EP1181553A1 - Procede et dispositif pour le comptage de cellules dans l'urine - Google Patents

Procede et dispositif pour le comptage de cellules dans l'urine

Info

Publication number
EP1181553A1
EP1181553A1 EP00959042A EP00959042A EP1181553A1 EP 1181553 A1 EP1181553 A1 EP 1181553A1 EP 00959042 A EP00959042 A EP 00959042A EP 00959042 A EP00959042 A EP 00959042A EP 1181553 A1 EP1181553 A1 EP 1181553A1
Authority
EP
European Patent Office
Prior art keywords
cells
added
urine sample
fixative
urine
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.)
Withdrawn
Application number
EP00959042A
Other languages
German (de)
English (en)
Inventor
Oddbj Rn Gjelsnes
Ystein R Nning
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.)
Optoflow AS
Original Assignee
Optoflow AS
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 Optoflow AS filed Critical Optoflow AS
Publication of EP1181553A1 publication Critical patent/EP1181553A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • 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

Definitions

  • the present invention regards a method and a device for counting bacteria and other micro-organisms in u ⁇ ne from a patient
  • the method and the device are very quick and accurate in terms of diagnosing cystitis
  • the technical area of the invention is medical diagnostics
  • the techniques used are covered by the areas of biochemistry/microbiology, optics, fluid mechanics, electronics and computer science
  • the novel aspects of the invention fall mainly within the subjects of biochemistry and microbiology
  • the invention seeks, through the method and device thereof, to solve the following problem
  • cystitis it takes a long time (one or more days) to count the number of bacteria m u ⁇ ne This because the bacteria must be cultivated on agar discs until they fomi macroscopic colonies than can be seen with the naked eye
  • the long wait required before a diagnosis can be made is unfortunate, as the patient is otten given antibiotics before a certain diagnosis has been made
  • the cells In order to be able to do this, the cells must be made fluorescent by adding special fluorochromes that attach to the cells In the flow cytometer, the cells are illuminated by a beam of light as they pass the measurement point in a liquid stream (thus flow cytometry) The instrument registers the light scatter and fluorescence from each individual cell The intensity of the scattered light is a function of among other things the size of the cell, and the intensity of the fluorescent light is a function of among other things the amount of substance made fluorescent (e g nucleic acids).
  • the concentration of cells is simply determined by counting the number of fluorescent particles in the sample. This may be grouped into different types of cells based on the size of the cells (light scatter) and the content of nucleic acids (fluorescence). There are also other quick methods of measuring bacteria in urine, however these are indirect and measure the presence of cellular metabolites (dipsticks).
  • US 5 693 484 regards a method of counting and classifying cells in urine.
  • a fluorescent dye is added to the urine sample, which dye attaches to the nucleic acids of the cells.
  • the cells are then illuminated with light at the blue and violet wavelengths, and analysed in a flow cytometer.
  • the cell walls of the bacteria act as a barrier against the surroundings.
  • the bacteria may have intracellular pumps that bring the dye out again.
  • the bacteria are considerably smaller than somatic cells, thus containing less of the cellular components that are to be stained.
  • the present invention provides a method and a device that are reliable and quicker than the known techniques.
  • the method consists of the following steps:
  • the urine sample from the patient is undiluted and is mixed with a fixative liquid so as to kill all the cells.
  • the fixative liquids that may be used must be such that they render the cellular membrane permeable for absorption of the dyes (fluorochromes) mentioned below.
  • Fixatives that may be used include ethanol, isopropanol and acetone, acetone being particularly preferred.
  • the mixture from point 1 has a buffer solution added to it, which is formulated so as to promote attachment of fluorochrome to the nucleic acids of the cells (DNA/RNA) (see point 3).
  • the buffer solution must prevent attachment to other cellular components.
  • the buffer that has been found to be the most optimal is the so-called TBE-buffer (90 mM Tris, 90 mM Borate, 2,5 mM EDTA, pH 8).
  • a fluorochrome is added to the mixture from point 2, which fluorochrome specifically attaches to the nucleic acids of the cells.
  • the present method may for instance involve the use of a monomer cyanine fluorochrome.
  • the mixture from point 3 is analysed in a device that measures light scatter and fluorescence from individual cells (e.g. a flow cytometer).
  • the excitation light has a wavelength (635 nm) such that auto-fluorescence from the cells is insignificant.
  • Steps 1 - 5 can be performed by a novel device according to the invention, such as appears in the accompanying schematic figure. More specifically, the invention regards a method for counting cells in a urine sample, characterised in that a fixative is added to and mixed with the urine sample; a buffer solution is added to the mixture; followed by a dye; the mixture is then analysed in a device that measures light scatter and fluorescence from individual cells; and the results are shown directly on a display.
  • the invention further regards a device for measuring cells in a liquid stream by means of flow cytometry, in particular bacteria in a urine sample, characterised in that it comprises pickup tubes for the urine sample, which tubes lead to one or more mixing chambers to which are also connected separate receptacles for the fixative and the staining solution that are added to the mixing chamber via adjustable multi-channel pumps; the mixing chamber is further connected to an optical flow cytometric cell that receives carrier liquid from a receptacle.
  • fluorescence is achieved by staining the bacteria.
  • the cellular membrane is broken down when the cell is fixed by a fixative liquid such as ethanol, isopropanol or preferably acetone.
  • the fixation also inactivates any eflux-pumps that may otherwise pump the dye back out of the cells. In this manner, the fluorochrome gains easy access to the intracellular components of the cells.
  • a further advantage is the fact that the method prevents auto-fluorescence by use of a dye that attaches specifically to nucleic acids and which is excited at light >500 nm (specifically 636 nm). The gain in fluorescence increases >10x upon attachment to the nucleic acids.
  • the method promotes specific attachment and reduces non-specific attachment by utilising special buffers, and the use of Tris-borate-EDTA, pH 8 has proven to be especially advantageous.
  • the device according to the invention which may be used to implement the method, is explained schematically in greater detail in Figure 1.
  • the device consists of a connection for inlet of urine from a sampling bottle 1.
  • the urine sample is sucked in by pump 2, and the sample is passed on to a mixing chamber or a reagent loop 5.
  • a fixative such as ethanol or acetone is introduced into the mixing chamber 5 by pump 4.
  • the staining solution is kept in receptacle 6 and is led to mixing chamber/reagent loop 8 by pump 7.
  • a common motor 18 can drive pumps 2, 4, 7.
  • the measurement of the urine sample in the flow cell is transferred to a data and control unit, where the results are shown on a display.
  • the results are presented on a display where the fluorescent cells appear separately with a different colour from that of non- fluorescent particles.
  • the total cell count is shown on the display. Cells in the lower size range from 0.5 to 2 ⁇ m are presented as bacteria.
  • the method and device according to the invention have a number of advantages over prior art, including the fact that they allow quicker and more reliable counting of bacteria in urine.
  • the analysis may take from one to several days, and may often require the sample to be sent away for analysis.
  • the results of the analysis are available on site in a matter seconds.
  • a great advantage of the device is the fact that it is automated. There is no manual handling of chemicals, which removes the risk of the operator being exposed to any chemicals that may be injurious to his or her health.
  • the device also ensures a reduced possibility of human e ⁇ ors and failures during the handling and treatment of the sample.
  • the cost per sample will be lower than that which is the case for the conventional methods of analysis that are in use today.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé et un dispositif pour le comptage de cellules dans l'urine, consistant à additionner à l'échantillon d'urine un agent de fixation, un tampon, et un colorant, et à analyser cet échantillon dans un appareil de mesure de la fluorescence.
EP00959042A 1999-09-01 2000-09-01 Procede et dispositif pour le comptage de cellules dans l'urine Withdrawn EP1181553A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
NO994228A NO994228D0 (no) 1999-09-01 1999-09-01 Metode og innretning for telling av celler i urin
NO994228 1999-09-01
PCT/NO2000/000286 WO2001016595A1 (fr) 1999-09-01 2000-09-01 Procede et dispositif pour le comptage de cellules dans l'urine

