WO1996029866A1

WO1996029866A1 - Histological processing device and method

Info

Publication number: WO1996029866A1
Application number: PCT/US1996/004457
Authority: WO
Inventors: Gregory M. Fahy
Original assignee: Organ, Inc.; Life Resuscitation Technologies, Inc.
Priority date: 1995-03-31
Filing date: 1996-04-01
Publication date: 1996-10-03
Also published as: AU5382396A

Abstract

In a device and method for histological processing of tissue samples, the tissue samples are placed in a sample chamber containing a volume of a first fluid. A volume of a second fluid is gradually introduced into the sample chamber at the same time that fluid is drained from the sample chamber. The flow of fluids into and out of the sample chamber is controlled so that the concentration of the fluid in the sample chamber gradually changes until the fluid in the chamber is comprised of almost 100 % of the second fluid. Diffusion causes the concentration of the fluid in the cells of the tissue samples to gradually change as the concentration of the surrounding fluid gradually changes.

Description

"HISTOLOGICAL PROCESSING DEVICE AND METHOD"

BACKGROUND OF THE INVENTION

The invention is related to apparatus and methods for histological processing of biological tissue samples.

Tissue samples that are to be examined under a microscope are often processed to replace the water or other fluids inside the tissue cells with a second fluid such as ethanol. The second fluid may be replaced with other fluids in a further processing steps.

Simply immersing a tissue sample in 100% ethanol would destroy most tissue samples because of the osmotic forces involved. To avoid damaging biological tissue samples it is necessary to gradually vary the concentration of the immersion bath. If a tissue sample has cells that contain a fluid mixture of fluids C1 and C2 having respective concentrations of C1% and C2%, it is generally desirable to avoid immersing the cells in a fluid bath that has a concentration ratio R of (C1% new +C2% new)/(C1% old + C2% old) of more than four. In other words, the total concentration of ethanol and salts should not be increased more than four-fold between equilibration steps.

To accomplish the replacement of naturally occurring water inside the cells of a tissue sample with ethanol, for example, the tissue sample is immersed in a series of water/ethanol fluid baths. Each succeeding water/ethanol fluid bath has a greater concentration of ethanol. Diffusion causes the higher concentration ethanol in the fluid bath to infiltrate the cells while the water leaves the cells. In a last step, the tissue sample is immersed in a 100% ethanol fluid bath. As a result of the fluid bath processing, the water in the cells of the tissue sample is replaced with ethanol. In the prior art, histological processing as described above may be accomplished manually or by a device which literally moves a tissue sample from one water/ethanol fluid bath to the next, each fluid bath having a different water/ethanol concentration, until the tissue sample is eventually immersed in a 100% ethanol bath. The processing may also be accomplished by a device that superfuses a tissue sample with fluid from a series of reservoirs, each reservoir containing a fluid having a different water/ethanol concentration, until the tissue sample is superfused with 100% ethanol. A typical prior art processing device which superfuses a tissue sample with fluid from a series of reservoirs is the Miles Scientific Tissue Tek III.

A prior art histological processing device that superfuses tissue samples with a series of varying concentration fluid baths is represented schematically in Figure 1. A description of the device will be given assuming that the device is using water/ethanol fluids of varying concentrations. A fluid storage cabinet 30 contains the fluids used to superfuse tissue samples and used fluid that is to be disposed of. Fluid reservoirs 32 are located inside the storage cabinet 30. Each reservoir 32 contains a water/ethanol fluid having a certain concentration. Each reservoir 32 is connected to a supply valve 34, which is capable of selecting the fluid from each reservoir 32 in turn. The outlet of the supply valve is connected to a first supply line 36 that is connected to a supply pump 38. The supply pump 38 is also connected to a second supply line 40 which delivers fluid to a sample chamber 42.

