WO1992002632A1 - Colorants fluorescents d'identification et d'enumeration de cellules viables dans le lait - Google Patents

Colorants fluorescents d'identification et d'enumeration de cellules viables dans le lait Download PDF

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Publication number
WO1992002632A1
WO1992002632A1 PCT/US1991/005377 US9105377W WO9202632A1 WO 1992002632 A1 WO1992002632 A1 WO 1992002632A1 US 9105377 W US9105377 W US 9105377W WO 9202632 A1 WO9202632 A1 WO 9202632A1
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cells
milk
dye
labeled
fluorescent
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PCT/US1991/005377
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Douglas Dean Redelman
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Sierra Cytometry
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01J—MANUFACTURE OF DAIRY PRODUCTS
    • A01J5/00—Milking machines or devices
    • A01J5/013—On-site detection of mastitis in milk
    • A01J5/0131—On-site detection of mastitis in milk by analysing the milk composition, e.g. concentration or detection of specific substances
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01J—MANUFACTURE OF DAIRY PRODUCTS
    • A01J5/00—Milking machines or devices
    • A01J5/013—On-site detection of mastitis in milk
    • A01J5/0131—On-site detection of mastitis in milk by analysing the milk composition, e.g. concentration or detection of specific substances
    • A01J5/0132—On-site detection of mastitis in milk by analysing the milk composition, e.g. concentration or detection of specific substances using a cell counter
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01J—MANUFACTURE OF DAIRY PRODUCTS
    • A01J5/00—Milking machines or devices
    • A01J5/013—On-site detection of mastitis in milk
    • A01J5/0135—On-site detection of mastitis in milk by using light, e.g. light absorption or light transmission
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/04—Dairy products

Definitions

  • Bovine mastitis, or inflammation of the udder, causes significant economic losses to the dairy industry amounting to approximately $2 billion per year in the
  • somatic cell counts caused by large numbers of shed epithelial cells or cell fragments, particularly late in lactation. Since the somatic cell count fails to distinguish between live and dead cells or between cell types, these cattle may be incorrectly perceived to have mastitis. Thus, the somatic cell count can produce both false negative-and false positive results. However, the somatic cell count can be automated, does not require highly trained technicians, and is not especially labor-intensive. Therefore, the somatic cell count is relatively inexpensive ($0.20-0.30 per test) and can be performed rapidly (180-220 samples per hour) making it capable of being used to screen large numbers of cattle on a regular basis. An improved mastitis test would measure the inflammatory cells that are actually characteristic of mastitis.
  • Granulocytes are the most significant in diagnosing mastitis.
  • Granulocytes are the first inflammatory cells present in mastitic milk and their continued presence indicates that the problem causing the mastitis has not been solved.
  • Granulocytes may also be accompanied by other leukocytes including lymphocytes and monocyte/macrophage type cells.
  • the detergent treatment lyses cells to render the nuclei available to the ethidiu bromide dye and also breaks up butterfat globules that can clog some types of instruments.
  • the detergent lysis also prevents the instrument from being able to distinguish different types of cells or to distinguish viable from dead cells.
  • early cases of mastitis in which the milk contains a small number of inflammatory cells can go undetected.
  • milk containing large numbers of normal epithelial cells but no inflammatory cells would be incorrectly identified as "mastitic”.
  • the instrument cannot detect a change in the types of cells present in milk, such as can occur during successful treatment of mastitis.
  • Flow cytometry i.e., the simultaneous measurement of one or more parameters for each individual cell in a single cell suspension
  • One type of single parameter flow cytometer the electronic particle counter as exemplified by the "Coulter Counter”
  • These instruments count "cells” by measuring an alteration in electric current flow caused by a particle as it passes between two electrodes. The magnitude of the alteration is related to particle size so that cells or particles of different sizes can be distinguished and enumerated.
  • the approach selected in accordance with this invention is to label the cells in milk with a dye that selectively is fluorescent in viable cells.
