CA2129535A1 - Method and materials for determining particle count in a flow cytometer - Google Patents
Method and materials for determining particle count in a flow cytometerInfo
- Publication number
- CA2129535A1 CA2129535A1 CA002129535A CA2129535A CA2129535A1 CA 2129535 A1 CA2129535 A1 CA 2129535A1 CA 002129535 A CA002129535 A CA 002129535A CA 2129535 A CA2129535 A CA 2129535A CA 2129535 A1 CA2129535 A1 CA 2129535A1
- Authority
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- Canada
- Prior art keywords
- particles
- cells
- specimen
- volume
- suspension
- 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.)
- Abandoned
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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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/528—Atypical element structures, e.g. gloves, rods, tampons, toilet paper
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/0038—Devices for taking faeces samples; Faecal examination devices
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N2001/002—Devices for supplying or distributing samples to an analysing apparatus
- G01N2001/005—Packages for mailing or similar transport of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
- G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration
-
- 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—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1477—Multiparameters
-
- 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—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1486—Counting the particles
Abstract
ABSTRACT OF THE DISCLOSURE
A method is provided to ascertain the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed. The method comprising mixing a suspension of a known quantity of particles having a first light scatter signal with a known volume of a cell specimen having a second light scatter signal different from the first signal to obtain a suspension having a concentration of particles per specimen volume, passing each of said particles and each cell in said cell specimen, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering pattern, counting the number of cells and the number of particles in a portion of said suspension, and determining the total number of cells per unit volume of specimen.
A method is provided to ascertain the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed. The method comprising mixing a suspension of a known quantity of particles having a first light scatter signal with a known volume of a cell specimen having a second light scatter signal different from the first signal to obtain a suspension having a concentration of particles per specimen volume, passing each of said particles and each cell in said cell specimen, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering pattern, counting the number of cells and the number of particles in a portion of said suspension, and determining the total number of cells per unit volume of specimen.
Description
WO93/1638j pcT/uss3/ol232 METHOD AND MAT~RIALS FOR DETERMINING
PARTICLE COUNT IN A FLOW CYTOMETER
This lnvention relates to a method for accurately .
determining the count per unit volume of particles suspended in a fluid containing two or more different particles. More particularly, the invention relates to 5 determining the blood cell count per unit specimen volume .
of blood cells in a flow cytometric system wherein the flow cytometer does not measure the volume of a suspension .. .
containing the specimen as it is b~ing analyzed and .
wherein the blood cells of interest are CD4 lymphocytes. : :~
In addition, this invention relates to the use of added particl.es as a quality control check during analysis of the sp~c~men. . .
~t-~ III or Human T-cell Leukemia virus Type III, now commonly reSerred to as Human Immunodeficiency Virus .
lS ("HI~") is recognized to be the causative agent for human immunode~iciency syndrome or AIDS. The chronic nature of ~IDS considered as approaching epidemic proportions in the United States and other countries is reflected by a ~ -surfeit of studies and ef~orts to develop diagnostic immunoassays ~or reliably and consistently detecting viral antigens and antibodies to such antigens in human peripheral blood. In substantial measure, monoclonal antibody technology has been recorded for developing such immunoassays.
HIV belongs to the retroviNs group of viruses.
Retroviruses carry a positive-stranded RNA and a special . .
enzyme called reverse transcriptase in its core which is used to convert viral RNA into DNA. This rever~es the classical process of cellular transcription in which DNA
is converted to RNA.
., , ~,.. .
.
W093/16385 PCT~US93/01232 ~ 1 2 ~ j ~ 3 It is known that the HIV binds to CD4 lymphocytes because the protein on the surface of CD4 lymphocytes -serves as a receptor or binding site for HIV ~Dalgleish A
et al., Nature 312:763-767 (1985)]. HIV also can bind to -and attack other cells, such as, monocytes, tissue macrophages, and cells in the brain, spinal cord and peripheral nerves. The life cycle of HIY calls for the virus entering the host patient through sexual activity or blood transfusion, for instance, and then binding to receptors on monocytes and lymphocytes. The virus penetrates the cell and sheds its envelope or protein coat 50 as to expose its viral RNA core. The reverse transcriptase converts the viral RNA core to DNA which is integrated into the host cell genome. New viral particles are produced in quantity until the membrane of the host cell is ruptured to release the new viral particles in the human blood system.
Diagnostic tests are commercially ava~lable at this time utilizing monoclonal antibodies to determine whether a person having the AIDS disease or has been immunologically exposed to the virus can be identified.
Diagnosis of the disease is complicated by the fact that extended periods of incubation are required before ~ymptoms of the disease are expressed. The highly infectious nature of the disease and the fact that its cure presently is not within scientific capability also increases the difficulty of investigating live virus and its adverse affect on the human immune system so that successful diagnostic tests can be developed.
Presently, absolute CD4 count is a generally accepted predictor of clinical HIV disease progression. Current methods for this measurement require multi-step sample w093/16385 PCT/US93/01232 J 3 j preparation and are known to have cumulative errors i-related to determiining both the proportion of CD4 lymphocytes and the absolute lymphocyte count. One method which attempted to solve this problem was presented in a "poster" at the Seventh International Conference on AIDS
in Florence, Italy, June 19, 1991. However, the disclosed method was incomplete since it did not teach nor enable one to practice the method.
Similarly, others have attempted to provide different lo methods and apparatuses to solve the general problem of determining the cell count per unit volume of particles suspended in a fluid containing undesirable particles.
One approach is described in U.S. Patent 4,989,978 to Groner. However, this method requires that a predetermined Xnown volume of a suspension of cells and undesirable particles be analyzed so that the volume of the undesirable particles may be subtracted from the original predetermined volume to provide an accurate count of cells per unit volume.
More generally, others have added a plurality of particle~, to various diluents in methods for testing and calibrating flow cytometry instruments. Although the prior art contains many such disclosures, two such examples are U.S. Patent 4,704,891 to Recktenwald, et al and U.S. Patent 4,331,862 to Ryan. In addition, by adding ~tandard particles to a cell suspension prior to i~nalysis, a "built in" fiducial mark is provided which can be used to interrelate distributions obtained under differing instrument conditions. According to Flow Cytometry and Sorting, John Wiley & Sons Publisher, 1979, this built in technique is taught by Jensen, et al, Multiparameter Flow Cytometry Applied Toward Diagnosis of Cer~ical Squamous WO93/16385 PCT/~S93/01232 1 t~ 9 i 3 j Cell Carcinoma. Proceedings of the Second International Conference on Automation o~ Cancer Cytology and Cell Image Analysis, May 6-7, 1977. However, these general references fail to address the problem of determination of cell count per specimen volume.
According to the invention there is provided a method to ascertain the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed, said method charactexized by the steps of:
a) mixing a known quantity of particles ha~ing a first light scatter signal with a known volume~of a cell specimen having a second light scatter signal different from the first light scatter signal to obtain a suspension having a concentration of particles per specimen volume;
b) passing each of said particles and cells in an unknown volume portion of said cell suspension, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering pattern; c) counting the number of cells and the number of particles in said unknown vOlUm8 portion of said ~uspension as is known;
and, d) determining the total number of cells per volume of the speci~en by correlating the number of particles and cells in said portion of the suspension to the known quantity of particles added to said cell specimen.
The present invention relates to a method for accurately determininq the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed and which doe.s not analyze the entire original known ~olume. ~he count of the cells is correlated to the count of the added discrete particles to , '~' W093/16385 PCr/US93/01232 ' ~:
determine the cell count per initial volume. The method employs a light scatter trigger to se~se the cells, and uses a two scale analysis of light scatter and fluorescence to count the cells of interest and the added discrete particles, as well as, to discriminate between the cells of interest and added discrete particl~s. In a more preferred embodiment, the cells of interest are labelled with a fluorescent monoclonal antibody.
In one embodiment, the method comprises mixing a suspension of a known quantity of discrete particles with a known volume of the cell specimen to obtain a suspension having a concentration of particles per specimen volume;
counting the number of cells and particles in a portion of said suspension; and determining the total nu~ber of cell per unit volume of the specimen. Said determination is obtained by correlating the ratio of said counted cells in said portion times the number of said known quantity of particles in the suspension to the specimen volume times the number o~ said counted particles in said portion.
In another embodiment, the method of this invention comprises mixing a suspension of a known quantity of particles having a first light acatter signal with a known volume of a cell specimen having a second light scatter signal different from the first light scatter signal to obtain a suspen6ion having a concentration of particles per specimen volume; passing each of said particles and cells in said cell suspension, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering pattern; counting the number of cells and the number of particles in a portion of said suspen~ion: and, determining the total number of cells per unit volume of the specimen by correlating the number of WO93/16385 PCT/~S93/01232 n ~ !) '3 particles and cells in said suspension to the known quar.tity of particles added to said cell specimen.
