US3896307A - Method for automatic differential leukocyte count - Google Patents

Method for automatic differential leukocyte count Download PDF

Info

Publication number
US3896307A
US3896307A US066560A US6656070A US3896307A US 3896307 A US3896307 A US 3896307A US 066560 A US066560 A US 066560A US 6656070 A US6656070 A US 6656070A US 3896307 A US3896307 A US 3896307A
Authority
US
United States
Prior art keywords
emissions
cells
blood
leukocyte
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US066560A
Inventor
Neil M Trowe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wheeler International Inc
Original Assignee
Wheeler International Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wheeler International Inc filed Critical Wheeler International Inc
Priority to US066560A priority Critical patent/US3896307A/en
Application granted granted Critical
Publication of US3896307A publication Critical patent/US3896307A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/011—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells with lysing, e.g. of erythrocytes
    • 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
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • 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
    • G01N2015/1477—Multiparameters

Definitions

  • Hematology is an important department of the clinical pathology laboratory. For many years pathologists have been concerned with various hematologic procedures. Among the earliest procedures was an examination of unstained blood; later the use of specific staining methods for the identification of various kinds of white cells was attempted. Then methods of absolute blood counting were introduced together with special tests such as, supravital staining and a method of differential counting of the various types of blood cells, introduced by Victor Schilling.
  • a slide is prepared of a drop of blood and the specimen then stained and dried.
  • the various types of leukocytes i.e. lymphocytes, eosinophils, basophils and monocytes
  • present different appearances when illuminated under a microscope and accordingly a trained technician is able to make a count.
  • a total of 100 leukocytes are counted in this manner and the number of each type of cell counted is reported in percentage form.
  • the prepared blood smear be made in such a way that there is a feathering of the smear down into a one cell thickness. Care must be taken to assure that the smear is made without the blood reaching the edge of the slide. If this does occur, the large cells are concentrated at the edge of the smear and the smaller cells are concentrated in the middle of the smear. An accurate differential on a smear of this type is impossible. In the staining technique itself, there are also many points at which error can occur.
  • the various types of white blood cells or leukocytes i.e. lymphocytes, monocytes, and granulocytes including neutrophils, basophils, and eosinophils each exhibit characteristic fluorescence and can be excited with ultraviolet radiation so each emits fluorescent radiation having a wave length characteristic of that particular type of cell.
  • the respective fluorescent emissions from the different types of white blood cells can be recorded in such a way as to permit the mechanical counting of the number of each type of cell in a given volume.
  • a blood sample which has been suitably diluted with about 40 50 parts by volume of say about a 1 percent saline solution is treated, for example with an appropriate lysing agent to destroy the erythrocytes present.
  • the treated specimen is introduced into a chamber or tube which narrows to a sufficiently small diameter, e.g. about microns, so that the passage of cells is in single file by one or more beams of ultraviolet monochromatic light of sufficient intensity to excite the cells to fluorescent emission.
  • These fluorescent emissions from the respective illuminated cells are segregated according to the wavelengths characteristic of each cell and counted on photomultiplier tubes.
  • Segregation of the fluorescent emissions from the respective cells according to wavelength can be effected by means of an analyzer monochrometor having photomultiplier tubes located at slits positioned to discriminate the wavelengths of the emissions of each of the different types of white blood cells being counted.
  • dicroic minors can also be arranged to receive and separate the respective wavelengths emitted from the cells.
  • the ultraviolet illumination from the exciter monochrometor causes the white blood cells to fluoresce and the fluorescence is detected by the photomultiplier tubes at the analyzer exit slits.
  • the output pulse from the photomultiplier tubes is passed to counters corresponding to the specific white cell being detected.
  • the outputs of each of the photomultipliers is passed to a total white cell counter. When the total white cell counter indicates a total of counts, all counters can be stopped, their counts now indicating the percentage abundance of each of the cell types.
  • EXAMPLE 0.1 c.c. of whole blood diluted in 4.9 c.cs of 0.9 percent aqueous sodium cloride solution was added to one drop of lysing agent to destroy the erythrocytes. After mixing thoroughly, the solution was poured into the mouth of a vessel provided with a release valve and a vacuum applied which caused the blood to pass into the testing chamber which narrowed to a 70 micron diameter orifice and then into a 70 micron tube which was placed at the exit slit of an exciter monochrometor.
  • the cells flowed through in single file and were illuminated by a monochromatic beam of light. At right angles to the path of the exciter beam was the entrance slit to an analyzer monochrometor. At the appropriate wavelength positions for each of the different types of white blood cells to be counted, photomultiplier tubes were located.
  • the ultraviolet illumination from the exciter monochrometor caused the white blood cells to fluoresce and the characteristic fluorescence of the individual e. registering the respective fluorescent emissions cells was detected by the individual photomultiplier from each leukocyte cell according to its wavetubes at the analyzer exit slits.
  • the output pulse from length on a photomultiplier tube; the photomultiplier tubes was passed to counters corref. by means of counters recording the number of fluosponding to the specific white cells being detected.
  • the outputs of each of the photomultipliers tubes from each type of leukocyte cell. was passed to a total white cell counter. When the total 2.
  • a method for making total and differential counts the actual percentage abundance of each of the cell of the leukocytes in blood which comprises the steps of: types. a. destroying the erythrocytes in a sample of the What is claimed is: blood cells; 1.
  • a method for making a differential count of leukob. subjecting said sample to ultra-violet radiation sufcytes in blood which comprises the steps of: ficient to cause fluorescent emissions of the leuko' a. preparing a dilute saline solution of the blood samcytes therein;