One or more tissue samples 44 may be mounted inside the sample chamber 42. A waste line 46 is connected to the sample chamber 42 to remove fluid from inside the sample chamber 42. The waste line 46 drains into a waste reservoir 50 located inside the storage cabinet 30. A waste valve 48 may be provided in the waste line 46 to control draining of fluid from the sample chamber 42 into the waste reservoir 50. The supply valve 34, the supply pump 38 and the waste valve 48 may all be computer controlled so that an automated program may accomplish the histological processing of tissue samples 44 in the sample chamber 42. In operation, a first water/ethanol fluid having a first concentration is pumped from a supply reservoir 32 into the sample chamber 42 by the supply pump 38. The waste valve 48 remains closed for a period of time until osmosis causes the concentration of the fluid inside the cells of the tissue samples 44 to approach the concentration of the fluid inside the sample chamber 42. Then the waste valve 48 is opened to drain the used fluid into the waste reservoir 50. Next, the waste valve 48 is again closed. A water/ethanol fluid from a second supply reservoir, which has a higher concentration of ethanol, is pumped into the sample chamber 42 by the supply pump 38. The waste valve remains closed for such a period of time that osmosis can again occur, causing the concentration of the fluid inside the cells of the tissue samples 44 to approach the concentration of the fluid in the sample chamber 42. Then the waste valve 48 is opened to drain the used fluid from the sample chamber 42 into the waste reservoir 50. The above described process is repeated until 100% ethanol is pumped into the sample chamber 42. The end result is that the cells of the tissue samples 44 are infused with 100% ethanol.

In a typical histological processing method, after the tissue has been infiltrated with 100% ethanol, it is transferred to a 100% xylene bath, and then into a 100% hot paraffin bath, all within the same chamber. The tissue is then retrieved for paraffin solidification in appropriate tissue containers. An exemplary sequence of water/ethanol solution volumes and concentrations used in the device are set forth in the table below. % v/v Ethanol nc. Volume of Vol. Equiv

Molarity Rattiioo Additive of 100% EtOH

0% 0 1 2000 ml 0 ml 70% 12.0 41 2000 ml 1400 ml 80% 13.8 1.15 2000 ml 1600 ml 95% 16.3 1.18 2000 ml 1900 ml 95% 16.3 1 2000 ml 1900 ml 100% 17.2 1.05 2000 ml 2000 ml 100% 17.2 1 2000 ml 2000 ml The total volume of solution consumed is 14 liters. The total volume of ethanol solution used is 12 liters. The total equivalent volume of absolute ethanol consumed is 10.8 liters.

A graph corresponding to the table shown above, and showing the ethanol concentration of the fluid bath surrounding the tissue samples over time is shown in Figure 2.

The prior art devices and methods described above have multiple drawbacks. First, they waste solution. The 2000 ml additive volume, reflecting the size of the sample chamber (which is intended to handle up to 200 tissue blocks), cannot be changed even if only one tissue block is being processed. As a result, ethanol consumption per procedure may be far higher than necessary. This increases the cost of purchasing ethanol, and the cost of disposing of the used fluid. In addition, the fire hazard of the used ethanol is maximized. Further, the consumption of 0% ethanol solution is also maximized. Second, prior art devices wastes space. The excessive amounts of used ethanol must occupy space in the form of several containers that take up room in a closed cabinet.

Third, the prior art devices are unnecessarily complicated, adding to the cost of the equipment. Each supply reservoir requires a separate withdrawal line. The supply valve must accommodate as many as 6-12 different supply reservoirs. In addition, a histological processing technician must inventory, replace, and handle all of the separate supply reservoirs . Fourth, prior art device are biologically suboptimum. The imposition of fixed steps of ethanol concentrations, as shown in Figure 2, produces osmotic forces that may damage the tissue and that are carried out in an undesirable manner for tissue integrity. As mentioned above, the biologically permissible concentration ratio R that can be imposed by adding a high concentration fluid to a previous lower concentration fluid with biological safety is about 4, including the contribution of the isotonic solutes used in addition to the transitional solvent.

As is evident from Figure 2, the initial immersion bath has a concentration far higher than desirable, which can cause tissue damage during the first immersion step. In fact, the initial immersion step may have a concentration ratio as high as 40, which is ten times the optimal ratio. In addition, the succeeding fluid baths fail to add further concentrations at rates that approach what is permissible, thus unnecessarily prolonging tissue exposure to the solvents and possibly damaging the tissue due to excessive exposure time.

Fifth, the prior art methods are unnecessarily time-consuming. The imposition of oddly-spaced, fixed steps, each of which must be attained prior to moving on to the subsequent step, and the lack of stirring, limit the rate at which solvent concentration can be increased without regard to underlying biological considerations.

