U.S. patent number 3,899,610 [Application Number 05/431,167] was granted by the patent office on 1975-08-12 for means for preparing cells for inspection.
Invention is credited to Donald E. Henry.
United States Patent |
3,899,610 |
Henry |
August 12, 1975 |
Means for preparing cells for inspection
Abstract
A process, and apparatus useful in carrying out the process, of
the type wherein a suspension of cells is agitated by convection
currents caused by the cooling effect of an evaporating liquid
suspension medium. The process is advantageously carried out in a
shallow well with wires or filaments or other such menisci-inducing
structure positioned to create a large perimeter of menisci which,
on breaking, supply kinetic energy effective to enhance the
cell-distribution pattern on the floor of well on completion of the
evaporation.
Inventors: |
Henry; Donald E. (Cambridge,
MA) |
Family
ID: |
23710767 |
Appl.
No.: |
05/431,167 |
Filed: |
January 7, 1974 |
Current U.S.
Class: |
427/2.11;
359/398; 427/372.2; 427/378; 428/210; 435/40.51 |
Current CPC
Class: |
G01N
1/28 (20130101); G02B 21/34 (20130101); Y10T
428/24926 (20150115) |
Current International
Class: |
G02B
21/34 (20060101); G01N 1/28 (20060101); G02b
021/34 () |
Field of
Search: |
;117/48,38,124R,124D,3
;23/253R,253TP ;350/95,93 ;424/3 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Beck, The Microscope, pp. 54 & 55 (1921)..
|
Primary Examiner: Gwinnell; Harry J.
Attorney, Agent or Firm: Kehoe; Andrew F. Cesari; Robert A.
McKenna; John F.
Claims
What is claimed is:
1. In a process for preparing cells for microscopic examination
comprising the steps of (a) forming a suspension of said cells in a
liquid suspension medium, and (b) forming a shallow pool of said
suspension on a slide, and (c) evaporating the liquid medium from
said suspension
the improvement comprising the steps of inducing menisci across the
surface of said pool as the liquid suspension medium evaporates,
utilizing the collapse of said menisci to avoid agglomeration and
piling up of said cells on said slide and thereby facilitate the
examination of said cells.
2. A process as defined in claim 1 wherein said pool has a depth of
at least 1/16 inch and wherein said cell population comprises a
volume not exceeding the volume of 200,000 cells per cc of 56
micron average particle diameter.
3. A process as defined in claim 1 wherein a radiant-heat absorbing
surface is positioned beneath said transparent slide to modify the
evaporation steps so cells are preferentially distributed in areas
which are congruent with areas of the slide beneath the pool which
are incongruent with said radiant-heat-absorbing surface.
4. A process as defined in claim 1 wherein said shallow pool is
formed by placing a wall-forming ring open at top and bottom
thereof on a transparent slide and placing said suspension in said
ring with a menisci-inducing material in contact with said
pool.
5. A process as defined in claim 4 wherein said ring and slide are
removably fastened together by liquid surface tension of liquid
between the bottom of said ring and top of said slide.
6. A process as defined in claim 4 wherein the surface of the ring
and the menisci-forming structure are selected to be wettable by
the suspension of cells.
7. A process as defined in claim 2 wherein a radiant-heat absorbing
surface is positioned beneath said transparent slide to modify the
evaporation steps so cells are preferentially distributed in areas
which are congruent with areas of the slide beneath the pool which
areas are incongruent with said radiant-heat-absorbing surface.
8. A process as defined in claim 2 wherein at least 12 inches of
menisci is formed per square inch of cell-display surface.
9. A process as defined in claim 2 wherein the menisci is formed by
a grid having holes of an average diameter of less than about 0.25
inch.
10. A process as defined in claim 1 wherein said cells are
dispersed over said slide at a unicellular depth.
11. A process as defined in claim 9 wherein said cells are
dispersed over said slide at an unicellular depth.