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

A differential count of leukocytes in blood is performed automatically by passing the individual blood cells through a constricted chamber while illuminating them with sufficient ultraviolet light to cause fluorescent emissions from each cell. The respective fluorescent emissions from the different types of leukocyte cells are each characteristic of the particular type of cell and can be automatically registered using photomultiplier tubes and counters.

Description

United States Patent Trowe METHOD FOR AUTOMATIC DIFFERENTIAL LEUKOCYTE COUNT Neil M. Trowe, Potomac, Md.
Wheeler International Inc., Washington, DC.
Filed: Aug. 24, 1970 Appl. No.: 66,560
Inventor:
Assignee:
References Cited UNITED STATES PATENTS 11/1968 Kamentsky 250/833 UV X [4 1 July 22,1975
3,497,690 2/1970 Wheeless, Jr. et al. 250/7] R 3,515,884 6/1970 lmadate 250/218 3,549,994 12/1970 Rothermel et al. 235/92 PC Primary Examiner-Archie R. Borchelt Attorney, Agent, or FirmCantor & Kraft [5 7 ABSTRACT A differential count of leukocytes in blood is performed automatically by passing the individual blood cells through a constricted chamber while illuminating them with sufficient ultraviolet light to cause fluorescent emissions from each cell. The respective fluorescent emissions from the different types of leukocyte cells are each characteristic of the particular type of cell and can be automatically registered using photomultiplier tubes and counters.
3 Claims, No Drawings METHOD FOR AUTOMATIC DIFFERENTIAL LEUKOCYTE COUNT This invention is concerned with a high speed, accurate and automated method for performing a differential analysis of white blood cells. More specifically, the present invention is concerned with determining the individual counts of the various types of white blood cells or leukocytes by registering their distinctive fluorescent emissions.
Hematology is an important department of the clinical pathology laboratory. For many years pathologists have been concerned with various hematologic procedures. Among the earliest procedures was an examination of unstained blood; later the use of specific staining methods for the identification of various kinds of white cells was attempted. Then methods of absolute blood counting were introduced together with special tests such as, supravital staining and a method of differential counting of the various types of blood cells, introduced by Victor Schilling.
Increasingly it has become important to be able to make rapid, accurate blood tests and while at least one generally accepted method has been derived to give hematrocrit, hemoglobin and total leukocyte counts in a rapid and reasonably accurate, semi-automated manner; the method for performing a differential count of the various kinds of leukocyte cells has improved only in respect to the staining techniques used. No method has heretofore been developed which would even partially automate the leukocyte differential and bring it to the level of performance of the hematrocrit, hemoglobin or total leukocyte count.
According to present techniques for making differential leukocyte counts, a slide is prepared of a drop of blood and the specimen then stained and dried. The various types of leukocytes (i.e. lymphocytes, eosinophils, basophils and monocytes) present different appearances when illuminated under a microscope and accordingly a trained technician is able to make a count. Normally, a total of 100 leukocytes are counted in this manner and the number of each type of cell counted is reported in percentage form.
Assuming that the drop of blood placed on the slide to make the smear is of a uniform size, it is imperative that the prepared blood smear be made in such a way that there is a feathering of the smear down into a one cell thickness. Care must be taken to assure that the smear is made without the blood reaching the edge of the slide. If this does occur, the large cells are concentrated at the edge of the smear and the smaller cells are concentrated in the middle of the smear. An accurate differential on a smear of this type is impossible. In the staining technique itself, there are also many points at which error can occur. One of the most common errors at this point is in using a buffer for the staining which is either too alkaline or too acid; the buffer and stain mixture must be neutral. If the buffer is either too acid or too alkaline, the possibility of determining the maturity of the leukocyte is greatly reduced because of the non-uniform staining of the nucleus.
Now in accordance with the present invention it has been found that both the total and differential count of leukocytes in blood can be quickly, efficiently, and accurately determined automatically without the need for technicians or other personnel visually observing the cells or otherwise manually making the determination and consequently with a great reduction in the possibility of error. i
More specifically, it has been found that the various types of white blood cells or leukocytes i.e. lymphocytes, monocytes, and granulocytes including neutrophils, basophils, and eosinophils each exhibit characteristic fluorescence and can be excited with ultraviolet radiation so each emits fluorescent radiation having a wave length characteristic of that particular type of cell. Further it has been found that using a spectrofluorometor, the respective fluorescent emissions from the different types of white blood cells can be recorded in such a way as to permit the mechanical counting of the number of each type of cell in a given volume.
