CA1312263C - Apparatus and method for analyses of biological specimens - Google Patents
Apparatus and method for analyses of biological specimensInfo
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- CA1312263C CA1312263C CA000541465A CA541465A CA1312263C CA 1312263 C CA1312263 C CA 1312263C CA 000541465 A CA000541465 A CA 000541465A CA 541465 A CA541465 A CA 541465A CA 1312263 C CA1312263 C CA 1312263C
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Abstract
ABSTRACT
AN APPARATUS AND METHOD FOR
ANALYSES OF BIOLOGICAL SPECIMENS
A kit for the quantitation of cell nuclei is described wherein the kit includes a stain and microscopic slides. Each slide has a reference cell objects and a specimen all area for receipt of specimen cells which are stained simultaneously with the reference cell objects.
AN APPARATUS AND METHOD FOR
ANALYSES OF BIOLOGICAL SPECIMENS
A kit for the quantitation of cell nuclei is described wherein the kit includes a stain and microscopic slides. Each slide has a reference cell objects and a specimen all area for receipt of specimen cells which are stained simultaneously with the reference cell objects.
Description
1 31 ~263 This invention relates to a method and apparatus for clinically testing ~u~d quantii~ing biological specimens such as ceDs through irnage analysis of the specimens.
~A~KGR~UND OEi r~ lNVl~T~ON
T`he present invention is directed to a quantitative testing apparatus and metllod S ` which may be used for a wide range of diagnostic testing and evaluation of various cells, tissues, or other materials,~alcen from the human body. The present mvention is directed to an appàratus (hereinafter the "lci~'r) and method used in image analysis using pattern recognition techniques to ana~ze and quantify ceD constituents or components which may be stained. This lcit and method are particularly useful and adaptable to the method and apparat~s clisclo~sed in applicant's U.S. Patent No. 4,741,Q43, issued April 26, 1988. In a preferred embodiment the method and the kit may be used in the measurement of cell~ar DNA for the purpo~se ot cancer diagnosis and prognosis.
As wi!l be explained in greater detail, the present invention is directed to providing equipment of a user interactive nature for ~Lse not or~y by researchers, but aLso by a pathologi~t in a laboratory and to low cast equipment which can be acquired by a typical pathalog~st laboratory.
The current state of the art in pathology laboratory is to estimate the content of cell constituents or components ~hereinafter "cell objects'~ such as constit~lents or components of DNA by the visual observation of the pa~ologist who observes plirnaril~ the shape ~md te~sture ~1) of the cell objects after staining For example, in connection with su~spected ,~
~A~KGR~UND OEi r~ lNVl~T~ON
T`he present invention is directed to a quantitative testing apparatus and metllod S ` which may be used for a wide range of diagnostic testing and evaluation of various cells, tissues, or other materials,~alcen from the human body. The present mvention is directed to an appàratus (hereinafter the "lci~'r) and method used in image analysis using pattern recognition techniques to ana~ze and quantify ceD constituents or components which may be stained. This lcit and method are particularly useful and adaptable to the method and apparat~s clisclo~sed in applicant's U.S. Patent No. 4,741,Q43, issued April 26, 1988. In a preferred embodiment the method and the kit may be used in the measurement of cell~ar DNA for the purpo~se ot cancer diagnosis and prognosis.
As wi!l be explained in greater detail, the present invention is directed to providing equipment of a user interactive nature for ~Lse not or~y by researchers, but aLso by a pathologi~t in a laboratory and to low cast equipment which can be acquired by a typical pathalog~st laboratory.
The current state of the art in pathology laboratory is to estimate the content of cell constituents or components ~hereinafter "cell objects'~ such as constit~lents or components of DNA by the visual observation of the pa~ologist who observes plirnaril~ the shape ~md te~sture ~1) of the cell objects after staining For example, in connection with su~spected ,~
-2- 1 ~1 22~3 cancer cells the pathologist observes the shape and texture of the cell objects and then classifies them into a normal category or into one of several abnormal cancer categories. These evaluations, however, are very subjective and can not differentiate and quantify smAll changes in DNA within individual cells or in very small populations of abnormal cells, which changes have clinical significance in the diagnosis and prognosis of cancer as discussed infra. Although there are commercially available general purpose flow cytometers, which are very expensive units and which can handle liquid blood specimens or tissue disaggregations, these cytometers are incapable of working on standard tissue sections and of using microscope slides which are the preferred specimen forms used in pathology laboratories. Additionally, an image analysis technique allows analysis of morphological features of cells such texture, in combination with size and shape of cell nuclei and alterations in nuclear-to-cytoplasmic ratios of cells whereas the ~low cytometer does not allow such analysis.
The progression of the skill-of-the-art techniques for testing in anatomology, surgery, and histopatholoyy has to date evolved to a primarily visual comparison of stain enhanced cells and tissues to human memory of previous examples~ New advances in measurement, ~i.e., U.S. Patent ~o. 4,741,043) to quantify and replace these current state-of-the-art~
subjective comparisons to past memory, require calibration of the measurement instruments. Movel calibration means are required, e.g., as compared to chemical analysis calibration (where the state-of-the~art for calibrated measurement is well developed), because the material, although it is being read by light transmission measurements as i~ chemical analysis, is actually presented to the meaurement instrument as a thin solid material, preserving cell and tissue morphology, on a transparent substrate. Methods, and the state-of-the-art techniques for calibrating ~uch readings after suitable quan~itative staining for specific cell or tissue parts, are essentially undeveloped and non-exi~tent. Adequate calibration, such as described in this invention, will revolutionize and transform testing in these laboratories from subjective to obiective. Such calibration preferably is on a test-by test basis, i.e., on the individual microscope slide for each specimen, because the tested objects are so incredibly small, e.g., measuring picograms of DNA in cells with nuclei on the order of l~O micrometers in size, that very small shifts in light transmission or subtle staining variations mak0 ~he measurement process too error pxone without such calibration.
The use of image analysis generally requires staining cell objects on a microscopic slide. The use of image analysis techniques and equipment and stained specimens by pathologists in a conventional pathology laboratory involves solving a number of problems, including variation of stain, variation of optical densities of stained cell objects and calibration of microscopic slides with stained objects thereon, all of which have been overcome by the present invention~
There are a number of available staining techniques which can be used. For example, with Azure A, Azure B, pararosanelin and methylene blue, Feulgen staining techniques may be used to st in DNA cell objects.
Proteins may be stained with congo red, hematoxylin, eosin, an eosin/hematoxylin combination, and methyl green~ Enzymes may be stained with diaminobenzadine or alkaline phosphatase; cell organelles may be stained with methylene blue; ribosomes with methylene blue stain; and mitochrodia with geimsa stain. Moreover, as used herein, stain includes counter s~ains such as methyl green. In breast cell cancer analysis some of .. . .. ... .... . . . . .. ..
The progression of the skill-of-the-art techniques for testing in anatomology, surgery, and histopatholoyy has to date evolved to a primarily visual comparison of stain enhanced cells and tissues to human memory of previous examples~ New advances in measurement, ~i.e., U.S. Patent ~o. 4,741,043) to quantify and replace these current state-of-the-art~
subjective comparisons to past memory, require calibration of the measurement instruments. Movel calibration means are required, e.g., as compared to chemical analysis calibration (where the state-of-the~art for calibrated measurement is well developed), because the material, although it is being read by light transmission measurements as i~ chemical analysis, is actually presented to the meaurement instrument as a thin solid material, preserving cell and tissue morphology, on a transparent substrate. Methods, and the state-of-the-art techniques for calibrating ~uch readings after suitable quan~itative staining for specific cell or tissue parts, are essentially undeveloped and non-exi~tent. Adequate calibration, such as described in this invention, will revolutionize and transform testing in these laboratories from subjective to obiective. Such calibration preferably is on a test-by test basis, i.e., on the individual microscope slide for each specimen, because the tested objects are so incredibly small, e.g., measuring picograms of DNA in cells with nuclei on the order of l~O micrometers in size, that very small shifts in light transmission or subtle staining variations mak0 ~he measurement process too error pxone without such calibration.
The use of image analysis generally requires staining cell objects on a microscopic slide. The use of image analysis techniques and equipment and stained specimens by pathologists in a conventional pathology laboratory involves solving a number of problems, including variation of stain, variation of optical densities of stained cell objects and calibration of microscopic slides with stained objects thereon, all of which have been overcome by the present invention~
There are a number of available staining techniques which can be used. For example, with Azure A, Azure B, pararosanelin and methylene blue, Feulgen staining techniques may be used to st in DNA cell objects.
Proteins may be stained with congo red, hematoxylin, eosin, an eosin/hematoxylin combination, and methyl green~ Enzymes may be stained with diaminobenzadine or alkaline phosphatase; cell organelles may be stained with methylene blue; ribosomes with methylene blue stain; and mitochrodia with geimsa stain. Moreover, as used herein, stain includes counter s~ains such as methyl green. In breast cell cancer analysis some of .. . .. ... .... . . . . .. ..
these stains are used with monoclonal antibodies which are estrogen receptors to breast cells. Antigen analysis may include the steps of binding of monoclonal antibodies to the specimen and control cell objects.
Later the monoclonal body may be conjugated with an enzyme stain. Also, the monoclonal antibody may be conjugated with a fluorescent material. Then the fluorescent stain may be excited at a wave length to induce the fluorescence and then this may be observed at another wave length at which fluorescent emission occurs. When the antibody is made for a particular virus, the contxol cell specimen objects may be txeated with a nucleic acid probe specific for the genome of the virus.
