US3819270A - Blood cell analyzer - Google Patents

Blood cell analyzer Download PDF

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US3819270A
US3819270A US00294245A US29424572A US3819270A US 3819270 A US3819270 A US 3819270A US 00294245 A US00294245 A US 00294245A US 29424572 A US29424572 A US 29424572A US 3819270 A US3819270 A US 3819270A
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radiation
cell
optical system
improvement
detectors
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T Hirschfeld
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Bio Rad Laboratories Inc
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Block Engineering Inc
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Priority to US00294245A priority Critical patent/US3819270A/en
Priority to GB4343073A priority patent/GB1424388A/en
Priority to AU60723/73A priority patent/AU477425B2/en
Priority to FR7334853A priority patent/FR2201064B1/fr
Priority to DE19732349271 priority patent/DE2349271A1/de
Priority to CA182,299A priority patent/CA984175A/en
Priority to CH1401273A priority patent/CH573600A5/xx
Priority to NL7313531A priority patent/NL7313531A/xx
Priority to JP48110908A priority patent/JPS505098A/ja
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Publication of US3819270A publication Critical patent/US3819270A/en
Assigned to BIO-RAD LABORATORIES, INC., A CORP. OF DE. reassignment BIO-RAD LABORATORIES, INC., A CORP. OF DE. MERGER (SEE DOCUMENT FOR DETAILS). EFFECTIVE DATE: 01/04/79 DELAWARE Assignors: BLOCK ENGINEERING, INC.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1497—Particle shape
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N2021/4704—Angular selective
    • G01N2021/4711—Multiangle measurement
    • G01N2021/4716—Using a ring of sensors, or a combination of diaphragm and sensors; Annular sensor

Definitions

  • ABSTRACT typically as a cone of radiation having a solid angle of much greater than 90. Such illumination serves to minimize variations, due to orientation, in
  • the classification by a pathologist of a population of blood cells is usually based upon five visual parameters determined microscopically;-the color, size and shape of the cell nucleus after appropriate staining, and the color and sizeof stained cytoplasm. While the tech nique provides quite good classification, it is usually limited to a population of a few hundred'cells. Despite its high degree of redundancy in cell identification, the technique can nevertheless be in serious error, because the limited populations may not provide a statistically reliable sampling. 1
  • rescent bands as well are both broad (on the order of 500 A) and restricted to a limited region of the spectrum (on the order of 10,000 A'wide). If dyes are to be used in combination, their spectra must be well separated a condition which is vastly more difficult in the case of fluorescent dyes, since both absorption and fluorescent bands of any dye used in combination must not interfere with the absorption and fluorescent bands of any of the other dyes in that combination.
  • a principal object of the present invention is therefore to provide a novel system for classifying a number of blood cells.
  • Another object of the invention is to provide a non-imaging measurement of cell parameters, compatible with standard hematological practice.
  • Yet another object of the present invention is to provide such a non-imaging measurement of blood cell parameters wherein data is preprocessed so that the use of large computers is avoided.
  • Yet another objects of the present invention are to provide such a measurement system which can be employed with blood cells moving in a flow stream; to provide such a systemwherein measurements are made with minimized orientation-caused error; to provide such a system wherein a shape factor of a cell or cell nucleus is determined from simultaneous measurement of the magnitudes of two different functions of shape; to provide such a system wherein the shape factor is determined from simultaneous measurement of magnitudes related respectively to the volume of the nucleus and the surface area of the nucleus; and to provide such a system wherein the shape factor is determined from simultaneous measurement of magnitudes related respectively to the effective thickness and the volume of the cell nucleus.
  • Yet another important object of the present invention is to provide a novel measurement system wherein measurements are made that minimizes variation caused by orientation whereby measurements of parameters of optically thick, dyed particles may be reproducibly made.
  • the invention accordingly comprises the apparatus possessing the construction, combination of elements and arrangement of parts, and the method comprising the several steps and relation of one or more of such steps with re spect to each of the others, all of which are exemplified in the following detailed disclosure, and the scope of the application of which will be indicated in the claims.
  • FIG. 1 is a schematic block diagram illustrating application of the invention to a system for obtaining a differential count among selected types of blood cells
  • FIG. 2 is a schematic diagram showing an exemplary optical system useful in part of the system'of FIG. 1;
  • FIG. 3 is a schematic diagram showing another exemplary optical system useful in connection with the system of FIG. 1',
  • FIG. 4 is a schematic diagram of an electrical circuit which is a modification of a portion of the system of FIG. 1 for alternative structure;
  • FIG. 5 is a schematic diagram of a vibration on the optical system employed to minimize orientation prob lems.