According to the present invention, a blood sample which has been suitably diluted with about 40 50 parts by volume of say about a 1 percent saline solution is treated, for example with an appropriate lysing agent to destroy the erythrocytes present. Subsequently the treated specimen is introduced into a chamber or tube which narrows to a sufficiently small diameter, e.g. about microns, so that the passage of cells is in single file by one or more beams of ultraviolet monochromatic light of sufficient intensity to excite the cells to fluorescent emission. These fluorescent emissions from the respective illuminated cells are segregated according to the wavelengths characteristic of each cell and counted on photomultiplier tubes. Segregation of the fluorescent emissions from the respective cells according to wavelength can be effected by means of an analyzer monochrometor having photomultiplier tubes located at slits positioned to discriminate the wavelengths of the emissions of each of the different types of white blood cells being counted. Alternatively, for example, dicroic minors can also be arranged to receive and separate the respective wavelengths emitted from the cells.
The ultraviolet illumination from the exciter monochrometor causes the white blood cells to fluoresce and the fluorescence is detected by the photomultiplier tubes at the analyzer exit slits. The output pulse from the photomultiplier tubes is passed to counters corresponding to the specific white cell being detected. In addition, the outputs of each of the photomultipliers is passed to a total white cell counter. When the total white cell counter indicates a total of counts, all counters can be stopped, their counts now indicating the percentage abundance of each of the cell types.
EXAMPLE 0.1 c.c. of whole blood diluted in 4.9 c.cs of 0.9 percent aqueous sodium cloride solution was added to one drop of lysing agent to destroy the erythrocytes. After mixing thoroughly, the solution was poured into the mouth of a vessel provided with a release valve and a vacuum applied which caused the blood to pass into the testing chamber which narrowed to a 70 micron diameter orifice and then into a 70 micron tube which was placed at the exit slit of an exciter monochrometor. The cells flowed through in single file and were illuminated by a monochromatic beam of light. At right angles to the path of the exciter beam was the entrance slit to an analyzer monochrometor. At the appropriate wavelength positions for each of the different types of white blood cells to be counted, photomultiplier tubes were located.
The ultraviolet illumination from the exciter monochrometor caused the white blood cells to fluoresce and the characteristic fluorescence of the individual e. registering the respective fluorescent emissions cells was detected by the individual photomultiplier from each leukocyte cell according to its wavetubes at the analyzer exit slits. The output pulse from length on a photomultiplier tube; the photomultiplier tubes was passed to counters corref. by means of counters recording the number of fluosponding to the specific white cells being detected. In 5 rescent emissions registered on the photomultiplier addition, the outputs of each of the photomultipliers tubes from each type of leukocyte cell. was passed to a total white cell counter. When the total 2. The method of claim 1, wherein the leukocyte cells white cell counter indicated a total of 100 counts, all are lymphocytes, monocytes and granulocytes. counters were stopped so that their counts indicated 3. A method for making total and differential counts the actual percentage abundance of each of the cell of the leukocytes in blood which comprises the steps of: types. a. destroying the erythrocytes in a sample of the What is claimed is: blood cells; 1. A method for making a differential count of leukob. subjecting said sample to ultra-violet radiation sufcytes in blood which comprises the steps of: ficient to cause fluorescent emissions of the leuko' a. preparing a dilute saline solution of the blood samcytes therein;
ple; c. separating the resulting emissions according to b. passing the dilute saline blood solution into a conwavelengths into lymphocytes, monocytes and stricted chamber so that the individual cells are disgranulocytes emissions sets; posed in a single array; d. registering the number of emissions for each set 0. subjecting the cells disposed in said constricted and for the sum of said sets; and
chamber in single file to sufficient ultraviolet radiae. stopping registration of said emissions when the tion as the cells pass through the chamber to cause total leukocyte count reaches 100; whereby said them to fluoresce; number of emissions for each set indicates the perd. segregating the fluorescent emissions from each centage abundance of said set relative to said total cell according to the characteristic wavelength of leukocyte count.
the fluorescent emission of that type of cell;
Disclaimer 3,896,307.Neil M Trowe, Potomac, Md. METHOD FOR AUTOMATIC DIF- FERENTIAL LEUKOCYTE COUNT. Patent dated July 22, 1975. Disclaimer filed Feb. 13, 1981, by the assignee, Wheeler International,
Inc. Hereby enters this disclaimer to claims 1-3, all the claims of said patent.
[Official Gazette April 14, 1981.]