Variation in the degree of staining of cell objects and the variation of the optical density of the s~ained cell objects presents a problem in the quantitation of the stained cell objects through image analysis. The staining of cell objects, such as the D~A
with Azure A, will vary substantially not only from slide to slide or from batch to batch by the same pathologist, but will vary substantially between different pathologists and different laboratories.
Because the image analysis equipment is measuring grey level or optical densities and because it is desired to provide a true actual weight of DNA per cell in picograms from optical density measurements from stained cell objects, it is i~portant to overcome the problem of different staining factors for different speci~ens.
Also, image anaIysis techniques use microscopes and optical lighting which are adjustable to provide different intensities of light when used by the pathologist. Trained researchers, in research laboratories may be equipped to adjust the optical intensity to the desired conditions for image analysis by image pattern techniques, but this generally will not be accomplished with the precision necessary in the . .
t 31 ~26:~
usual pathology laboratory. Thus, there is a need to overcome the problem of this optical density and staining variable.
Heretofore in cell analysis, an inexpensive and simple quantitation of cell objects has not been available. For example, except for those using only ~ore expensive and sophisticated equipment, relative comparisons of data which are a function of cell object content have been only available to workers studying the proliferation of cell objects~ In the case of DNA, absolute values of D~A content of cell nuclei in terms of picograms has not been readily available to laboratory workers for uses such as cell object cycle analysis. Moreover in connection with DNA, this i6 clinically significant in the diagnosis and prognosis of cancer. The analysis of the DNA content of cells has been shown to be of value in the assessment of proliferations of benign and malignant cells. Abnormal DNA content (aneuploidy) has been observed consistently in numerous cancers ~uch as prostate, colon, cervical, breast, and bladderO Also, preliminary data indicates that assessment of aneuploidy has pro~nostic value. In addition the presence o~ an increased number o~ cells which are synthesizing DNA ( BO called "S p~ase" cells~
has been shown to relate the extent of tumor cell proliferation, in some cases.
The method and kit of this invention coupled with any apparatus of carrying out image analysis of cell obiects after staining, using pattern recognition techniques (such as t~e apparatus disc70sed in U.S. Patent No. 4,741,043 ), permit a ~orker to readily and inexpensively not only detect minute alterations in cell objects including DNA, but also to measure and quantify the amount of cell objects as an aid to statistical analysis in research and patient diagnosis and treatment.
6 ~312263 The present invention overcomes the problem of high costs heretofore associated with computerized equipment used for image analysis; and to this end, the present invention is an interactive system in which the pathologist performs a number of tasks including the selection, preparation, placement and staining of cell~
on microscopic slides. The pathologist is provided with the kit of the invention which includes stain and slides both of which are especially prepared and calibrated.
The slide includes reference cells to aid in the diagnosis of the specimen cell objects and to assi~t in overcoming the staining density problem above-described. The present invention also permits location of cell objects for examination as to their morphology and preserve~ their location for a later analysis or corroborating analysis by a second pathologist when so desired. With respect to nuclei, measurements may be obtained as to area in microns, total nuclear optical density or nuclear ~ass in picograms, average nuclear optical density, nuclear texture, and deviation of the nuclear shape from being a round nucleus. Also, a number of such measurements may be made of the cell cytoplasm.
When the kit of this invention is used for cell analysis, tissue and cell specimens are applied to a ~lide which then is stained with a specific stain that combines proportionately with the cell objects which generally essentially renders invisible the remainder of the cell so that the image analysis measures the cell o~ject conten~ such as DNA or protein which is concentrated principally at the nucleus of the cellO
The stain associates with the cell object to provide a detailed nuclear structure and pat~ern which may be visually observed and interpreted by the pathologist using an apparatus for image analysis. In connection with DNA analyses for diagnosis and progno~is of cancer, the amount of D~A in the malignant cells generally i~
... ~ .. . ... . . . . . .
~7 1 3 1 2263 substantially greater than that for normai cells because the malignant cells usually are dividing and replicating rapidly or the malignant cells have abnormal numbers of chromosomes-or have defective chromosomes.
The kit of the invention comprises a microscopic slide which includes a reference area and a specimen cell object area for receipt of specimen cells. The reference axea contains a reference ~eans for simultaneous staining for a predetermined time with the specimen cells or cell objects after the specimen cells or cell objects are applied to the specimen cell object area of the slide. According to the invention this simultaneous staining of the reference means and specimen cell objects with a stain of predetermined concentration permits a self-calibration of the slide as hereinafter described. The kit also includes one or more containers of stain and may include a container of rinse sulfonating agent for addition to a rinse used in preparation of the slide for microscopic image analysis. The amount of stain in the kit affects the optical density of the reference means and the specimen cell objects. This is an important aspect of the inventionO After the staining the optical density Gf the reference means (and the specimen cell objects if they contain the matexial being investigated and measured such as DNA) will be a linear function of stain concentration per unit of material (such as stain concentr~tion per cell object if the material being stained are cells) only over a select range of stain concentrations per stained cell object. Except or this linear portion, a curve of a plot of optical density versus stain concentxation per cell will not be linear and not provide readily measurable or understood differences in optical densities with changes in stain concentrations per ~ell object. This is important to cell analysis. In cancer diagnosis and progno~is observation of varying D~A content by virtue of -8- 1 31 22~3 differing stain content and the resulting differing optical densities will be more readily detected and understood if the variation of optical density to stain ~oncentxation per cell is linear. Quantitation of D~A, 5 however, i5 only an example and analysis o any cell object by optical density and will be more readily understood if the optical density of the cell object is linear. Hence it is important that the stain in the kit be provided in an effective amount of stain to provide an optical density to the reference means after staining for a predetermined amount of time such that the optical density of the reference means will be a substantially linear function of the stain concentration of the reference means after staining. The same is true of the specimen cell object if the object contains the material being referenced by the reference material.
The reference means for staining contains or constitutes any reference material which combines with stain proportionately to the combination of stain with the cell objects being analyzed. In connection with DNA
analysis, the reference material may be trout blood cell erythrocytes, chicken red blood cells, dried D~A or cultured cell lines which reproduce themselves such as lymphoblastoid cells. In connection with proteins or enzymes the reference material may be any material containing a known amount of protein or enzymes to which an analysis is being directed.
The stain of the kit also may include a stain sulfonating agent. The stain sulfonating agent and rinse sulfonating a~ent are used in conjunction with acidic aqueous solutions of stain and rinseO Preparation of the slide frequently contemplates putting the stain in an acidic aqueous solution and then staining the reference means as well a the specimen cell objects with the aquevus stain solution~ After the materials on the slide are stained, they are rinsed with a solution which also frequently is an acidic aqueous solution. In such cases ~ 31 2263 ~9 consistent reproducible re~ults demand 6tains and rin~es having pHs within consistent relatively narrow ranges. A
sulfonating agent which is compatible with the stain aids in binding the Azure A to the hydrolyzed DNA.
In an alternate embodiment of the invention, the microscopic slide of the Xit includes an optical densi~y reference area which area includes a material which has a predetermined known optical density to calibrat~ the microscopic slide with the instrument being used to study the specimen cell objects. Without the optical density reference area, the slide in conjunction with the kit described herein is self calibrating without regard to certain other variables that may change from analysis to analysis. These include variations in thickness and type of glass used in the slide as well as variations in temperature and humidity conditions encountered during analysis and which could affect analysiQ.
The method of the invention permits the quantitation of specimen cell objects by comparing thP
optical density of the stained specimen cell objects with the optical density of the stained reference material having known amounts of material to be quantified~ For example, in connection with DNA quantitation, trout blood cell erythrocytes are known to have 5.6 picograms of DNA. Use of these cells as a reference material will permit the calculation of the DNA content of specimen cell objects in terms of absol~te DNA weiyht when such specimen cell objects are simultaneously prepared and stained and the optical densities of the stained referencP material and specimen cell objects are compared. This calculation and computer program relative thereto are described in my application Serial No. 539,834 filed June 1~, 1987. In breast cell analysis for the quantitation of estrogen t 3 1 2263 receptors cultured breast cancer cells or tissue sections of oryanic material e~g. endometriun may be used as reference cells and as a source of reference cell objects.
In connection with the quantitation of nuclear DNA, the method of the invention includes providing a slide with a reference area and a specimen cell object area; providing a reference material in the reference area, the reference material having physical characteristics which include a known amount of DNA and permit association of the reference material with a stain which is proportional to an association of the stain with DNA; providin~ specimen cell objects in the specimen cell object area; simultaneously staining the reference material and the specimen cell objects with a stain in aqueous solution of stain for a predeter~ined amount of time, the stain in aqueous solution being in an effective amount to provide the reference material with an optical density after staining which will be a substantially linear function of stain concentration of the reference material; measuring the optical density of the reference materials after staining; measuring the optical density of the specimen cell objects after staining and determining the quantitative amount of D~A
in the specimen cell objects from the measured optical densities.
The kit and method of the invention permits an easy and inexpensive detection of minute alterations in specimen cell objectfi. In connection with cell object alterations in DNA content, this is done by providing a real and accurate measurement of the DNA in picograms.
The invention also permits measurement and quantification of the amount of DNA and relates it to stored statistical analyses to aid in the diagnosis.
More specifically, the invention in conjunction with my inventions disclosed and described in my U.S. Patent No. 4,741,043 and copending Canadian Serial No. 539,834 !
:' ' in respect to DNA analysis allows an iteratlve analysis of specime~
population cells and provides a histogram or display of the population distribution of the cells with respect to their DN~ content and with respect to a standard DNA for normal cells so that subtle shifts in population distribution can be readily understood. To this end cell nuclei images are not only acquired and stored but the data therefrom can be integrated with statistical data to provide multi-variate analysis, discrimination of cell&, histograms, and scattergrams of cells or cell populations.
