Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Mechanical measurement system
Reexamination Certificate
2000-03-10
2003-04-01
Nguyen, Judy (Department: 2861)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Mechanical measurement system
Reexamination Certificate
active
06542833
ABSTRACT:
TECHNICAL FIELD
The present invention concerns a new method of checking the performance of a flow cytometer, the flow cytometer comprising means delivering electrical pulse signals indicating the presence of particles, such as somatic cells or bacteria. The invention is specifically dedicated to milk testing and more specifically to determination of the number of somatic cells in milk. Further according to the present invention the method allows for inspection and correction for any agglomeration of particles in a standard sample used for checking the performance of the flow cytometer. Further the invention concerns an apparatus comprising data processing equipment arranged to execute the above method, as well as a stannard kit for application in the apparatus.
BACKGROUND ART
Flow cytometers are instruments used for analysis of particles suspended in a fluid, e.g. biological cells in body fluids, such as somatic cells in milk. Briefly, a flow cytometer comprises a liquid flow system and an optical system intersecting each other in a flow cuvette, as well as an electronic system, detecting fluorescence or scatter originating from particles passing through the cuvette. Preferably, the fluid sample flows in a single, thin string inside the cuvette thereby allowing any particles present to be counted one by one. Further, in a preferred embodiment the thin string is surrounded by a sheath fluid. Thanks to the sheath fluid, casual impurities, present in the fluid and having a size larger than the particles or cells to be counted and accordingly larger than the size of the string of fluid sample, will not cause clogging. They might have done so if flowing in a liquid channel as narrow as the thin inner string.
The method according to the invention is specifically developed for flow cytometers applied for fast determination of the number of cells in a milk sample or a milk product and more specifically for the fluorescent type of flow cytometers, in which the cells or particles are all stained by a fluorescent dye, which reacts fluorescent when exposed to illumination. A fluorescence signal of adequate size, i.e. above noise, is considered to indicate the passage of a particle or cell. In the preferred embodiment of the flow cytometer, the presently preferred agent or dye is Ethidium Bromide. The present type of flow cytometer has only one channel, i.e. it is optimised to detect only one kind of fluorescence or scatter. However, flow cytometers may have a plurality of channels, each dedicated to a specific dye.
The somatic cell count is considered a measure of the milk quality (high quality milk has a low cell count). Accordingly the cell count can be applied by the dairy when setting the price according to which the farmer is paid for the delivered milk. In order to ensure correct payment to the farmer and to reveal any milk of too low quality, a proper functioning of the flow cytometer is crucial.
In order to obtain accurate and reproducible results flow cytometers must be aligned and calibrated. The light ray from the illumination source must hit the stream of particles and the detector optics must focus on the particle stream when the illuminated particles exhibit fluorescence. Also the gain and characteristics of the electronic circuits processing the detected signals and the applied mathematics must produce a number indicating the true number of particles in the stream with a sufficient accuracy and reproducibility.
Possible Reasons for Poor Performance
Any defects or maladjustment of flow rates (or clogging) in the flow system and/or defects or misalignment of the optical system may cause the count to either decrease or increase compared to the true value.
Prior Art
U.S. Pat. No. 5,093,234 (Schwartz) discloses a method of aligning, compensating and calibrating a flow cytometer for analysis of samples and a microbead standard kit therefore. The method can be applied to multi-channel flow cytometers. The method includes running test measurements on standard kits; adjusting fluorescence channel PMT voltages and gain to position resulting dot plot or histogram near the origin of the axis of each of the fluorescence channels; and setting boundary levels in each channel to indicate fluorescence intensity. The standard kit comprises a blank and/or an auto-fluorescent microbead population and at least two series of calibrated microbead populations labelled with fluorescent dye(s). U.S. Pat. No. 5,084,394 (Vogt et al) discloses a similar method for corrective calibration of a flow cytometry using a mixture of fluorescent microbeads and cells. These methods provide for an advanced, sophisticated and qualitative analysis of single cells such as lymphocytes in blood.
The present method concerns a “performance check”, i.e. a monitoring of functional performance, of a single channel flow cytometer for counting somatic cells in milk. The instrument is specifically designed for fast sample handling and counting, allowing about 500 samples pr hour to be counted. This kind of flow cytometer counts the cells based upon measuring only one parameter, such as green or red fluorescence. The instrument is not intended for qualitative studies of the cells. The performance check method is based on the use of a standard and/or calibration fluid comprising only one type of particles or microbeads which are unstained until they enter the process according to the present invention. Thereby the standard fluid samples are as simple as possible. The standard samples are very stable and adequate for a long term shelf life, i.e. a great number of standard samples may be stored by the user for months or years for future use, such as a regular performance check every morning or when ever necessary.
Besides a thorough check of the operation of the flow cytometer the standard fluid could also be applied for a calibration of the flow cytometer.
SUMMARY OF THE INVENTION
The present invention provides a method of checking the performance of a flow cytometer instrument, in which instrument the number of particles or cells in a fluid flow are counted by providing data representing a PHA diagram (Pulse Height Analysis), of registered pulses. The invention is characterised by—providing a lot of standard samples, including only one type of substantially uniform microbeads,—providing data representing an optimal (desired) PHA diagram (PHA
0
) of the pulses registered, when measuring a standard sample from the lot on a reference instrument, said data being stored in a memory in the instrument itself, or a memory in data processing means connected to the instrument, or in means from which the data may be imported into the instrument or into the data processing means connected to the instrument,—measuring a standard sample on the instrument to be checked,—providing data representing a PHA diagram (PHAS) for the pulses registered during the measurement of the standard sample on the instrument to be checked,—comparing the present PHA diagram (PHAS) to the optimal PHA diagram (PHA
0
),—and analysing and/or evaluating said data in order to determine any poor or faulty operation of the instrument. By this method the user can check the instrument regularly, and the user can readily be informed of any precautions to be taken. Preferably the microbeads in the lot are unstained until the enter the instrument. The use of unstained microbeads are specific favourable in that also the staining process in the instrument is controlled when measuring the standard sample.
Preferably at least one of the following parameters are calculated: a particle count, a plurality of particle counts on the same sample, a standard deviation, s, and/or Coefficient of Variation, CV, based on (at least two, preferably three) repeated/consecutive measurements on the same sample and substantially at the same time, a signal mean value, and a signal width, i.e. the width of the bell-curve in the PHA-diagram—the corresponding data for the standard sample of the standard fluid measured on a reference instrument. i.e. the optimum values of said data, being provided w
Birch & Stewart Kolasch & Birch, LLP
Foss Electric A/S
Nguyen Judy
LandOfFree
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