  • a dye sufficiently selective for viable cells would allow measuring the fluorescence after depositing the cells on a filter, concentrating them by centrifugation, or other techniques that permit measuring the fluorescent signal associated with the cells.
  • the dyes that have been used in the previously reported flow cytometric tests to identify cells in milk namely acridine orange and CMFDA, are not suitable for these types of tests. In the case of acridine orange, it labels nucleic acid in cell fragments and would not distinguish viable from dead cells.
  • CMFDA although generally considered a "vital" dye that selectively labels only viable cells, was found to produce substantial- fluorescence in dead cells. Therefore, since neither of the previously reported flow cytometric procedures for identifying viable cells in milk appeared to be suitable, other types of dyes and labeling procedures that have not previously been applied to cells in milk were considered.
  • CMFDA 5(and-6)carboxy-4• ,5'- dimethylfluorescein diacetate
  • CFDA the related compound 5(and-6)-carboxyfluorescein diacetate
  • SFDA 5(and-6)-sulfofluorescein diacetate
  • a second AM esterified dye namely the Ca 2+ indicator dye Indo-1 AM, also labels cells with high efficiency.
  • Indo-1 AM has not been as thoroughly tested, it appears it may also be useful in analyzing cells in milk.
  • fluorescent dyes esterified with the acetoxymethyl group may be generally useful in analyzing cells in milk.
  • Esterified derivatives of fluorescent dyes that are able to enter cells and be de-esterified in situ to a charged fluorescent form thus are a category of dyes that includes individual dyes which can be used in this invention.
  • a specific dye in this category to be useful in this invention it must be able to label viable inflammatory cells in milk at least 10-fold more intensely than dead cells or other cell types in milk.
  • the suitability of any specific member of this category can be determined by measuring the fluorescence it produces in an aliquot of leukocytes isolated from blood that is suspended in buffered saline and allowed to stand at room temperature for sufficient time to cause cell death in a portion of the cells in the suspension.
  • Two esterase-dependent dyes, namely FDA- and BCECF-AM meet these criteria. Preliminary tests indicate that the calcium-sensitive dye Indo-1 AM is also satisfactory.
  • esterase-dependent vital dyes including CFDA, CMFDA, and SFDA, have been shown to be unsatisfactory because they fail to discriminate .. adequately between viable inflammatory cells and other cells in milk. 2. Nucleic acid binding dyes.
  • fluorescent dyes that bind to and label nucleic acids can be divided into three categories: a) Dyes that do not effectively cross intact cell membranes. Dyes of this type, as typified by ethidium bromide and propidium iodide, are usually charged molecules. When added to cell suspension containing intact viable cells, dead cells with damaged membranes, and fragments of cells and cell nuclei, these dyes selectively label the dead cells and the cell fragments. Therefore, nucleic acid binding fluorescent dyes of this type are not suitable for use in the process of this invention. b) Dyes that are able to enter live cells and fluorescently label nucleic acids without further chemical conversion.
  • HED is a reduced form of ethidium that has altered fluorescence excitation and emission properties and that is able to enter viable cells. Within viable cells, intracellular oxidation converts HED back to ethidium which can then bind to nucleic acid and label it fluorescently. HED has been ur.ed to label several types of viable cells selectively, including sperm cells (Ericsson SA et al. Gamete Res. 22:355-368 1989. Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two-flow cytometric systems.) but it had not previously been tested with cells in milk.
  • sperm cells Ericsson SA et al. Gamete Res. 22:355-368 1989. Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two-flow cytometric systems.
  • HED has now been tested with cells from milk and demonstrated to label viable cells with high fluorescence intensity, i.e., at least 10,000 fold brighter than the normal autofluorescence level. Furthermore, HED discriminates between viable cells and dead cells at least as effectively as the esterase-dependent dyes FDA or BCECF-AM. Therefore, HED has established that this third category of nucleic acid binding dyes is suitable in this invention. Other nucleic acid binding fluorescent dyes can also be chemically modified to fit the criteria listed above to meet the required labeling criteria and are suitable for this invention.