Claims (3)

1. A method for making a differential count of leukocytes in blood which comprises the steps of: a. preparing a dilute saline solution of the blood sample; b. passing the dilute saline blood solution into a constricted chamber so that the individual cells are disposed in a single array; c. subjecting the cells disposed in said constricted chamber in single file to sufficient ultraviolet radiation as the cells pass through the chamber to cause them to fluoresce; d. segregating the fluorescent emissions from each cell according to the characteristic wavelength of the fluorescent emission of that type of cell; e. registering the respective fluorescent emissions from each leukocyte cell according to its wavelength on a photomultiplier tube; f. by means of counters recording the number of fluorescent emissions registered on the photomultiplier tubes from each type of leukocyte cell.
2. The method of claim 1, wherein the leukocyte cells are lymphocytes, monocytes and granulocytes.
3. A method for making total and differential counts of the leukocytes in blood which comprises the steps of: a. destroying the erythrocytes in a sample of the blood cells; b. subjecting said sample to ultra-violet radiation sufficient to cause fluorescent emissions of the leukocytes therein; c. separating the resulting emissions according to wavelengths into lymphocytes, monocytes and granulocytes emissions sets; d. registering the number of emissions for each set and for the sum of said sets; and e. stopping registration of said emissions when the total leukocyte count reaches 100; whereby said number of emissions for each set indicates the percentage abundance of said set relative to said total leukocyte count.
US066560A 1970-08-24 1970-08-24 Method for automatic differential leukocyte count Expired - Lifetime US3896307A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US066560A US3896307A (en) 1970-08-24 1970-08-24 Method for automatic differential leukocyte count

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US066560A US3896307A (en) 1970-08-24 1970-08-24 Method for automatic differential leukocyte count

Publications (1)

Publication Number Publication Date
US3896307A true US3896307A (en) 1975-07-22

Family

ID=22070273

Family Applications (1)

Application Number Title Priority Date Filing Date
US066560A Expired - Lifetime US3896307A (en) 1970-08-24 1970-08-24 Method for automatic differential leukocyte count

Country Status (1)