SUMMARY OF THE INVENTION The invention is a device and method of performing histological processing of biological tissue samples wherein the tissue samples are immersed in a fluid bath that has a controlled, gradually varying concentration.

This can be accomplished by first immersing a tissue sample in a sample chamber containing a certain volume of a first fluid, such as fixative, then gradually adding a second fluid, such as ethanol. The fluid level in the sample chamber is controlled by allowing fluid to escape from the sample chamber through a waste line connected to the sample chamber. The added second fluid is mixed with the first fluid directly in the sample chamber. As more and more of the second fluid is added to the sample chamber, the concentration of the second fluid gradually increases. The concentration ratio R becomes irrelevant because step changes in concentration are avoided, which produces the equivalent of low acceptable R values at all times. After a period of time, almost all of the first fluid will have escaped from the sample chamber through the waste line, and the fluid in the sample chamber will be almost 100% of the second fluid.

To ensure that the fluid in the cells of a tissue sample is entirely replaced with the second fluid, a device and method of practicing the invention may also make use of additional processing steps wherein the tissue sample is immersed in a fluid bath of 100% of the second fluid. This may be accomplished using auxiliary supply reservoirs filled with the second fluid. After the process described above is complete, the fluid in the sample chamber may be completely drained. Next, the sample chamber may be partially filled with a volume of the second fluid, such as 100% ethanol, from an auxiliary supply reservoir. This will ensure that the tissue sample is immersed in a fluid bath containing 100% of the second fluid. In addition, a device embodying the present invention may include additional fluid reservoirs having additional fluids. After a tissue sample has been infused with a second fluid, the device can repeat the processing steps to replace the second fluid in the tissue sample with a third fluid. These steps can be repeated for a number of different fluids until the tissue sample is infused with the final desired fluid. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will be described with reference to the following drawing figures wherein like elements are identified with like reference numbers, and wherein:

Figure 1 is a schematic diagram of a prior art histological processing apparatus;

Figure 2 is a graph showing the concentration of ethanol over time in a sample chamber of a prior art histological processing device;

Figure 3 is a schematic diagram of a histological processing device embodying the present invention;

Figure 4 is a graph showing the concentration of ethanol over time in a sample chamber of a histological processing device embodying the present invention;

Figure 5 is a graph showing the concentration of ethanol over time in a sample chamber of a histological processing device embodying the present invention;

Figure 6 is a graph showing the concentration of ethanol over time in a sample chamber of a histological processing device embodying the present invention; and

Figure 7 is a schematic diagram of another histological processing device embodying the present invention. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the description which follows, histological processing devices and methods embodying the present invention are discussed with reference to replacing water in the cells of a tissue sample with ethanol. These fluids are used as examples only and are not intended to be limiting. Any fluids could be used in the devices and methods of the present invention. In addition, although only a single processing is described, the histological processing could be repeated a number of times to replace fluid within the cells of a tissue sample with a series of different liquids such as ethanol, xylene, hot paraffin, and other fluids. A schematic diagram of a histological processing device embodying the present invention is shown in Figure 3. The fluid storage cabinet 30 contains a first fluid supply reservoir 52, and a second fluid supply reservoir 54. The first and second fluid supply reservoirs 52, 54 may be arranged as concentric cylinders. The first and second fluid supply reservoirs are connected to a supply valve 34. The supply valve 34 is connected to a supply line 56, which leads to the sample chamber 42. The sample chamber 42 has an area for mounting one or more tissue samples 44. The tissue samples 44 may be mounted inside a wire mesh cage 58, or a similar structure to immobilize the tissue samples 44. A stir table 60 having a stir bar 62, may be mounted on the floor or side wall of the sample chamber 42. Alternatively, a simple stirring rod or blade may be mounted inside the sample chamber 42 on a rotating shaft of a motor. The stir bar helps to facilitate mixing of fluids in the sample chamber. A first waste line 46 is connected to the sample chamber 42 to allow fluids to be drained from the sample chamber 42. The first waste line 46 is connected to a waste valve 64. In alternate embodiments, the waste valve 64 may be replaced with a waste pump to actively pump fluid from the sample chamber 42. A second waste line 66 is connected to the waste valve 64. The second waste line 66 leads to a waste fluid reservoir 50 in the fluid storage cabinet 30.

