US3899610A - Means for preparing cells for inspection - Google Patents

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 menisciinducing 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.

Description

United States Patent Henry Aug. 12, 1975 [54] MEANS FOR PREPARING CELLS FOR 3,580,658 5/1971 Swanson 350 93 3,736,042 5/1973 Markovits et a1 350/95 INSPECTION [76] Inventor: Donald E. Henry, 983 Memorial Dr., Cambridge, Mass. 02138 [22] Filed: Jan. 7, 1974 21 Appl. No.: 431,167

[52] US. Cl. 427/2; 350/93; 350/95; 424/3; 427/372; 427/378; 428/210 [51] Int. Cl. G02b 21/34 [58] Field of Search 117/48, 38, 124 R, 124 D, 117/3; 23/253 R, 253 TP; 350/95, 93; 424/3 [56] References Cited UNITED STATES PATENTS 2,090,914 8/1937 Porter 350/95 2,302,830 11/1942 Axelrad 350/95 2,342,409 2/1944 Levy 424/13 2,965,219 12/1960 Rhodin 350/95 UX 3,031,924 5/1962 Lamal 350/95 3,141,548 7/1964 Newby 350/95 X OTHER PUBLICATIONS Beck, The Microscope, pp. 54 & 55 (1921).

Primary Examiner-Harry J. Gwinnell Attorney, Agent, or Firm-Andrew F. Kehoe; Robert A. Cesari; John F. McKenna [5 7] 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 menisciinducing 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.

11 Claims, 5 Drawing Figures PATENTEU M1121 2 I975 FIG.4

MEANS FOR PREPARING CELLS FOR INSPECTION 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 fromthe 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 depositeda 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 nonspherical 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 wellknown 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 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 filtera 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 labo ratory 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 wholecell-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 meniscipromoting 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 35 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 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 crosscontamination.

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 oneeighth of an inch, the lower wires 17 are about onesixteenth 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 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 menisciinducing 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 widthzdepth ratio should be at least 1 to l.

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.

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 radiantheat 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 menisciinducing 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 radiantheat 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 celldisplay surface.

9. A process as defined in claim 2 wherein the rne-v nisci 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.

Claims (11)

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) FOMING 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 POCESS AS DEFINED IN CLAIM I WHEREIN A RADIANT-HEAT ABSORBING SURFACE IS POSITIONED BENEATH SAID TRANSPRENT SLIDE TO MODIFY THE EVAPORATION STEPS SO CELLS ARE PREFERENTIALLY DISTRIBUTED IN REAS WHICH ARE CONGRUENT WITH AREAS OF THE SLIDE BENEATH THE POOL WHICH ARE INCOGRUENT 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.

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