  • Fluorescent dyes can be chemically reduced such that they have altered fluorescence properties and/or are..able to enter viable cells. Inside viable cells, these modified dyes can be oxidized back to the fluorescent form. The resulting fluorescence intensity of the cell is directly related to the activity of the intracellular oxidative process. Dyes of this type are particularly useful in accordance with this invention to detect inflammatory cells because inflammatory cells, especially granulocytes and macrophages, have elevated levels of intracellular oxidative activity. Furthermore, granulocytes stimulated with agents such as phorbol myristate acetate (PMA) produce as much as 30-fold higher levels of intracellular oxidative activity in a manner of minutes. Thus, cells incubated with these dyes alone or in the presence of PMA will produce different levels of fluorescence depending upon the number of granulocytes in the population.
  • PMA phorbol myristate acetate
  • the esterified dye enters cells where it is de-esterified by intracellular esterases to the charged but nonfluorescent DCHF. Oxidative processes then convert DCHF to the charged fluorescent DCF that remains trapped in the cell.
  • This dye has been used to measure the oxidative response of granulocytes isolated from human blood, Bass DA, et al., J. Immuonol. 130:1910-1917 (1983), from bovine milk, Salgar SK, et al. FASEB Journal 3:A320 Abstract #546, 1989, or from bovine blood or milk. Redelman D,
  • Membrane potential sensitive dyes comprise another category of dyes that have been used to measure viable cells. These dyes are charged fluorescent molecules which accumulate in intact cells because of the electrochemical potential that exists across the cell membranes. Shapiro, et al., Proc. Nat. Acad. Sci. USA 76:5728, 1979. have shown that these dyes could be used in flow cytometric tests to measure membrane potential. These dyes have also formed the basis of a test to identify blood leukocytes. See U.S. 4,343,782.
  • the fluorescence intensity of cells incubated with membrane potential sensitive dyes is related to the membrane polarization.
  • Mammalian cells have an electrochemical potential across their membranes primarily because of differences in the intra- and extracellular concentrations of Na * and K' ions.
  • Dead cells or cells with damaged membranes do not have such an electrochemical potential so they do not accumulate these dyes and hence label less intensely.
  • processes that alter membrane potential by causing alterations in the content of intracellular ions affect the fluorescence intensity of cells incubated with these dyes. For example, normal granulocytes stimulated with PMA depolarize and lose fluorescence intensity, but not to the level of dead or damaged cells. Since dyes of this sort have been used to detect viable cells isolated from blood, Shapiro HM, U.S. Patent 4,343,782. they were tested with cells from milk in order to determine if they would be suitable. Cells from milk were tested with
  • the esterase dependent dyes in the first category label appropriately if the cells are incubated with the dye at room temperature for 10-20 minutes.
  • a dye in the second category that selectively labels nucleic acid in viable cells it is necessary to incubate the cells with the dye at physiologic temperature for 10-20 minutes.
  • the cells In order to detect the cells labeled with the fluorescent dyes, the cells must be illuminated with light of the appropriate excitation wavelength. The labeled cells will then fluoresce and emit light at a longer wavelength. The emitted light may be detected qualitatively or quantitatively by using a photomultiplier tube or other light,sensing device. These measurements can be made in a flow cytometer which permits measuring individual cells in the milk sample. Alternatively, these measurements can be made after depositing the cells on a filter, after centrifuging them, or after using other conventional techniques to separate the cells from the milk and/or to concentrate the cells into a small volume of liquid or onto a solid surface. The dyes and labeling conditions have been selected so that it is not necessary to use flow cytometry for analysis.
  • the tests that comprise this invention are designed to detect viable cells in milk and/or to distinguish different cell types in milk. Therefore, a number of variables that can affect the number of cells recovered, the types of cells recovered, and/or the viability of the cells recovered were examined. These variables were- examined with the established quantitative technique of labeling with CMFDA and measuring the labeled cells by flow cytometry.