Description
BACKGROUND OF THE INVENTION
There is a considerable problem in preparing samples of
physiological specimens for visual inspection, e.g., for inspection
through a microscope. These problems arise primarily from the
difficulty of obtaining a uniform distribution of the cells on the
surface to be inspected. Often the distributing problems are the
result of excessive agglomeration of cells deposited from an
evaporating liquid medium.
Because of this distribution problem, a microscopist must exercise
unusual care in making sure he has looked at all areas where cells
are deposited-- a mere discontinuity in cell coverage on a slide
will not be indicative that there is no further sample in the
scanning path being inspected. Moreover, because the cells are not
well distributed on the sample surface, a larger surface must be
inspected to assure that, say, a diseased kind of cell has not been
segregated in a "clump" of cells in one corner of the slide being
inspected.
Over and above the problems associated with distribution per se, it
would be desirable to have non-spherical cells orient or arrange
themselves so that they will be more clearly definable in three
dimensions. Prior art preparations of cells on slides have not
contributed any such orientation of the cells.
One well-known technique used in display of cells for inspection is
the taking of the sample manually, wiping it onto a glass slide
with another glass slide, dipping one of the glass slides in
alcohol to fix the smear, sending the fixed smear to the laboratory
where it will be stained with whatever dye is appropriate, covering
the dyed smear in the laboratory for the first time and then
sending it on to another lab station for visual inspection under
the microscope. The microscopist has to run up and down with the
microscope over the entire sample inspecting the cells which are
distributed very badly in agglomerates, clumps, whatever, over the
entire surface. The entire procedure lends itself to a high risk of
contamination and an attendant risk that very significant cell
members of the population will be overlooked, and it takes a lot of
time on the part of the microscopist.
Generally the foregoing procedure is being replaced by the
following improved procedure:
The doctor takes a sample and instead of smearing it, he puts it
into a test tube and sends it to the lab. The lab spins it down,
pours off the supernatant liquid and adds some absolute alcohol to
the cell suspension.
The cell suspension is analyzed. It may be analyzed by resuspending
it and pouring it onto a slide from which the alcohol will
evaporate leaving the cells on the slide. This second procedure is
a big improvement over the first procedure primarily because you
get a better distribution of cells on the slide, and you run a
considerable smaller risk of contamination.
There is a third procedure, one utilizing filter means, which has
become very popular over the last 10 years or so. This third
procedure came into being primarily because there was a major need
to concentrate a small number of cells so that they could be
studied. One example of the problem that was faced by the
laboratories was frequently receiving spinal tap samples which had
so few cells that the laboratory technician could not visually see
them. A spinal tap is often such that it can't be repeated again in
a short time, and, therefore, it is very important that the cells
that are in a spinal tap sample be so concentrated that they can be
seen under the microscope. In this filter-type procedure the
material is put into absolute alcohol after being spun down much as
it is in the second procedure described above. But in the filter
process, the material is put through a filter to concentrate the
cells in the filter matrix. The filter is then stained and put on a
slide. Next, formalin is used to dissolve the filter leaving the
stained cells in "concentrated" position. This procedure was a
definite advance in the art, and there has been a considerable
commercial success. There is now a further modification of that
process, which has an improved filter in that it does not leave a
grey background which is left by the action of the formalin on the
earlier filter-- a substantial drawback to the earlier filter
system. However, in both the newer and earlier filter process there
is still clumping and piling of cells. Moreover, these filter-type
processes do take a substantial amount of laboratory time to
implement.
Finally, there is a substantial improvement in this art described
in the aforesaid Ser. No. 406,251 filed on Oct. 15, 1973 by the
instant inventor. That application utilized the agitation induced
by evaporation to achieve good cell distribution. However, the
improvement whereby menisci could be used to enhance the cell
distribution pattern was not disclosed in the earlier application.
The invention disclosed below relates to this improvement.