The use of this method permits the cell specimen to be subjected to one or more volumetric dilutions caused by the processing of the cells. More specifically, providing the original cell specimen volume is known, the cell specimen may be lysed, have cells of interest tagged with s~ain or monoclonal antibodies, or be subject to such ~ -other process which would dilute the original cell specimen volume to an unknown volume.
In addition, the inv~ntion further provides a quality control method to insure that the operating parameters of the counting instrument is within specification 50 that it accurately senses all of the desired particles, while simultaneously determining the count per unit volume of a cell suspension.
By using fluorescent partioles with the cell specimen, the flow cytometer can u~e light scatter triggering for the parameter set so that the particles and cells are analyzed by light scatter and fluorescence.
Providing that the cells and particles are aensed by light scatter and fluorescence during analysis, then one has adequate assurance that the flow cytometeF has been properly operating during the analysis of the specimen.
In a preferred embodiment, the method ~urther comprises the additional stepæ of correlating eaid light scattering pattern and fluorescence signal of the particles to a predetermined ratio which represents a threshold of minimum instrument performance and adjusting, if the predetermined ratio has not been attained, the operation of said instrument, while the particles are within the incident beam of light, until the measured d wo 93/163~5 PCI/US93/OlZ32 ~23~
ratio reaches said predetermined ratio whereby the instrument is analyzing both particles and cells in said cell suspension.
In order to determine the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed, it is necessary to begin with a known volume of the specimen. The accuracy of determining the cell count per unit volume will depend, in part, upon the accuracy of the measurement of the specimen volume. ~ ~
In order to expeditiously obtain the cel? count of ~;
cells of interest per unit volume, it is preferred that the whole blood is lysed to remove the erythrocytes by such means as known to those skilled in the art. These means include hypotonic lysis, acid lysis, as well as, the use of magnetic particles or microspheres coated with an antibody for sorting and separating the selected population from a ~eterogeneous cell population. The only ~O limiting fact in the selection o~ the method of lysis is that it should preserve the integrity of the leukocytes.
More specifically, in a pre~erred embodiment of this invention, the preferred method of lysis of the whole blood is by the COULTER Immuno-Prep EPICS~ Leukocyte Preparation System, distributed by Epics Division of Coulter Corporation (COULTER and EPICS are trademarks of Coulter Corporation, Hialeah, Florida). As known to those skilled in the art, this preferred method of lysis is a hypotonic lysis.
Although the flow cytometer is capable of di~stinguishing between leukocytes of interest and erythrocytes in the whole blood, evaluation of a whole w093/16385 PCT/~JS93101232 212~ -~3~
blood sample would require an extended time to analyze when using a light scatter trigger. The extended time is due to the cytometer having to analyze both the red blood cells as well as the white blood cells. More specifically, the white blood cell count in normal blood is 5,000 to ll,000 per microliter (~L) with a lymphocyte value of 20 to 40% and the red blood cells are 4,000,000 to 5,000,000 cells per microliter (~L). Therefore, in order to calculate a lymphocyte count of 5,000, approximately lO million to 30 million cells will be analyzed.
After lysing the whole blood, the remaining leukocytes are then identified by such means as known to those skilled in the art. Sùch identification means includes stains, ligands, probes, and monoclonal antibodies. The selection of the stain, ligand, probe, or monoclonal antibody enables the enumeration of the selected leukocyte of interest to be identified and i `
accurately counted in an expeditious manner.
Having determined the initial volume of the cell specimen, a known number of discrete particles are then added to the blood speci~en which has optionally been lysed, and in which the labelled leukocytes of interest have been identified.
~oth biological and plastic discrete particles known to those ~killed in the art may be used in the present invention. The discrete particles should ~e~inert with respect to the carrier in which they are diluted and with respect to the test probe of the counting instrument. In addition, the discrete particles should be stable at conventional working temperatureæ of 0-30C. Unstable ~ -particles have a tendency to shift in their histogram WO93/16385 PCT/~IS93101232 positioning, and contribute imprecision to the determination of cells per specimen volume. An important feature of the present invention is that the particles do not need to have characteristics of a like nature to the cells of interest. More specifically, the particles are not required to replicate the volume, size, fluorescence, light scatter, impedance, or Coulter opacity of the cells of interest. Using particles having different characteristics than the cells, eliminates any overlapping of cell count to cause erroneous or inaccurate results.
Preferably, the density of the discrete particle should approximate that of the cell. More specifically, the density of the particle should be greater than 1.0 grams per milliliter. This is advantageous because if the lS density is substantially disproportionate to the cells in the specimen, the discrete particles will have a tendency ~ ' to settle in the specimen if not analyzed immediately. As such, if the discrete particles are substantially disproportionate to the density of the cells, a non-uniform mixture might be analyzed which could contribute to an inaccurate determination of cell count ;
per unit ~olume. Therefore, if the density of the particle differs from the density of the cell specimen by greater than 20%, then the cell-particle suspension should be analyzed within 30 minutes of preparation. More preferably, the density of the particle should differ from the cells by less than 5%, and the cell-particle suspension should be analyzed within 4 hours of preparation.
The preferred type of particles for the present invention are plastic microbeads. Such beads may be fabricated with substantially uniform diameter, and have W093/1638~ PCT/US~3/01232 ,~ , 3 j surface characteristics which are suitable for surface binding of fluorophores or other marking agents.
Substantially spherical beads may be formed having a diameter between 0.5 and 20 microns. While the beads are S usually made in solid form, they may be hollow inside and could be vesicles or other microcarriers. Moreover, the beads do not have to be perfect spheres in order to function in accordance with the present invention.
Plastic materials such as polystyrene, polyacrylamide a~d other latex materials may be employed for fabricating the beads, and other plastic materials such as polyvinylchloride, polypropylene and the like may also be utilized.
When plastic particles are used, they should be ~ -treated to prevent the plastic particles from adhering to the cells in the cell-particle specimen and to prevent the plastic particles from aggregating with each other. These ~ `
two impediments need to be resolved 60 that the particles remain discrete ~nd are susceptible of accurate differentiation.
To prev~nt the plastic particles from adhering to the cells of interest, the particles may be coated with such substances which will counteract this tendency. Such substances include bovine serum albumin, glycolipid, polyvinylpyrrolidone or other substances which would change the bior~activity of the plastic particle so that it would not attach to the cells of interest. For the purposes of this invention, it is preferred to use a bovine serum albumin solution.
It is believed that the aggregation of the plastic particles is caused by the surface charge of the discrete plastic particle. It has been found that this obstacle , ~ - , - , , -, ; -, , , ; . . . .- -. .. .. .:.
WO93/16385 P~/US93/01232 `` h ~ 3 ~
11 , ".~' may be resolved by the aforementioned coating process.
But in addition, the pH of the carrier appears to contribute to the resolution of the aggregation problem.
Moreover, it is further believed that the use of a non-ionic surfactant would also contribute to eliminating thisproblem.
The carrier used as the storage and suspension medium for the particles must be chemically and physically compatible with the selected particle material and with the type of counting instrument. Thus, for use in those counting instruments which use the change in electrical conductivity to calculate count, the carrier must be electrically conductive. Those skilled in the art who use countiny instruments employing the principle of electrical conductivity appreciate that the conductivity of the carrier will greatly affect the indicated count of the instruments. Thus, a standard carrier conductivity has ~`
been adopted and is the conductivity of 0.8% equivalent ~ `
molarity of codium chloride. `
On the other hand, for those counting instruments which utilize the light scattering principle, the carrier must be optically conductive (or transparent, i.e., having a very low absorption coefficient). However, those skilled in the art who use counting i~struments employing the pr~nciple of light scattering appreciate that when using certain plastic particles, such as DNA check beads, there is a tendency for them to aggregate. As noted previously, it is believed that the aggregation is caused by electrostatic attraction of the discrete plastic particles which may be related to the salt concentration in the carrier. Therefore, when using such particles in a W093/16385 PCTlUS93/Ot232 light scattering instrument, the carrier should not cause aggregation of the plastic particles.
In a preferred embodiment of the invention, the carrier should have a density equal to or greater than the density of the added discrete particle. Preferably, the carrier density is between O to 15% and more preferably o to 5% greater than the discrete particle density. If the density of the carrier is substantially disproportionate to the discrete particle, then the particles will have a tendency t~ either settle to the bottom or float to the t~p of the carrier solution resulting in a non-homogeneous -~ ;
particle suspension. A homogeneous particle suspension is desirable to facilitate the procedure to add a known count of the particles to the cell specimen.
More preferably, the carrier comprises an aqueous solution of glycerol and most preEerably comprises between 17 to 21% by volume of the aqueous solution. However, as known to those skilled in the art, other ~nown media may be used to increase the carrier density, such as sucrose, Ficoll-Hypaque~R (manufactured by Pharmacia L.K.B.