To conduct histological processing of tissue samples with the device shown in Figure 3, the tissue samples 44 are first placed in the wire mesh cage 58. Next, the sample chamber is filled to a certain level with a first fluid, such as fixative.

The first fluid supply reservoir 52 is filled to a certain level with a second fluid, such as ethanol. It may be necessary to also prime the fluid supply line 56 with the second fluid. In this embodiment of the invention, the fluid level in the sample chamber should be at the same height as the fluid level in the first fluid supply reservoir 52.

To begin the mixing process, the stir bar 62 is activated, and the supply valve 34 is operated to connect the first fluid supply reservoir 52 with the sample chamber, through the supply line 56. Because the fluid levels are equal, however, little or none of the second fluid in the first fluid supply reservoir 52 will flow into the sample chamber 42. The waste valve 64 (or waste pump) is then operated to allow fluid from within the sample chamber 42 to flow out into the waste fluid reservoir 50 through the first and second waste lines 46, 66. As fluid escapes the sample chamber 42, and the fluid level in the sample chamber 42 drops, fluid from the first fluid supply reservoir 52 will be drawn by gravity into the sample chamber 42. As the fluid level in the sample chamber continues to drop, the concentration of the second fluid will gradually rise in chamber 42. As the fluid level in the sample chamber 42 approaches the bottom, almost all of the fluid in the supply chamber will comprise the second fluid.

The device allows the concentration of the second fluid to gradually rise over a period of time. As the mixing and draining occurs, diffusion will cause the mixed fluid in the sample chamber 42 to infiltrate the cells of the tissue samples 44, while water also leaves the tissue samples 44 by osmosis. As a result, a sudden immersion in a high concentration of the second fluid may be prevented to avoid damaging the tissue samples.

Assuming the fluid originally in the sample chamber 42 is mostly water, and the fluid in the first fluid supply reservoir is ethanol, a graph showing the concentration of ethanol in the sample chamber is shown in Figure 4. As can be seen in Figure 4, the concentration of ethanol gradually rises until it is essentially 100%.

It should be noted that element 64 will normally be a pump acting at constant speed. When element 64 is a computer-controlled valve, this valve should be put onto an appropriate duty cycle, not simply opened up. Purely passive fluid flow through valve 64 will often be too rapid at first and too slow later, or too rapid at all times.

In alternate embodiments the supply valve 34 and the waste valve 64 can be controlled to vary the flow rates into and out of the sample chamber 42. The valve control can be used to establish a concentration profile like the one shown in Figure 5, wherein the rate of change of the concentration of ethanol (i.e. , the slope of the line) varies at different times. In addition, by simply closing the waste valve 64 for short periods of time during the process, the concentration can be held steady for a period of time, as shown in Figure 6, to allow tissue equilibration to occur.

To ensure that the fluid in the cells of the tissue samples 44 is completely replaced with the second fluid, one or more additional processing steps may be performed after all fluid from the sample chamber 42 and the first fluid reservoir 52 has drained into the waste fluid reservoir 50. In the additional processing steps, the waste valve 64 is closed, and the supply valve 34 is operated to allow a portion of the volume of the second fluid in the second fluid supply reservoir 54 to flow into the sample chamber 42. After a period of time the waste valve 64 is opened and the second fluid is drained into the waste fluid reservoir 50. This additional processing step may be repeated a number of times to ensure that all the fluid in the cells of the tissue samples 44 is the second fluid.

In a device embodying the invention, there is no requirement for multiple fluid supply reservoirs having fluids of different concentrations. This greatly simplifies the apparatus, and the operation of the apparatus. In addition, by controlling the height of the fluids in the sample chamber and the fluid supply reservoirs, the fluids can flow between the supply reservoirs, the sample chamber and the waste fluid supply reservoir under the influence of gravity. This allows the entire operation to be accomplished by operating two simple valves, thus eliminating the need for any pumps. The volume of the first fluid in the sample chamber, and the volume of the second fluid in the fluid supply reservoir can be varied to obtain desired concentration mixing results.