SUMMARY OF THE INVENTION
It is a principal object of the invention to provide an improved
process for the preparation, for inspection, of samples of
physiological specimens, especially whole-cell-containing
specimens.
It is another object of the invention to provide novel apparatus
especially useful in carrying out the aforesaid improved
process.
Another object of the invention is to provide an improved process
utilizing evaporation -- induced agitation to dispense cellular
samples.
A more particular object of the invention is to provide an improved
process, and improved apparatus for use therein, for distributing
and orienting cells on a surface to which they will adhere and
wherein they may be advantageously displayed and preserved.
It is an object to provide apparatus and a process which will
achieve the above objects and will do so in a greatly reduced time
period and with greatly improved, microscopically apparent, cell
definition.
A further object of the invention is to provide means to facilitate
identical treatment of different cellular samples and thereby
facilitate comparisons of such samples.
Another object of the invention is to provide a superior means for
concentrating dilute cellular samples so that they may be studied
optically.
Other objects of the invention will be obvious to those skilled in
the art on reading the present application.
The above objects have been achieved by utilizing a well-forming
retaining wall (which is referred to hereinafter as a "ring" but
which may be of any convenient shape as will be readily evident to
one skilled in the art on reading this disclosure) in conjunction
with menisci-promoting structure. A glass slide or other convenient
receiving surface is placed at the bottom of the well and provides
receiving surface for the cells. The advantage of the invention
seems to relate to the phenomenon whereby menisci form between the
menisci-promoting structure (which is conveniently a wire grid
positioned in or above the well and at a selected distance from the
aforesaid glass slide) and the cell suspension which is falling
because of evaporation of the suspension medium. When the menisci
collapse simultaneously, they release sufficient energy to
materially contribute to the degree of distribution of cells on the
surface of the glass slide or other substrate.
To achieve optimum performance, the height of the menisci should be
adjusted to break at a point where a minimal coverage of the cell
suspension on the bottom of the slide will be achieved. Were the
depth of suspension excessive when the menisci breaks, the
advantages of this invention would be lost; albeit, the residual
evaporative action would result in such improvements as were
achievable with the process and apparatus disclosed in Ser. No.
406,251.
In the usual circumstance the height of the meniscus will break at
a time when the suspension will just be approaching depletion by
evaporation at the cell-display surface. The menisci-promoting
structure will be conveniently positioned to achieve a distance of
from between about 0.05 to about 0.1 inches of menisci above the
cell-display surface. The exact position will, of course, depend on
the surface tension of the suspension and the wettability of the
menisci-promoting structure with the particular suspending
medium.
In preferred embodiments of the invention the wetted perimeter of
the menisci-forming structure will be at least about 8 inches per
square inch of cell-display surface. However, it is advantageous to
have at least 12 inches or more per square inch of cell-display
surface. The area defined by holes in a menisci-promoting grid are
advantageously less than 0.25 inch in average diameter, but a
substantial beneficial effect can be achieved by doing away with
one entire set of menisci-inducing bars (say the warp or woof wires
of a woven screen).
The materials of construction can be selected broadly. However,
since menisci-promotion is desirable, wettable surfaces such as
aluminum, stainless steel and glass are preferable for use with
polar suspending media. However, plastics may also be utilized
beneficially in some circumstances. Paraffin wax, which could be
dissolved in subsequent processing of the cells is also useful for
making a menisci promoting grid.
The menisci-promoting structure can be placed over, or set within,
the retaining ring. It can be molded into a single part with the
ring. Various methods of manufacture are available and all are
believed to be suitable, albeit some may not be economically
feasible.
It is notable that the process of the invention can actually
achieve an extremely good dispersion of cells which have been so
numerous that form a coating of a unicellular depth wherein the
cells are in cell-to-cell contact on the display surface. In the
normal course of cytological investigation, a smaller number of
cells will be deposited.
