WO1995013540A1 - Procede de comptage absolu de cellules rares - Google Patents

Procede de comptage absolu de cellules rares Download PDF

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Publication number
WO1995013540A1
WO1995013540A1 PCT/US1994/012952 US9412952W WO9513540A1 WO 1995013540 A1 WO1995013540 A1 WO 1995013540A1 US 9412952 W US9412952 W US 9412952W WO 9513540 A1 WO9513540 A1 WO 9513540A1
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Prior art keywords
cells
sample
beads
fluorescence
labelled
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PCT/US1994/012952
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English (en)
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Leon Terstappen
Chia-Huei Chen
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Becton Dickinson And Company
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Priority to EP95901205A priority Critical patent/EP0685071A4/fr
Priority to JP7513998A priority patent/JP2680931B2/ja
Publication of WO1995013540A1 publication Critical patent/WO1995013540A1/fr

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    • 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
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • 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
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells

Definitions

  • This invention relates to a method for determining the absolute number of rare cells in a sample of cells, and more particularly relates to a method for determining the absolute number of progenitor cells in a sample of peripheral blood or bone marrow cells by means of flow cytometry.
  • CD4 + cell counting became more important in the diagnosis and monitoring of the disease.
  • absolute CD4 counts were attempted by multiplying a leukocyte count by the percentage of lymphocytes (both as determined by a hematology analyzer (e.g., Coulter Counter) with the percentage of CD4 + as determined by flow cytometry. See NCCLS Guidelines Report. This approach suffers from large imprecision due to well recognized variations in precision between hematology instruments.
  • Other approaches involve the use of a syringe delivery system coupled to a flow cytometer where a known sample volume is delivered to the instrument and the number of events in that sample volume is counted.
  • Still other approaches involve the addition of a known number of fluorescent beads to a known sample volume and then counting the number of beads and events per unit of time. The ratio of beads to cells provides the number of cells per unit volume. See U.S. Pat. No. 4,110,604.
  • Civin In between very rare cells and cell populations that exceed 5%, there are cell populations that are "rare" for which absolute counting is becoming increasingly important.
  • Civin described a population of pluripotent lympho-hematopoietic cells which were substantially free of mature lymphoid and myeloid cells.
  • Civin also described an antigen, MY-10 (also known as CD34) and a monoclonal antibody (of the same name) thereto, which was present on these cells.
  • MY-10 also known as CD34
  • a monoclonal antibody of the same name
  • Civin described a number of therapeutic uses for progenitor cells. Many of these uses involve the transplantation of CD34 + cells into the bone marrow of a patient having undergone ablative therapy for the purpose of not only reconstituting the patient's hematopoietic system but also avoiding graft versus host disease. Still other therapies involve the transplantation of CD34 + cells into peripheral blood.
  • leukocytes are collected from peripheral blood, cord blood or bone marrow and the CD34 + cells are separated from the remainder of the cells in the sample using either CD34 monoclonal antibodies in a positive selection step or in combination with one or more other antibodies to deplete non-progenitor cells.
  • the cells may be harvested from an allogeneic source or from the patient. If obtained from the patient, the patient first may be given growth factors, such as GM-CSF or G-CSF, prior to harvesting in order to boost the number of circulating progenitor cells.
  • Means for performing the collection process include fluorescence activated cell sorting (see, e.g., U.S. Pat. No.s 3,826,364 and 5,030,002), biotin/avidin columns (see, e.g., U.S. Pat. No.s 5,215,927, 5,225,353, and 5,240,856) and magnetic bead based systems (see, e.g., WO91/09141 and Kato et al . , Cytometry, 14:384 (1993)) .
  • Systems comprising flow cytometers, data gathering and analysis hardware and software, and reagents are sold by a number of commercial entities including Becton Dickinson Immunocytometry Systems ("BDIS”) .