  • milk was collected from mastitic cows at the beginning of milking and then again at the end of milking.
  • Milk collected at the start of milking was relatively enriched in granulocytes whereas milk collected at the end of milking was relatively enriched in mononuclear cells.
  • all cell types were present in both types of samples.
  • the tests employed in this invention depend on the cells in the milk being viable. Therefore, variables that could affect the viability of the cells in the milk after collection were compared.
  • the stability of cells in milk analyzed at various times after collection was determined. Mastitic milk was collected, kept at room temperature and analyzed at various times from approximately 30 minutes to 8 hours after collection, or after standing 20-24 hours after collection. Milk samples analyzed within approximately 8 hours after collection produced comparable results. Milk samples allowed to stand overnight before analysis had increased numbers of disintegrating granulocytes and comparably reduced numbers of intact granulocytes. Therefore, inflammatory cells are best detected if milk samples were analyzed within 8 hours of collection.
  • the second variable was the stability of cells after they are labeled.
  • the question of stability was particularly important with cells labeled with FDA since it is known that the intracellular fluorescein "leaks" from viable cells.
  • cells were separated from milk by centrifugation and labeled with either FDA or CMFDA. These cells were then kept at 0-4 * C and analyzed by flow cytometry after various times. After 4 hours, there was a 10-38% loss in fluorescence intensity, but no appreciable difference between cells labeled with FDA or with CMFDA. Therefore, cells may be kept at 0-4* C for several hours after labeling before analysis without appreciable loss of fluorescence intensity.
  • the isotonic saline sheath fluid (American Scientific Products, McGaw Park, IL 60085, Catalog No. B-3157-12A) contained EDTA and no Ca 2+ or Mg + .
  • the BSS contained both Ca 2+ and Mg 2+ , each at a concentration of about 1.2mM.
  • the membrane potential sensitive dye 3',3'- dihexyloxacarbocyanine iodide (Di0C 6 (3), Molecular Probes, Catalog No. D-273) was solubilized in DMSO at 1.0 mM and additionally diluted in ethanol to produce a 10 ⁇ M solution.
  • Another membrane potential sensitive dye, Di- 4-ANEPPS (Molecular Probes, Catalog No. D-1129) , was solubilized in ethanol at 1.0 or 0.10 mM.
  • the activation of intracellular oxidative activity was measured with 2' ,7'-dichloro-dihydrofluorescein diacetate (DCHF-DA, Molecular Probes, Catalog No.
  • Leukocytes from human and bovine peripheral blood and cells from mastitic bovine milk were examined. Venous blood was collected from normal human donors into heparinized Vacutainer tubes. To prepare leukocytes from human blood, 4-8 ml of blood in a 15 ml conical centrifuge tube was underlain with an equal volume of Histopaque 1077 (Sigma Chemical, Catalog No. 1077-1) and centrifuged for 10-15 minutes at room temperature at 1000 rpm (IEC No. 269 rotor) .
  • Histopaque 1077 Sigma Chemical, Catalog No. 1077-1
  • the cells at the plasma- Histopaque interface i.e., the peripheral blood mononuclear cells (PBM)
  • PBM peripheral blood mononuclear cells
  • the layer of Histopaque above the erythrocytes containing the polymorphonuclear neutrophils (PMN) was diluted with BSS or with sheath fluid, centrifuged, and the washed pellet of cells resuspended in a volume approximately equal to that of the initial blood volume.
  • Venous blood from Holstein dairy cattle was also collected into heparinized Vacutainer tubes, and leukocytes were prepared by a modification of the procedures used for obtaining human leukocytes.
  • Heparinized bovine blood (2-3 ml) was diluted with an equal volume of sheath fluid, underlain with a volume of Histopaque equal to the volume of the diluted blood, -and centrifuged for 20 minutes at 2000 rpm.
  • the PBM were prepared as described above for human cells and resuspended in a volume of BSS or sheath fluid equivalent to the total volume of the original diluted blood.
