upon said capillary in a plurality of beam spots so as to illuminate within the capillary tube a plurality of columnar regions of known volume;
• a light collector positioned proximate said capillary and configured to gather a fluorescent emission caused by said means for generating a beam of light and further configured to transmit the fluorescent emission in a retrobeam;
• a light detector responsive to the fluorescent emission, said detector being positioned away from said capillary and positioned to be impinged by the retrobeam from said light collector;
• and
• a spatial filter positioned between said light collector and said detector, the spatial filter disposed to intercept the retrobeam and configured to admit only a portion of the retrobeam to said detector so as to limit light detected to a single columnar region illuminated by a single beam spot and confined to an interior depth dimension of each columnar region.