  • CD34 + cells are present within the material to be transplanted. Generally, it is believed that 1 x IO 6 CD34+ cells per kilogram of body weight are necessary in any donor material to have a reasonable likelihood of successful engraftment. In other cases (i.e., where cytokines are used to stimulate progenitor cell production prior to harvesting) , it is important to know the level of progenitor cells in the blood so as to time the harvest of those cells from the donor with maximum production. Thus, it is important to be able to accurately measure the absolute number of progenitor cells (or cells in subsets thereof) . Flow cytometry provides a convenient means for rapidly and accurately counting cells.
  • the invention comprises reagents and methods for accurately determining the absolute numbers of rare cells
  • the method has particular utility in determining the number of CD34 + cells in a sample and subsets of CD34+ cells such as CD34 + /CD38-/HLA-DR + cells.
  • the method comprises the steps of: a) labelling cells in a sample with a composition comprising 1) one or more fluorescent markers that react differentially react with all of the cells in the sample, 2) one or more fluorescent markers that selectively react with the rare cells, and 3) a known number of fluorescent beads, wherein all of the fluorescent markers have emission spectra that are distinguishable; and b) analyzing the labelled cells by means of flow cytometry comprising the steps of 1) setting a fluorescence threshold on the fluorescence emitted by the cells labelled with the marker of a)l) so as to include all labelled cells and beads in a data analysis set, 2) analyzing the cells in the set that meet or exceed the threshold for one or more of light scatter and fluorescence emissions, 3) using the data recorded to discriminate between and among
  • the method comprises the steps of: a) labelling cells in a sample with a composition comprising 1) one or more monoclonal antibodies, labelled with a first fluorochrome, that di ferentially react with all cell populations in the sample, 2) one or more monoclonal antibodies, labelled with a fluorochrome that has an emission spectra that- is distinguishable from the first fluorochrome, that selectively react with rare cells, and 3) a known number of fluorescent beads; and b) analyzing the labelled cells by means of flow cytometry comprising the steps of 1) setting a fluorescence threshold on the fluorescence emitted by the cells labelled with the fluorescent dye of a)l) so as to include all labelled cells and beads in a data analysis set, 2) analyzing the cells in the set that meet or exceed the threshold for one or more of light scatter and fluorescence emissions, 3) using the data recorded to discriminate between and among the various cell populations in the sample and beads, 4) counting the number of beads and rare cells
  • the composition may be added to the analysis tube before or after the cell sample is added.
  • each of the elements of the composition may be added separately or in sub-combinations. It is preferred to provide the elements as a unitary composition.
  • the beads used in the practice of this invention should have certain properties. First, they should be small (i.e., between 0.2 ⁇ m and 20 ⁇ m, 2 ⁇ m are preferred) so as to stay suspended in the mixture and not settle any faster than the cells in the sample. Second, they should be made of a material that avoids clumping or aggregation. Third, they should be fluorescent and/or of such size so as to be distinguishable from the cells in the sample.
  • Fluorescence can be achieved by selecting the material that comprises the micropaxticle to be autofluorescent or it can be made fluorescent by being tagged with a fluorescent dye. If fluorescence is used, the fluorescence of the beads must be such that in one fluorescence channel it is sufficiently greater than noise from background so as to be distinguishable and also must be distinguishable in other fluorescence channel (s) from the fluorescent dye(s) used as part of the fluorescent marker(s) . One log difference between the dye(s) and "the bead fluorescence is sufficient.
  • Beads having these properties may be selected from the group consisting of fixed chicken red blood cells, coumarin beads, liposomes containing a fluorescent dye, fluorescein beads, rhodamine beads, fixed fluorescent cells, fluorescent cell nuclei, microorganisms and other polymeric beads tagged with a fluorescent dye. Beads of the latter type are commercially available from Molecular Probes, Inc. and Flow Cytometry Standards Corp.