In general, the process of the invention comprises the dispersion
of a cellular sample in a liquid, which is volatile at a test
temperature of 25.degree.- 35.degree. C.
In order to allow good mixing action and to avoid agglomeration and
piling up of cells on the substrate, it is advantageous to keep the
cell population of the suspension from being excessive. What is
excessive in terms of cell populations per unit volume depends on
the depth of the suspension over the substrate and the size of the
cell. It is convenient to use an empirical model comprising cells
of 56-micron diameter and a model cell suspension depth of 0.125
inch to define such a limit. In this situation, the cell population
should not exceed about 200,000 cells per cc of suspension. As
larger cells are used the maximum number of cells should be
decreased, as smaller cells are used, it may be increased
shomewhat. Normally, the total number of cells will be between 100
and 140,000 cells per cc.
The above-described suspension is poured into a shallow well with a
glass slide or some other such manipulative surface-forming means
forming the bottom of the well. The walls of the well are suitably
formed by a retainer ring which is placed on the slide surface in
sufficiently close contact to effect a seal with the surface of,
say the glass slide. This sealing contact can be assured in several
ways including such conventional means as clamps, springs and
mechanical brackets. A machined steel surface is entirely suitable
because it provides some porosity at the interface between the
slide and ring. A bit of liquid can seep into the interstices at
this interface and effectively seal the ring into position on the
slide so that inadvertent movement of the ring is avoided during
handling even in the absence of other mechanical locking or sealing
means.
One application of the invention is that two cellular samples may
be treated exactly the same, in terms of physical and chemical
processing, say on two different areas of the same slide but
without danger of cross-contamination.
In general, a ring-slide combination wherein the well is of about
0.5 inch diameter and 0.125 inch deep its entirety convenient.
However, rectangular wells are particularly advantageous because
they are best adapted for the scanning paths built into microscopes
and like equipment.
In any event, it has been found that when the alcohol evaporated
from a suspension is constrained in such a well as described above,
the cooling inherent in the evaporation, together with the
temperature-stabilizing effect of the retaining ring material about
the edge of the evaporating liquid, sets up a substantially thermal
agitation that tends of agitate the cells in circulating eddy
currents during substantially the entire period of the evaporation.
In the process of the invention agitation is very active in the
meniscis until such time as the liquid is largely evaporated and
the menisci break with the resulting dispersive effect on the
cells. Cells that are acicular (or at least those that deviate
substantially from a spherical shape) tend to be buoyed up so that
such cells are left "standing on end" so to speak. Thus cells of
varying shapes tend to be more clearly identifiable because of this
three-dimensional effect of the process.
The slide material, i.e. the bottom of the well, should be formed
of glass (or its optical eqivalent, as will be obvious to anyone
skilled in the microscope). The rings, or wells, of the well can be
formed of any convenient substance.
In the above description, the use of the term "rings" and other
descriptive words describing or connoting circular configurations
are used. It will be understood by all skilled in the art that
circular wells are conventional and convenient but these
illustrations in no way intended to omit other shapes such as
rectangles, elipses, triangles or whatever may be convenient. The
use of a circular well is illustrative only.
ILLUSTRATIVE EXAMPLE OF THE INVENTION
In this application and accompanying drawings there is shown and
described a preferred embodiment of the invention and suggested
various alternatives and modifications thereof, but it is to be
understood that these are not intended to be exhaustive and that
other changes and modifications can be made within the scope of the
invention. These suggestions are selected and included for purposes
of illustration in order that others skilled in the art will more
fully understand the invention and the principles thereof and will
be able to modify it in a variety of forms, each as may be best
suited in the condition of a particular case.
In the drawings:
FIG. 1 is plan view of a fluid distribution apparatus subject of
the instant invention.
FIG. 2 is a section in elevation of the apparatus of FIG. 1.
FIG. 3 is a schematic diagram indicative of patterns of liquid
during evaporation of a sample from a device such as shown in FIG.
1.