  • FIG. 6 is a schematic elevational diagram, partly in cross-section, showing a fragment of a preferred optical system for an orientation insensitive measuring system.
  • the system of the invention herein disclosed is com-- patible with standard hematology laboratory practice, and does not require cell fractionation or selective lysis and requires few solutions.
  • the sample is not destroyed and is available for subsequent microscopic examination or other diagnostic tests.
  • the present invention permits the direct assignment of cells to the major cell fractions. This reduces error, since no counts are derived as a remainder when two other counts are differenced, as for example, in a scheme which characterizes monocytes as being nongranulocytes which are also non-lymphocytes.
  • function as used herein is intended to be interpreted in the mathematical sense to mean a variable, the value or magnitude of which is determined by a second variable.
  • different functions is intended then to mean a plurality of functions wherein the laws of dependence on the second variables are different.
  • the present invention generally is a system comprising means for substantially simultaneously measuring the magnitudes of two different shape-dependent functions of the cell or its nucleus and means for determining a relationship between the magnitudes.
  • the property of sphericity can be considered to be the desired shape factor.
  • any two of these shape dependent functions can be expressed by a ratio where n and m are exponents selected so that d, which is size dependent primarily, vanishes.
  • n and m are exponents selected so that d, which is size dependent primarily, vanishes.
  • the shape factor y for a cellular nucleus having a perfectly spherical shape will be a readily computed limit quite independent of the value of d. Any departures measured from such limit are measures of variation of the shape of the nucleus from sphericity and hence a determination of an aspect of shape.
  • means are provided for deriving a magnitude which is proportional to the volume of a cell nucleus, preferably by measuring fluorescent re-emission of light absorbed by the nuclear DNA, and simultaneously deriving a magnitude related to the nuclear surface area, preferably by measuring the light scattering from the nucleus.
  • Means are also provided for then determining a shape factor which is proportional to a ratio of the volumetrically related magnitude to the surface related magnitude.
  • the two magnitudes derived are respectively related to other shape-dependent factors, particularly the apparent thickness and the volume of a cell nucleus, and a shape factor is obtained from a ratio of these two functions. Measurement here can be achieved from light absorption or by fluorescent re-emission. The effective thickness is measured by examining the self-shadowing reduction in the measured volume-at wavelengths where the particle is optically thick. Such reduction arises from the non-linearity of the transmission-thickness relationship.
  • the system of FIG. 1 includes source 20 of a sample of blood cells.
  • Source 20 is connectable through conduit 22 and valve 24 to a mixing chamber 26.
  • the latter also is connectable through conduit 28 and valve 30 to supply 32 of a dye or stain bath.
  • the output of chamber 26 is coupled to an input of dialyzer 34, another input of the latter being connectable through conduit 36 and valve 38 to a reservoir or supply 40 of dialysis solution.
  • One output of dialyzer is for discharge of dialyzed stain, the other output of the dialyzer being connected as one input to dilution chamber 42.
  • Another input to chamber 42 is connectable through conduit 44 and valve 46 to supply 48 of diluent fluid.
  • the diluent from supply 48 is preferably selected (and metered by valve 46) to provide a number of basic qualities for purposes of this invention.
  • the diluent should, when added in metered proportion to the stained cell suspension from dialyzer 34, provide matching of the indices of refraction of the cell cytoplasm and the mixed fluids; because it is highly desirable to maintain substantially laminar flow through the flowcell at a high rate of flow, the diluent should also be selected so that, when added in metered proportion to the cell suspension, it will adjust the fluid viscosity to permit high speed laminar flow.
  • the diluent also should be selected so that, when added to the cell suspension it will attain an optimum osmotic pressure with respect to the cells to maintain their stability.
  • pumps may be provided to yield the high rate of flow through the flow cell. In such case, the diluent may also serve to adjust osmotic pressure and thereby compensate for the high static pressure caused by the pumps.
  • the fluid output from dilution chamber 42 is connected to pump 50, and the output of the latter in turn is connected preferably to a central injector nozzle 51 of a sheathed-stream flow cell 52.
  • the latter typically can be of the design disclosed in Advances in Automated Analysis, Technicon International Congress 1970, Volume l Clinical (1971) on pages 454-455 of the article by Alex M. Saunders et al, entitled A Rapid Automated System for Differentiating and Counting White Blood Cells.”
  • the annular space 53 around central injector 51 is connected to the output of second pump 54.