Country Link
US (1) US3896307A (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4122348A (en) * 1976-05-21 1978-10-24 Elscint, Ltd. Method of and apparatus for classifying biological cells
US4284412A (en) * 1979-07-13 1981-08-18 Ortho Diagnostics, Inc. Method and apparatus for automated identification and enumeration of specified blood cell subclasses
US4325706A (en) * 1980-08-15 1982-04-20 Ortho Diagnostic Systems Inc. Automated detection of platelets and reticulocytes in whole blood
WO2003006956A3 (en) * 2001-07-11 2003-09-25 Nat Health Lab Service Cell enumeration
US20060073470A1 (en) * 2002-05-30 2006-04-06 Naohiro Noda Method of counting microorganisms or cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3413464A (en) * 1965-04-29 1968-11-26 Ibm Method for measuring the nucleic acid in biological cells after enhancement in an acidic solution
US3497690A (en) * 1967-09-21 1970-02-24 Bausch & Lomb Method and apparatus for classifying biological cells by measuring the size and fluorescent response thereof
US3515884A (en) * 1967-05-17 1970-06-02 Toa Electric Co Ltd Detecting and counting apparatus for particles suspended in a liquid
US3549994A (en) * 1963-12-12 1970-12-22 Coulter Electronics Automatic method and apparatus for obtaining different dilutions from blood or the like samples and processing the same by fluid handling and electronics to obtain certain nonelectric parameters

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3549994A (en) * 1963-12-12 1970-12-22 Coulter Electronics Automatic method and apparatus for obtaining different dilutions from blood or the like samples and processing the same by fluid handling and electronics to obtain certain nonelectric parameters
US3413464A (en) * 1965-04-29 1968-11-26 Ibm Method for measuring the nucleic acid in biological cells after enhancement in an acidic solution
US3515884A (en) * 1967-05-17 1970-06-02 Toa Electric Co Ltd Detecting and counting apparatus for particles suspended in a liquid
US3497690A (en) * 1967-09-21 1970-02-24 Bausch & Lomb Method and apparatus for classifying biological cells by measuring the size and fluorescent response thereof

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4122348A (en) * 1976-05-21 1978-10-24 Elscint, Ltd. Method of and apparatus for classifying biological cells
US4284412A (en) * 1979-07-13 1981-08-18 Ortho Diagnostics, Inc. Method and apparatus for automated identification and enumeration of specified blood cell subclasses
US4325706A (en) * 1980-08-15 1982-04-20 Ortho Diagnostic Systems Inc. Automated detection of platelets and reticulocytes in whole blood
WO2003006956A3 (en) * 2001-07-11 2003-09-25 Nat Health Lab Service Cell enumeration
US20040197768A1 (en) * 2001-07-11 2004-10-07 National Health Laboratory Service Cell enumeration
US20070275413A1 (en) * 2001-07-11 2007-11-29 National Health Laboratory Service Enumeration of CD4+lymphocytes
US7670793B2 (en) 2001-07-11 2010-03-02 National Health Laboratory Service Enumeration of CD4+ lymphocytes
US7892551B2 (en) 2001-07-11 2011-02-22 National Health Laboratory Service Antibody kit for enumeration of CD4+ lymphocytes
US20060073470A1 (en) * 2002-05-30 2006-04-06 Naohiro Noda Method of counting microorganisms or cells

Similar Documents

Publication Publication Date Title
US3916197A (en) Method and apparatus for classifying biological cells
US4599307A (en) Method for elimination of selected cell populations in analytic cytology
CA2207396C (en) Method for rapid and simultaneous analysis of nucleated red blood cells
US3864571A (en) Method and Apparatus for Automatically, Identifying and Counting Various Cells in Body Fluids
CA1309326C (en) Method of classifying leukocytes by flow cytometry and reagents used in the method
JP6029657B2 (en) Nucleated red blood cell analysis system and method
US5928949A (en) Reagent and method for classifying leukocytes by flow cytometry
EP0483116B1 (en) Method of classifying leukocytes by flow cytometry and reagents used in the method
JPS61195358A (en) Method of analyzing accessory cell population of corpuscle
JP6104233B2 (en) Leukocyte analysis system and method
US9797824B2 (en) Method for hematology analysis
EP2630492B1 (en) Internal focus reference beads for imaging cytometry
JPH0447265B2 (en)
US3896307A (en) Method for automatic differential leukocyte count
CN114441480B (en) Nuclear erythrocyte analysis device and analysis method
EP0259833B1 (en) Reagent and method for classifying leukocytes by flow cytometry
JPH01320464A (en) Method and agent for discrimination and measurement of leucocyte
Steinkamp Identification of Single Cells by Fluorescent and Darkfield Optical Techniques
Holm et al. Flow Cytometry
ITFI940105A1 (en) FLOW CYTOFLUORIMETRIC METHOD FOR DETERMINING THE LEUCOCYTARY FORMULA AND CYTOMETER TO IMPLEMENT THIS METHOD