In alternate embodiments the sample chamber 42 and the fluid supply chambers 52 and 54 may be located at different heights, and/or a pump may be installed in the supply line to deliver appropriate amounts of the second fluid into the sample chamber 42. In addition, a pump may be installed in the first and/or second waste lines 46, 66 to actively pump fluid from the sample chamber 42 to the waste fluid reservoir 50.

A device embodying the present invention may be completely computer controlled by microprocessor 100, connected to the various valves and pumps in the system. In another embodiment, as shown in Figure 7, the tissue samples 44 may be located in a recessed area 70 at the bottom of the sample chamber 42. In this embodiment, the mixing of the first and second fluids may be accomplished in the reservoir 52 by a fluid flow path 73, which may act under the influence of gravity alone to create a specified concentration profile dictated by computer control of valve and/or pump 72. The premixed, constantly varying concentration fluid may then simply flow over the tissue samples 44 and then into the waste fluid supply chamber 50 via the first and second waste fluid lines 46, 66 and the waste valve 64. In this embodiment, tissue chamber 42 remains almost empty during gradual concentration elevation and is needed only if flooding of the tissue with large volumes of secondary fluids, such as xylenes and paraffin, is desired. Accordingly, the drain for chamber 42 may be located in recess 70 to maintain liquid volume in 42 as low as possible during gradual concentration elevation in 70. This embodiment may also include an additional fluid supply reservoir 80 filled with a third fluid. The additional fluid supply reservoir may be used to process the tissue samples 44 again to replace the second fluid (for example, nearly 100% ethanol) in the tissue samples 44 with an additional pure fluid of the type found in reservoir 54 to ensure full equilibration and removal of water. Of course, the processing steps could be repeated any number of times for additional fluids. In the prior art example given above, the mandatory volume of pure ethanol consumed is 10.8 liters. A device embodying the present invention could accomplish the same histological processing with 4 liters or less of pure ethanol. This represents a savings of 63% in comparison to the prior art. Thus, the current invention reduces operating costs involving ethanol by about two- thirds while making the procedure faster, simpler, and biologically superior. Osmotic/diffusional stress is always small, and there is no need to deal with or to drain intermediate solutions. The speed of gradient progress can be programmed simply by changing rates at which the fluids flow through the supply and waste lines, and concentration ramps that include pauses are easily accommodated. In each of the embodiments described above, air or an inert gas or the like is allowed to enter the fluid supply chambers as the fluid is drained. Accordingly, a one way valve may be installed that allows air to enter, but which prevents fumes from the fluid supply reservoirs from escaping to the atmosphere.

In an alternate embodiment, the second fluid may be supplied from closed variable volume flexible plastic bags. This would simplify handling of the fluids, and would prevent escape of fumes from open fluid supply reservoirs. Similarly, the sample chamber 42 may comprise a variable volume bag that is partially filled with a biologically inert gas. This would prevent fumes from rising out of the sample chamber and would prevent the tissue samples from being exposed to damage from evaporation when the fluid is drained from the sample chamber. When sample chamber 42 is a flexible bag, this bag may include a "Zip-lok" type sealing provision for introducing and removing the tissues, and may contain inlet and outlet tubes for ready connection to lines 56, 46, etc.

While this invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the embodiments of the invention, as set forth herein, are intended to be illustrative and not limiting. Various changes may be made without departing from the spirit and scope of the invention as defined in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for histological processing of a tissue sample, comprising the steps of: immersing the tissue sample in a fluid bath; gradually varying the proportions of first and second fluids that comprise the fluid bath so that the tissue sample is immersed in gradually increasing proportions of the second fluid.

2. The method of claim 1, wherein the step of immersing the tissue sample in fluid bath comprises the step of immersing the tissue sample in a volume of a first fluid in a sample chamber; and wherein the step of gradually varying the proportions of first and second fluids that comprise the bath comprises the steps of: adding a volume of a second fluid to the sample chamber over a period of time; and reducing the total volume of fluid in the sample chamber simultaneously with the addition of the volume of the second fluid to the sample chamber so that a mixture of the first and second fluids in the sample chamber has gradually changing proportions of the first and second fluids over time.

3. The method of claim 2, further comprising the steps of: draining all of the first and second fluids from the sample chamber after the volume of the second fluid has been added to the sample chamber; and immersing the tissue sample in a second volume of the second fluid.

4. The method of claim 2, further comprising the step of controlling a rate at which the volume of the second fluid is added to the sample chamber to control the proportions of the first and second fluids in the sample chamber.

5. The method of claim 4, further comprising the step of controlling a rate at which the total volume of fluid in the sample chamber is reduced to control the proportions of the first and second fluids in the sample chamber.

6. The method of claim 2, further comprising the step of controlling a rate at which the total volume of fluid in the sample chamber is reduced to control the proportions of the first and second fluids in the sample chamber.

7. The method of claim 2, wherein the step of adding a volume of a second fluid to the sample chamber over a period of time comprises allowing a volume of the second fluid to drain into the sample chamber under the influence of gravity.

8. The method of claim 2, wherein the step of reducing the total volume of fluid in the sample chamber comprises allowing the fluid in the sample chamber to drain from the sample chamber under the influence of gravity.

9. The method of claim 2, wherein the step of adding a volume of a second fluid to the sample chamber over a period of time comprises pumping a volume of the second fluid from a supply reservoir into the sample chamber at a predetermined rate.

10. The method of claim 9, further comprising the step of varying the rate at which fluid is pumped into the sample chamber according to a predetermined profile to control the proportions of the first and second fluids in the sample chamber

11. The method of claim 9, wherein the step of reducing the total volume of fluid in the sample chamber comprises pumping fluid out of the sample chamber at a predetermined rate.

12. The method of claim 2, wherein the step of reducing the total volume of fluid in the sample chamber comprises pumping fluid out of the sample chamber at a predetermined rate.

13. The method of claim 12, further comprising the step of varying the rate at which fluid is pumped out of the sample chamber according to a predetermined profile to control the proportions of the first and second fluids in the sample chamber.

14. The method of claim 1, wherein the step of immersing the tissue sample in a fluid bath comprises the steps of: placing the tissue sample in a flow through sample chamber; creating a mixed fluid flow by continuously mixing proportions of first and second fluids from separate sources; and introducing the mixed fluid flow into the sample chamber so that the mixed fluid continuously flows over the tissue sample and flows out of the sample chamber; and wherein the step of gradually varying the proportions of first and second fluids that comprise the fluid bath comprises: gradually varying the proportions of the first and second fluids in the mixed fluid flow so that the tissue sample is immersed in gradually increasing proportions of the second fluid.

15. The method of claim 14, further comprising the step of controlling the proportions of the first and second fluids in the mixed fluid flow according to a predetermined profile.

16. A device for histological processing of a tissue sample, comprising: means for immersing the tissue sample in a fluid bath; and means for gradually varying the proportions of first and second fluids that comprise the fluid bath until the fluid bath comprises approximately only the second fluid.

17. A device for histological processing of a tissue sample, comprising: a sample chamber for holding a tissue sample and a volume of a first fluid; a fluid supply reservoir for holding a volume of a second fluid; a fluid supply valve or pump connected to the fluid supply reservoir for controlling a flow of the second fluid out of the fluid supply reservoir; a fluid supply line connected to the fluid supply valve or pump and the sample chamber for conveying the second fluid from the fluid supply valve or pump to the sample chamber; and a waste valve or pump connected to the sample chamber for controlling a flow of fluid out of the sample chamber; wherein the second fluid in the fluid supply reservoir can flow into the sample chamber and mix with the volume of the first fluid in the sample chamber at the same time that fluid from within the sample chamber flows out of the sample chamber through the waste valve so that the proportions of the first and second fluids in the sample chamber gradually change and approach a condition wherein all the fluid in the sample chamber is the second fluid.

18. The device of claim 17, further comprising a stirring mechanism in the sample chamber for mixing the first and second fluids together.

19. The device of claim 17, further comprising a second fluid supply reservoir for holding a second volume of the second fluid, wherein the second fluid supply reservoir is connected to the fluid supply valve or pump, and wherein after all of the first fluid and the second fluid from the first fluid supply reservoir have flowed out of the sample chamber, at least a portion of the second fluid from the second fluid supply reservoir can be introduced into the sample chamber through the fluid supply valve or pump and the fluid supply line.

20. The device of claim 17, further comprising a pump connected to the waste line for pumping fluid out of the sample chamber.