  • the inputs to the latter is connected to a supply 56 of sheath fluid. Injector 51 and space 53 are disposed the fluid carrier becomes many times smaller than the dye concentration on a typical cell.
  • the sample is then diluted in chamber 62 with diluent-from supply 48 to provide adequate separation between blood cells in flow cell 52. It is possible that thedilution alone may be adequate to reduce the solution concentration of dye, in which case the dialyzer may be eliminated. It is desirable to eliminate the dialyzer, asit accoiints for a major portion of, the totaltime required for measuring the parameters of a sample (the time between samples can, of course, be much smaller than the time required persample). 3 j j
  • the diluted sample is next pumped by pump 50 through injector 51 into the measuring flowcell 52.
  • the sample stream is confined by a fluid sheath provided by pump 54 of liquid from supply 56in order to obtain a narrow, rapidly flowing sample stream.
  • flow cell 52 is constructed so that fluid is introduced in one stream through'injectornozzle 51 and in an annular stream,surrounding the first stream, by pump '54 into annular space 53.
  • the velocities of the central sample stream and the annular or sheath stream are controlled such that laminar flow conditions are established at the junction of the two streams, hencethe two streams will move-together with the sheath stream effectively constricting the sample stream.
  • the sheath fluid provided from source of supply 56 preferably is selected to provide the requisite viscosity which will permit laminar flow underthe head pressure provided by pump 54. It should also be selected so that there is close matching of refractive indices between the sheath and sample fluidls.
  • The'diluent from supply 48 and the sheath, fluid 56 may be the same if desired although the requirements for the two need not be identical.
  • the salt additive is usually and preferably simply NaCl.
  • the flow cell preferably has circular cross section and has the largest diameter adjacent the tip of injector nozzle 51, being tapered down stream from that point. To obtain a desirable center sample stream of microns in diameter, typically, the flow cell will be tapered down to an internal diameter of about 200 microns.
  • the blood cells are transmitted along the central stream in single file, at high speeds.
  • the flow cell thus described then essentially confines the blood cells to a narrow stream wherein the blood cells move each through a particular point substantially one at a time and therefore each can be examined in sequence. Further because the center-stream confines the blood cells'to a substantially axial-flow, the latter motion of the blood cells is sharply limited and hence the cells will remain well within focus of an optical system.
  • the system of the invention includes an electro-optical subsystem which is shown'schemati'cally in FIG. 1.
  • the subsystem preferably includes one or more optical devices 60 such'as lenses, mirrors and the like for illuminating separate portions of flow cell 52 with radiation from one or more sources; shown generically as spectral source 62.
  • a detection device 64 typically associated with each optical device 60 for converting selected parameters of radiation from a corresponding device 60 into an electrical signal.
  • FIG. 1 Forsimplicity in exposition only one detection device 64 is shown in FIG. 1 as electrically connected to other equipment.
  • a typical detection device as shown in FIG. 2 includes radiation source 62A andan optical system, essentially an inverted microscope having eyepiece lens 66 disposed adjacent source 62A and objective lens 68 disposed adjacent one side of flow cell 52. Positioned intermediate lenses 66 and 68 is a mask 70 having a plurality of apertures such as slots 72 therein.
  • the microscope formed of lenses 66 and 68 is so positioned that radiation from source 62A, formed into a plurality V of discrete apparent sources by lens 66 and mask 70 is viscosity of both of the fluids, as well as the osmotic pressure produced across the surface of sample cells which may be suspended in or associated with the fluids.
  • the fluids are preferably aqueous solutionsjilntaining both additives which are polymeric and ad i lves which'are salts.
  • the control of refractive index is established by adjusting the concentration of the polymer in the solution. For a given concentration of polymer, the viscosity of the fluid can be adjusted by selectirlg an appropriate degree of polymerization (i.e.
  • the fluid may include a complementary dissolved salt, the concentration of which will serve to adjust the osmotic pressure to some desired value.
  • polymeric additives for ent and sheath fluids are polyethylene glycol and'the like, and blood plasma extenders such as dextran, poly use in the dilu-.
  • objective lens 68 focused by objective lens 68 at a like plurality of spots distributed axially substantially along the center of the center stream in flow cell 52.
  • lens 74 Disposed on the opposite side of flow cell 52 and typically on the common optic axis of lenses 66 and 68 ia another lens 74, typically a microscope type objective which has an equal orhigher numerical aperture than lens 68, and therefore is capable of accepting all of the radiation transmitted through lens 68 from source 62A.