8iotechnology), and Percoll~ (manufactured by Pharmacia L.K.B. Biotechnology). When DNA check beads are the particles of choice, the carrier will have a density within 5% difference of 1.03 grams/milliliter.
In order to maximize the life of the particles and carrier and to prevent further aggregation of the particles, it is important that the pH of the carrier be ~ ~
compatible with the particles and other additives in the ~;
carrier. For example, it has been found that a pH falling generally within the range of 6.0 to 8.0 is acceptable and that a preferable range is from 6.9 to 7.3 for use with particles of a latex material. If the pH of the carrier WO93/16385 PCT/US93/01232 ~;
3 ) 3 j is either too high or too low, it has been found that the latex particles may aggregate, thereby diminishing the known total number of particles and further increasing the size of the aggregates to perhaps a size that would fall outside of the ability of the instrument to count the particles. It is noted that the term aggregation is used broadly and also includes the formation of doublets or triplets of particles which, it should be appreciated, would be as detrimental as the formation of much larger aggregates of particles.
In addition, it is also desirable to include in the carrier a small, but sufficient concentration of a preservative to prevent the growth of bacteria or fungi in the carrier or on the particles. More particularly, the preservative is preferably a fungicide and/or a bactericide which does not increase background noise by either creating floculations or adding fluorescence.
Although ~ny addit~ve which i8 compatible wlth the other components of the carrier and with the particle material is acceptable, it is preferred that the preservative be selected Srom the group comprising organic aldehydes, including monaldehydes such as formaldehyde, or dialdehydes ~uch as glutaraldehyde: alcohols, including organic alcohol, such as aliphatic and cylic alcohols, such as phenol, toluene, ethanol, and propanol; and sodium fluoride and sodium azide. More preferably, the additive selected is formaldehyde and is used in an amount within the range of about 0.5% to 2.5% and preferably 1.0% by -volume of the carrier liguid.
It is believed that the stability of the carrier can be enhanced if an appropriate surfactant is added thereto.
However, the selected surfactant must not de}eteriously 2 1 2 9 i ~
affect the cells of interest. Preferably, such a a, .
surfactant is non-ionic type and is present in the minimum amounts necessary so that the aggregation of particles is minimized. Too great a percentage of surfactant in the carrier results in the formation of bubbles, as well as, causes cell degradation, while too little surfactant does not provide the desired degree of stability. -The preferred procedure to add a Xnown count of particles to the cell specimen comprises pipetting a selected volume of the carrier containing the discrete particles. The accuracy of determining the cell count per unit volume will depend, in part, upon the accuracy of the -volumetric measurement of this particle suspension. ~y this method, the number of beads that can be added to the cell specimen is known within one percent. However, any method known to those skilled in the art to add a known count of particles to the cell specimen may be employed.
The unknown volume portion of the cell specimen and particles is analyzed by a method which is effective to -determine the nu~ber of dîscrete particles and the number of cells of interest. The method comprises a light scatter triggering followed by light scatter and fluorescence measurements in a flow cytometer.
In typical flow cytometry instruments, cells and Z5 other discrete particles are caused to flow in a liquid stream so that each cell and particle, preferably one at a time, passes through a sensing region which measures one or more of its physical or chemical characteristics.
The sensing methods include electrical impedance related to particle volume based upon the well known Coulter Principle; ~luorescence: light absorption: light scatter; light extinction fluorescence polarization and WO 93/16385 ~'C r/l,S93/U1232 r .~
fluorescence signal waveform. Any one or more of these sensing methods may be used to activate a particular parameter set of analysis.
For purposes of the ensuing discussion, and for exemplary purposes, reference is made to a flow cytometer wherein, the liquid flow stream of cells and particles is ensheathed in a sheath fluid so that the cells and pa~ticle stream may be hydrodynamically focused as it flows through an incident beam of light normally directed ;-at right angles to the stream of cell and particle flow.
As the cells and particles pass through the beam of light, light characteristics associated with each cell and particle triggers a particular parameter set of analysis which may optimally be selected by the instrument operator. Although fluorescence and light scatter are optical signals, when used as a trigger for a parameter set, each has a different function and effect.
When a light scatter trigger is employed, each cell and particle passing through the incident beam of light is sen6ed by junction silicon photodiodes. Since light scatter iB affected by pnrticle size, shape, density, stain uptake, and granularity, one or more of these criteria may be used to activate cell analysis. As such, cell analysis may be activated by requiring the cell and particle to meet an upper or lower threshold of these criteria.
When a fluorescence trigger is employed, only those cells ~nd particles having a fluorescence is sensed by photomultiplier tubes which converts light signals to electrical signals. The fluorescent cells and particles absorb the incident beam of light and emit a fluorescent signal at a light wavelength different from the incident WO93/l6385 PCT/US93/01232 ~129~}3c~
source. Fluorescence i5 affected by the uniformity of illumination and signal intensity. Illumination uniformity is related to the stability of the light source and its spat~al intensity distribution. Fluorescence signal intensity depends upon a variety of instrumental characteristics, included are the excitation light intensity, sample stream flow velocity, the illumination source and excitation spectra of the dye, quantum efficiency Df the dye, collection and transmission efficiency of the light detection optics and the sensitivity of the photodetectors. As such, cell and particle analysis may be activated by requiring the particle to meet an upper or lower threshold of fluorescence signal intensity.
For the purposes of this invention, the method of sensing the particles comprises a light scatter trigger.
This will enable all particles in the suspension to be analyzed by the parameter set selected. Moreover, a light scatter trigger is not dependent upon the signal intensity of a fluorescence stain which can vary on a cell by cell basis. More particularly, the use of a light scatter trigger will provide a two-fold benefit.
First, when the cells are analyzed, a light scatter trigger will provide the ability to differentiate among the three types of leukocytes in the cell specimen, and in addition, by the use of markers or tags, to detect selected cluster designation of lymphocytes.
Secondly, the use of a light scatter trigger enables an in situ quality control of the instrument performance.
~ore specifically, the instrument will initially sense the cells and discrete particles by light scatter and depending upon their characteristics, will trigger a W093/~6385 PCT/US93/01232 2 :1 2 3; ~ ~
certain parameter set of measurements. Such parameter set will include the simultaneous measurement of the light scatter and one or more other sensing properties. ~his ;~
has the advantage that all particles would be detected which would pro~ide an accurate representation of the particles and cells in the sample suspension.
In addition, a light scatter trigger has the further advantage that during the analysis of the sample suspension, the instrument operator can have assurance that the instrument is operating within performance specifications and analyzing the sample suspension. More particularly, while the sample suspension is being analyzed, the technician can confirm, and if necessary adjust, the alignment of the instrument by its ability to detect and report in the proper position in the histogram the known discrete particles and the amplification of the signal intensity of the known discrete particles.
These parameters are not apparent during an analysis of a sample suspension which contains a limited number of cells o~ interest or which i5 of limited quantity or which contains contaminates which are detected ~y the instrument. In essence, the operator is able to validate the analy~is by focusing upon the measurements during analysis to determine that the machine is properly functioning.
In a most preferred embodiment, the sample suspension is analyzed by a simultaneous measurement of light scatter and fluorescence to provide significantly more accurate sensin~ of the cells than the measurement of only light scatter or only fluorescence. Thus, when using fluorescent particles and fluorescent markers for CD4 lymphocytes in the cell suspension which has ~een lysed of red blood cells, quality control can b~ assured by triggering on light scatter of the discrete particles and the cells of interest and confirming detection with light -scatter and fluorescence. Correlating these two signals S enable assurance of uniformity of illumination, si~nal intensity and improvement in the discrimination between the cells of interest and discrete particles.
In the most preferred embodiment of this invention, the cell-particle suspension will be further analyzed to differentiate the three major populations of leukocytes, (i.e., lymphocytes, monocytes and granulocytes), as well as, determine the CM lymphocytes in a single assay. In this embodiment, fluorescence triggering is not merited because not all cells and particles of interest will have a fluorescence intensity susceptible to accurate and reproducible measurement.
The measurement of particles and cells occurs after the suspension has reached a steady state of flow. This will allow for hydrodynamic instabilities of the suspension Slow to be eliminated. These instabilities are most apparent at the sta~t ~nd end of analysis when the particles and cells may either be tightly or loosely grouped together. Moreover, during steady state flow, the suspension is substantially free of air bubbles which has a tendency to impede measurements. When the frequency of particl~ distribution is nearly equal, a steady flow rate is achieved. This provides the most accurate interval for determining the cell count per unit volume resulting in an precision greater than 95S.
The features and advantages of the present invention are more fully shown with respect to the following non-limiting examples.
W093/16385 PCT/US93/Olt32 7 ~ . 3 `.i ~ ~
.
PI.