Accordingly, a general object of the invention is to provide a new and improved apparatus and methoA
for analyzing cells or other biological materials by using image analysis techniques.
A further object of the invention is to provide a new and improved kit which includes a stain and a slide or support for specimen cell objects which slide has a reference means or cell objects thereon wherein the stain with the slide permits calibration of the slide for image analysis of the slide in conjunction with image analysi~ equipment.
Another object of the invention is to provide a new and improved apparatus and method for making a ploidy analysis o cells using image pattern recognition equipm~nt.
These and other objects and advantages of the invention will become apparent from the following description taken in connection with the accompanying drawings.
DETAILED DESCRIPTION OF THE DRAWINGS
FIGURE l is a perspective view of a kit in accordance with the invention.
FIGURE 2 is a view of a specimen slide or support constructed in accordance wit~ the invention.
f~ `
.
-12- ~ 3 1 22~3 FIGURE 3 is a plan view of a slide with materials thereon for control cells, specimen cells, ~ight calibration, reference location, and integrity checking.
FIGURE 4 is a cross sectional view taken along the line 3-3 in FIGURE 3.
FIGURE 5 is a post staining schematic plot of optical density versus stain concentration per cell.
FIGURE 6 is a histogram of control cell ploidy calibration made in accordance with the invention.
FIGURE 7 is a histogram of a summary report of cell ploidy distribution in accordance with the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
As sXown in the drawings for purposes of illustration, the kit and method of the invention will generally be used in conjunction wit~ an apparatus or automatically analyzing "cell objects". The latter term is used herein to be generic to cells, including but not limited to blood cells or cells taken from tumors, or the like, which are prepared so that their nuclei may be observed. In the case of the quantitation of DNA, the cells are prepared using a Feulgen staining reaction such that DNA in the cell nuclei may be observed.
Hydrochloric acid hydrolyzes ribose - nucleic acid bonds to the ~NA to give aldehyde sugar residues. A stain such a Azure A then couples via the Schiff reaction to the sugar aldehydes to give a blue-violet col~rO Other stains such as Azure B, paralosinalin and methylene blue may be used in lieu of Azure A. Moreover, the present invention is not ~nly useful for the staining study of DNA for ploidy analysis and blood cell analysis, but also can be used to analyze pap smear cells, monoclonal antibodies conjugated to stains and used as cell markers, and other infectious diseases which can be diagnosed by DNA probes for viruses; and as previously 1 3 1 22~3 stated, can be used for the study and quantitation of proteins, enzymes, cell organelles, riboso~es and mitochrondia stained with a compatible stain and rinse.
As shown in FIGURE 1, the kit 2 includes a container 4 comprising one or more bottles or vials of stain material 6, one or more boxes 8 of microscopic slides 10 and one or more bottles or vials of rinse sulfonating agent 12. The container has a base 14, lid 16 hinged to the base and a latch which includes latch members 18 and 20 which frictionally and snappingly engage the lid and base respectively to close the container. The base of the container has a cushion 22 of sponge, foam or the like with molded or cut inserts 24~ The inserts have the shape o~ the vials or boxes of slides and are adapted to hold the bottles and boxes in a fixed position without damage during transport of the kit.
Turning now to FIGURE 2, the microscopic or specimens slide 10 may ~e of any size or shape, but because of the familiarity of lab technicians and pathologists with glass slides used with microscopes, it is preferred that a slide 10 be an actual microscope slide of glass which typically measures 3 inch by 1 inch. The illustrated s}ide 10 shown in FIGURE 2 has a preprinted border 26 which defines a reference area within which are located the reference means for staining such as reference cell objects 28. The reference means in this illustrated embodiment o the invention, are trout cell erythrocytes, i.e., trout red blood cells, of known size and shape and DNA content, which is about 5.6 picograms of DNA. The reference cell objects may be other types of cells having dark centers or nuclei which stain well, ~uch as chicken blood cells having a D~A content of 2.51 picograms; they may be artifacts deposited on the slide, which may or may not '5 have cell shapes, or the cell objects 28 may be well known plastic beads of a predetermined size which will react with a particular fluorescent stain or enzyme stain. The reference cell objects will vary from test to test and the present invention i5 not limited to any particular test or cell objects therefor.
The slide also includes a specimen cell object area 30 for receipt of specimen cell objects 32 which are~ in this instance, cells from a slice of tissue ~such a tumor tissue), or a needle aspirate of tumor tissue or monolayer of blood cells or other cells, at the area 30 on the slide.
In the preferred embodiment of the invention, the slide also includes an optical density reference area which preferably is a printed mark such as a cross 3~ on the slide. This area has a material with a predeter~ined known optical density which can be used as a reference to calibrate an instrument analyzing the slide. As will be explained in greater detail hereinafter, a histogram and instructions are provided to the operator from an instruction control logic to the operator as described in my application Serial No. 794,937 filed November 4, 1985 and the operator manually adjusts the optical light intensity until the desired intensity is obtained for the optical density reference material, and the background light. The system logic as described in my U. S. Patent No.
25 4,741,043 also is calibrated with the optical density reference material to read the proper optical density of objects.
The slide also may include an optical integrity pattern 36 as a safeguard to the integrity of the system. This pattern provides an integrity check or identification from the slide 10 by analyzing a predetermined and preixed optical pattern on the slide which is read and measured as to gray levels and physical dimensions before the analyzing may be begun.
35 Herein, the optical integrity pattern may be in ~he form of initials CAS located above the control cell objects as seen in FIGURE 2. Manifestly, the integrity check may be the cross 34 of the optical end or any other material on the slide 10.
The kit and slide is useful for later analy is o~ the specimen cell object6 32 on the slide 10; and to aid in the recall of cell images stored in memory or to allow the operator or another person to return to a given cell for a second review thereo at a later time.
To this end after the slide lO has been secured on a microscope stage, a certain location on the slide, such as the center 38 of the cross 34, is noted as the zero-zero X-Y referen~e point; then the location registers for the X and Y distances are zeroed at this point so that subsequently all cell locations may have a specific X and Y coordinate address from the center 3~
of the cross. A further easy location to find with the adjustment with the ~icroscope stage iB a corner such as the right hand lower corner 39 of the box border 40 within which are located the reference cell objects 28.
Herein, the box border 40 is printed on the slide and it also may be used for optical density calibration rather than the special cro~s 34. On the other hand, by suitable logic and control, any point on the slide and microscope stage at which the classification operation begins may be taken as the zero X and Y location with the location registers for the X and Y coordinates being æeroed initially at this location and then providing a readout for each cell location from this zeroed location.
The slide may urther include locator strips on the slide to facilitate location of specific cell objects. The particular X and Y location for each specimen cell may be obtained by the use of conventional stepping motor techniques which are well known in the art and which are relatively expensive. In a preferred embodiment of the slide the X and Y locations are easily determined for any given location with an X direction . ... . . . ~ . . . .. . ... .. .
sensing strip 40 which may be fastened to the underside of the microscope slide lO for movement with the slide past a sensing read head secured to a stationary part of the microscope and which reads a sensing scale on the ~ensing strip and provides a digital output to an interface electronic which provides the X coordinate in digital numbers to an instrument control logic for storing in memory and for display. Likewise, a similar strip 42 is fastened to the slide for movement in the Y direction with the stage past a read head which is secured to a stationary part of the microscope so that the read head may read the indicia on the Y strip 42 to provide a digital readout to the interface electronic which supplies digital signals to the instrument control logic for storage of the Y coordinate and for showing the Y
coordinate on the video monitor adjacent the X
coordinate. The system can be reversed with the read heads fastened to the stage for movement therewith with the scale strips 40 and 42 being mounted stationary to provide digital readouts as the heads move thereacross.
The illustrated and preferred strips and heads commonly used as instrument feelers gages, or the like, sold under the trademark "SYLVAC" using magnetic strips and magnetic read heads.
The stain material contained in the bottles 6 of the kit includes stain and may include a sulfonating agent. The stain is not only compatible with the ~pecimen cell objects and reference cell objects, but is an amount effective for providing an optical density to the reference cell objects and specimen cell objects upon application of an aqueous solution of the stain for a predetermined time which optical density is substantially a linear functi~n of stain concentration per stained reference cell object As shown in FIGURE 5, aftQr ~ell objects are stained with an aqueous solution of stain, a curve of a plot of optical density versus stain concentration stain per stained cell object is substantially linear as at 46 over a select range of stain concentration per cell~
Prior to use the stain i8 generally mixed with 0.1 HCL
aqveous sQlution water and then applied to the cell objects under study for a predetermined amount of time.
Thereafter the slide is rinsed and studied. Keeping the amount of stain after it is mixed with water such that for a given staining time range the staining solution provides the concentration of stain associated with the cell objects along the substantially linear portion of the curve of FIGURE 5 is an important aspect of the invention. Keeping the stain concentration per stained cell object a substantially linear function of optical density facilitates a determination of stain concentration per stained cell object from optical density generally will facilitate the measurement of optical densities and differences in optical densities over relatively small differences in stain concentration per cell obiect. These factors are important in quantitation of cell constituents or components from stain ccn_~entration which is in turn measured by optical density. In the case of Azure A and the quantitation of DNA, the concentration of stain in the aqueous staining - solution is in the range of from about 4.9 to about 5.1 mg/ml for a staining time in the range of from about 115 to 120 minutes, the concentration of the stain pre~erably being about 5 mg/ml for a s~aining time of about 120 minutesO
The aqueous solution of Azure A stain applied to the slide will be acidic. In the case of staining DNA with Azure A, the aqueous ~olution of stain has a pH
of 2.7 controlled with 0.1 N hydrochloric acid.