  • each aperture 72 in mask Disposed between lens 74 and its focal planeis apertured diaphragm 76. Apertu'res 78 in the latter are disposed such that light originating from each aperture 72 in mask is substantially focused through a corresponding aperture 78 in diaphragm 76. Disposed on the opposite side of each aperture 78 from lens 74 are optiparticular wavelengths as desired. Positioned to detect the radiation transmitted by each of filters 80 are corresponding ones of detectors 82. The lattertypically can 7 be photodiodes with extremely fast rise times, or photocells of other known types.
  • the source can be means for providing a specified spectrum, such as a xenon high intensity lamp, with or without a selected output filter. It is quite important to set up a number of images (corresponding to the number of apertures 72 in mask 70) and that these images be rather close to one another axially along the center of flow cell 52. This structure serves to minimize the time for a single cell to go from one image or lightspot to the next and to activate corresponding detectors 82, thereby serving to minimize consequences of the errors in the velocity of the cells traversing flow cell 52. This minimization of velocity error is particularly important in instances where it is desired to correlate successive readings of the same cell so as to characterize the cell according to several different measurements.
  • FIG. 2 To measure scattering, a typical system is shown in FIG. 3 as including source 623, apertured mask 71 and lens 68. These elements are positioned to focus the radiation from the aperture in mask 71 as a spot centered within flow cell 52.
  • the system of FIG. 3 also includes lens 74, screen 75 and detector 82.
  • Screen 75 is simply an opaque screen with an annular opening 77 therein. Opening 77 of course surrounds an opaque center.
  • lens 74, screen 75 and detector 82 are so positioned with respect to one another and to flow cell 52, that light scattered from a particle in flow cell 52 over some given range of angles will be detected by detector 82.
  • the signals X and Y each represent a different shape-dependent function of a particular cell occasioned by an appropriate selection of input radiation to the cell, the selected output radiation from the cell, the position of the input radiation to the cell and the position of the detector and detector optics with respect to the output radiation from the cell.
  • Line 84 is coupled to provide the signal X to the input of function element 86 which is capable of generating from signal X an output signal which is an exponential in the form X"" where n and m are values selected according to the type of shape-dependent functions that X and Y may respectively be.
  • Function element 86 may be any of a number of known types of electronic elements.
  • such an element may be a diode function generator the output current of which is an arbitrary function of an input voltage.
  • diode function generators are commercially available as Model SPFX-N/P circuits currently sold by Teledyne Philbrick/Nexus, Dedham, Massachusetts and described in Teledyne Philbrick/Nexus Bulletin EEM, File No. 1100.
  • ratiometric device 88 is a wellknown device capable of accepting a pair of different inputs and for providing an output which is a ratio of those inputs.
  • the output of ratiometer 88 is typically in a formflX)/f(Y) where X is X""". It will be appreciated that the output of the ratiometer is therefore some shape factor for the particle.
  • ratiometer 88 will often not be simultaneous if the corresponding detectors are triggered in sequence.
  • the two inputs should be time correlated, as by introducing a delay line into line 84 or by employing known sample-and-hold techniques. Such refinements have been omitted from the drawings for the sake of simplicity in exposition and because such expedients are so well known in the art.
  • FIG. 1 In order to employ the shapefactor or output signal from ratiometric device 88 for classifying blood cells, a typical circuit is shown in FIG. 1 wherein the output from ratiometric device 88 is connected in parallel to respective first inputs of a group of comparators 90, 92 and 94. Each such comparator includes a second input which is connected to a respective corresponding source, 96, 97 and 98 of reference signals. The output of comparators 90, 92 and 94 are respectively connected to counters 100, 101 and 102. Comparators 90, 92 and 94 are of the type known as threshholding comparators for providing an output pulse only when the input signal is greater than (or less than as the case may be) the amplitude of the reference signal provided from a corresponding reference source. Such comparators are also well known in the art and need not further be described here.
  • the shape factor y is derived as the ratio of any two of a number of different shape dependent functions. I-Ience, as a blood cell traverses flow cell 52, at least two shape dependent functions of that cell are then measured.
  • the mean effective thickness of the nucleus of the cell and the nuclear volume can be selected as the two desired shape dependent functions, the mean effective thickness of the nucleus of the cell and the nuclear volume.
  • the two desired shape dependent functions the mean effective thickness of the nucleus of the cell and the nuclear volume.