  • the PMN were obtained by hypotonic lysis of the erythrocyte- PMN pellet.
  • the pellet of cells (about 1 ml) was resuspended in sheath fluid to a total volume of 4 ml.
  • the RBCs were then lysed by adding 8 ml of water and mixing. After 30 seconds, 3 ml of 3.40% (w/v) NaCl, i.e., 4x concentrated, was added to restore normal tonicity.
  • the PMN were pelleted and resuspended in BSS or sheath fluid in a volume equivalent to the total volume of the original diluted blood. Milk samples were collected in several ways for analysis.
  • the induction of oxidative activity was detected with cells loaded with DCHF-DA or with CDDF-DA. These cells, suspended in BSS or in buffered saline sheath fluid, were incubated with 1.0 l/ml of the stock solution of DCHF-DA or CDDF-DA for 20 minutes at room temperature. Aliquots (0.5 ml) of these loaded cells were then added to tubes containing 5 ⁇ l of DMSO, ethanol, or fMLP or PMA in these solvents, and incubated at 37° for 20 minutes. The activated cells were kept on ice until the flow cytometric analysis was completed. Changes in membrane potential were detected with DiO.C 6 (3) .
  • Cells were incubated with 5 ⁇ l/ml of the 10 ⁇ K DiOC 6 (3) stock solution for 20-40 minutes at room temperature. The cells were then added to stimulators as described above, incubated for 5 minutes at 37*, and kept on ice until flow cytometric analysis.
  • the FACStar Plus is a laser-based instrument with a different type of fluidic system that employs a jet-in-air stream instead of an enclosed system, as used in the FACS Analyzer. Instead of an electronic volume sensor, the FACStar Plus uses low angle forward light scatter to estimate volume. Effects of Milk Collection and Storage Conditions on the Cells Recovered
  • Pre- and post-milking samples were collected from individual quarters before and after regular milking to determine if milk sampling procedures affected the types of cells recovered. Aliquots of these samples were centrifuged, the cells were labeled with CMFDA, and examined by flow cytometry. The pre-milking samples contained relatively more granulocytes, and the post- milking samples contained relatively more lymphocytes and monocyte-macrophage-type cells. Both types of milk samples contained a complete spectrum of the cells.
  • Example 1 Cells Labeled with Esterase-Sensitive Dyes
  • CMFDA Cells Labeled with Esterase-Sensitive Dyes
  • Viable cells labeled with BCECF-AM or with FDA had fluorescence intensities at least 1000-fold higher than the natural autofluorescence and 100-fold higher than the signals produced by dead or damaged cells. These dyes can be used with other types of instruments to screen milk samples in order to detect those with elevated numbers of viable cells.
  • Example 2 Cells Labeled with Nucleic Acid Binding Dyes
  • Dihydroethidium is a unique dye that can be used to label selectively the nucleic acid of viable cells. When tested with cells from milk, it had at least three significant advantages in identifying and/or enumerating viable cells.
  • HED labeled viable cells with a very high signal to noise ratio.
  • the fluorescence intensity of viable cells after incubation with HED was as much as 10,000 fold higher than the intensity of autofluorescence.
  • the fluorochrome in viable cells is strongly bound.
  • the reduced, non-charged HED is incorporated into viable cells where it is converted by intracellular oxidation to the charged ethidium ion.
  • the intracellular ethidium binds to nucleic acid with very high affinity.
  • viable cells are selectively labeled with a dye that binds tightly to intracellular materials.
  • the spectral properties of ethidium produced by intracellular oxidation of HED offers several useful features. The chief advantage is that ethidium has a very large Stokes shift making it relatively easy to select optical filters that discriminate efficiently between the excitation and emission wavelengths. Therefore, HED can be used to identify viable cells either by flow cytometric techniques, as has been demonstrated, or by other procedures more suitable for processing large numbers of samples.
  • phorbol esters such as PMA
  • the phorbol esters are capable of activating granulocytes and causing an increase in the intracellular oxidative activity.