Frequently after cell objects on the ~lide are stained such as with an acidic aqueous stain solution, excess stain is rinsed therefrom with an acidic aqueous rinse solution. In the case o~ staining DNA with A7uxe .
A and thereafter rinsing it, the a~ueous rinse sollltion is preferably controlled with 0.05 N hydrochloric acid to a pH of about 2.~5. The rinse solution will also contain a rinse sulfonating agent in an amount that the stain to rinse sulfonating ratio is in the range of from about 1.7 to about 2.
In a preferred form of the invention the slide box 8 includes a base 48 and lid 50, each slide box containing five microscopic slides maintained in juxtaposition in side-by-side spaced relation.
Longitudinal ribs run the length of the two opposite sides of slide box base to maintain the slides in their position within the box. The ribs are separated sufficiently to permit the microscopic slides to slide therebetween and be held by the ribs at the longitudinal edges of the slides such that the slides are in juxtaposition. An aperture 52 in the lid permits the aspiration of stain into a closed box by a hypodermic needle or the like such that the box may be used for application of stain to the slidesfin lieu of coplin jars.
In connection with the analysis of DNA with Azure A reagent, a kit in the preferred embodiment includes seven vials each containing 375 mg of A~ure A
(Certified) with 1.5 g o~ K2S205 stain sulfonating agent. Each vial is sufficient for preparation of 75 ml of Azure A reagent solution to provide a concentration of 5 mg/ml of Azure A, as hereinafter described. qhe kit also includes seven vials of rinse sulfonating agent which is 1/5 g of K2S205 and five boxes of xlide~.
Each vial of rinse sulfonating agent when the K2S205 is mixed with water and hydrochloric acid is sufficient to make 300 ml of rinse reagent 801ution. The five boxes or containers each with five m;croscopic slides include DNA reference cell objects which are trout red blood cells.
The kit may further include a bottle for the aqueous stain solution marked to indicate 75 ml of volume for preparation of the aqueous acidic Azure A
colution; and a bottle for rinse solution marked to a volume 300 ml for preparation of 300 ml of acidic rinse solution. Materials used with the kit and method of the invention, but not necessarily supplied with the kit, include 0.05 ~ HCl, 5~ HC1 and three 75 ml coplin jar According to the invention in respect to quantitation of D~A using the Feulgen staining reaction, preparation of the slides using the kit preferably is as follows.
Cytologic specimens (e~g. cytospin preparations, touch preparations, fine needle aspirates, smears and smear preparations) are air dried for about 3 to about 5 minutes and fixed in 10~ neutral buffered formalin for about 5 minutes. Then the slides are air dried for about 3 to about 5 minutes. The formalin fixed, air dried slides may be stored at room temperature until stained.
Standard paraffin ti~sue sections following placement on microscope slide and warming in an oven at 63C for l hour, are deparaffinized using standard procedures (e.g. xylenes, absolute ethanol, 95~ ethanol, 80% ethanol, ~0% ethanol, then distilled water).
Following the final water rinse, the slides are ready for the Feul~en staining procedures with the kit.
Staining should be carried out within about 2 hours after completion of deparaffinization.
The aqueous Azure A solution is prepared from the kit by transferring the entire contents of one Azure A vial into one Azure A solution bottle marked to a volume of 75 ml. The bottle is filled to the line with 0.1 N hydrochlQric acid, closed tightly and shaken well. The bottle is kept closea tightly and permitted to stand for two hours at room temperature (18-20C).
It is again shaken before use to assure that reagen~ ;s mixed and is completely dissolved.
.
1 3`1 2263 The acid aqueous rinse solution is prepared by transferring the entire contents (1/5 g of K2S205) of one rinse sulfonating agent vial into a rinse bottle marked to a volume of 300 ml. The rinse bottle marked to 300 ml is filled to the mark with 0.05 N hydrochloric acid. The container is closed tightly and mixed until the rinse buffer is completely dissolved. Both aqueous Azure A and rinse solutions are stable for 4 to 6 hours when stored at room temperature (1~ to 28C). Both the A~.ure A and rinse solutions can be used for staining up to 2 sets of slides or ten slides providing the second set is completed within 6 hours fxom when the solutions were made.
Further an acid hydrolysis solution (75 ml) which is 5 N hydrochloric acid solution is prepared ~or prepara~ion of the cell objects via a hydrolysis reaction of the D~A as described above. This 5 hydrochloric acid solution has a pH of .5.
The slides are stained and prepared according to the following procedure.
1. A. For Cytologic Material The slides are fixed in 10% by weight neutral buffered formalin for 5 minutes at room temperature~
B. For Paraffin Embedded Sections Paraffin sections following deparaffinization may go directly to hydrochloric acid step (see step 2).
2. I'he slides are placed in a coplin jar containing 5 N hydrochloric acid for about 60 to about 75 minutes~
3. The slides are transferred from the coplin jar containing the hydrochloric acid solution directly to a coplin jar containing Azure A solution and stain for about 2 hours.
. . .. .. ,.. . . ~ .. ..... . .. . .... . . .
-21~
4. Three coplin jars, each filled with the rinse solution are readied for use. The slides are placed into the firs~ coplin jar containing rinse solution and permitted to S contact the rinse for about 5 minutes. The slides then are moved to the second coplin jar filled with rinse solution and are permitted to stand or about 5 minutes.
The slides are then moved to the third ~oplin jar filled with rinse solution and again are permitted to stand for about S
minutes (that is, 3 rinses at 5 minutes each).
5. The slides then are washed for about 5 minutes in xunning distilled water.
Later the monoclonal body may be conjugated with an enzyme stain. Also, the monoclonal antibody may be conjugated with a fluorescent material. Then the fluorescent stain may be excited at a wave length to induce the fluorescence and then this may be observed at another wave length at which fluorescent emission occurs. When the antibody is made for a particular virus, the contxol cell specimen objects may be txeated with a nucleic acid probe specific for the genome of the virus.
Variation in the degree of staining of cell objects and the variation of the optical density of the s~ained cell objects presents a problem in the quantitation of the stained cell objects through image analysis. The staining of cell objects, such as the D~A
with Azure A, will vary substantially not only from slide to slide or from batch to batch by the same pathologist, but will vary substantially between different pathologists and different laboratories.
Because the image analysis equipment is measuring grey level or optical densities and because it is desired to provide a true actual weight of DNA per cell in picograms from optical density measurements from stained cell objects, it is i~portant to overcome the problem of different staining factors for different speci~ens.
Also, image anaIysis techniques use microscopes and optical lighting which are adjustable to provide different intensities of light when used by the pathologist. Trained researchers, in research laboratories may be equipped to adjust the optical intensity to the desired conditions for image analysis by image pattern techniques, but this generally will not be accomplished with the precision necessary in the . .
t 31 ~26:~
usual pathology laboratory. Thus, there is a need to overcome the problem of this optical density and staining variable.
Heretofore in cell analysis, an inexpensive and simple quantitation of cell objects has not been available. For example, except for those using only ~ore expensive and sophisticated equipment, relative comparisons of data which are a function of cell object content have been only available to workers studying the proliferation of cell objects~ In the case of DNA, absolute values of D~A content of cell nuclei in terms of picograms has not been readily available to laboratory workers for uses such as cell object cycle analysis. Moreover in connection with DNA, this i6 clinically significant in the diagnosis and prognosis of cancer. The analysis of the DNA content of cells has been shown to be of value in the assessment of proliferations of benign and malignant cells. Abnormal DNA content (aneuploidy) has been observed consistently in numerous cancers ~uch as prostate, colon, cervical, breast, and bladderO Also, preliminary data indicates that assessment of aneuploidy has pro~nostic value. In addition the presence o~ an increased number o~ cells which are synthesizing DNA ( BO called "S p~ase" cells~
has been shown to relate the extent of tumor cell proliferation, in some cases.
The method and kit of this invention coupled with any apparatus of carrying out image analysis of cell obiects after staining, using pattern recognition techniques (such as t~e apparatus disc70sed in U.S. Patent No. 4,741,043 ), permit a ~orker to readily and inexpensively not only detect minute alterations in cell objects including DNA, but also to measure and quantify the amount of cell objects as an aid to statistical analysis in research and patient diagnosis and treatment.
6 ~312263 The present invention overcomes the problem of high costs heretofore associated with computerized equipment used for image analysis; and to this end, the present invention is an interactive system in which the pathologist performs a number of tasks including the selection, preparation, placement and staining of cell~
on microscopic slides. The pathologist is provided with the kit of the invention which includes stain and slides both of which are especially prepared and calibrated.
The slide includes reference cells to aid in the diagnosis of the specimen cell objects and to assi~t in overcoming the staining density problem above-described. The present invention also permits location of cell objects for examination as to their morphology and preserve~ their location for a later analysis or corroborating analysis by a second pathologist when so desired. With respect to nuclei, measurements may be obtained as to area in microns, total nuclear optical density or nuclear ~ass in picograms, average nuclear optical density, nuclear texture, and deviation of the nuclear shape from being a round nucleus. Also, a number of such measurements may be made of the cell cytoplasm.
When the kit of this invention is used for cell analysis, tissue and cell specimens are applied to a ~lide which then is stained with a specific stain that combines proportionately with the cell objects which generally essentially renders invisible the remainder of the cell so that the image analysis measures the cell o~ject conten~ such as DNA or protein which is concentrated principally at the nucleus of the cellO
The stain associates with the cell object to provide a detailed nuclear structure and pat~ern which may be visually observed and interpreted by the pathologist using an apparatus for image analysis. In connection with DNA analyses for diagnosis and progno~is of cancer, the amount of D~A in the malignant cells generally i~
... ~ .. . ... . . . . . .