  • These wavelength regions can be selected by corresponding choices of filters 80and the spectral output of source 62A of FIG. 2. Self-shadowing will therefore make the ratio of these two measurements a non-linear, thickness-dependent value. This ratio of measurement provides a measure of optical depth and therefore of the mean effective thickness of the nucleus. In such case, the circuit of FIG.
  • ratiometric circuit 104 should be modified as shown in FIG. 4 to provide ratiometric circuit 104 connected to lines 84 and for determining the ratio of X/Y.
  • the output of ratiometric circuit 104 is then connected as the input to function element 86.
  • the output of element 86 can be considered as flZ) or Z"""; the output of ratiometric circuit can be considered as the term flX)/f(Y).
  • the output of ratiometric device 88 will be f (Z')/f(Y) where Z Z"''.
  • the measurement done in the wavelength region where the nucleus is optically thin provides the signal Y which is proportional to the nuclear volume. This latter measurement is correlated in device 9 88 ashe'retofore described with the thickness data from function element 86 to give a shape describing factor.
  • Measurements for an optically thick absorber are usually orientation dependent, i.e. even if the radiation incident on the cell is of constant amplitude, theextent of absorption noted by the detector depends markedly on the spatial orientation of the nuclear components unless for example the latter are grouped symmetrically as a sphere.
  • the cell should be illuminated by a system from a number of different directions (referred to the cell itself or as seen by the cell) the system being arranged so that the sum of the signals corresponding to the observed cellular or nuclear cross-section is substantially independent of orientation, i.e. is substantially invariant.
  • the system being arranged so that the sum of the signals corresponding to the observed cellular or nuclear cross-section is substantially independent of orientation, i.e. is substantially invariant.
  • three individual sources 162A, 1628 and 162C are provided and disposed to diy rect three corresponding light beams along mutually orthogonal paths to intersect at a common point 163 preferably disposed on the longitudinal axis of flowcell 52.
  • three detection devices 164A, 1648 and 164C are disposed to detect radiation emitted by, for example, the nucleus of a cell located at point 163 responsively respectively toirradiation bymeans from sources 162A, 162B and 162C.
  • the detection devices being adapted to provide electrical output signals responsively to radiation incident thereon, all have their output terminals connected to summing device 166.
  • the signal fromdevice 166 the sum of all the inputs, will be substantially invariant for a particular aggregate of particles of given shape and size located at point 163 regardless of the orientation of the particle of aggregate.
  • the beams from sources 162A, 1623 and 162C need not intersect but can illuminate successively a blood cell traversing flowcell52 assuming that the orientation of that blood cell with respect to the sources does not materially change.
  • the output signals from detection devices 164A,- ll64B and l6dC can be suitably delayed or stored so as to be summed later.
  • means such as a spiralled input channel.
  • flowcell 52 for introducing a known rotation about one or more axes to blood cells traveling down the axis of the flowcell.
  • the blood cell can at successive intervals see the light beam from at least two, and preferably three, mutually orthogonal directions.
  • a simple detector responsive to correspondingly successive levels of radiation outputs from the blood cell will suffice to feed a device which ultimately will sum the successive signals.
  • a shape factor can also be obtained by instead measuring the nuclear volume and the nuclear surface and correlating these measurements to obtain a shape factor.
  • the nuclear volume asnoted, is best determined photometrically throughmeasurements of its DNA content.
  • the nuclear volume may also be determined by measuring the fluorescent reemissionof the absorbed light. This may be preferablev in some cases because to obtain good signal-to-noise ratio in absorption measurements a substantial fraction of the light must be absorbed. For fluroescent measurements, on the other hand, the same signal-to-noise ratio can be obtained with much smaller signals.
  • the surface related magnitude can be measured in terms of light scattering from the molecular structure of the nucleus where the particles are in the order of a few wavelengths in size. Because the extent of scattering produced per unit particle volume increases as the particle becomes smaller an assemblage of small particles will scatter more light than a single particle of the same body. However, in such case spurious scattering must be eliminated; Such spurious scattering usually arises from three sources: (1 scattering from the blood cell cytoplasm which may be eliminated by matching the index of refraction of the fluid carrying the blood cell to the cytoplasm by the techniques heretofore described; (2) scattering from the blood cell membrane which is particularly strong for fairly thick membranes such as are found in erythrocytes; and (3) colloid scattering from the whole cell and surrounding solvent.
  • Scattering from erythrocytes can be eliminated by providing a detector 64 which will detect hemoglobin, and by using the output of such detector to gate out any signals arising from erythrocytes.
  • Colloid scattering is very strongly concentrated in the forward direction and thus the provision of detector optics of the type to avoid detecting wholly forward scattered light will serve to minimize the effect of colloid scattering.