  • Granulocytes from bovine or human blood that were labeled with DCHF-DA increased in fluorescence intensity by 10- 30-fold when stimulated with PMA.
  • the carboxy derivative, CDDF-DA labeled granulocytes less efficiently and produced a relatively small increment of fluorescence when stimulated with PMA.
  • Cells separated from milk by centrifuga ion and' labeled with Di0C 6 (3) could be differentially identified by flow cytometry about as well as cells labeled with CMFDA.
  • cells in whole milk could also be labeled with Di0C 6 (3) and identified using the FACStar flow cytometer.
  • These dyes provided a poorer signal-to- noise ratio in attempts to discriminate between live and dead cells.
  • dyes such as Di0C 6 (3) can also measure the degree of membrane depolarization, and hence the degree of activation of granulocytes.
  • the fluorescence intensity of cells labeled with DiOC 6 (3) is directly related to membrane potential. It was found that granulocytes and lymphocytes from bovine and human blood labeled with comparable intensity with DiOC 6 (3) .
  • the blood granulocytes from both species increased in fluorescence when hyperpolarized by exposure to valinomycin. These cells decreased in fluorescence when treated with stimulatory amounts of PMA.
  • the milk granulocytes neither hyperpolarized with valinomycin, nor did they change in intensity when exposed to PMA.
  • the fluorescence intensity of the milk granulocytes was essentially the same as granulocytes from blood after stimulation with PMA. These results indicate that granulocytes from mastitic milk had been activated in vivo. These tests confirmed the results measuring intracellular oxidation, as described above.

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Abstract

Procédé amélioré de détection, d'identification et/ou d'énumération de cellules viables dans le lait de bovin, dans lequel des cellules viables sont sélectivement marquées à l'aide d'un colorant fluorescent et ensuite identifiées et/ou comptées. On utilise dans ce procédé des colorants dépendant de l'estérase, des colorants de liaison d'acides nucléiques, ainsi que des colorants détectant une activité d'oxydation intracellulaire.
PCT/US1991/005377 1990-07-30 1991-07-30 Colorants fluorescents d'identification et d'enumeration de cellules viables dans le lait WO1992002632A1 (fr)

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WO1997017463A1 (fr) * 1995-11-08 1997-05-15 Pioneer Hi-Bred International, Inc. Procedes de determination de la viabilite de cultures microbiennes
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WO2015016763A1 (fr) * 2013-07-29 2015-02-05 Delaval Holding Ab Procédé de préparation d'un échantillon de lait, et dispositif conçu pour être utilisé lors de la préparation d'un échantillon de lait
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US9624463B2 (en) 2013-11-04 2017-04-18 Charles River Laboratories, Inc. Filtration system and use thereof
EP3080341A4 (fr) * 2013-12-11 2017-05-17 Analiza, Inc. Dispositifs et méthodes de détermination et/ou d'isolation de cellules telles que les cellules f tales ou les cellules cancéreuses circulantes
US10324036B2 (en) 2012-05-02 2019-06-18 Charles River Laboratories, Inc. Porous planar cell capture system
US10718757B2 (en) 2017-04-11 2020-07-21 MEP Equine Solutions LLC Method for the rapid and convenient detection and enumeration of neutrophils in biological samples
US10928405B2 (en) 2013-12-11 2021-02-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells

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Cited By (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017463A1 (fr) * 1995-11-08 1997-05-15 Pioneer Hi-Bred International, Inc. Procedes de determination de la viabilite de cultures microbiennes
EP0948639A4 (fr) * 1996-11-01 2001-12-19 Coulter Int Corp Identification de cellules blastiques dans une preparation de leucocytes
US8081312B2 (en) 1997-05-05 2011-12-20 Chemometec A/S Method and a system for determination of particles in a liquid sample
US8125643B2 (en) 1997-05-05 2012-02-28 Chemometec A/S Method and a system for determination of particles in a liquid sample
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