~7 1 3 1 2263 substantially greater than that for normai cells because the malignant cells usually are dividing and replicating rapidly or the malignant cells have abnormal numbers of chromosomes-or have defective chromosomes.
The kit of the invention comprises a microscopic slide which includes a reference area and a specimen cell object area for receipt of specimen cells. The reference axea contains a reference ~eans for simultaneous staining for a predetermined time with the specimen cells or cell objects after the specimen cells or cell objects are applied to the specimen cell object area of the slide. According to the invention this simultaneous staining of the reference means and specimen cell objects with a stain of predetermined concentration permits a self-calibration of the slide as hereinafter described. The kit also includes one or more containers of stain and may include a container of rinse sulfonating agent for addition to a rinse used in preparation of the slide for microscopic image analysis. The amount of stain in the kit affects the optical density of the reference means and the specimen cell objects. This is an important aspect of the inventionO After the staining the optical density Gf the reference means (and the specimen cell objects if they contain the matexial being investigated and measured such as DNA) will be a linear function of stain concentration per unit of material (such as stain concentr~tion per cell object if the material being stained are cells) only over a select range of stain concentrations per stained cell object. Except or this linear portion, a curve of a plot of optical density versus stain concentxation per cell will not be linear and not provide readily measurable or understood differences in optical densities with changes in stain concentrations per ~ell object. This is important to cell analysis. In cancer diagnosis and progno~is observation of varying D~A content by virtue of -8- 1 31 22~3 differing stain content and the resulting differing optical densities will be more readily detected and understood if the variation of optical density to stain ~oncentxation per cell is linear. Quantitation of D~A, 5 however, i5 only an example and analysis o any cell object by optical density and will be more readily understood if the optical density of the cell object is linear. Hence it is important that the stain in the kit be provided in an effective amount of stain to provide an optical density to the reference means after staining for a predetermined amount of time such that the optical density of the reference means will be a substantially linear function of the stain concentration of the reference means after staining. The same is true of the specimen cell object if the object contains the material being referenced by the reference material.
The reference means for staining contains or constitutes any reference material which combines with stain proportionately to the combination of stain with the cell objects being analyzed. In connection with DNA
analysis, the reference material may be trout blood cell erythrocytes, chicken red blood cells, dried D~A or cultured cell lines which reproduce themselves such as lymphoblastoid cells. In connection with proteins or enzymes the reference material may be any material containing a known amount of protein or enzymes to which an analysis is being directed.
The stain of the kit also may include a stain sulfonating agent. The stain sulfonating agent and rinse sulfonating a~ent are used in conjunction with acidic aqueous solutions of stain and rinseO Preparation of the slide frequently contemplates putting the stain in an acidic aqueous solution and then staining the reference means as well a the specimen cell objects with the aquevus stain solution~ After the materials on the slide are stained, they are rinsed with a solution which also frequently is an acidic aqueous solution. In such cases ~ 31 2263 ~9 consistent reproducible re~ults demand 6tains and rin~es having pHs within consistent relatively narrow ranges. A
sulfonating agent which is compatible with the stain aids in binding the Azure A to the hydrolyzed DNA.
In an alternate embodiment of the invention, the microscopic slide of the Xit includes an optical densi~y reference area which area includes a material which has a predetermined known optical density to calibrat~ the microscopic slide with the instrument being used to study the specimen cell objects. Without the optical density reference area, the slide in conjunction with the kit described herein is self calibrating without regard to certain other variables that may change from analysis to analysis. These include variations in thickness and type of glass used in the slide as well as variations in temperature and humidity conditions encountered during analysis and which could affect analysiQ.
The method of the invention permits the quantitation of specimen cell objects by comparing thP
optical density of the stained specimen cell objects with the optical density of the stained reference material having known amounts of material to be quantified~ For example, in connection with DNA quantitation, trout blood cell erythrocytes are known to have 5.6 picograms of DNA. Use of these cells as a reference material will permit the calculation of the DNA content of specimen cell objects in terms of absol~te DNA weiyht when such specimen cell objects are simultaneously prepared and stained and the optical densities of the stained referencP material and specimen cell objects are compared. This calculation and computer program relative thereto are described in my application Serial No. 539,834 filed June 1~, 1987. In breast cell analysis for the quantitation of estrogen t 3 1 2263 receptors cultured breast cancer cells or tissue sections of oryanic material e~g. endometriun may be used as reference cells and as a source of reference cell objects.
In connection with the quantitation of nuclear DNA, the method of the invention includes providing a slide with a reference area and a specimen cell object area; providing a reference material in the reference area, the reference material having physical characteristics which include a known amount of DNA and permit association of the reference material with a stain which is proportional to an association of the stain with DNA; providin~ specimen cell objects in the specimen cell object area; simultaneously staining the reference material and the specimen cell objects with a stain in aqueous solution of stain for a predeter~ined amount of time, the stain in aqueous solution being in an effective amount to provide the reference material with an optical density after staining which will be a substantially linear function of stain concentration of the reference material; measuring the optical density of the reference materials after staining; measuring the optical density of the specimen cell objects after staining and determining the quantitative amount of D~A
in the specimen cell objects from the measured optical densities.
The kit and method of the invention permits an easy and inexpensive detection of minute alterations in specimen cell objectfi. In connection with cell object alterations in DNA content, this is done by providing a real and accurate measurement of the DNA in picograms.
The invention also permits measurement and quantification of the amount of DNA and relates it to stored statistical analyses to aid in the diagnosis.
More specifically, the invention in conjunction with my inventions disclosed and described in my U.S. Patent No. 4,741,043 and copending Canadian Serial No. 539,834 !
:' ' in respect to DNA analysis allows an iteratlve analysis of specime~
population cells and provides a histogram or display of the population distribution of the cells with respect to their DN~ content and with respect to a standard DNA for normal cells so that subtle shifts in population distribution can be readily understood. To this end cell nuclei images are not only acquired and stored but the data therefrom can be integrated with statistical data to provide multi-variate analysis, discrimination of cell&, histograms, and scattergrams of cells or cell populations.
Accordingly, a general object of the invention is to provide a new and improved apparatus and methoA
for analyzing cells or other biological materials by using image analysis techniques.
A further object of the invention is to provide a new and improved kit which includes a stain and a slide or support for specimen cell objects which slide has a reference means or cell objects thereon wherein the stain with the slide permits calibration of the slide for image analysis of the slide in conjunction with image analysi~ equipment.
Another object of the invention is to provide a new and improved apparatus and method for making a ploidy analysis o cells using image pattern recognition equipm~nt.
These and other objects and advantages of the invention will become apparent from the following description taken in connection with the accompanying drawings.
DETAILED DESCRIPTION OF THE DRAWINGS
FIGURE l is a perspective view of a kit in accordance with the invention.
FIGURE 2 is a view of a specimen slide or support constructed in accordance wit~ the invention.
f~ `
.
-12- ~ 3 1 22~3 FIGURE 3 is a plan view of a slide with materials thereon for control cells, specimen cells, ~ight calibration, reference location, and integrity checking.
FIGURE 4 is a cross sectional view taken along the line 3-3 in FIGURE 3.
FIGURE 5 is a post staining schematic plot of optical density versus stain concentration per cell.
FIGURE 6 is a histogram of control cell ploidy calibration made in accordance with the invention.
FIGURE 7 is a histogram of a summary report of cell ploidy distribution in accordance with the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
As sXown in the drawings for purposes of illustration, the kit and method of the invention will generally be used in conjunction wit~ an apparatus or automatically analyzing "cell objects". The latter term is used herein to be generic to cells, including but not limited to blood cells or cells taken from tumors, or the like, which are prepared so that their nuclei may be observed. In the case of the quantitation of DNA, the cells are prepared using a Feulgen staining reaction such that DNA in the cell nuclei may be observed.
Hydrochloric acid hydrolyzes ribose - nucleic acid bonds to the ~NA to give aldehyde sugar residues. A stain such a Azure A then couples via the Schiff reaction to the sugar aldehydes to give a blue-violet col~rO Other stains such as Azure B, paralosinalin and methylene blue may be used in lieu of Azure A. Moreover, the present invention is not ~nly useful for the staining study of DNA for ploidy analysis and blood cell analysis, but also can be used to analyze pap smear cells, monoclonal antibodies conjugated to stains and used as cell markers, and other infectious diseases which can be diagnosed by DNA probes for viruses; and as previously 1 3 1 22~3 stated, can be used for the study and quantitation of proteins, enzymes, cell organelles, riboso~es and mitochrondia stained with a compatible stain and rinse.
As shown in FIGURE 1, the kit 2 includes a container 4 comprising one or more bottles or vials of stain material 6, one or more boxes 8 of microscopic slides 10 and one or more bottles or vials of rinse sulfonating agent 12. The container has a base 14, lid 16 hinged to the base and a latch which includes latch members 18 and 20 which frictionally and snappingly engage the lid and base respectively to close the container. The base of the container has a cushion 22 of sponge, foam or the like with molded or cut inserts 24~ The inserts have the shape o~ the vials or boxes of slides and are adapted to hold the bottles and boxes in a fixed position without damage during transport of the kit.
Turning now to FIGURE 2, the microscopic or specimens slide 10 may ~e of any size or shape, but because of the familiarity of lab technicians and pathologists with glass slides used with microscopes, it is preferred that a slide 10 be an actual microscope slide of glass which typically measures 3 inch by 1 inch. The illustrated s}ide 10 shown in FIGURE 2 has a preprinted border 26 which defines a reference area within which are located the reference means for staining such as reference cell objects 28. The reference means in this illustrated embodiment o the invention, are trout cell erythrocytes, i.e., trout red blood cells, of known size and shape and DNA content, which is about 5.6 picograms of DNA. The reference cell objects may be other types of cells having dark centers or nuclei which stain well, ~uch as chicken blood cells having a D~A content of 2.51 picograms; they may be artifacts deposited on the slide, which may or may not '5 have cell shapes, or the cell objects 28 may be well known plastic beads of a predetermined size which will react with a particular fluorescent stain or enzyme stain. The reference cell objects will vary from test to test and the present invention i5 not limited to any particular test or cell objects therefor.