PreParation of Plasti~ M'cros~heres Materi~ls:
1. Plastic microspheres having a fluorescence intensity susceptible of measurement 2. Glycerol, ACS
PARTICLE COUNT IN A FLOW CYTOMETER
This lnvention relates to a method for accurately .
determining the count per unit volume of particles suspended in a fluid containing two or more different particles. More particularly, the invention relates to 5 determining the blood cell count per unit specimen volume .
of blood cells in a flow cytometric system wherein the flow cytometer does not measure the volume of a suspension .. .
containing the specimen as it is b~ing analyzed and .
wherein the blood cells of interest are CD4 lymphocytes. : :~
In addition, this invention relates to the use of added particl.es as a quality control check during analysis of the sp~c~men. . .
~t-~ III or Human T-cell Leukemia virus Type III, now commonly reSerred to as Human Immunodeficiency Virus .
lS ("HI~") is recognized to be the causative agent for human immunode~iciency syndrome or AIDS. The chronic nature of ~IDS considered as approaching epidemic proportions in the United States and other countries is reflected by a ~ -surfeit of studies and ef~orts to develop diagnostic immunoassays ~or reliably and consistently detecting viral antigens and antibodies to such antigens in human peripheral blood. In substantial measure, monoclonal antibody technology has been recorded for developing such immunoassays.
HIV belongs to the retroviNs group of viruses.
Retroviruses carry a positive-stranded RNA and a special . .
enzyme called reverse transcriptase in its core which is used to convert viral RNA into DNA. This rever~es the classical process of cellular transcription in which DNA
is converted to RNA.
., , ~,.. .
.
W093/16385 PCT~US93/01232 ~ 1 2 ~ j ~ 3 It is known that the HIV binds to CD4 lymphocytes because the protein on the surface of CD4 lymphocytes -serves as a receptor or binding site for HIV ~Dalgleish A
et al., Nature 312:763-767 (1985)]. HIV also can bind to -and attack other cells, such as, monocytes, tissue macrophages, and cells in the brain, spinal cord and peripheral nerves. The life cycle of HIY calls for the virus entering the host patient through sexual activity or blood transfusion, for instance, and then binding to receptors on monocytes and lymphocytes. The virus penetrates the cell and sheds its envelope or protein coat 50 as to expose its viral RNA core. The reverse transcriptase converts the viral RNA core to DNA which is integrated into the host cell genome. New viral particles are produced in quantity until the membrane of the host cell is ruptured to release the new viral particles in the human blood system.
Diagnostic tests are commercially ava~lable at this time utilizing monoclonal antibodies to determine whether a person having the AIDS disease or has been immunologically exposed to the virus can be identified.
Diagnosis of the disease is complicated by the fact that extended periods of incubation are required before ~ymptoms of the disease are expressed. The highly infectious nature of the disease and the fact that its cure presently is not within scientific capability also increases the difficulty of investigating live virus and its adverse affect on the human immune system so that successful diagnostic tests can be developed.
Presently, absolute CD4 count is a generally accepted predictor of clinical HIV disease progression. Current methods for this measurement require multi-step sample w093/16385 PCT/US93/01232 J 3 j preparation and are known to have cumulative errors i-related to determiining both the proportion of CD4 lymphocytes and the absolute lymphocyte count. One method which attempted to solve this problem was presented in a "poster" at the Seventh International Conference on AIDS
in Florence, Italy, June 19, 1991. However, the disclosed method was incomplete since it did not teach nor enable one to practice the method.
Similarly, others have attempted to provide different lo methods and apparatuses to solve the general problem of determining the cell count per unit volume of particles suspended in a fluid containing undesirable particles.
One approach is described in U.S. Patent 4,989,978 to Groner. However, this method requires that a predetermined Xnown volume of a suspension of cells and undesirable particles be analyzed so that the volume of the undesirable particles may be subtracted from the original predetermined volume to provide an accurate count of cells per unit volume.
More generally, others have added a plurality of particle~, to various diluents in methods for testing and calibrating flow cytometry instruments. Although the prior art contains many such disclosures, two such examples are U.S. Patent 4,704,891 to Recktenwald, et al and U.S. Patent 4,331,862 to Ryan. In addition, by adding ~tandard particles to a cell suspension prior to i~nalysis, a "built in" fiducial mark is provided which can be used to interrelate distributions obtained under differing instrument conditions. According to Flow Cytometry and Sorting, John Wiley & Sons Publisher, 1979, this built in technique is taught by Jensen, et al, Multiparameter Flow Cytometry Applied Toward Diagnosis of Cer~ical Squamous WO93/16385 PCT/~S93/01232 1 t~ 9 i 3 j Cell Carcinoma. Proceedings of the Second International Conference on Automation o~ Cancer Cytology and Cell Image Analysis, May 6-7, 1977. However, these general references fail to address the problem of determination of cell count per specimen volume.
According to the invention there is provided a method to ascertain the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed, said method charactexized by the steps of:
a) mixing a known quantity of particles ha~ing a first light scatter signal with a known volume~of a cell specimen having a second light scatter signal different from the first light scatter signal to obtain a suspension having a concentration of particles per specimen volume;
b) passing each of said particles and cells in an unknown volume portion of said cell suspension, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering pattern; c) counting the number of cells and the number of particles in said unknown vOlUm8 portion of said ~uspension as is known;
and, d) determining the total number of cells per volume of the speci~en by correlating the number of particles and cells in said portion of the suspension to the known quantity of particles added to said cell specimen.
The present invention relates to a method for accurately determininq the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed and which doe.s not analyze the entire original known ~olume. ~he count of the cells is correlated to the count of the added discrete particles to , '~' W093/16385 PCr/US93/01232 ' ~:
determine the cell count per initial volume. The method employs a light scatter trigger to se~se the cells, and uses a two scale analysis of light scatter and fluorescence to count the cells of interest and the added discrete particles, as well as, to discriminate between the cells of interest and added discrete particl~s. In a more preferred embodiment, the cells of interest are labelled with a fluorescent monoclonal antibody.
In one embodiment, the method comprises mixing a suspension of a known quantity of discrete particles with a known volume of the cell specimen to obtain a suspension having a concentration of particles per specimen volume;
counting the number of cells and particles in a portion of said suspension; and determining the total nu~ber of cell per unit volume of the specimen. Said determination is obtained by correlating the ratio of said counted cells in said portion times the number of said known quantity of particles in the suspension to the specimen volume times the number o~ said counted particles in said portion.
In another embodiment, the method of this invention comprises mixing a suspension of a known quantity of particles having a first light acatter signal with a known volume of a cell specimen having a second light scatter signal different from the first light scatter signal to obtain a suspen6ion having a concentration of particles per specimen volume; passing each of said particles and cells in said cell suspension, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering pattern; counting the number of cells and the number of particles in a portion of said suspen~ion: and, determining the total number of cells per unit volume of the specimen by correlating the number of WO93/16385 PCT/~S93/01232 n ~ !) '3 particles and cells in said suspension to the known quar.tity of particles added to said cell specimen.
The use of this method permits the cell specimen to be subjected to one or more volumetric dilutions caused by the processing of the cells. More specifically, providing the original cell specimen volume is known, the cell specimen may be lysed, have cells of interest tagged with s~ain or monoclonal antibodies, or be subject to such ~ -other process which would dilute the original cell specimen volume to an unknown volume.
In addition, the inv~ntion further provides a quality control method to insure that the operating parameters of the counting instrument is within specification 50 that it accurately senses all of the desired particles, while simultaneously determining the count per unit volume of a cell suspension.
By using fluorescent partioles with the cell specimen, the flow cytometer can u~e light scatter triggering for the parameter set so that the particles and cells are analyzed by light scatter and fluorescence.
Providing that the cells and particles are aensed by light scatter and fluorescence during analysis, then one has adequate assurance that the flow cytometeF has been properly operating during the analysis of the specimen.
In a preferred embodiment, the method ~urther comprises the additional stepæ of correlating eaid light scattering pattern and fluorescence signal of the particles to a predetermined ratio which represents a threshold of minimum instrument performance and adjusting, if the predetermined ratio has not been attained, the operation of said instrument, while the particles are within the incident beam of light, until the measured d wo 93/163~5 PCI/US93/OlZ32 ~23~
ratio reaches said predetermined ratio whereby the instrument is analyzing both particles and cells in said cell suspension.
In order to determine the total number of cells per unit volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed, it is necessary to begin with a known volume of the specimen. The accuracy of determining the cell count per unit volume will depend, in part, upon the accuracy of the measurement of the specimen volume. ~ ~
In order to expeditiously obtain the cel? count of ~;
cells of interest per unit volume, it is preferred that the whole blood is lysed to remove the erythrocytes by such means as known to those skilled in the art. These means include hypotonic lysis, acid lysis, as well as, the use of magnetic particles or microspheres coated with an antibody for sorting and separating the selected population from a ~eterogeneous cell population. The only ~O limiting fact in the selection o~ the method of lysis is that it should preserve the integrity of the leukocytes.
