WO2001016595A1

WO2001016595A1 - Method and device for counting cells in urine

Info

Publication number: WO2001016595A1
Application number: PCT/NO2000/000286
Authority: WO
Inventors: Oddbjørn GJELSNES; Øystein RØNNING
Original assignee: Optoflow As
Priority date: 1999-09-01
Filing date: 2000-09-01
Publication date: 2001-03-08
Also published as: AU7043100A; EP1181553A1; NO994228D0

Abstract

The invention regards a method and a device for measuring the number of cells in urine. A fixative, a buffer and a dye are added to the urine sample, which is then analysed in a device for measuring fluorescence.

Description

Method and device for counting cells in urine

The present invention regards a method and a device for counting bacteria and other micro-organisms in uπne 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 involves detecting bacteria by means of light scatter and fluorescence with an improved signal-to-noise ratio when compared with prior art

Persons suffering from cystitis have cells in their urine that should not normally be there These cells in the urine may be bacteria and fungus, as well as the patient's own cells (somatic cells), such as leukocytes or epithelial cells

The invention seeks, through the method and device thereof, to solve the following problem In the case of cystitis, it takes a long time (one or more days) to count the number of bacteria m uπne 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

Attempts have been made to solve this problem by counting the bacteria and other cells (lymphocytes and epithelial cells) directly in the urine by using specially designed cell counting devices (flow cytometers) 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 (number of cells per ml urine) 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).

The main problem associated with prior art that makes use of plate counting is the time it takes. The problem with today's flow cytometers is that they are not good enough at routinely measuring bacteria in urine, which are small in comparison with somatic cells (lymphocytes, epithelial cells).

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 method according to US 5 639 484 functions satisfactorily with somatic cells, but does not work well with bacteria. This is, among other things, due to the following facts:

Using violet/blue excitation light results in auto-fluorescence, which causes the signal-to-noise ratio to be reduced at low fluorescence intensities (as in the case of bacteria). - It is more difficult for live bacteria to absorb dye than it is for somatic cells, for several reasons.

Firstly, the cell walls of the bacteria act as a barrier against the surroundings.

Secondly, the bacteria may have intracellular pumps that bring the dye out again.

Thirdly, the bacteria are considerably smaller than somatic cells, thus containing less of the cellular components that are to be stained.

As a result of this, the fluorescence intensity per cell is low. 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:

1. 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.

2. 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). At the same time, 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).

3. 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.

4. 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.

5. The results are presented on a display that shows the fluorescent particles (cells) appearing separately (different colour) from particles without fluorescence, while displaying the absolute count. Cells in the lower size range (0.5 - 2μm) are assumed to be bacteria.

6. 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.

According to the method of the invention, 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.

After the mixing has been completed in chamber 8, biological and chemical waste is separated out in a separate receptacle 10. The mixture of the urine sample, the fixative and the staining solution is sent on to the flow cell 11, in which the optical detection takes place. Light scatter is detected using MICROCYTE (Norwegian, European, US patent, pending Japan). For detection in the flow cell, use is made of a carrier liquid from receptacle 12. The amount and velocity of the carrier liquid 12 is adjusted by means of e.g. a throttle valve 9. Following detection of the sample in the flow cell 1 1, it is sent to waste container 14 by pump 13, which is connected to motor 17. This waste consists mainly of water with a very low content of biological material and chemicals.

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. In addition, the total cell count is shown on the display. Cells in the lower size range from 0.5 to 2 μm 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.

Using today's conventional plate technique, in which cultivated colonies of bacteria must be determined and counted using the naked eye, the analysis may take from one to several days, and may often require the sample to be sent away for analysis. By using the method and the device of the invention, 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 eπors and failures during the handling and treatment of the sample.

By using the method and the device of the invention, the cost per sample will be lower than that which is the case for the conventional methods of analysis that are in use today.

Claims

C l a i m s 1.

A method for counting cells in a urine sample, c h a r a c t e r i s e d i n 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 analysed in a device that measures light scatter and fluorescence from individual cells; and the results are shown directly on a display.

2.

A method according to Claim 1 , c h a r a c t e r i s e d i n that the fixative is of the type that renders the cellular membrane permeable, and may be acetone, ethanol or isopropanol, preferably acetone.

3.

A method according to Claim 1, c h a r a c t e r i s e d i n that the buffer solution promotes attachment to the nucleic acids of the cells, and that it is preferably a TBE-buffer consisting of 90 mM Tris, 90 mM Borat, 2,5 mM EDTA, pH 8.

4.

A method according to Claims 1 - 2, c h a r a c t e r i s e d i n that the dye used is a fluorochrome that specifically attaches to the nucleic acids of the cells, and that it is a monomer cyanine fluorochrome, preferably TOPRO-3.

5. A method according to Claims 1 - 4, c h a r a c t e r i s e d i n that the mixture is analysed in a device that measures light scatter and fluorescence from the individual cells, such as a flow cytometer.

6.

A method according to Claims 1-5, c h a r a c t e r i s e d i n that the analyses are performed at a wave length >500, preferably at 635 nm.

7.

A device for measuring cells in a liquid stream by means of flow cytometry, particularly bacteria in a urine sample, c h a r a c t e r i s e d i n that it comprises pickup tubes for a urine sample (1), which tubes lead to one or more mixing chambers (5, 8) to which are also connected separate receptacles for a fixative (3) and a staining solution (6) that are added to the mixing chamber (5, 8) via adjustable multi-channel pumps (2, 4, 7, 9), the mixing chamber further being connected to an optical flow cytometric cell (11) to which is added a earner liquid from receptacle (12).