The slide also includes a specimen cell object area 30 for receipt of specimen cell objects 32 which are~ in this instance, cells from a slice of tissue ~such a tumor tissue), or a needle aspirate of tumor tissue or monolayer of blood cells or other cells, at the area 30 on the slide.
In the preferred embodiment of the invention, the slide also includes an optical density reference area which preferably is a printed mark such as a cross 3~ on the slide. This area has a material with a predeter~ined known optical density which can be used as a reference to calibrate an instrument analyzing the slide. As will be explained in greater detail hereinafter, a histogram and instructions are provided to the operator from an instruction control logic to the operator as described in my application Serial No. 794,937 filed November 4, 1985 and the operator manually adjusts the optical light intensity until the desired intensity is obtained for the optical density reference material, and the background light. The system logic as described in my U. S. Patent No.
25 4,741,043 also is calibrated with the optical density reference material to read the proper optical density of objects.
The slide also may include an optical integrity pattern 36 as a safeguard to the integrity of the system. This pattern provides an integrity check or identification from the slide 10 by analyzing a predetermined and preixed optical pattern on the slide which is read and measured as to gray levels and physical dimensions before the analyzing may be begun.
35 Herein, the optical integrity pattern may be in ~he form of initials CAS located above the control cell objects as seen in FIGURE 2. Manifestly, the integrity check may be the cross 34 of the optical end or any other material on the slide 10.
The kit and slide is useful for later analy is o~ the specimen cell object6 32 on the slide 10; and to aid in the recall of cell images stored in memory or to allow the operator or another person to return to a given cell for a second review thereo at a later time.
To this end after the slide lO has been secured on a microscope stage, a certain location on the slide, such as the center 38 of the cross 34, is noted as the zero-zero X-Y referen~e point; then the location registers for the X and Y distances are zeroed at this point so that subsequently all cell locations may have a specific X and Y coordinate address from the center 3~
of the cross. A further easy location to find with the adjustment with the ~icroscope stage iB a corner such as the right hand lower corner 39 of the box border 40 within which are located the reference cell objects 28.
Herein, the box border 40 is printed on the slide and it also may be used for optical density calibration rather than the special cro~s 34. On the other hand, by suitable logic and control, any point on the slide and microscope stage at which the classification operation begins may be taken as the zero X and Y location with the location registers for the X and Y coordinates being æeroed initially at this location and then providing a readout for each cell location from this zeroed location.
The slide may urther include locator strips on the slide to facilitate location of specific cell objects. The particular X and Y location for each specimen cell may be obtained by the use of conventional stepping motor techniques which are well known in the art and which are relatively expensive. In a preferred embodiment of the slide the X and Y locations are easily determined for any given location with an X direction . ... . . . ~ . . . .. . ... .. .
sensing strip 40 which may be fastened to the underside of the microscope slide lO for movement with the slide past a sensing read head secured to a stationary part of the microscope and which reads a sensing scale on the ~ensing strip and provides a digital output to an interface electronic which provides the X coordinate in digital numbers to an instrument control logic for storing in memory and for display. Likewise, a similar strip 42 is fastened to the slide for movement in the Y direction with the stage past a read head which is secured to a stationary part of the microscope so that the read head may read the indicia on the Y strip 42 to provide a digital readout to the interface electronic which supplies digital signals to the instrument control logic for storage of the Y coordinate and for showing the Y
coordinate on the video monitor adjacent the X
coordinate. The system can be reversed with the read heads fastened to the stage for movement therewith with the scale strips 40 and 42 being mounted stationary to provide digital readouts as the heads move thereacross.
The illustrated and preferred strips and heads commonly used as instrument feelers gages, or the like, sold under the trademark "SYLVAC" using magnetic strips and magnetic read heads.
The stain material contained in the bottles 6 of the kit includes stain and may include a sulfonating agent. The stain is not only compatible with the ~pecimen cell objects and reference cell objects, but is an amount effective for providing an optical density to the reference cell objects and specimen cell objects upon application of an aqueous solution of the stain for a predetermined time which optical density is substantially a linear functi~n of stain concentration per stained reference cell object As shown in FIGURE 5, aftQr ~ell objects are stained with an aqueous solution of stain, a curve of a plot of optical density versus stain concentration stain per stained cell object is substantially linear as at 46 over a select range of stain concentration per cell~
Prior to use the stain i8 generally mixed with 0.1 HCL
aqveous sQlution water and then applied to the cell objects under study for a predetermined amount of time.
Thereafter the slide is rinsed and studied. Keeping the amount of stain after it is mixed with water such that for a given staining time range the staining solution provides the concentration of stain associated with the cell objects along the substantially linear portion of the curve of FIGURE 5 is an important aspect of the invention. Keeping the stain concentration per stained cell object a substantially linear function of optical density facilitates a determination of stain concentration per stained cell object from optical density generally will facilitate the measurement of optical densities and differences in optical densities over relatively small differences in stain concentration per cell obiect. These factors are important in quantitation of cell constituents or components from stain ccn_~entration which is in turn measured by optical density. In the case of Azure A and the quantitation of DNA, the concentration of stain in the aqueous staining - solution is in the range of from about 4.9 to about 5.1 mg/ml for a staining time in the range of from about 115 to 120 minutes, the concentration of the stain pre~erably being about 5 mg/ml for a s~aining time of about 120 minutesO
The aqueous solution of Azure A stain applied to the slide will be acidic. In the case of staining DNA with Azure A, the aqueous ~olution of stain has a pH
of 2.7 controlled with 0.1 N hydrochloric acid.
Frequently after cell objects on the ~lide are stained such as with an acidic aqueous stain solution, excess stain is rinsed therefrom with an acidic aqueous rinse solution. In the case o~ staining DNA with A7uxe .
A and thereafter rinsing it, the a~ueous rinse sollltion is preferably controlled with 0.05 N hydrochloric acid to a pH of about 2.~5. The rinse solution will also contain a rinse sulfonating agent in an amount that the stain to rinse sulfonating ratio is in the range of from about 1.7 to about 2.
In a preferred form of the invention the slide box 8 includes a base 48 and lid 50, each slide box containing five microscopic slides maintained in juxtaposition in side-by-side spaced relation.
Longitudinal ribs run the length of the two opposite sides of slide box base to maintain the slides in their position within the box. The ribs are separated sufficiently to permit the microscopic slides to slide therebetween and be held by the ribs at the longitudinal edges of the slides such that the slides are in juxtaposition. An aperture 52 in the lid permits the aspiration of stain into a closed box by a hypodermic needle or the like such that the box may be used for application of stain to the slidesfin lieu of coplin jars.
In connection with the analysis of DNA with Azure A reagent, a kit in the preferred embodiment includes seven vials each containing 375 mg of A~ure A
(Certified) with 1.5 g o~ K2S205 stain sulfonating agent. Each vial is sufficient for preparation of 75 ml of Azure A reagent solution to provide a concentration of 5 mg/ml of Azure A, as hereinafter described. qhe kit also includes seven vials of rinse sulfonating agent which is 1/5 g of K2S205 and five boxes of xlide~.
Each vial of rinse sulfonating agent when the K2S205 is mixed with water and hydrochloric acid is sufficient to make 300 ml of rinse reagent 801ution. The five boxes or containers each with five m;croscopic slides include DNA reference cell objects which are trout red blood cells.
The kit may further include a bottle for the aqueous stain solution marked to indicate 75 ml of volume for preparation of the aqueous acidic Azure A
colution; and a bottle for rinse solution marked to a volume 300 ml for preparation of 300 ml of acidic rinse solution. Materials used with the kit and method of the invention, but not necessarily supplied with the kit, include 0.05 ~ HCl, 5~ HC1 and three 75 ml coplin jar According to the invention in respect to quantitation of D~A using the Feulgen staining reaction, preparation of the slides using the kit preferably is as follows.
Cytologic specimens (e~g. cytospin preparations, touch preparations, fine needle aspirates, smears and smear preparations) are air dried for about 3 to about 5 minutes and fixed in 10~ neutral buffered formalin for about 5 minutes. Then the slides are air dried for about 3 to about 5 minutes. The formalin fixed, air dried slides may be stored at room temperature until stained.
Standard paraffin ti~sue sections following placement on microscope slide and warming in an oven at 63C for l hour, are deparaffinized using standard procedures (e.g. xylenes, absolute ethanol, 95~ ethanol, 80% ethanol, ~0% ethanol, then distilled water).
Following the final water rinse, the slides are ready for the Feul~en staining procedures with the kit.
Staining should be carried out within about 2 hours after completion of deparaffinization.
The aqueous Azure A solution is prepared from the kit by transferring the entire contents of one Azure A vial into one Azure A solution bottle marked to a volume of 75 ml. The bottle is filled to the line with 0.1 N hydrochlQric acid, closed tightly and shaken well. The bottle is kept closea tightly and permitted to stand for two hours at room temperature (18-20C).
It is again shaken before use to assure that reagen~ ;s mixed and is completely dissolved.
.