More specifically, in a pre~erred embodiment of this invention, the preferred method of lysis of the whole blood is by the COULTER Immuno-Prep EPICS~ Leukocyte Preparation System, distributed by Epics Division of Coulter Corporation (COULTER and EPICS are trademarks of Coulter Corporation, Hialeah, Florida). As known to those skilled in the art, this preferred method of lysis is a hypotonic lysis.
Although the flow cytometer is capable of di~stinguishing between leukocytes of interest and erythrocytes in the whole blood, evaluation of a whole w093/16385 PCT/~JS93101232 212~ -~3~
blood sample would require an extended time to analyze when using a light scatter trigger. The extended time is due to the cytometer having to analyze both the red blood cells as well as the white blood cells. More specifically, the white blood cell count in normal blood is 5,000 to ll,000 per microliter (~L) with a lymphocyte value of 20 to 40% and the red blood cells are 4,000,000 to 5,000,000 cells per microliter (~L). Therefore, in order to calculate a lymphocyte count of 5,000, approximately lO million to 30 million cells will be analyzed.
After lysing the whole blood, the remaining leukocytes are then identified by such means as known to those skilled in the art. Sùch identification means includes stains, ligands, probes, and monoclonal antibodies. The selection of the stain, ligand, probe, or monoclonal antibody enables the enumeration of the selected leukocyte of interest to be identified and i `
accurately counted in an expeditious manner.
Having determined the initial volume of the cell specimen, a known number of discrete particles are then added to the blood speci~en which has optionally been lysed, and in which the labelled leukocytes of interest have been identified.
~oth biological and plastic discrete particles known to those ~killed in the art may be used in the present invention. The discrete particles should ~e~inert with respect to the carrier in which they are diluted and with respect to the test probe of the counting instrument. In addition, the discrete particles should be stable at conventional working temperatureæ of 0-30C. Unstable ~ -particles have a tendency to shift in their histogram WO93/16385 PCT/~IS93101232 positioning, and contribute imprecision to the determination of cells per specimen volume. An important feature of the present invention is that the particles do not need to have characteristics of a like nature to the cells of interest. More specifically, the particles are not required to replicate the volume, size, fluorescence, light scatter, impedance, or Coulter opacity of the cells of interest. Using particles having different characteristics than the cells, eliminates any overlapping of cell count to cause erroneous or inaccurate results.
Preferably, the density of the discrete particle should approximate that of the cell. More specifically, the density of the particle should be greater than 1.0 grams per milliliter. This is advantageous because if the lS density is substantially disproportionate to the cells in the specimen, the discrete particles will have a tendency ~ ' to settle in the specimen if not analyzed immediately. As such, if the discrete particles are substantially disproportionate to the density of the cells, a non-uniform mixture might be analyzed which could contribute to an inaccurate determination of cell count ;
per unit ~olume. Therefore, if the density of the particle differs from the density of the cell specimen by greater than 20%, then the cell-particle suspension should be analyzed within 30 minutes of preparation. More preferably, the density of the particle should differ from the cells by less than 5%, and the cell-particle suspension should be analyzed within 4 hours of preparation.
The preferred type of particles for the present invention are plastic microbeads. Such beads may be fabricated with substantially uniform diameter, and have W093/1638~ PCT/US~3/01232 ,~ , 3 j surface characteristics which are suitable for surface binding of fluorophores or other marking agents.
Substantially spherical beads may be formed having a diameter between 0.5 and 20 microns. While the beads are S usually made in solid form, they may be hollow inside and could be vesicles or other microcarriers. Moreover, the beads do not have to be perfect spheres in order to function in accordance with the present invention.
Plastic materials such as polystyrene, polyacrylamide a~d other latex materials may be employed for fabricating the beads, and other plastic materials such as polyvinylchloride, polypropylene and the like may also be utilized.
When plastic particles are used, they should be ~ -treated to prevent the plastic particles from adhering to the cells in the cell-particle specimen and to prevent the plastic particles from aggregating with each other. These ~ `
two impediments need to be resolved 60 that the particles remain discrete ~nd are susceptible of accurate differentiation.
To prev~nt the plastic particles from adhering to the cells of interest, the particles may be coated with such substances which will counteract this tendency. Such substances include bovine serum albumin, glycolipid, polyvinylpyrrolidone or other substances which would change the bior~activity of the plastic particle so that it would not attach to the cells of interest. For the purposes of this invention, it is preferred to use a bovine serum albumin solution.
It is believed that the aggregation of the plastic particles is caused by the surface charge of the discrete plastic particle. It has been found that this obstacle , ~ - , - , , -, ; -, , , ; . . . .- -. .. .. .:.
WO93/16385 P~/US93/01232 `` h ~ 3 ~
11 , ".~' may be resolved by the aforementioned coating process.
But in addition, the pH of the carrier appears to contribute to the resolution of the aggregation problem.
Moreover, it is further believed that the use of a non-ionic surfactant would also contribute to eliminating thisproblem.
The carrier used as the storage and suspension medium for the particles must be chemically and physically compatible with the selected particle material and with the type of counting instrument. Thus, for use in those counting instruments which use the change in electrical conductivity to calculate count, the carrier must be electrically conductive. Those skilled in the art who use countiny instruments employing the principle of electrical conductivity appreciate that the conductivity of the carrier will greatly affect the indicated count of the instruments. Thus, a standard carrier conductivity has ~`
been adopted and is the conductivity of 0.8% equivalent ~ `
molarity of codium chloride. `
On the other hand, for those counting instruments which utilize the light scattering principle, the carrier must be optically conductive (or transparent, i.e., having a very low absorption coefficient). However, those skilled in the art who use counting i~struments employing the pr~nciple of light scattering appreciate that when using certain plastic particles, such as DNA check beads, there is a tendency for them to aggregate. As noted previously, it is believed that the aggregation is caused by electrostatic attraction of the discrete plastic particles which may be related to the salt concentration in the carrier. Therefore, when using such particles in a W093/16385 PCTlUS93/Ot232 light scattering instrument, the carrier should not cause aggregation of the plastic particles.
In a preferred embodiment of the invention, the carrier should have a density equal to or greater than the density of the added discrete particle. Preferably, the carrier density is between O to 15% and more preferably o to 5% greater than the discrete particle density. If the density of the carrier is substantially disproportionate to the discrete particle, then the particles will have a tendency t~ either settle to the bottom or float to the t~p of the carrier solution resulting in a non-homogeneous -~ ;
particle suspension. A homogeneous particle suspension is desirable to facilitate the procedure to add a known count of the particles to the cell specimen.
More preferably, the carrier comprises an aqueous solution of glycerol and most preEerably comprises between 17 to 21% by volume of the aqueous solution. However, as known to those skilled in the art, other ~nown media may be used to increase the carrier density, such as sucrose, Ficoll-Hypaque~R (manufactured by Pharmacia L.K.B.
8iotechnology), and Percoll~ (manufactured by Pharmacia L.K.B. Biotechnology). When DNA check beads are the particles of choice, the carrier will have a density within 5% difference of 1.03 grams/milliliter.
In order to maximize the life of the particles and carrier and to prevent further aggregation of the particles, it is important that the pH of the carrier be ~ ~
compatible with the particles and other additives in the ~;
carrier. For example, it has been found that a pH falling generally within the range of 6.0 to 8.0 is acceptable and that a preferable range is from 6.9 to 7.3 for use with particles of a latex material. If the pH of the carrier WO93/16385 PCT/US93/01232 ~;
3 ) 3 j is either too high or too low, it has been found that the latex particles may aggregate, thereby diminishing the known total number of particles and further increasing the size of the aggregates to perhaps a size that would fall outside of the ability of the instrument to count the particles. It is noted that the term aggregation is used broadly and also includes the formation of doublets or triplets of particles which, it should be appreciated, would be as detrimental as the formation of much larger aggregates of particles.
In addition, it is also desirable to include in the carrier a small, but sufficient concentration of a preservative to prevent the growth of bacteria or fungi in the carrier or on the particles. More particularly, the preservative is preferably a fungicide and/or a bactericide which does not increase background noise by either creating floculations or adding fluorescence.
Although ~ny addit~ve which i8 compatible wlth the other components of the carrier and with the particle material is acceptable, it is preferred that the preservative be selected Srom the group comprising organic aldehydes, including monaldehydes such as formaldehyde, or dialdehydes ~uch as glutaraldehyde: alcohols, including organic alcohol, such as aliphatic and cylic alcohols, such as phenol, toluene, ethanol, and propanol; and sodium fluoride and sodium azide. More preferably, the additive selected is formaldehyde and is used in an amount within the range of about 0.5% to 2.5% and preferably 1.0% by -volume of the carrier liguid.
It is believed that the stability of the carrier can be enhanced if an appropriate surfactant is added thereto.