FIGS. 4 and 5 show other grid-like structures useful with apparatus
formed according to the invention.
FIG. 1 illustrates a cell-distribution apparatus 12 constructed
according to the invention. The apparatus comprises a retaining
wall 14 and a grid formed of upper wires 16 and lower wires 17.
These wires are about 0.03 inch in diameter and the areas 19
defined by their intersection are about one-sixth of an inch on a
side. The height of retaining wall 14 is about one-eighth of an
inch, the lower wires 17 are about one-sixteenth of an inch from
the glass slide 21 on which apparatus 12 will ordinarily be placed
when in use.
FIGS. 2 and 3 illustrate the ability of wires to provide a great
deal of linear menisci-forming perimeter 30 as liquid 32
evaporates.
FIGS. 4 and 5 show different grid structures, one a
foraminous-metal structure 23, the other a woven plastic screen
25.
The following is an example of one highly useful embodiment of the
invention, one in which cells obtained from a vaginal washing are
prepared for microscopic inspection:
The cellular sample is asperated and placed in a tube containing an
alcohol saline mixture, sent to a laboratory where it is spun down
on a centrifuge. The supernatant liquid is discarded; the residual
cellular material is suspended in ethanol. This much of the
procedure was practiced in the art before the work described in
this application or in the aforesaid Ser. No. 406,251.
The ethanol suspension is diluted to a cell concentration of 70,000
per cc. A quantity of about 0.4 ml is placed in a steel well as
described in FIGS. 1 and 2. The room temperature is 75.degree. F.
This causes evaporation of the ethanol liquid suspending medium
from the sample. The residue on the slide is adhesively bound
thereto, but has an excellent cellular distribution and excellent
cell orientation, as is apparent after the slide has been stained
and subjected to microscopic examination.
In normal daylight conditions as are generally experienced in many
laboratories, an effective preferential distribution of the cells
is achieved merely by putting a good radiant-heat-receptive (black,
or dark-colored) surface on the bottom of the glass slide. In
general, a black-inked surface preferentially reduces the
deposition of cells on the surface of the slide just above the
black-inked surface. So, for example, a black-inked perimeter
placed on a cardboard and just below the outer circumferential area
just inside the walls of the well will result not only in
diminishing or overcoming any agglomeration of cells due to the
meniscus, but will reduce the number of cells in the area below the
number achievable with a non-meniscus developing combination of
wall and suspension medium. Thus the invention provides means to
form uniform cell dispersons in predetermined geometric patterns
related to black-inked (radiant-heat-absorbing) surface.
The process of evaporation can be further accelerated by the use of
evaporation-accelerating means utilizing a menisci-forming
structure. This is believed to be because of some heat removed by
the menisci-inducing structure itself. Moreover, a cell suspension
medium taking 26 minutes to evaporate in a relatively stagnant air
environment can be evaporated with favorable results in a small
fraction of that time when a stream of air is blown across the
well.
By "pool" used in the claims is meant a body of liquid having
sufficient depth to allow the thermal agitating described above.
Usually a minimum depth of about 0.05 inch is required in the area
over which the cells are to be distributed and a width:depth ratio
should be at least 1 to 1.
The use of the term "radiant-heat-absorbing" surface in the claims
is meant to describe a dark-colored surface or other surface which
absorbs heat-producing wavelengths of light. However, the term is
not used in the precise functional sense. It has not been
determined whether such a surface functions because of its
radiation or its absorption of heat. Such surfaces are known to
function well as both emitters and absorbers of radiant heat
energy. Moreover, it will be obvious to one reading this
application that the black-body can be placed at either surface of
the transparent slide and, indeed can be integral with (i.e.,
attached to as painted or bonded onto) or separate from the
surface.
It is to be understood that the following claims are intended to
cover all of the generic and specific features of the invention
herein described, and all statements of the scope of the invention
which, as a matter of language, might be said to fall
therebetween.
* * * * *