1 3`1 2263 The acid aqueous rinse solution is prepared by transferring the entire contents (1/5 g of K2S205) of one rinse sulfonating agent vial into a rinse bottle marked to a volume of 300 ml. The rinse bottle marked to 300 ml is filled to the mark with 0.05 N hydrochloric acid. The container is closed tightly and mixed until the rinse buffer is completely dissolved. Both aqueous Azure A and rinse solutions are stable for 4 to 6 hours when stored at room temperature (1~ to 28C). Both the A~.ure A and rinse solutions can be used for staining up to 2 sets of slides or ten slides providing the second set is completed within 6 hours fxom when the solutions were made.
Further an acid hydrolysis solution (75 ml) which is 5 N hydrochloric acid solution is prepared ~or prepara~ion of the cell objects via a hydrolysis reaction of the D~A as described above. This 5 hydrochloric acid solution has a pH of .5.
The slides are stained and prepared according to the following procedure.
1. A. For Cytologic Material The slides are fixed in 10% by weight neutral buffered formalin for 5 minutes at room temperature~
B. For Paraffin Embedded Sections Paraffin sections following deparaffinization may go directly to hydrochloric acid step (see step 2).
2. I'he slides are placed in a coplin jar containing 5 N hydrochloric acid for about 60 to about 75 minutes~
3. The slides are transferred from the coplin jar containing the hydrochloric acid solution directly to a coplin jar containing Azure A solution and stain for about 2 hours.
. . .. .. ,.. . . ~ .. ..... . .. . .... . . .
-21~
4. Three coplin jars, each filled with the rinse solution are readied for use. The slides are placed into the firs~ coplin jar containing rinse solution and permitted to S contact the rinse for about 5 minutes. The slides then are moved to the second coplin jar filled with rinse solution and are permitted to stand or about 5 minutes.
The slides are then moved to the third ~oplin jar filled with rinse solution and again are permitted to stand for about S
minutes (that is, 3 rinses at 5 minutes each).
5. The slides then are washed for about 5 minutes in xunning distilled water.
6. The slides are dehydrated in absolute ethanol for about 5 minutes to prepare slides for coverslipping.
7. The slides are cleared in xylene for about 5 minutes.
8. The lides are mounted with permount and a coverslip.
The presence of dark blue staining in the nuclei of the control cells in the calibration area of the slide is evidence of proper performance of the reagent~. The Feulgen reaction will produce specific blue staining o~
nuclear DNA. ~ucleoli, if present, and cytoplasm should show no staining. Normal human cells have a D~A content equal to 83~ of the amount found in the reference cell objects in the reference cell object area, i.e. 7.18 programs. Malignant cells may show normal, increased, or occasionally decreased amounts of DNA. Proliferating (S phase) cells show increased amounts of DNA compared to the main D~A peak for that cell type.
After calibration of the apparatus disclosed in my u. S. Patent No . 4, 741~ 043 with the optical density reference area of the image analysis apparatus, in respect to a slide prepared in the above described procedure, a control progr~m logic requests a reference cell object calibration function as shown in the histogram of FIGURE 6. During this control cell calibration, the operator moves the microscope slide to shit the reference cell objects 28 into view on a monitoring screen. When an individual stained reference cell object 28 is within a reference area the summed optical density for that stained reference cell object is measured and stored. After a suitable nu~ber of stained reference cell objects have been analyzed, an analyst will be provided with a histogram such as shown in FIGURE 7 such as on a video monitor which shows an analyst the control cell object ploidy distribution as having a relative quantity of DNA. Internally within an instrument control logic, as described in my application Serial No. PCTtUS86/02409 filed of even date, the model value of the histogram of individually summed optical density values actually measured for the control cell objects are compared to a predetermined standard or reference amount of DNA which the control cells are known to have. The actual summed optical density found by the operator is divided into the stored reference D~A
value to provide a factor by which to adjust fox deviation of the stain ~rom a perfect staining for which the internal reference level has been set up.
The analyst may now begin cell data acquisition for the DNA ploidy analysisO The analyst will select a number of field locations along the specimen cell object area 30 for analysis. The analyst will move the microscope slide to move into view specimen cell objects to be analyzed for DNA content as well as for cell morphology if desired. The analyst will classify the cell in a manner similar to that disclosed in my application PCT/US86/02409 and in U.S. Patent 4~453,266 to give summed optical density for the specimen cell object i~e., a stained cell object nucleus, as well a its area, its roundness, and other classification information. A histogram may then be provided which provides DNA content~ Generally the analyst will select a number of cell objects in each field or area and then will move the microscope stage to position a number of different field~ of specimen cell objects into view and to take and analyze a number of these specimen cell objects until he feels he has a representative sample.
This permits the making of a hi~togram, such as shown in FIG. 7 which shows the number of cells of a particular DNA content and shows the D~A conten~ averages for each of the reference peaks. The data may also be stored internally within a computer logic for later recall and comparison with data of any new specimen from the same patient for analysis of the patient's progxess or regression.
After staining and image analysis of the stained slides, the control cells should give a single main peak indicating the position of the DNA content of normal human cells~ Shifts of the main DNA peaX on unknown samples indicate an abnormal D~A content.
Skewing of the main peak to the right especially with production of a second peak with 2 times the DNA conten~
of the first peak indicates a proli~erating cell popula~ion. 50-100 cells ~hould be counted or non-prolifer~ting populations and 100-200 cells for proliferating populations for reasonable accuracy.
Thus, from the above it will be seen that control cell ad specimen cell objects may be stained with methyl green to counterstain for diaminoben~idine (DAB) so that nuclei may be first isolated by specific wavelengths of light by imaging techniques; and, then the monodonal antibody conjuga~ed with DAB may be shown up by a second wavelength of light imaging technique~
.. . .. . .. .
-24- 1312~63 The present invention is not limited to t~e above described embodiments but extends to cover other embodiments, not shown or described, but falling with the ambit of the appended claims.
.
:
:
': '
The presence of dark blue staining in the nuclei of the control cells in the calibration area of the slide is evidence of proper performance of the reagent~. The Feulgen reaction will produce specific blue staining o~
nuclear DNA. ~ucleoli, if present, and cytoplasm should show no staining. Normal human cells have a D~A content equal to 83~ of the amount found in the reference cell objects in the reference cell object area, i.e. 7.18 programs. Malignant cells may show normal, increased, or occasionally decreased amounts of DNA. Proliferating (S phase) cells show increased amounts of DNA compared to the main D~A peak for that cell type.
After calibration of the apparatus disclosed in my u. S. Patent No . 4, 741~ 043 with the optical density reference area of the image analysis apparatus, in respect to a slide prepared in the above described procedure, a control progr~m logic requests a reference cell object calibration function as shown in the histogram of FIGURE 6. During this control cell calibration, the operator moves the microscope slide to shit the reference cell objects 28 into view on a monitoring screen. When an individual stained reference cell object 28 is within a reference area the summed optical density for that stained reference cell object is measured and stored. After a suitable nu~ber of stained reference cell objects have been analyzed, an analyst will be provided with a histogram such as shown in FIGURE 7 such as on a video monitor which shows an analyst the control cell object ploidy distribution as having a relative quantity of DNA. Internally within an instrument control logic, as described in my application Serial No. PCTtUS86/02409 filed of even date, the model value of the histogram of individually summed optical density values actually measured for the control cell objects are compared to a predetermined standard or reference amount of DNA which the control cells are known to have. The actual summed optical density found by the operator is divided into the stored reference D~A
value to provide a factor by which to adjust fox deviation of the stain ~rom a perfect staining for which the internal reference level has been set up.
The analyst may now begin cell data acquisition for the DNA ploidy analysisO The analyst will select a number of field locations along the specimen cell object area 30 for analysis. The analyst will move the microscope slide to move into view specimen cell objects to be analyzed for DNA content as well as for cell morphology if desired. The analyst will classify the cell in a manner similar to that disclosed in my application PCT/US86/02409 and in U.S. Patent 4~453,266 to give summed optical density for the specimen cell object i~e., a stained cell object nucleus, as well a its area, its roundness, and other classification information. A histogram may then be provided which provides DNA content~ Generally the analyst will select a number of cell objects in each field or area and then will move the microscope stage to position a number of different field~ of specimen cell objects into view and to take and analyze a number of these specimen cell objects until he feels he has a representative sample.
This permits the making of a hi~togram, such as shown in FIG. 7 which shows the number of cells of a particular DNA content and shows the D~A conten~ averages for each of the reference peaks. The data may also be stored internally within a computer logic for later recall and comparison with data of any new specimen from the same patient for analysis of the patient's progxess or regression.
After staining and image analysis of the stained slides, the control cells should give a single main peak indicating the position of the DNA content of normal human cells~ Shifts of the main DNA peaX on unknown samples indicate an abnormal D~A content.
Skewing of the main peak to the right especially with production of a second peak with 2 times the DNA conten~
of the first peak indicates a proli~erating cell popula~ion. 50-100 cells ~hould be counted or non-prolifer~ting populations and 100-200 cells for proliferating populations for reasonable accuracy.
Thus, from the above it will be seen that control cell ad specimen cell objects may be stained with methyl green to counterstain for diaminoben~idine (DAB) so that nuclei may be first isolated by specific wavelengths of light by imaging techniques; and, then the monodonal antibody conjuga~ed with DAB may be shown up by a second wavelength of light imaging technique~
.. . .. . .. .
-24- 1312~63 The present invention is not limited to t~e above described embodiments but extends to cover other embodiments, not shown or described, but falling with the ambit of the appended claims.
.
:
:
': '
Claims (36)
1. A kit for the quantitaion of cell nuclei comprising:
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference cell area containing a reference means for staining, the reference means having predetermined physical characteristics which are detectable after staining; and one or more containers of stain material; the stain material including stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers being in an effective amount to provide an optical density to the reference means after staining for a predetermined time such that the optical density of the reference means will be substantially a linear function of stain concentration of the reference means after staining.