  • the scattering behavior of small particles of arbitrary shape and size in the order of a few wavelengths is exceedingly complex.
  • the shape dependent factor sought has no particular linear or accuracy requirement, so that as long as scattering efficiency grows for smaller particles, one can differentiate between a large particle of given volume and an assemblage of smaller particles having the same total volume. Therefore, the only requirement for the sizedependent function based on scattering is that it have a slope which does not change in sign.
  • a broadband source ie a source which provides radiation covering at least one octave of wavelengths at amplitudes above some minimum level.
  • the detector employed should be also responsive substantially across the bandwidth of the source. By employing such a broadband source and broadband detector one achieves smoothing of the scattering function'preferably a curve with substantially invariant sign of its slope.
  • a nuclear shape factor can be determined for each white blood cell traversing flow cell 52.
  • a nuclear shape factor can be determined for each white blood cell traversing flow cell 52.
  • the magnitude'(in comparators 90, 92 and 94) of each such factor against a corresponding different reference magnitude from a corresponding reference source, one then can arbitrarily count those shape factors which for example represent very spherical, very unspherical and medium sphericity" as an example of an arbitrary classification scheme.
  • Such classification is of course not limited necessarily to any number of classes. Indeed, the outputs of the comparators need not be directly counted but can be gated to counters according to correlation with yet other aspects of the blood cells as may be determined for example by yet another detector devices 64.
  • a first optical system for illuminating said cell with radiation
  • a second optical system having a numerical aperture equal to or greater than said first system, for gathering radiation emitted from said cell due to radiation incident thereon from said firstoptical system;
  • detection means for producing a signal corresponding to the total radiation gathered by said second optical system, said first optical system being arranged for providing illumination to said cell from a plurality of different directions as seen by said cell so that said signal is substantially independent of the orientation of said cell.
  • said second optical system comprises a plurality of optical light gathering devices corresponding in number to said light beams and each disposed to gather radiation from said cell due to the incidence on the latter of a respective one of said beams.
  • said detection means comprises a like plurality of detectors each being substantially sensitive to radiation gathered by a respective one of said light gathering devices so as to provide a corresponding signal, said improvement including means for summing all signals from said detectors.
  • said first optical system comprises means for directing radiation of a predetermined wavelength band in at least one cone of greater than to at least one focal spot disposed substantially on the axis of said flowstream,
  • said detection means being substantially sensitive to radiation across said band for providing a signal corresponding to detected radiation.
  • said means for directing radiation comprises a single refractor for directing a plurality of said cones of radiation to a corresponding plurality of focal spots disposed adjacent but separated from one another along the axis of said flowcell,
  • said detection means comprises a plurality of corresponding detectors substantially sensitive to radiation arising at respective ones of said focal spots, and
  • said second optical system comprises means for gathering radiation from each said focal spot and directing the gathered radiation from each spot to a respective one of said detectors.

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US00294245A 1972-10-02 1972-10-02 Blood cell analyzer Expired - Lifetime US3819270A (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
US00294245A US3819270A (en) 1972-10-02 1972-10-02 Blood cell analyzer
GB4343073A GB1424388A (en) 1972-10-02 1973-09-17 Blood cell analyzer
AU60723/73A AU477425B2 (en) 1972-10-02 1973-09-26 Blood cell analyzer
FR7334853A FR2201064B1 (sl) 1972-10-02 1973-09-28
DE19732349271 DE2349271A1 (de) 1972-10-02 1973-10-01 Vorrichtung zum ermitteln von parametern von blutzellen