However, the selected surfactant must not de}eteriously 2 1 2 9 i ~
affect the cells of interest. Preferably, such a a, .
surfactant is non-ionic type and is present in the minimum amounts necessary so that the aggregation of particles is minimized. Too great a percentage of surfactant in the carrier results in the formation of bubbles, as well as, causes cell degradation, while too little surfactant does not provide the desired degree of stability. -The preferred procedure to add a Xnown count of particles to the cell specimen comprises pipetting a selected volume of the carrier containing the discrete particles. The accuracy of determining the cell count per unit volume will depend, in part, upon the accuracy of the -volumetric measurement of this particle suspension. ~y this method, the number of beads that can be added to the cell specimen is known within one percent. However, any method known to those skilled in the art to add a known count of particles to the cell specimen may be employed.
The unknown volume portion of the cell specimen and particles is analyzed by a method which is effective to -determine the nu~ber of dîscrete particles and the number of cells of interest. The method comprises a light scatter triggering followed by light scatter and fluorescence measurements in a flow cytometer.
In typical flow cytometry instruments, cells and Z5 other discrete particles are caused to flow in a liquid stream so that each cell and particle, preferably one at a time, passes through a sensing region which measures one or more of its physical or chemical characteristics.
The sensing methods include electrical impedance related to particle volume based upon the well known Coulter Principle; ~luorescence: light absorption: light scatter; light extinction fluorescence polarization and WO 93/16385 ~'C r/l,S93/U1232 r .~
fluorescence signal waveform. Any one or more of these sensing methods may be used to activate a particular parameter set of analysis.
For purposes of the ensuing discussion, and for exemplary purposes, reference is made to a flow cytometer wherein, the liquid flow stream of cells and particles is ensheathed in a sheath fluid so that the cells and pa~ticle stream may be hydrodynamically focused as it flows through an incident beam of light normally directed ;-at right angles to the stream of cell and particle flow.
As the cells and particles pass through the beam of light, light characteristics associated with each cell and particle triggers a particular parameter set of analysis which may optimally be selected by the instrument operator. Although fluorescence and light scatter are optical signals, when used as a trigger for a parameter set, each has a different function and effect.
When a light scatter trigger is employed, each cell and particle passing through the incident beam of light is sen6ed by junction silicon photodiodes. Since light scatter iB affected by pnrticle size, shape, density, stain uptake, and granularity, one or more of these criteria may be used to activate cell analysis. As such, cell analysis may be activated by requiring the cell and particle to meet an upper or lower threshold of these criteria.
When a fluorescence trigger is employed, only those cells ~nd particles having a fluorescence is sensed by photomultiplier tubes which converts light signals to electrical signals. The fluorescent cells and particles absorb the incident beam of light and emit a fluorescent signal at a light wavelength different from the incident WO93/l6385 PCT/US93/01232 ~129~}3c~
source. Fluorescence i5 affected by the uniformity of illumination and signal intensity. Illumination uniformity is related to the stability of the light source and its spat~al intensity distribution. Fluorescence signal intensity depends upon a variety of instrumental characteristics, included are the excitation light intensity, sample stream flow velocity, the illumination source and excitation spectra of the dye, quantum efficiency Df the dye, collection and transmission efficiency of the light detection optics and the sensitivity of the photodetectors. As such, cell and particle analysis may be activated by requiring the particle to meet an upper or lower threshold of fluorescence signal intensity.
For the purposes of this invention, the method of sensing the particles comprises a light scatter trigger.
This will enable all particles in the suspension to be analyzed by the parameter set selected. Moreover, a light scatter trigger is not dependent upon the signal intensity of a fluorescence stain which can vary on a cell by cell basis. More particularly, the use of a light scatter trigger will provide a two-fold benefit.
First, when the cells are analyzed, a light scatter trigger will provide the ability to differentiate among the three types of leukocytes in the cell specimen, and in addition, by the use of markers or tags, to detect selected cluster designation of lymphocytes.
Secondly, the use of a light scatter trigger enables an in situ quality control of the instrument performance.
~ore specifically, the instrument will initially sense the cells and discrete particles by light scatter and depending upon their characteristics, will trigger a W093/~6385 PCT/US93/01232 2 :1 2 3; ~ ~
certain parameter set of measurements. Such parameter set will include the simultaneous measurement of the light scatter and one or more other sensing properties. ~his ;~
has the advantage that all particles would be detected which would pro~ide an accurate representation of the particles and cells in the sample suspension.
In addition, a light scatter trigger has the further advantage that during the analysis of the sample suspension, the instrument operator can have assurance that the instrument is operating within performance specifications and analyzing the sample suspension. More particularly, while the sample suspension is being analyzed, the technician can confirm, and if necessary adjust, the alignment of the instrument by its ability to detect and report in the proper position in the histogram the known discrete particles and the amplification of the signal intensity of the known discrete particles.
These parameters are not apparent during an analysis of a sample suspension which contains a limited number of cells o~ interest or which i5 of limited quantity or which contains contaminates which are detected ~y the instrument. In essence, the operator is able to validate the analy~is by focusing upon the measurements during analysis to determine that the machine is properly functioning.
In a most preferred embodiment, the sample suspension is analyzed by a simultaneous measurement of light scatter and fluorescence to provide significantly more accurate sensin~ of the cells than the measurement of only light scatter or only fluorescence. Thus, when using fluorescent particles and fluorescent markers for CD4 lymphocytes in the cell suspension which has ~een lysed of red blood cells, quality control can b~ assured by triggering on light scatter of the discrete particles and the cells of interest and confirming detection with light -scatter and fluorescence. Correlating these two signals S enable assurance of uniformity of illumination, si~nal intensity and improvement in the discrimination between the cells of interest and discrete particles.
In the most preferred embodiment of this invention, the cell-particle suspension will be further analyzed to differentiate the three major populations of leukocytes, (i.e., lymphocytes, monocytes and granulocytes), as well as, determine the CM lymphocytes in a single assay. In this embodiment, fluorescence triggering is not merited because not all cells and particles of interest will have a fluorescence intensity susceptible to accurate and reproducible measurement.
The measurement of particles and cells occurs after the suspension has reached a steady state of flow. This will allow for hydrodynamic instabilities of the suspension Slow to be eliminated. These instabilities are most apparent at the sta~t ~nd end of analysis when the particles and cells may either be tightly or loosely grouped together. Moreover, during steady state flow, the suspension is substantially free of air bubbles which has a tendency to impede measurements. When the frequency of particl~ distribution is nearly equal, a steady flow rate is achieved. This provides the most accurate interval for determining the cell count per unit volume resulting in an precision greater than 95S.
The features and advantages of the present invention are more fully shown with respect to the following non-limiting examples.
W093/16385 PCT/US93/Olt32 7 ~ . 3 `.i ~ ~
.
PI.
PreParation of Plasti~ M'cros~heres Materi~ls:
1. Plastic microspheres having a fluorescence intensity susceptible of measurement 2. Glycerol, ACS
3. Mannitol, ACS
4. Bovine Serum Albumin (~SA) (35% solution) 5. Formaldehyde (36%), ACS ~-6. Distilled, deionized water Procedure:
1. Suspend the plastic microspheres in 0.1% BSA + 1%
Mannitol in water and incubate with roc~ing for 60 minutes at ambient conditions.
2. Centrifuge the plastic microspheres in a swinging bucket centrifuge at 1000 RPM (200xG), 5 minutes, at ambient conditions.
3. Remove and discard the supernate.
4. Re6usp~nd the plastic microspheres in 0.1% BSA +
1% Mannitol in water and incubate with rocking for 60 minutes at ambi¢nt conditions.
5. R~peat steps 2, 3, and 4, except rock overnight.
6. Repeat steps 2 and 3.
1. Suspend the plastic microspheres in 0.1% BSA + 1%
Mannitol in water and incubate with roc~ing for 60 minutes at ambient conditions.
2. Centrifuge the plastic microspheres in a swinging bucket centrifuge at 1000 RPM (200xG), 5 minutes, at ambient conditions.
3. Remove and discard the supernate.
4. Re6usp~nd the plastic microspheres in 0.1% BSA +
1% Mannitol in water and incubate with rocking for 60 minutes at ambi¢nt conditions.
5. R~peat steps 2, 3, and 4, except rock overnight.
6. Repeat steps 2 and 3.
7. Resuspend the plastic microspheres with 50 parts 0.1% BSA + 1% Mannitol + 1.0~ Formaldehyde + 20% Glycerol in water (bottling solution) to every one part plastic microspheres.
8. Transfer to a glass bottle and sonicate for 5 minutes.
W093/16385 PCT/US93/Ot232 ; 3 9. Prepare serial dilutions in bottling solution such that 10,000 plastic microspheres (+/- 100) is obtain in 100 ~
~A~PhE 2 Pre~aration and Anal~sis of Sam~le $usPension for CD4_~YmPhoc~te Determination 1. 100 ~1 of whole blood specimen is added to a 12x75 mm polypropylene tube.