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference cell area containing a reference means for staining, the reference means having predetermined physical characteristics which are detectable after staining; and one or more containers of stain material; the stain material including stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers being in an effective amount to provide an optical density to the reference means after staining for a predetermined time such that the optical density of the reference means will be substantially a linear function of stain concentration of the reference means after staining.
2. A kit as recited in Claim 1 wherein the kit further contains a rinse sulfonating agent.
3. A kit as recited in Claim 1 wherein the reference means includes a monoclonal antibody associated therewith.
4. A kit as recited in Claim 2 wherein the stain includes Azure A in an effective amount to provide an aqueous solution with a concentration of Azure A in a range from about 4.9 to about 5.1 mg/ml when mixed with a .1 HCl aqueous solution.
5. A kit as recited in Claim 4 wherein the stain material further includes a stain sulfonating agent.
6. A kit as recited in Claim 3 wherein the stain includes an enzyme stain.
7. A kit as recited in Claim 5 wherein the rinse sulfonating agent is K2S2O5.
8. A kit as recited in Claim 7 wherein the stain sulfonating agent is K2S2O5.
9. A kit as recited in Claim 1 wherein the stain is selected from the group consisting of Azure A, Azure B, pararosinalin, methylene, congo red, hematoxylin, eosin, methyl green, diaminobenzadine, alkaline phosphatase, methylene blue, geimsa and mixtures thereof.
10. A kit as recited in Claim 1 wherein the microscopic slide further includes an optical density reference area, the optical density reference area including a material having a predetermined known optical density to calibrate the slide with an instrument being used to study the specimen cells.
11. A kit as recited in Claim 4 wherein the microscopic slide further includes an optical density reference area, the optical density reference area including a material having a predetermined known optical density to calibrate the slide with an instrument being used to study the specimen cells.
12. A kit for the quantitation of nuclear DNA
by light microscopy, the kit comprising:
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with DNA;
one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of Azure A, Azure B, pararosinalin, methylene and mixtures thereof in an effective amount to provide an optical density to the reference means after staining for a predetermined time such that the optical density of the reference means will be substantially a linear function of stain concentration of the reference means after staining.
by light microscopy, the kit comprising:
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with DNA;
one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of Azure A, Azure B, pararosinalin, methylene and mixtures thereof in an effective amount to provide an optical density to the reference means after staining for a predetermined time such that the optical density of the reference means will be substantially a linear function of stain concentration of the reference means after staining.
13. A kit as recited in Claim 12 further comprising a rinse sulfonating agent.
14. A kit as recited in Claim 13 wherein the stain is Azure A in an effective amount to provide an aqueous solution with concentration of Azure A in a range of from about 4.9 to about 5.1 mg/ml when the Azure A is mixed with a .1 HCl aqueous solution.
15. A kit as recited in Claim 14 wherein the stain material further includes a stain sulfonating agent.
16. A kit as recited in Claim 14 wherein the rinse sulfonating agent is K2S2O5.
17. A kit as recited in Claim 12 wherein the microscopic slide further includes an optical density reference area, the optical density reference area including a material having a predetermined known optical density to calibrate the slide with an instrument being used to study the specimen cells.
18. A kit as recited in Claim 14 wherein the microscopic slide further includes an optical density reference area, the optical density reference area including a material having a predetermined known optical density to calibrate the slide with an instrument being used to study the specimen cells.
19. A kit as recited in Claim 12 wherein the reference means includes a reference material which is selected from the group consisting of trout blood cell erythrocytes, chicken blood cell erythrocytes, dried DNA, cultured cell lines, and mixtures thereof.
20. A kit as recited in Claim 14 wherein the reference means includes a reference material which is selected from the group consisting of trout blood cell erythrocytes, chicken blood cell erythrocytes, dried DNA
and mixtures thereof.
and mixtures thereof.
21. A kit as recited in Claim 15 wherein the reference means includes a reference material which is selected from the group consisting of trout blood cell erythrocytes, chicken blood cell erythrocytes, dried DNA
and mixtures thereof.
and mixtures thereof.
22. A method for the quantitation of nuclear DNA of specimen cell objects, the method comprising:
providing a slide with a reference area and a specimen cell object area:
providing a reference material in the reference area, the reference material having physical characteristics, the characteristics including a known amount of DNA and permitting association of the reference material with a stain which is proportional to an association of the stain with DNA;
providing specimen cell objects in the specimen cell object area;
simultaneously staining the reference material and the specimen cell objects with a stain in aqueous solution, the stain in aqueous solution being in an effective amount to provide the reference material with an optical density after staining which will be a substantially linear function of stain concentration of the reference material;
measuring the optical density of the reference materials after staining;
measuring the optical density of the specimen cell objects after staining; and determining the quantitative amount of DNA, various cultured cell lines in the specimen cell objects from the measured optical densities.
providing a slide with a reference area and a specimen cell object area:
providing a reference material in the reference area, the reference material having physical characteristics, the characteristics including a known amount of DNA and permitting association of the reference material with a stain which is proportional to an association of the stain with DNA;
providing specimen cell objects in the specimen cell object area;
simultaneously staining the reference material and the specimen cell objects with a stain in aqueous solution, the stain in aqueous solution being in an effective amount to provide the reference material with an optical density after staining which will be a substantially linear function of stain concentration of the reference material;
measuring the optical density of the reference materials after staining;
measuring the optical density of the specimen cell objects after staining; and determining the quantitative amount of DNA, various cultured cell lines in the specimen cell objects from the measured optical densities.
23. A kit for the quantitation of nuclear DNA
by light microscopy, the kit comprising:
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with DNA;
one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of Azure A, Azure B, pararosinalin, methylene and mixtures thereof in an effective amount to provide an aqueous solution having a stain concentration in a range of from about 4.9 to about 5.1 mg/ml upon preparation of the aqueous solution for staining the reference means and specimen cell objects.
by light microscopy, the kit comprising:
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with DNA;
one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of Azure A, Azure B, pararosinalin, methylene and mixtures thereof in an effective amount to provide an aqueous solution having a stain concentration in a range of from about 4.9 to about 5.1 mg/ml upon preparation of the aqueous solution for staining the reference means and specimen cell objects.
24. A kit as recited in Claim 23 further comprising a rinse sulfonating agent.
25. A kit as recited in Claim 24 wherein the stain is Azure A in an effective amount to provide an aqueous solution with concentration of Azure A in a range of from about 4.9 to about 5.1 mg/ml when the Azure A is mixed with a .1 HCl aqueous solution.
26. A kit as recited in Claim 23 wherein the stain material further includes a stain sulfonating agent.
27. A kit as recited in Claim 24 wherein the rinse sulfonating agent is K2S2O5.
28. A kit as recited in Claim 23 wherein the microscopic slide further includes an optical density reference area, the optical density reference area including a material having a predetermined known optical density to calibrate the slide with an instrument being used to study the specimen cells.
29. A kit for the quantitation of cell nuclei comprising:
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference cell area containing a reference means for staining, the reference means having predetermined physical characteristics which are detectable after staining; and one or more containers of stain material;
the stain material including stain for simultaneous stain application to the specimen cell objects and the reference means.
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference cell area containing a reference means for staining, the reference means having predetermined physical characteristics which are detectable after staining; and one or more containers of stain material;
the stain material including stain for simultaneous stain application to the specimen cell objects and the reference means.
30. A kit as recited in Claim 29 wherein the kit further contains a rinse sulfonating agent.
31. A kit as recited in Claim 30 wherein the stain includes Azure A in an effective amount to provide an aqueous solution with a concentration of Azure A in a range from about 4.9 to about 5.1 mg/ml when mixed with a .1 HCL aqueous solution.
32. A kit as recited in Claim 31 wherein the stain material further includes a stain sulfonating agent.
33. A kit for use in breast cell analysis by light microscopy, the kit comprising:
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with nuclear material;
one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of methyl green, hematoxlyn, acridine orange and mixtures thereof in an effective amount to provide an aqueous solution having a stain concentration of about 3.33 mg/ml upon preparation of the aqueous solution for staining the reference means and specimen cell objects.
a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with nuclear material;
one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of methyl green, hematoxlyn, acridine orange and mixtures thereof in an effective amount to provide an aqueous solution having a stain concentration of about 3.33 mg/ml upon preparation of the aqueous solution for staining the reference means and specimen cell objects.
34. A kit as recited in Claim 33 further comprising a rinse sulfonating agent.
35. A kit as recited in Claim 34 wherein the stain is methyl green.
36. A kit as recited in Claim 35 wherein the stain material further includes a stain sulfonating agent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| USUS86/02411 | 1986-11-04 | ||
| PCT/US1986/002411 WO1987002803A1 (en) | 1985-11-04 | 1986-11-04 | An apparatus and method for analyses of biological specimens |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1312263C true CA1312263C (en) | 1993-01-05 |
Family
ID=22195708
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000541465A Expired - Fee Related CA1312263C (en) | 1986-11-04 | 1987-07-07 | Apparatus and method for analyses of biological specimens |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1312263C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110082902A (en) * | 2019-04-09 | 2019-08-02 | 湖南莱博赛医用机器人有限公司 | A kind of glass slide with lens wiping paper and cell analysis equipment |
-
1987
- 1987-07-07 CA CA000541465A patent/CA1312263C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110082902A (en) * | 2019-04-09 | 2019-08-02 | 湖南莱博赛医用机器人有限公司 | A kind of glass slide with lens wiping paper and cell analysis equipment |
| CN110082902B (en) * | 2019-04-09 | 2024-04-09 | 湖南莱博赛医用机器人有限公司 | Glass slide with mirror wiping paper and cell analysis equipment |
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