  • Fluorescent markers may comprise fluorescent dyes, such as nucleic acid dyes, and/or monoclonal antibodies (or other reagents that have a high binding affinity for a particular target molecule) that are labelled with a fluorochrome.
  • Fluorochromes for monoclonal antibodies may be selected from the group consisting of fluorescein isothiocyanate ("FITC”), phycoerythrins (“PE”), allophycocyanin (“APC”), peridinin chlorophyll complex protein ("PerCP”, BDIS), CY3 and CY5 (Biological Detection Systems, Inc.), Texas Red (Molecular Probes, Inc.) and tandem conjugates thereof, such as PE/CY5. See U.S. Pat. No.s 4,542,104 and 4,520,110.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrins
  • APC allophycocyanin
  • PerCP peridinin chlorophyll complex protein
  • CY3 and CY5 Biological Detection Systems, Inc.
  • Texas Red Molecular Probes, Inc.
  • Fluorescent dyes that exhibit a Stokes shift when reacted with nucleic acids include Thiazole Orange (BDIS) , 7-amino-actinomycin D (Sigma) , SY-III-8 (Molecular Probes, Inc.), LDS-751 (Molecular Probes, Inc.), and dyes described in U.S. Pat. No.s 4,544,546, 4,945,171 and 5,066,580. It is understood that the fluorescence of the beads, if any, and fluorescent markers -should be such that each of their peak emission spectra are distinguishable, preferably without compensation.
  • Means for discriminating between the cell populations and beads in the sample comprise both cell analyzers (e.g., FACScan brand flow cytometer, BDIS) and cell sorters (e.g., FACSort brand flow cytometer, BDIS) .
  • a threshold is set according to manufacturer's directions for fluorescence emitted from one of the fluorescent markers. This may be a nucleic acid dye or a fluorochrome used in conjunction with a monoclonal antibody to differentially label all of the cells. (In an alternative embodiment, multiple thesholds may be set in an "A and B" or "A but not B” configuration.) For each event that exceeds the fluorescence threshold(s) , one or more light scatter and fluorescence emission parameters are recorded. (See U.S.
  • Samples of cells can be obtained from any tissue source, such sources included peripheral blood, cord blood, bone marrow, thymus, spleen or lymph node. Sources of cells from peripheral blood and bone marrow are particularly applicable to the methods of this invention.
  • the composition may be modified to replace the fluorescently labelled antibody specific for the rare cells with a fluorescently labelled irrelevant isotpye control.
  • the sample may be split into aliquots with one being stained with the isotype composition and the other with the standard composition. The former aliquot then can be analyzed first to determine background fluorescence levels prior to analysis with the second aliquot.
  • the method has particular utility in counting CD34 + cells (and subsets thereof) from peripheral blood, cord blood or bone marrow.
  • the method comprises the steps of: a) labelling cells from a sample with a composition comprising 1) a nucleic acid dye that will selectively react with nucleated cells, 2) a monoclonal antibody labelled with a first fluorochrome that differentially reacts with mature lymphoid, neutrophil, erythroid and monocytic cells and weakly with progenitor cells, 3) a CD34 antibody labelled with a second fluorochrome, and 4) a known number of fluorescent beads; and b) analyzing the labelled cells by means of flow cytometry comprising the steps of 1) setting a fluorescence threshold on the fluorescence emitted by the nucleic acid dye so as to include all nucleated cells and beads, 2) analyzing the cells and beads in the sample that meet or exceed the threshold for light scatter and fluorescence emissions, 3) using fluorescence emissions and scatter data recorded to discriminate between
  • Thiazole Orange and/or SY-III-8 are preferred nucleic acid dyes. SY-III-8 is especially preferred.
  • CD45 is a preferred antibody for differentially reacting with all leukocytes.
  • a tandem conjugate of PE/CY5 is preferred as one fluorochrome and PE is preferred as the other fluorochrome. Nile Red beads (Molecular Probes, Inc.) are preferred.
  • the nucleic acid dye may be eliminated and the threshold set on the fluorescence emitted by the cells labelled with the first fluorochrome.
  • rare cell populations that may be counted by this method include antigen specific B or T lymphocytes and specific effector cells (e.g., cytotoxic T cells) .
  • FIG. 1 comprises two dot plots of lysed normal peripheral blood to which 50,000 fluorescent beads were added and then analyzed by means of flow cytometry wherein 1A is a plot of transformed orthogonal light scatter versus forward light scatter and IB is a plot of log fluorescence emissions at 564-606 nm versus log fluorescence emissions at 515-545 nm.
  • FIG. 2 comprises two dot plots of lysed normal peripheral blood treated as in FIG. 1 to which the nucleic acid dye SY-III-8 also was added wherein 2A is a plot of transformed orthogonal light scatter versus log fluorescence for events collected using a light scatter threshold and 2B is a plot of transformed orthogonal light scatter versus log fluorescence for events collected using a fluorescence threshold.
  • FIG. 3 comprises two dot plots of lysed abnormal peripheral blood treated as in FIG. 2 to which the CD45 PE/CY5 also was added for events collected using a fluorescence threshold wherein 3A is a plot of log SY-III-8 fluorescence versus forward light scatter and 3B is a plot of transformed orthogonal light scatter versus log PE/CY5 fluorescence.
  • FIG. 4 comprises a series of six dot plots of lysed normal peripheral blood treated as in FIG. 3 for events collected using a fluorescence threshold to which either an IgGi PE control antibody also was added (3A, 3C and 3E) or a CD34 PE antibody was added (3B, 3D and 3F) wherein 3A and #B are plots of log PE fluorescence versus log SY-III-8 fluorescence, 3C and 3D are plots of transformed orthogonal light scatter versus forward light scatter and 3E and 3F are plots of transformed orthogonal light scatter versus log PE/CY5 fluorescence.
  • 3A and #B are plots of log PE fluorescence versus log SY-III-8 fluorescence
  • 3C and 3D are plots of transformed orthogonal light scatter versus forward light scatter
  • 3E and 3F are plots of transformed orthogonal light scatter versus log PE/CY5 fluorescence.
  • FIG. 5 comprises histograms showing the number of CD34 + cells per ⁇ l in 5 replicate analyses of two different lysed peripheral blood samples ("low” CD34 counts closed box, "high” CD34 counts open box) treated as in FIG. 4 with a CD34 PE antibody for events collected using a fluorescence threshold wherein 5A represents one sample treated once and analyzed five times and 5B represents one sample split into five aliquots each aliquot treated separately and then analyzed.
  • FIG. 6 comprises a series of eight dot plots of lysed leukopheresis samples to which have been added 50,000 fluorescent beads, SY-III-8, CD45 PE/CY5 and IgGi PE (6A-6D) or 50,000 fluorescent beads , SY-III-8, CD45 PE/CY5 and CD34 PE (6E-6H) wherein 6A and 6E are plots of transformed orthogonal light scatter versus forward scatter, 6B and 6F are plots of transformed orthogonal light scatter versus log PE/CY5 fluorescence, 6C and 6G are plots of log PE versus log SY-III-8 fluorescence, and 6D and 6H are plots of log PE versus log PE/CY5 fluorescence.
  • Bone marrow aspirates, peripheral blood and leukopheresis samples were collected from patients and normal donors. Heparin was used as an anticoagulant for the bone marrow samples and EDTA ⁇ 3 as an anticoagulant for peripheral blood samples. For each test, 50 ⁇ l of peripheral blood, bone marrow or leukopheresis sample was used.
  • the samples were incubated with lO ⁇ l of control or test reagent and 50,000 freeze-dried 2.49 ⁇ m Nile Red beads.
  • the control and test reagent mixture consisted of a nucleic acid dye (i.e., 5ng SY-III-8, Molecular Probes, Inc.), a PE/CY5 tandem conjugate of C45 (lOOng clone HLe-1, BDIS) and a PE conjugate of CD34 (lOOng clone HPCA-2, BDIS) in the test reagent or IgG ⁇ control antibody (50ng, BDIS) in the control reagent.
  • a mixture of Thiazole Orange and SY-III-8 was used. Samples were incubated for* 20 minutes at room temperature. After incubation, the sample was diluted with 0.5ml of a red cell lysing solution (FACSLysing solution, BDIS) .
  • FACSLysing solution red cell lysing solution
  • SY-III-8 is a nucleic acid dye with a high preference for DNA over RNA.
  • Thiazole Orange is a similar type dye and also has a preference for DNA over RNA but not to the same degree. The use of Thiazole Orange to label nucleated cells is described in U.S. Pat. No. 4,883,867.
  • SY-III-8 is excitable at 488nm and its emission spectra is distinguishable from that of PE/CY5 and PE.
  • Flow cytometric analysis was performed on a FACScan brand flow cytometer (BDIS) equipped with an Argon laser (488nm) . Data acquisition was performed with LYSIS II brand data acquisition software (BDIS) .
  • FIG. 1 shows the light scatter and fluorescence properties of the beads. The beads are depicted red, the cells green and debris gray. The data was analyzed and showed that 9.8% of the events were beads and 28.6% of the events were cells. Knowing that 50,000 beads were added to 50 ⁇ l of blood, the absolute 'number of cells is calculate 'to be 2.9 x IO 3 per ⁇ l. Because of the poor separation between debris and cells at the light scatter threshold, however, this number may not be accurate and, as importantly, may vary from sample to sample or instrument to instrument. Thus, merely adding fluorescent beads to a sample does not necessarily result in accurate absolute counting.
  • nucleic acid dye may be added to the sample preparation.
  • a nucleic acid dye may be added to the sample preparation.
  • Using the combination of light scatter and nucleic acid content adds an additional dimension over the method disclosed in FIG. 1.
  • FIG. 2A shows a plot of orthogonal light scatter versus log fluorescence with the threshold set on orthogonal light scatter. Debris, cells and beads are colored as in FIG. 1. Data analysis of the sample revealed that beads accounted for 9.6% of the events, nucleated cells 34.1% and non-nucleated debris 56.3%.
  • FIG. 2B shows that the percentage of beads was 22.8% while the percentage of nucleated cells was 77.0% of the events measured.
  • the absolute number of nucleated cells was determined to be 3.55 x IO 3 per ⁇ l whereas in FIG. 2B the absolute number of cells was determined to be 3.38 x IO 3 ⁇ l.
  • the use of light scatter as the event threshold therefore, introduced a 5% error.
  • current methods of immunofluorescence measurements using a nucleic acid dye, fluorescent beads and a single immunofluorescent marker for the cell population of interest is appropriate.
  • FIG. 3 illustrates a peripheral blood sample from a chemotherapy patient to which the composition used in FIG. 2 was added.
  • CD45 PE/CY5 also was added to the composition. Beads were colored red, monocytes blue, lymphocytes green, neutrophils and eosinophils yellow and nucleated red cells purple.
  • the cell population colored gray contains progenitor cells among which are CD34+ cells, plasma cells and basophilic granulocytes.
  • Data analysis for events exceeding the SY-III-8 threshold shows that in a plot of transformed orthogonal light scatter versus log PE/CY5 fluorescence, each of the cell populations falls into a defined region including progenitor cells.
  • monocytes were 12.5% of the events (0.77 x IO 3 per ⁇ l)
  • lymphocytes were 15.8% of the events (0.98 x IO 3 per ⁇ l)
  • neutrophils and eosinophils were 50.2% of the events (3.1 x IO 3 per ⁇ l)
  • nucleated red cells were 3.5% of the events
  • progenitor cells were 1.75% of the events (0.11 x IO 3 per ⁇ l) .
  • CD34 monoclonal antibodies are commercially available from a number of sources including BDIS.
  • FIG. 4 a sample of peripheral blood was split into two aliquots and treated as in FIG. 3; however, to one aliquot, a PE labelled IgG_ isotype control was added to the composition while in the other aliquot a PE labelled CD34 antibody was added.
  • FIG.s 4A, 4C and 4E show the control aliquot while FIG. 4B, 4D and 4F show the aliquot with CD34 PE.
  • Cells and beads are colored as in FIG. 3 with CD34 + cells colored black.
  • the arrow points to the expected location of the CD34 + cells which is confirmed in FIG. 4F.
  • CD34 + cells from FIG. 4F comprise only about 30% of the progenitor cells identified in the space marked by the arrow in FIG.
  • the mean absolute number of CD34 + cells from the "low” sample was 37.2 per ⁇ l (CV 10.4%) and the mean absolute number of cells from the "high” sample was -399.1 per ⁇ l (CV 8.2%) .
  • the mean percentage of CD34 + cells from the "low” sample was 0.2% (CV 4.4%) and the mean percentage of cells from the "high” sample was 1.2% (CV 6.0%) .
  • the mean absolute number of nucleated cells in the "low” sample was 24.6 x IO 3 per ⁇ l (CV 4.7%), and the mean absolute number of nucleated cells in the" high” sample was 34.1 x IO 3 per ⁇ l (CV 3.8%) .
  • the mean absolute number of CD34 + cells from the "low” sample was 39.7 per ⁇ l (CV 9.7%), and the mean absolute number of cells from the "high” sample was 322.2 per ⁇ l (CV 9.0%) .
  • the mean percentage of CD34 + cells from the "low” sample was 0.2% (CV 13.3%) and the mean percentage of cells from the "high” sample was 1.1% (CV 9.3%) .
  • the mean absolute number of nucleated cells in the "low” sample was 26.7 x IO 3 per ⁇ l (CV 3.8%), and the mean absolute number of nucleated cells in the" high” sample was 29.8 x IO 3 per ⁇ l (CV 5.5%) .