CA182,299A CA984175A (en) 1972-10-02 1973-10-01 Blood cell analyzer
CH1401273A CH573600A5 (sl) 1972-10-02 1973-10-01
NL7313531A NL7313531A (sl) 1972-10-02 1973-10-02
JP48110908A JPS505098A (sl) 1972-10-02 1973-10-02

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CA (1) CA984175A (sl)
CH (1) CH573600A5 (sl)
DE (1) DE2349271A1 (sl)
FR (1) FR2201064B1 (sl)
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NL (1) NL7313531A (sl)

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US3941479A (en) * 1974-09-26 1976-03-02 G. D. Searle & Co. Use of modulated stimulus to improve detection sensitivity for signals from particles in a flow chamber
JPS5537998A (en) * 1978-09-06 1980-03-17 Ortho Diagnostics Method of and apparatus for detecting blood plasma plate in all blood
FR2445962A1 (fr) * 1979-01-02 1980-08-01 Coulter Electronics Procede et dispositif pour mesurer la reradiation d'une particule dans des systemes a chambre d'ecoulement
FR2445959A1 (fr) * 1979-01-02 1980-08-01 Coulter Electronics Procede et dispositif pour mesurer la dispersion de la lumiere dans des systemes de detection de particules et miroir compose a utiliser dans ce but
US4338024A (en) * 1980-05-02 1982-07-06 International Remote Imaging Systems, Inc. Flow analyzer and system for analysis of fluids with particles
WO1987005108A1 (en) * 1986-02-12 1987-08-27 Combustion Engineering, Inc. An in situ particle size measuring device cross-reference to related applications
FR2636429A1 (fr) * 1988-09-09 1990-03-16 Canon Kk Appareil de mesure de particules
US5116125A (en) * 1990-10-31 1992-05-26 Biophos Medical Ab Fertility analyzer
WO1993016368A1 (en) * 1992-02-12 1993-08-19 Cambridge Consultants Limited Particle measurement system
WO1996014737A1 (en) * 1994-11-14 1996-05-23 Cerus Corporation Treating red blood cell solutions with anti-viral agents
US5844685A (en) * 1996-07-30 1998-12-01 Bayer Corporation Reference laser beam sampling apparatus
US5872627A (en) * 1996-07-30 1999-02-16 Bayer Corporation Method and apparatus for detecting scattered light in an analytical instrument
US5999256A (en) * 1992-02-12 1999-12-07 Cambridge Consultants Limited Particle measurement system
US6177441B1 (en) 1995-06-05 2001-01-23 Cerus Corporation Treating red blood cell solutions with anti-viral agents
US6359683B1 (en) * 2000-04-27 2002-03-19 Becton, Dickinson And Company Method for determining the volume of particles suspended in liquids
US6473172B1 (en) 2000-09-20 2002-10-29 International Remote Imaging Systems, Inc. Flow cell and method of operating therefor
US7016523B1 (en) * 1999-04-21 2006-03-21 Hiroyuki Ogawa Method for observing object by projection, method for detecting microorganisms and projection detecting system
US20060147347A1 (en) * 2002-04-19 2006-07-06 Leslie Leonard Method and apparatus for improved light collection
US20080205739A1 (en) * 2007-02-23 2008-08-28 Visiongate, Inc. Fluid focusing for positional control of a specimen for 3-d imaging
US20160041083A1 (en) * 2013-03-15 2016-02-11 Iris International, Inc. Hematology systems and methods
US10888804B2 (en) * 2016-01-22 2021-01-12 Korea Advanced Institute Of Science And Technology Method for separating and washing of microparticles via a stratified coflow of non-Newtonian and Newtonian fluids
US20230185067A1 (en) * 2020-03-18 2023-06-15 Refeyn Ltd Methods and apparatus for optimised interferometric scattering microscopy

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US3992096A (en) * 1975-08-04 1976-11-16 Minnesota Mining And Manufacturing Company Detecting system
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EP2572182A2 (de) * 2010-05-18 2013-03-27 Partec GmbH Anordnung zum messen der optischen eigenschaften von partikeln einer dispersion

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US3669542A (en) * 1969-10-09 1972-06-13 Coulter Electronics Liquid borne particle sensor
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Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3941479A (en) * 1974-09-26 1976-03-02 G. D. Searle & Co. Use of modulated stimulus to improve detection sensitivity for signals from particles in a flow chamber
JPS5537998A (en) * 1978-09-06 1980-03-17 Ortho Diagnostics Method of and apparatus for detecting blood plasma plate in all blood
JPS6351268B2 (sl) * 1978-09-06 1988-10-13 Ooso Daiagunosuteitsuku Shisutemuzu Inc
FR2445962A1 (fr) * 1979-01-02 1980-08-01 Coulter Electronics Procede et dispositif pour mesurer la reradiation d'une particule dans des systemes a chambre d'ecoulement
