Note: Descriptions are shown in the official language in which they were submitted.
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
,~
-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
3 1 3 1 2263
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
.. . .. ... .... . . . . .. ..
1 31 2263
-4-
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
--10--
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
!
:' '
1312263
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
-13-
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
1 31 2263
-14-
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
1 3 1 2263
--15--
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
. ... . . . ~ . . . .. . ... .. .
-16- 1 3 1 2263
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~
1312263
-17-
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
.
1 3 1 2263
-18-
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
1 3 1 2263
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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
-20-
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.
. . .. .. ,.. . . ~ .. ..... . .. . .... . . .
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-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
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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
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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.
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