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Abstract

Procédé de comptage absolu de cellules rares (p. ex. < 5 %) d'un échantillon de cellules. Le procédé recourt à la cytométrie de flux et à un mélange de réactifs comprenant des perles fluorescentes, un ou plusieurs marqueurs fluorescents réagissant différemment selon les populations de cellules de l'échantillon et un ou plusieurs marqueurs fluorescents réagissant sélectivement avec les cellules rares. Ledit procédé est particulièrement utile pour le comptage des cellules parentes.
PCT/US1994/012952 1993-11-12 1994-11-10 Procede de comptage absolu de cellules rares WO1995013540A1 (fr)

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EP95901205A EP0685071A4 (fr) 1993-11-12 1994-11-10 Procede de comptage absolu de cellules rares
JP7513998A JP2680931B2 (ja) 1993-11-12 1994-11-10 希少細胞の絶対数を数える方法

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JP4566299B2 (ja) * 1998-04-09 2010-10-20 シスメックス株式会社 赤芽球の分類計数方法
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4751188A (en) * 1982-10-15 1988-06-14 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Method for the simultaneous quantitative determination of cells and reagent therefor
US5047321A (en) * 1988-06-15 1991-09-10 Becton Dickinson & Co. Method for analysis of cellular components of a fluid
US5137809A (en) * 1987-11-09 1992-08-11 Becton, Dickinson And Company Method to determine the composition of bone marrow samples

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4751188A (en) * 1982-10-15 1988-06-14 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Method for the simultaneous quantitative determination of cells and reagent therefor
US5137809A (en) * 1987-11-09 1992-08-11 Becton, Dickinson And Company Method to determine the composition of bone marrow samples
US5047321A (en) * 1988-06-15 1991-09-10 Becton Dickinson & Co. Method for analysis of cellular components of a fluid

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* Cited by examiner, † Cited by third party
Title
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