FR2445959A1 (fr) * 1979-01-02 1980-08-01 Coulter Electronics Procede et dispositif pour mesurer la dispersion de la lumiere dans des systemes de detection de particules et miroir compose a utiliser dans ce but
US4338024A (en) * 1980-05-02 1982-07-06 International Remote Imaging Systems, Inc. Flow analyzer and system for analysis of fluids with particles
WO1987005108A1 (en) * 1986-02-12 1987-08-27 Combustion Engineering, Inc. An in situ particle size measuring device cross-reference to related applications
FR2636429A1 (fr) * 1988-09-09 1990-03-16 Canon Kk Appareil de mesure de particules
US5116125A (en) * 1990-10-31 1992-05-26 Biophos Medical Ab Fertility analyzer
US5999256A (en) * 1992-02-12 1999-12-07 Cambridge Consultants Limited Particle measurement system
WO1993016368A1 (en) * 1992-02-12 1993-08-19 Cambridge Consultants Limited Particle measurement system
US6171777B1 (en) 1994-11-14 2001-01-09 Cerus Corporation Treating blood or blood product with a compound having a mustard and a nucleic acid binding moiety
US20020182581A1 (en) * 1994-11-14 2002-12-05 Cerus Corporation Treating blood or blood products with compounds which have a mustard, aziridinium or aziridine group and a nucleic acid binding group
AU712034B2 (en) * 1994-11-14 1999-10-28 Cerus Corporation Treating red blood cell solutions with anti-viral agents
US5559250A (en) * 1994-11-14 1996-09-24 Steritech, Inc. Treating red blood cell solutions with anti-viral agents
US6143490A (en) * 1994-11-14 2000-11-07 Cerus Corporation Treating blood or blood product with a compound having a mustard and a nucleic acid binding moiety
WO1996014737A1 (en) * 1994-11-14 1996-05-23 Cerus Corporation Treating red blood cell solutions with anti-viral agents
US5691132A (en) * 1994-11-14 1997-11-25 Cerus Corporation Method for inactivating pathogens in red cell compositions using quinacrine mustard
US6410219B1 (en) 1994-11-14 2002-06-25 Cerus Corporation Treating blood or blood products with compounds which have a mustard, azirdinium or aziridine group and a nucleic acid binding group
US6177441B1 (en) 1995-06-05 2001-01-23 Cerus Corporation Treating red blood cell solutions with anti-viral agents
US5844685A (en) * 1996-07-30 1998-12-01 Bayer Corporation Reference laser beam sampling apparatus
US5872627A (en) * 1996-07-30 1999-02-16 Bayer Corporation Method and apparatus for detecting scattered light in an analytical instrument
US7016523B1 (en) * 1999-04-21 2006-03-21 Hiroyuki Ogawa Method for observing object by projection, method for detecting microorganisms and projection detecting system
US6359683B1 (en) * 2000-04-27 2002-03-19 Becton, Dickinson And Company Method for determining the volume of particles suspended in liquids
US6473172B1 (en) 2000-09-20 2002-10-29 International Remote Imaging Systems, Inc. Flow cell and method of operating therefor
US20060147347A1 (en) * 2002-04-19 2006-07-06 Leslie Leonard Method and apparatus for improved light collection
US20080205739A1 (en) * 2007-02-23 2008-08-28 Visiongate, Inc. Fluid focusing for positional control of a specimen for 3-d imaging
US7867778B2 (en) 2007-02-23 2011-01-11 Visiongate, Inc. Fluid focusing for positional control of a specimen for 3-D imaging
US9702806B2 (en) * 2013-03-15 2017-07-11 Iris International, Inc. Hematology systems and methods
US20160041083A1 (en) * 2013-03-15 2016-02-11 Iris International, Inc. Hematology systems and methods
US20170370820A1 (en) * 2013-03-15 2017-12-28 Iris International, Inc. Hematology systems and methods
US10060846B2 (en) * 2013-03-15 2018-08-28 Iris International, Inc. Hematology systems and methods
US11525766B2 (en) 2013-03-15 2022-12-13 Iris International, Inc. Dynamic range extension systems and methods for particle analysis in blood samples
US11543340B2 (en) 2013-03-15 2023-01-03 Iris International, Inc. Autofocus systems and methods for particle analysis in blood samples
US10888804B2 (en) * 2016-01-22 2021-01-12 Korea Advanced Institute Of Science And Technology Method for separating and washing of microparticles via a stratified coflow of non-Newtonian and Newtonian fluids
US20230185067A1 (en) * 2020-03-18 2023-06-15 Refeyn Ltd Methods and apparatus for optimised interferometric scattering microscopy

Also Published As

Publication number Publication date
FR2201064A1 (sl) 1974-04-26
GB1424388A (en) 1976-02-11
JPS505098A (sl) 1975-01-20
NL7313531A (sl) 1974-04-04
FR2201064B1 (sl) 1977-05-27
AU6072373A (en) 1975-03-27
DE2349271A1 (de) 1974-04-18
CA984175A (en) 1976-02-24
CH573600A5 (sl) 1976-03-15

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