2. 10 ~1 of a CD4 monoclonal antibody reagent used ~`
to label the CD4 lymphocyte cells is added to the tube to form a suspension and said suspension is vortexed.
3. Incubation is continued for 10 minutes at ambient conditions.
4. The suspension is lysed to remove erythrocytes.
5. Add 100,000 plastic microspheres which have been treated a~ describ¢d in Example 1.
6. The sample is again vortexed to 50% of tube height.
7. Immediately before analysis, vortex again to 50%
of tube height for 5 seconds.
8. The sample suspension is then analyzed by flow cytometric methods known to those skilled in the art in an EPICS~ Profile Elite Flow Cytometer and the count of CD4 lymphocytes per unit specimen volume is obtained.
WOs3/16385 PCT/US93/01232 21,'..9.~3 ~AMPL~ 3 Pre~aration and Analvsis of Sam~le susPension for Cultured Leukemic Ce~Ll~
The method of Example 2 is repeated except the whole blood is replaced with lOO ~1 of cultured leukemic cells and the CD4 monoclonal antibody reagent is replaced with a propidium iodine reagent.
After incubation for twenty minutes to allow iodine binding to non-viable cells, steps 4 through 8 of Example 2 are repeated. The results obtained are the number of viable cultured cells per unit specimen volume.
ESAM~LB
Pre~aration and An~lysis of ~amP
Sus~ension ~ Re~iculocvtes 15The method of ~xample 2 is repeated except instead of using lOO ~1 of whole blood, only 2 ~1 is used, and the CD4 reagent is replaced with a reticulocyte reagent, such as thiazole orange. After incubation for approximately 10 -minutes, steps 5 through a of Example 2 are repeated. The results obtained are the number of reticulocytes per unit speci~en volume.
While the invention has been descrlbed with reference to specific embodiments and compositions, it will be appreciated that numerous modifications, variations and 2S embodiments are possible, as being within the spirit and scope of the invention.
. .
V., Oh~ R~h. ~ A . ' ' ~ '
W093/16385 PCT/US93/Ot232 ; 3 9. Prepare serial dilutions in bottling solution such that 10,000 plastic microspheres (+/- 100) is obtain in 100 ~
~A~PhE 2 Pre~aration and Anal~sis of Sam~le $usPension for CD4_~YmPhoc~te Determination 1. 100 ~1 of whole blood specimen is added to a 12x75 mm polypropylene tube.
2. 10 ~1 of a CD4 monoclonal antibody reagent used ~`
to label the CD4 lymphocyte cells is added to the tube to form a suspension and said suspension is vortexed.
3. Incubation is continued for 10 minutes at ambient conditions.
4. The suspension is lysed to remove erythrocytes.
5. Add 100,000 plastic microspheres which have been treated a~ describ¢d in Example 1.
6. The sample is again vortexed to 50% of tube height.
7. Immediately before analysis, vortex again to 50%
of tube height for 5 seconds.
8. The sample suspension is then analyzed by flow cytometric methods known to those skilled in the art in an EPICS~ Profile Elite Flow Cytometer and the count of CD4 lymphocytes per unit specimen volume is obtained.
WOs3/16385 PCT/US93/01232 21,'..9.~3 ~AMPL~ 3 Pre~aration and Analvsis of Sam~le susPension for Cultured Leukemic Ce~Ll~
The method of Example 2 is repeated except the whole blood is replaced with lOO ~1 of cultured leukemic cells and the CD4 monoclonal antibody reagent is replaced with a propidium iodine reagent.
After incubation for twenty minutes to allow iodine binding to non-viable cells, steps 4 through 8 of Example 2 are repeated. The results obtained are the number of viable cultured cells per unit specimen volume.
ESAM~LB
Pre~aration and An~lysis of ~amP
Sus~ension ~ Re~iculocvtes 15The method of ~xample 2 is repeated except instead of using lOO ~1 of whole blood, only 2 ~1 is used, and the CD4 reagent is replaced with a reticulocyte reagent, such as thiazole orange. After incubation for approximately 10 -minutes, steps 5 through a of Example 2 are repeated. The results obtained are the number of reticulocytes per unit speci~en volume.
While the invention has been descrlbed with reference to specific embodiments and compositions, it will be appreciated that numerous modifications, variations and 2S embodiments are possible, as being within the spirit and scope of the invention.
. .
V., Oh~ R~h. ~ A . ' ' ~ '
Claims (5)
1. A method to ascertain a total number of cells per volume of a cell specimen in an apparatus which does not measure the volume of a suspension containing the specimen as it is being analyzed, said method characterized by the steps of:
a. mixing a known quantity of particles having a first light scatter signal with a known volume of a cell specimen having a second light scatter signal different from the first light scatter signal to obtain a suspension having a concentration of particles per specimen volume, b. passing each of said particles and cells in an unknown volume portion of said cell suspension, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering signal c. counting the number of cells and the number of particles in said unknown volume portion of said suspension by using a flow cytometer with a light scatter trigger, and d. determining the total number of cells per volume of the specimen by correlating the number of particles and cells in said portion of the suspension to the known quantity of particles added to said cell specimen using light scatter measurements and fluorescent measurements.
a. mixing a known quantity of particles having a first light scatter signal with a known volume of a cell specimen having a second light scatter signal different from the first light scatter signal to obtain a suspension having a concentration of particles per specimen volume, b. passing each of said particles and cells in an unknown volume portion of said cell suspension, in turn, through a light beam, each of said particles and cells producing at least one forward light scattering signal c. counting the number of cells and the number of particles in said unknown volume portion of said suspension by using a flow cytometer with a light scatter trigger, and d. determining the total number of cells per volume of the specimen by correlating the number of particles and cells in said portion of the suspension to the known quantity of particles added to said cell specimen using light scatter measurements and fluorescent measurements.
2. A method to ascertain a total number of cells per volume of a cell specimen in an apparatus which does not measure volume of a suspension containing the specimen as it is being analyzed, said method characterized by the steps of:
a. mixing a known quantity of particles with a known volume of a cell specimen to obtain a suspension having a concentration of particles per specimen volume, said particles remaining separate from the cells in said suspension, b. counting the number of cells and the number of particles in an unknown volume portion of said suspension by using a flow cytometer with a light scatter trigger, and c. determining the total number of cells per volume of the specimen by correlating the number of particles and cells in said unknown volume portion of the suspension to the known quantity of particles added to said cell specimen using light scatter measurements and fluorescent measurements.
a. mixing a known quantity of particles with a known volume of a cell specimen to obtain a suspension having a concentration of particles per specimen volume, said particles remaining separate from the cells in said suspension, b. counting the number of cells and the number of particles in an unknown volume portion of said suspension by using a flow cytometer with a light scatter trigger, and c. determining the total number of cells per volume of the specimen by correlating the number of particles and cells in said unknown volume portion of the suspension to the known quantity of particles added to said cell specimen using light scatter measurements and fluorescent measurements.
3. The method of claims 1 or 2, characterized wherein said particles have a density less than 20% difference than the cells in the cell specimen.
4. The method of claims 1 or 2 which further includes maintaining operating parameters of a counting apparatus within specification during the determination of the total number of cells per volume of a cell specimen and wherein said particles have fluorescence at a known wavelength;
further characterized by (e) correlating said light scattering signal and fluorescence signal of the particles to a predetermined ratio which represents a threshold of minimum apparatus performance; and (f) adjusting, if the predetermined ratio has not been attained, the operation of said apparatus, while the particles are within the incident beam of light, until the measured ratio reaches said predetermined ratio.
further characterized by (e) correlating said light scattering signal and fluorescence signal of the particles to a predetermined ratio which represents a threshold of minimum apparatus performance; and (f) adjusting, if the predetermined ratio has not been attained, the operation of said apparatus, while the particles are within the incident beam of light, until the measured ratio reaches said predetermined ratio.
5. The method of claim 1, characterized by wherein said particles have been treated to prevent them from attaching to the cells in the cell specimen.