Publications (1)

Publication Number Publication Date
EP1181553A1 true EP1181553A1 (fr) 2002-02-27

Family

ID=19903722

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00959042A Withdrawn EP1181553A1 (fr) 1999-09-01 2000-09-01 Procede et dispositif pour le comptage de cellules dans l'urine

Country Status (4)

Country Link
EP (1) EP1181553A1 (fr)
AU (1) AU7043100A (fr)
NO (1) NO994228D0 (fr)
WO (1) WO2001016595A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4759438B2 (ja) * 2006-05-17 2011-08-31 シスメックス株式会社 尿中有形成分分析装置

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5301685A (en) * 1989-01-10 1994-04-12 Guirguis Raouf A Method and apparatus for obtaining a cytology monolayer
JP3070968B2 (ja) * 1991-05-14 2000-07-31 シスメックス株式会社 尿中の細胞分析用試薬及び方法
US5545535A (en) * 1993-04-13 1996-08-13 Molecular Probes, Inc. Fluorescent assay for bacterial gram reaction
US5563070A (en) * 1993-05-28 1996-10-08 Omron Corporation Method of counting reticulocytes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0116595A1 *

Also Published As

Publication number Publication date
NO994228D0 (no) 1999-09-01
WO2001016595A1 (fr) 2001-03-08
AU7043100A (en) 2001-03-26

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