Applications Claiming Priority (2)
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|---|---|---|---|
| US07/836,638 US5451525A (en) | 1992-02-14 | 1992-02-14 | Method and materials for determining particle count in a flow cytometer |
| US07/836,638 | 1992-02-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2129535A1 true CA2129535A1 (en) | 1993-08-15 |
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ID=25272392
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|---|---|---|---|
| CA002129535A Abandoned CA2129535A1 (en) | 1992-02-14 | 1993-02-11 | Method and materials for determining particle count in a flow cytometer |
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|---|---|
| US (1) | US5451525A (en) |
| EP (1) | EP0627080B1 (en) |
| JP (1) | JP3328690B2 (en) |
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| CA (1) | CA2129535A1 (en) |
| DE (1) | DE69328296T2 (en) |
| FI (1) | FI943732A (en) |
| NO (1) | NO942989L (en) |
| WO (1) | WO1993016385A1 (en) |
Families Citing this family (32)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5691204A (en) * | 1995-04-21 | 1997-11-25 | Abbott Laboratories | Compositions and methods for the rapid analysis of reticulocytes |
| US5882933A (en) * | 1995-06-08 | 1999-03-16 | Coulter International Corp. | Method for determination of leukocytes and hemoglobin concentration in blood |
| US5942443A (en) | 1996-06-28 | 1999-08-24 | Caliper Technologies Corporation | High throughput screening assay systems in microscale fluidic devices |
| DE69737513T2 (en) * | 1996-04-25 | 2007-12-13 | Genicon Sciences Corp., San Diego | PARTICULAR LABEL USING ANALYTASSAY |
| US6790366B2 (en) * | 1996-06-07 | 2004-09-14 | Immunivest Corporation | Magnetic separation apparatus and methods |
| US6890426B2 (en) * | 1996-06-07 | 2005-05-10 | Immunivest Corporation | Magnetic separation apparatus and methods |
| US7666308B2 (en) * | 1996-06-07 | 2010-02-23 | Veridex, Llc. | Magnetic separation apparatus and methods |
| US5993665A (en) * | 1996-06-07 | 1999-11-30 | Immunivest Corporation | Quantitative cell analysis methods employing magnetic separation |
| US6136182A (en) * | 1996-06-07 | 2000-10-24 | Immunivest Corporation | Magnetic devices and sample chambers for examination and manipulation of cells |
| US6660159B1 (en) | 1996-06-07 | 2003-12-09 | Immunivest Corporation | Magnetic separation apparatus and methods |
| US20060000722A1 (en) * | 1996-06-28 | 2006-01-05 | Caliper Life Sciences, Inc. | High throughput screening assay systems in microscale fluidic devices |
| CN1173776C (en) * | 1996-06-28 | 2004-11-03 | 卡钳技术有限公司 | High-throughput screening assay systems in microscale fluidic devices |
| EP0979409B1 (en) | 1997-02-20 | 2006-12-27 | The Regents of the University of California | Plasmon resonant particles, methods and apparatus |
| US6852252B2 (en) | 1997-03-12 | 2005-02-08 | William Marsh Rice University | Use of metalnanoshells to impede the photo-oxidation of conjugated polymer |
| US6344272B1 (en) * | 1997-03-12 | 2002-02-05 | Wm. Marsh Rice University | Metal nanoshells |
| US5976994A (en) * | 1997-06-13 | 1999-11-02 | Regents Of The University Of Michigan | Method and system for locally annealing a microstructure formed on a substrate and device formed thereby |
| US6428811B1 (en) | 1998-03-11 | 2002-08-06 | Wm. Marsh Rice University | Temperature-sensitive polymer/nanoshell composites for photothermally modulated drug delivery |
| US6699724B1 (en) * | 1998-03-11 | 2004-03-02 | Wm. Marsh Rice University | Metal nanoshells for biosensing applications |
| AU3146800A (en) | 1999-03-05 | 2000-09-28 | De Danske Kvaegavlsforeninger | Determination of sperm concentration and viability for artificial insemination |
| JP3817091B2 (en) * | 1999-07-02 | 2006-08-30 | テルモ株式会社 | Cell number measuring device |
| JP4606543B2 (en) * | 2000-04-13 | 2011-01-05 | パナソニック株式会社 | Method for confirming amount of solution to be measured and measuring system control method in optical property measuring apparatus |
| US6784981B1 (en) * | 2000-06-02 | 2004-08-31 | Idexx Laboratories, Inc. | Flow cytometry-based hematology system |
| WO2002068932A2 (en) | 2001-02-23 | 2002-09-06 | Genicon Sciences Corporation | Methods for providing extended dynamic range in analyte assays |
| US7299135B2 (en) * | 2005-11-10 | 2007-11-20 | Idexx Laboratories, Inc. | Methods for identifying discrete populations (e.g., clusters) of data within a flow cytometer multi-dimensional data set |
| WO2010135627A1 (en) | 2009-05-21 | 2010-11-25 | Intellicyt | System and method for separating samples in a continuous flow |
| US20110129864A1 (en) * | 2009-12-02 | 2011-06-02 | Idexx Laboratories, Inc. | Method for analyzing lymph node aspirate using multi-angle light scatter flow cytometer |
| WO2012054783A2 (en) * | 2010-10-21 | 2012-04-26 | Nexcelom Bioscience Llc | Internal focus reference beads for imaging cytometry |
| US8975595B2 (en) * | 2013-04-12 | 2015-03-10 | Becton, Dickinson And Company | Automated set-up for cell sorting |
| KR102289358B1 (en) * | 2013-10-10 | 2021-08-11 | 에이껜 가가꾸 가부시끼가이샤 | Feces sampling container |
| EP4343304A2 (en) | 2018-03-30 | 2024-03-27 | IDEXX Laboratories, Inc. | Flow cytometer, laser optics assembly thereof, and methods of assembling the same |
| BR112022025798A2 (en) | 2020-06-17 | 2023-01-10 | Idexx Lab Inc | FLOW CYTOMETER OF A BLOOD ANALYZER AND METHOD TO DETECT RETICULOCYTES AND GRANULOCYTES |
| CN115436237B (en) * | 2022-09-22 | 2023-08-01 | 东莞理工学院 | Particle swarm size distribution determining method, system, equipment and storage medium |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4331862A (en) * | 1979-02-23 | 1982-05-25 | Ryan Wayne L | Method for calibrating a particle counting machine and a calibration standard therefor |
| US4499052A (en) * | 1982-08-30 | 1985-02-12 | Becton, Dickinson And Company | Apparatus for distinguishing multiple subpopulations of cells |
| US4751179A (en) * | 1984-05-31 | 1988-06-14 | Coulter Electronics, Inc. | Method and reagents for differential determination of four populations of leukocytes in blood |
| US4661913A (en) * | 1984-09-11 | 1987-04-28 | Becton, Dickinson And Company | Apparatus and method for the detection and classification of articles using flow cytometry techniques |
| US5073498A (en) * | 1984-12-24 | 1991-12-17 | Caribbean Microparticles Corporation | Fluorescent alignment microbeads with broad excitation and emission spectra and its use |
| US4989978A (en) * | 1986-04-04 | 1991-02-05 | Technicon Instruments Corporation | Method and apparatus for determining the count per unit volume of particles |
| US4704891A (en) * | 1986-08-29 | 1987-11-10 | Becton, Dickinson And Company | Method and materials for calibrating flow cytometers and other analysis instruments |
| US4888290A (en) * | 1987-11-06 | 1989-12-19 | Coulter Corporation | Monoclonal antibody specific to HIV antigens |
| IE76732B1 (en) * | 1990-08-07 | 1997-11-05 | Becton Dickinson Co | One step test for absolute counts |
-
1992
- 1992-02-14 US US07/836,638 patent/US5451525A/en not_active Expired - Lifetime
-
1993
- 1993-02-11 AT AT93905022T patent/ATE191561T1/en not_active IP Right Cessation
- 1993-02-11 CA CA002129535A patent/CA2129535A1/en not_active Abandoned
- 1993-02-11 DE DE69328296T patent/DE69328296T2/en not_active Expired - Lifetime
- 1993-02-11 WO PCT/US1993/001232 patent/WO1993016385A1/en active IP Right Grant
- 1993-02-11 EP EP93905022A patent/EP0627080B1/en not_active Expired - Lifetime
- 1993-02-11 AU AU36170/93A patent/AU667344B2/en not_active Ceased
- 1993-02-11 BR BR9305886A patent/BR9305886A/en not_active Application Discontinuation
- 1993-02-11 JP JP51428893A patent/JP3328690B2/en not_active Expired - Lifetime
-
1994
- 1994-08-12 FI FI943732A patent/FI943732A/en unknown
- 1994-08-12 NO NO942989A patent/NO942989L/en unknown
- 1994-08-13 KR KR1019940702808A patent/KR950700542A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| NO942989D0 (en) | 1994-08-12 |
| DE69328296T2 (en) | 2000-12-21 |
| US5451525A (en) | 1995-09-19 |
| AU3617093A (en) | 1993-09-03 |
| BR9305886A (en) | 1997-08-19 |
| JPH07503795A (en) | 1995-04-20 |
| ATE191561T1 (en) | 2000-04-15 |
| FI943732A (en) | 1994-10-12 |
| DE69328296D1 (en) | 2000-05-11 |
| EP0627080A1 (en) | 1994-12-07 |
| EP0627080A4 (en) | 1996-11-20 |
| EP0627080B1 (en) | 2000-04-05 |
| KR950700542A (en) | 1995-01-16 |
| WO1993016385A1 (en) | 1993-08-19 |
| NO942989L (en) | 1994-10-11 |
| JP3328690B2 (en) | 2002-09-30 |
| FI943732A0 (en) | 1994-08-12 |
| AU667344B2 (en) | 1996-03-21 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |