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Publication number | US4745771 A |
Publication type | Grant |
Application number | US 07/038,840 |
Publication date | 24 May 1988 |
Filing date | 13 Apr 1987 |
Priority date | 23 Aug 1983 |
Fee status | Lapsed |
Publication number | 038840, 07038840, US 4745771 A, US 4745771A, US-A-4745771, US4745771 A, US4745771A |
Inventors | John G. Linner, Stephen A. Livesey |
Original Assignee | Board Of Regents, The University Of Texas System |
Export Citation | BiBTeX, EndNote, RefMan |
Patent Citations (26), Non-Patent Citations (10), Referenced by (31), Classifications (9), Legal Events (7) | |
External Links: USPTO, USPTO Assignment, Espacenet | |
This application is a continuation-in-part of copending, commonly assigned U.S. patent application Ser. No. 942,172 filed Dec. 16, 1986, now U.S. Pat. No. 4,742,690; which in turn is a continuation-in-part of copending, commonly assigned U.S. patent application Ser. No. 777,083 filed Sept. 17, 1985, now abandoned, which in turn was a continuation of commonly assigned U.S. patent application Ser. No. 676,855 filed Nov. 30, 1984, now U.S. Pat. No. 4,567,847; which in turn was a continuation-in-part of commonly assigned U.S. patent application Ser. No. 525,626 filed Aug. 23, 1983, now U.S. Pat. No. 4,510,169.
1. Field of the Invention
This invention relates to apparatus and the method for preparing biological tissue samples for ultrastructural analysis or other medical use, i.e. transplantation, by avoiding significant modification of the ultrastructure of tissue during preparation of the samples themselves. It is well known in the medical arts that to examine tissue samples, and determine the cellular structure and function thereof, the tissue must be "fixed" prior to the application of nearly all analytical methodologies.
The terms "biological samples," "tissue samples," and "biological tissue" are used throughout this disclosure to refer to samples that can be ultrarapidly cooled by the method and apparatus of this invention. The terms are used interchangeably and are not intended as a limitation on the functional capability of the method or apparatus disclosed herein. The terms should be understood to include small tissue samples appropriate for microscopic examination and larger tissue masses such as corneas which are appropriate for transplantation. The terms should be understood to include any material composed of one or more cells, either individual or in complex with any matrix or in association with any chemical; and to include any biological or organic material and any cellular subportion, product or by-product thereof. The terms should be further understood to include without limitation sperm, eggs, embryos, blood components and other cellular components. The contemplated utility of the apparatus of this invention is not limited to specific types or sizes of tissue, rather it should be understood to refer to any tissue made up from cells. The apparatus of this invention can be designed or adapted to any size, shape or type of cellular tissue. Therefore, the terms "tissue" and "tissue samples" are used interchangeably and are not limiting on the uses to which the method and apparatus of this invention can be placed.
Although the examination of tissue by microscopes or related magnifying apparatus has been practiced for many years, there has been an inherent problem in preparing tissue for use with contemporary high resolution analytical microscopes, such as the STEM electron microscopes, which permit the examination of sample constituents via X-ray analysis at powers of from 500× to 500,000× with point to point resolution of 2 to 3 Angstrom units.
Specifically, it is difficult to interpret the results of tissue analysis while concomitantly assessing the extent of various artifacts produced during the tissue preparation processes. It is thus essential that artifacts be avoided wherever possible. The term "artifact" refers to a product of artificial character due to extraneous agency. Another problem results from physical shrinkage of the tissue sample itself when subjected to the extreme, but necessary for successful preparation, procedures extant in current dogma. In most currently used tissue preparation steps, tissue shrinkage is in the order of 40% to 50%. This shrinkage inevitably results in alteration of ultrastructure and massive rearrangement of infrastructural resolution. The net result of this is ultrastructural translation damage and inaccurate detail in descriptions via existing analytical procedures.
During the so-called "Golden Age of Morphology" the predominant underlying goal in qualitative and quantitative microscopy has been an aesthetically pleasing image. This goal is readily attainable with the fixation methods and apparatus which are currently available. However, it has become essential that the aesthetically pleasing image, which is produced by the preparation process, also yield a tissue sample which accurately reflects the true condition of tissue in the living organism, i.e., approaching the "living state." This is the problem which the apparatus of this invention addresses and solves. Magnification apparatus which are currently available for analytical use are technically more advanced than are current tissue preparation techniques which have been previously employed. The method of this invention results in the preparation of tissue samples which are readily usable on known magnification and analytical apparatus.
Although the primary thrust of this application is in the preparation of tissue samples for analysis by current magnification apparatus, the invention is not intended to be so limited. More specifically, the "preparation" of tissue should be understood to refer to preparation of tissue for analysis as well as the cryofixation of tissue in anticipation of transplantation, modification, in vitro or in vivo cellular growth, fertilization, animated suspension or the more typical resin impregnation, setting, infiltration and analysis. The apparatus of this invention can be used to prepare tissue for any medical or analytical procedure without the ultrastructural damage previously thought to be inevitable in cryopreparation.
The apparatus of this invention is to be distinguished from contemporary freeze-drying apparatus. Freeze-drying is a technique which is well known in the art together with the equipment necessary to implement such freeze drying. See, for example, U.S. Pat. No. 4,232,453. Although in certain freeze-drying techniques liquid nitrogen is used as a cooling medium, the tissue or sample itself does not attain such temperature. Freeze-drying typically contemplates sample temperatures of -50° C. to -80° C. In contrast, the cryopreparation of this invention contemplates sample temperatures of -120° C. or below. Therefore, for purposes of this application the terms "cryopreparation" and "cryofixation" are used in distinction to conventional "freeze drying" technology (-50° C. to -80° C.).
The extreme low temperatures and vacuums used by the apparatus and method of this invention have generated unique problems not associated with freeze-drying apparatus. For example, sealing devices such as squeezable O-rings made from elastomeric material do not function effectively at cryopreparation temperatures and vacuums. Therefore, it is necessary that cryopreparation apparatus be designed to seal various structures at the extremes of temperature and pressure encountered, i.e., the sample chamber to the rest of the apparatus, outside the liquid nitrogen environment. This is but one example of problems which have been encountered in the design of and which are unique to cryopreparation apparatus.
The vacuum levels disclosed and used in the apparatus of this invention cannot be achieved safely with prior art freeze drying equipment. Typical of previous methods for drawing vacuums in freeze drying methods and apparatus is the above-mentioned U.S. Pat. No. 4,232,453 which discloses the use of molecular sieves in glass containers. Molecular sieves in easily compromised containers cannot be used safely to create and maintain the required vacuum levels to achieve the partial pressures required for sublimation of water at the anticipated temperatures (-120° C. or below) created by the apparatus of the disclosed invention.
2. The Prior Art
The most common prior art method for preparation of tissue samples for analysis is by means of chemical fixation and organic solvent dehydration. Inherent in prior art processes is the concomitant artifact creation, sample shrinkage and resultant damage to and modification of tissue characteristics. These tissue characteristic modifications, whether in the form of artifacts or the like, require interpretation by the individual or apparatus analyzing or evaluating the sample. This introduces, in many instances, an unsatisfactory risk of error.
Chemical fixation is a well known technique and has served the analytical biologist well for many years and undoubtedly will continue to do so in certain limited applications. However, as the use of tissue sample analysis becomes more complex and the use of such analysis becomes more widespread, alternatives to chemical fixation are demanded. This is especially true as advances are being made in the magnification and analytical apparatus which are available. It is necessary that tissue preparation methods and the apparatus necessary to prepare tissue samples be equally advanced as the analytical tools, i.e., electron microscopes, which are being used to analyze the samples. Obviously, if the technology for tissue sample preparation is inferior to microscopy technology, then the advanced microscopes cannot be used to full advantage by the morphologist or other tissue examiner.
Similarly, it is essential that cryopreparation methods and apparatus develop concurrently with other medical technology, i.e., surgical transplant techniques, bio-engineering and biogenetics. In short, cryopreparation is an essential intermediate step in evolving processes using or analyzing cells or tissue. If cryopreparation apparatus does not evolve then the thrust of medical technology into unexplained and unexplored medical arts will be blunted. The apparatus of this invention represents one component of the cryopreparation breakthrough that will permit research into the use and preparation of biological tissue to keep pace with other advances in medical technology. Importantly, the apparatus of this invention represents an improvement to the apparatus disclosed and claimed in commonly assigned, copending U.S. patent application Ser. No. 942,172, filed Dec. 16, 1986, which is hereby incorporated by reference.
The most common alternative to chemical fixation and organic solvent dehydration is freeze-drying cryofixed samples. Freeze-drying following cryofixation is a well documented and well known technique for tissue preservation. It has several advantages. Freeze-drying results in a near-instantaneous arrest of cellular metabolism. There is also a stabilization and retention of soluble cell constituents through elimination of solvent contact with the sample. These are significant advantages to cryofixation freeze-drying that have resulted in a great deal of research in attempting to apply cryofixation and freeze-drying techniques to known tissue preparation processes.
Unfortunately, freeze-drying technology inherently possesses a number of disadvantages relevant to tissue preparation methodologies. The primary disadvantage in currently available freeze-drying techniques and apparatus is the inherent formation of ice crystals. As can be readily appreciated, the formation of ice crystals destroys the ultrastructural integrity of the tissue sample being reviewed. The image is distorted and the cytoplasm becomes reticulated. The formation of ice crystals in the sample can also result in a change in pH within microcompartments of the tissue (eutectic formation) which possibly can result in abnormal tertiary conformation of macromolecules. There is also the possibility that proteins will denature and precipitate. These are but a few of the disadvantages which are inherent in the freeze-drying process.
This general topic is discussed in some detail together with other prior art methods in an article entitled Freezing and Drying of Biological Tissues for Electron Microscopy, Louis Terracio and Karl G. Schwabe, published in The Journal of Histochemistry and Cytochemistry, Volume 29, No. 9 at pp. 1021-1028 (1981). Problems associated with artifact formation are described in Understandinq the Artefact Problem in Freeze-Fracture Replication: A Review. The Royal Microscopial Society, (1982) at pp. 103-123.
A general principle found applicable to freezing techniques, which has demonstrated utility in the preparation of tissue samples, is that as the cooling rate increases, tissue fluids can be vitrified without the separation of water to extracellular spaces. It has been postulated that regardless of the rate of cooling, ice crystals may still be formed, but as the cooling rates increase the size of the intracellular ice crystals decrease. The small size or absence of ice crystals at high freeze rates is of course a substantial advantage in morphology retention as this results in minimal artifact creation and minimal ultrastructural damage during tissue dehydration. The apparatus of this invention requires the rapid supercooling of tissue samples to the vitreous phase in less than one second followed by dehydration of the tissue sample while in the state of reduced partial pressure of water vapor, all without substantial ultrastructural damage to the tissue cells.
Historically, the criteria by which the techniques for rapid supercooling have been judged was not the cooling rate of the system but simply the temperature of the environment in which the tissue was frozen. Thus, the term rapid supercooling has been applied to any system in which the supercooling agent has a temperature of -150° C. or below. The effectiveness of a cooling system is dependent upon the rate at which heat is removed from the sample. Heat transfer is dependent not only on the temperature of the freezing system but also of its physical and thermal characteristics, as well as the size and thermal characteristics of the tissue.
The most commonly used technique for rapid supercooling is to immerse or "quench" the sample in a fluid cooling bath. The most commonly used fluids for quenching are liquid nitrogen, isopentane, propane and fluorocarbons such as Freon 12 and Freon 22. Although liquid nitrogen is generally regarded as an ideal quenching fluid due to its low temperature (31 196° C.), there are inherent disadvantages in the use of liquid nitrogen due to the occurrence of tissue surface film boiling caused at least in part by the low heat of vaporization of liquid nitrogen. Film boiling is a characteristic of liquid nitrogen that inhibits the heat transfer rates by actually insulating the sample.
An alternate prior method for rapid supercooling is freezing on the polished surface of a chilled metal block. This typically involves opposing the tissue sample to a polished flat metal surface by pressing it firmly against the surface of the metal. Silver and copper are typically used as the polished metal blocks. This method is designed to take advantage of the high thermal conductivities and heat capacities of these metals when cooled to liquid nitrogen or liquid helium temperatures. The critical step in chilling on the surface of a metal is making firm contact with the dry, chilled metal surface with no rotational, translational or rebounding motion. Certain commercially available apparatus having known utility in the medical arts address and provide "bounce-free" freezing. Credit for the development of this apparatus is generally accorded to Dr. Alan Boyne of the University of Maryland School of Medicine.
There has recently been verification that there is a direct correlation between cooling rate and ultrastructural preservation in quenching fluids. As the freezing rate increases over the range of 100° C. to 4100° C. per second (liquid nitrogen-propane), there is a corresponding decrease in the size of ice crystals formed and thus an improvement in morphological preservation. Use of such quenching fluids or other supercooling apparatus to vitrify a tissue sample in less than 1 second is preferred.
The critical steps in the subsequent tissue preparation process are invariably stimulated sublimation dehydration of the supercooled tissue fluids, which have recently been described as a stimulated "molecular distillation" process. Once the appropriate supercooling method has been chosen and implemented, it is sometimes necessary to further process the tissue for microscopic evaluation, since electron microscopes or other magnification apparatus that allow the viewing of frozen hydrated specimens are not readily available. Thus, dehydration is an essential step in the preparation of biological tissue samples for storage and a step which oftentimes results in the destruction via reticulation of the infrastructure and ultrastructure of the tissue. Tissue cell destruction from dehydration not only impairs analysis by magnification apparatus but also adversely affects the functional characteristics and viability of tissue masses being used, i.e. transplanted.
In certain prior drying techniques, the tissue sample was not entirely solidified due to eutectic formation as the cellular fluid solutes were concentrated in bound water compartments. This transfer of solute occurs while the materials are in the fluid state when slow cooling is employed. When rapid cooling techniques are used, unique procedures which are distinct from those characteristic of freeze-drying, must be employed in the dehydration step. Problems result from the fact that dehydration must take place (the water must be removed) in the solid rather than the liquid state, i.e., via sublimation. An alternate procedure which has been used successfully is stimulated molecular distillation. Stimulated molecular distillation refers to a process in which the amount of energy in the antibonding orbitals of surface molecules is elevated, enabling them to escape to the gas phase and not be recaptured by the solid phase.
In the prior art, the freeze substitution approach has involved the removal of tissue water by substituting a solvent or solvent-fixative mixture for the solid phase water at -50° to -80° C. This introduces less severe solvent phase separation and chemical alteration artifacts to a tissue sample than past routine chemical fixation methodologies. From a practical standpoint freeze-drying is complicated by the requirement that the tissue sample be warmed to increase the vapor pressure of the supercooled water and allow sublimation to proceed in a reasonable period of time. The increased temperature, in addition to increasing vapor pressure can produce a series of physical events leading to the expansion of ice crystals and concomitant damage to the ultrastructural morphology of the tissue sample. Many of the physical events which occur during the warming process have to do with transitions in the physical state of the water which is present. Changes which are typically encountered are glass transition, devitrification and recrystallization with an ensuing series of crystal lattice configuration transitions.
Thus it can be appreciated that freeze-drying technology and cryopreparation techniques present an exceptional opportunity for the preparation of tissue samples for morphological examination. However, inherent in the use of freeze-drying techniques are problems associated with dehydration and fixation of samples. These are the problems which are overcome by the process and apparatus of this invention.
The cryopreparation process of this invention has demonstrated an extraordinary application in the transplanting of corneal tissue. Prior to this invention attempts to transplant corneas which involved a necessary freezing or freeze-drying of the corneas after removal from the donor invariably resulted in a clouded cornea upon transplanting. This physical condition of the transplanted cornea was caused by crystal formation in the cornea itself and concomitant damage to the stroma. Use of the apparatus of this invention has enabled ophthalmologists to cryoprepare corneas and to then transplant those corneas to recipients with virtually negligible clouding or crystal formation. The ability to so transplant corneas represents an exceptional advantage to the process of this invention as well as a medical breakthrough in corneal transplant surgery.
One advantage of the apparatus of this invention is the ability to cryoprepare tissue without overt disruption or destruction of the morphological characteristics of the ultrastructure of tissue cells. The apparatus of this invention permits the cryopreparation of tissue by dehydrating tissue maintained in the solid, vitreous phase without creating unnecessary artifacts which restrict interpretation by conventional analytical apparatus.
FIG. 1 is a schematic flow diagram of a method associated with the use of the apparatus of this invention.
FIG. 2 is a schematic representation of the cryopreparation apparatus of which this invention is one component.
FIG. 3 is an exploded schematic drawing of the portion of the apparatus of this invention connecting the vacuum means to the sample chamber.
FIG. 4 is an exploded schematic drawing of the sample chamber and sample holder components of the cryopreparation apparatus associated with this invention.
FIG. 5 is a schematic view of the sample holder of this invention.
FIG. 6 is a schematic view of the tissue reservoir cover used in the sample holder of this invention.
FIG. 7 is a cross-sectional side view of the sample holder of this invention.
FIG. 8 is a cross-sectional view of the sample holder of this invention taken at the plane defined by arrows 7--7 in FIG. 5.
This invention relates to apparatus and the method for the cryopreparation of biological tissue samples. The apparatus includes components for implementing the stimulated dehydration of biological tissue under severely depressurized conditions. A depressurized, vitrified tissue sample is brought to equilibrium at a temperature of less than -123° C. and preferably less than -140° C. An especially preferred equilibrium temperature is -178° C. The tissue sample is then dehydrated while maintained in a state of equilibrium. After removal of tissue water, the tissue sample is optionally infiltrated with a degassed resin followed by polymerization of the resin to form an embedded tissue sample. In other applications of the apparatus and method of this invention the dehydrated tissue sample can be used, i.e. transplanted, without any infiltration or degassing steps.
The apparatus of this invention includes a sample holder for retaining vitrified biological tissue. The sample holder and the vitrified tissue are maintained at cryogenic temperatures while the tissue sample is being dehydrated. Ultra-high vacuum means are used to depressurize the atmosphere of the sample holder to permit the desired sublimation; equilibration and dehydration procedures.
The apparatus of the invention is used in combination with conventional apparatus to vitrify biological tissue. The preferred vitrifying apparatus is a metal rod adapted to transform the tissue to the vitreous phase at a temperature of -123° C. or below. The vitrified tissue is inserted in a sample holder which is fittably received by a sample chamber which in turn can be inserted or withdrawn from a cryogenic bath.
The ultra high vacuum assembly used to depressurize the sample chamber provides a pressure of from 1×10-8 mbar, to 1×10-10 mbar. The ultra-high vacuum assembly is removably attached to the sample chamber.
In practice the apparatus of this invention is used to cryoprepare biological tissue for analysis or other medical end use, i.e. transplantation. The apparatus is adaptable to an infinite variety of tissue shapes, sizes and configurations. The apparatus of this invention results in the cryopreparation of biological tissue resulting in a final product whose ultrastructure is substantially unmodified and which is ready for analysis and end uses which have been heretofore impossible in the medical arts.
In the apparatus of this invention it is a fundamental prerequisite that the desired tissue is obtained. Tissue samples are collected by a variety of means, i.e., surgical extraction, withdrawn blood samples, binders and any of a variety of other techniques which are well known and conventional. The particular method of obtaining the biological sample is not limiting on the apparatus of this invention. However, the preparation of the tissue sample in the apparatus of this invention is enhanced if the tissue sample is processed as soon after excising as is possible.
The preparation of the tissue sample takes place immediately as it is received. The tissue sample cannot be retained in a fixative, i.e., formaldehyde, or another biologically active stabilizing solution, in an attempt to maintain the sample during shipping, storage or other necessary operations. It is also critical that the sample not be routinely frozen or otherwise physically or chemically modified prior to preparation according to the method of this invention. The sample may later be physically sectioned or otherwise physically prepared for long-term storage in apparatus or use with various currently available commercial analytical apparatus.
In one application of the apparatus of this invention a tissue sample is prepared for analysis. The preferred optimum biological sample for preparation in the apparatus of this invention is a fresh one cubic millimeter biopsy sample. This sample must be vitrified as soon as possible. By vitrifying or vitrification it is intended to make reference to a process which results in cryofixation of the sample which is different from "frozen." In the process of vitrifying, the cooling apparatus which is used renders the sample in the vitreous phase such that soluble and insoluble moities contained in the tissue sample are not disturbed, translated, or altered nor are they concentrated (as eutectics). By definition, a vitrified liquid will shatter when undergoing a shear stress, e.g., window glass. The vitreous phase involves the conversion of liquid water into an amorphous or "glass" phase. This is accomplished by rapidly supercooling the tissue sample by opposing it "bounce-free" onto the highly polished (mirror-like) condensate-free surface of a metal rod maintained at about -196° C. These operations have been discussed previously in the prior art section of this disclosure. It is preferred that such rapid-supercooling be completed in less than one second. For purposes of this application, the terms "vitreous" or "vitrified" should be understood to refer to not only true vitrified tissue, but tissue wherein conditions of cryopreparation are such that resolvable ice crystals do not form within the time period for preparation and analysis or other ultimate end use.
Of particular utility in the process and apparatus of this invention is a "bounce-free" freezing apparatus which has been identified in association with Dr. Alan Boyne of the University of Maryland School of Medicine. In this freezing apparatus, a copper block is used to vitrify the tissue sample. This vitrification in conjunction with a supercooled fluid such as liquid nitrogen, helium, propane or the various freons will cause the tissue sample fluids to supercool to the amorphous state before and/or without the formation of noticeable or resolvable cell water ice crystals. It is desirable in the preferred embodiment that the now vitrified tissue sample be maintained at a temperature of less than about -120° C. and preferably less than -140° C. during storage and transfer operations prior to removal of the tissue water.
Temperature control is essential to prevent ice crystallization. It is thought that ice crystallization begins to occur at about -123° C. This is, however, dependent on the chemical constituents of the cellular water. Applicant has therefore selected -140° C. as the preferred temperature. It should be understood that the desired result is to maintain the temperature below that at which ice begins to crystallize and that -123° C. and -140° C. have been selected based on current experimentation. Therefore, for purposes of this application, the preferred tissue sample temperature to be maintained is below -123° C. while the more preferred temperature is below -140° C. and the most preferred temperature is -196° C. or below.
Depending on the anticipated time lag between supercooling of the sample and dehydration of the sample, it may be stored submerged in a liquid nitrogen dewar. Once the sample has been dried and embedded properly it may be stored virtually indefinitely without cytoplasmic reticulation or other forms of cellular catabolism which will cause modifications and crystal lattice transitions resulting in undesirable artifacts which render the tissue uninterpretable as analytical data.
After vitrifying, and while maintaining the tissue sample at a temperature of less than -140° C. it is transferred via a specimen transport and fed to a specimen holder in vacuo. The specimen holder (also commonly referred to as a sample holder) is maintained in a temperature controlled container. The container and specimen holder are both preferably maintained at temperatures below -140° C. In the most preferred embodiment of this invention, liquid nitrogen temperatures of -196° C. are maintained. The reason that -140° C. is preferred is that pure water, existing in the vitreous phase when at liquid nitrogen temperatures, will begin to initiate cubic ice crystallization at -123° C. As discussed in the prior art section of this disclosure, ice crystallization causes ultrastructural damage, i.e., reticulation to the morphology of tissue samples.
Next, the atmosphere surrounding the tissue sample, specimen holder and container is depressurized. This is typically done by drawing a vacuum on the sample holder with conventional mechanical vacuum apparatus. The vacuum is drawn to a level of 3×10-9 mbar. in less than 300 minutes. In other embodiments of this invention, the vacuum which is drawn is from 1×10-8 mbar. to 1×10-10 mbar. accomplished in less than 300 minutes. These pressures remain at approximately 3×10-9 mbar. throughout the remainder of the prescribed routine until all the tissue water has been removed. Throughout equilibration of the system (10-100 hours), the specimen temperature is maintained by liquid nitrogen or other suitable cooling means while the vacuum is being drawn and maintained.
At this time the tissue sample is at ultra low pressure and exceptionally low equilibrium cryo-temperature. After equilibration is obtained (with equilibrium temperature below -140° C. and preferably at -178° C.), the vitreous water which is found in the tissue sample will begin to sublime as energy equal to the heat of sublimation is intermittently and incrementally supplied to the sublimation front found in the tissue. This is a slow process but one which is critical to the preparation of the sample. It is an important requirement that the sample be permitted enough time to allow it to reach equilibrium after each addition of energy. By equilibrate it is meant that the temperature of the tissue sample no longer changes over a 1 to 5 hour time period and preferably a 2 to 4 hour time interval. In a typical tissue preparation process the sample is rapidly vitrified to -196° C. and maintained below -140° C. during storage/transfer to the sample holder in the sublimation (drying) apparatus. After appropriate equilibration time the equilibrium temperature will be somewhere between -140° C. and -196° C. During this entire equilibration process a critical ultra-low pressure is maintained at 3×10-9 mbar. or below.
After the equilibration process, it would take an exceptional length of time for any appreciable amount of water to evaporate from the sample if no energy (heat) of sublimation were added to the system. Estimates are in terms of years for the water to evaporate at temperatures and pressures which are associated with the method of this invention. Therefore, in the most preferred embodiment of this invention, a secondary energy source (heating) is added to excite or stimulate the sublimating water molecules without causing damage to the ultrastructure of the dry tissue sample. Radiant photon energy, having a particular wavelength, is thought to be an especially useful approach to accomplish this goal. Sublimation energy via microwave, laser systems and magnetic energy are also appropriate. The most preferred secondary source is the nuclear magnetic resonance or electron spin resonance approach in combination with the above. At equilibrium, the temperature of the tissue will not change unless the ambient parameters of the immediate environment (radiant energy predominates, i.e., room temperature is 27° C.) change. This is the general identification of the end point of system equilibrium.
Subsequent to the tissue sample reaching equilibrium, it is necessary to remove the supercooled solid water and/or presently unresolvable ice crystals (20 nanometers diameter or less) which have formed in the tissue during the vitrification operation. This portion of the dehydration process is absolutely critical and is the step where most potential disruption and reticulation of the ultrastructure in the tissue will express itself. This is accomplished by gradually replacing the energy of sublimation in the sample by minimal increments of stimulated energy per hour. The optimal condition is to have no tissue temperature increases.
By so raising the thermal energy equivalent to the latent heat of sublimation all of the solid water, whether micro-ice crystals or amorphous supercooled water, is effectively removed from the tissue sample by the surrounding cryosystem. This drying may be accomplished at temperatures between -150° C. and -80° C. This regimen of greater temperature latitude will provide variable results and is possible due to elevation of devitrification temperatures by the solutes that are dissolved in cell water at varying concentrations. With appropriate instrumentation, i.e. residual gas analyzers, it is possible to determine when all cell water has been removed. At that point, the energy increase can be accelerated to produce a final specimen temperature 3° C. above room temperature (28° C.-30° C.). Thus, with this instrumentation a significant advantage in the process of this invention is obtained.
The now dehydrated tissue sample has been permitted to reach room temperature plus 3° C. Even though reaching room temperature the vacuum is maintained at the original exceptionally ultra-low levels as has the temperature surrounding the sample. Room temperature for purposes of this application should be understood to be approximately 24° C.-27° C. There may logically be variations in this temperature level.
A person of ordinary skill in the art can readily appreciate that control of temperature throughout the processes of vitrification, equilibration, sublimation and dehydration are essential. The precise temperatures at which the tissue is maintained and the rate that the tissue temperature is changed are crucial although varied for different cellular structures. A typical routine for a cell mass such as a cornea would require the initial The sample is immediately heated to -150° C. in approximately 4 hours. During the equilibrium, sublimation, dehydration stage the tissue sample is heated from -150° C. to -70° C. in 60 hours (rate=1.333° C./hr.). The drying process begins at approximately -119° C. and is completed before devitrification at -80° C. The sample is then heated from -70° C. to +25° C. in 4 hours. Generally the sample is heated to slightly above room temperature to prevent water condensation from invading the sample.
At this juncture, the investigator has the option of exposing the tissue to osmium vapors for approximately one hour to provide contrast enhancement via electron density. This may be omitted if proven to be deleterious to the moiety of interest or if the ultimate goal is clinical use. The osmium vapor is removed by recrystallization by cryoprecipitation. In other established fixation processes, paraformaldehyde and/or gluteraldehyde in buffer solution is used. These materials are typically referred to as chemical-fixative materials. The most preferred material which is typically added is osmium tetraoxide. This material will enhance the resolution and contrast of the various constituents of the tissue for the various analytical apparatus which might be used to interpret the tissue sample.
For samples prepared for analysis a degassed resin is then added to the tissue while still maintaining the depressurized condition. This is typically referred to as resin infiltration and results in an embedded tissue sample. Resins which have shown utility in past methods are equally applicable to the method of this invention. See for example U.S. Pat. Nos. 3,679,450; 4,100,158; 4,120,991 and 4,278,701.
Subsequent to these steps the tissue sample and resin are brought to atmospheric pressure by slowly admitting air through the resin port. The embedded tissue sample which has resulted from the resin application process is removed and the resin is polymerized at its prescribed temperature. The particular method of polymerization is largely dependent on the resin that is used. Typically, the tissue sample is polymerized by heat application in an oven for 12 hours. A normal temperature would be 60° C., but may be as low as -80° C. if necessary. It is essential that the polymerization step be accomplished without damage to the tissue ultra-structure.
Following polymerization the tissue sample can then be stored at room temperature, thin sectioned, stained or further prepared for other analysis. However, having been dehydrated in the fashion disclosed by this invention the sample is maintained in a cryofixed state which is readily interpretable by conventional ultramicrotomes and electron microscopes and provides the basis for exceptionally meaningful analysis of tissue samples with a significant alteration of and reduction of artifacts concomitantly reducing or eliminating past constraints thought to be ubiquitous in fixation and/or tissue preparation for visual analysis.
The actual relating of structure to function in these biological tissues is done by routine ultrathin sectioning with an enormous expansion of applicable staining methods heretofore deemed unapproachable via conventional electron microscopy, (i.e., immunological analysis of any soluble moieties, sugars, lipids and soluble proteins), enzyme cytochemistry, X-ray dispensive STEM analysis, tissue transplant preparations, microprobe analysis, autoradiography (especially of soluble compounds) and pharmaceutical preparations.
Other apparatus are available for the execution of this hierarchy, but none have produced the result expected as they do not incorporate in totality the required, defined parameters discussed earlier. The apparatus which is used in the practice of the method of this invention is illustrated schematically in FIGS. 2 through 8.
The rapid freezing attained by the apparatus of the Alan Boyne type is preferred to the practice of the process of this invention. Liquid nitrogen and other types of quenching baths in conjunction with chilled metal applications are used in the process of this invention to the extent they provide the vitrified phase of cell water in less than one second. A liquid nitrogen quenching bath is used to lower and maintain the temperature of the tissue sample which is included in the tissue holder. It should be noted that while the tissue sample is maintained in the liquid nitrogen condition, it is necessary that tubulation access the various staining and fixation materials which are optionally preferred in the process of this invention, as well as the various resins which are ultimately used to embed the tissue samples of this invention prior to polymerization. Again, each of these functions is illustrated schematically in the attached figures. However, it should be understood that these are not intended to be limiting features of this invention but merely illustrative of available technology.
In designing the apparatus or in selecting the apparatus for use in the method of this invention, it is necessary to understand the effects of the exceptionally low temperatures and pressures on various materials. For that reason, portions of the apparatus of this invention used to treat the material while in the vitrified state are typically made from stainless steel. Other materials may well be equally viable. Likewise, portions of the apparatus of this invention are made from or coated with Teflon®, a Dupont manufactured material which consists in a major portion of tetrafluorans.
FIG. 2 illustrates schematically the entire apparatus associated with this invention, sometimes referred to as the Linner Apparatus™ or the Linner Process™. As shown in FIG. 2, the apparatus is broadly categorized into a control panel 10 and the remainder of the apparatus used to vitrify, sublime and equilibrate the tissue. Microprocessor 11 of control panel 10 controls a turbomolecular pump 30. The control by microprocessor 11 is primarily of the revolutions per minute at which the components of the turbomolecular pump 30 are rotated and the temperature of the two main bearings in the turbomolecular pump.
Digital vacuum gauge 12 of control panel 10 is connected to the apparatus in several places. In addition, the digital vacuum gauge 12 is attached to mechanical pumps to provide digital readings of both the low vacuum caused by the mechanical pumps and the ultra high vacuum caused by the turbomolecular pump.
The next component of control panel 10 is a residual gas analyzer 13. Residual gas analyzer 13 functions by reading the partial pressure of each gas in the sample chamber 90. Included in the analyzer 13 is a quadrapole mass spectrometer. This instrument can read the atomic weight of each gas present in the sample chamber 90. In addition, residual gas analyzer 13 is used to determine the water vapor levels in the chamber which can be used to determine the end point for dehydration.
Microprocessor 14 is the component of control panel 10 used to read and control the temperature of the tissue samples in sample holder 100 (see FIG. 4). Microprocessor 14 reads the temperature of the metal supporting the tissue sample in sample holder 100 and does not contact the sample itself. The programmable features of microprocessor 14 enable the implementation of a temperature control function as well as a temperature monitoring function.
Component 15 of control panel 10 is a chart recorder for microprocessor 14. Chart recorder 15 provides a graphic illustration of temperatures measured by microprocessor 14.
Mechanical pumps 20 (backing pump) and 21 (rough pump) are located in the control panel 10 as well as in conjunction with the main apparatus. Mechanical pump 20 is activated to draw the backing vacuum on the turbomolecular pump system. The initial vacuum is typically 1×10-3 mbar. The mechanical pump 20 is also connected to molecular sieve trap 22 to trap any hydrocarbons that may be going back to the turbomolecular pump 30 from the mechanical pump 20. It is essential that no hydrocarbons reach the turbomolecular pump 30. The mechanical pump 20 and the molecular sieve trap 22 are arranged in series so that no hydrocarbons can bypass molecular sieve trap 22.
Molecular sieve trap 22 is connected to turbomolecular pump 30 by T connection 23. Low vacuum gauge head 24 extends from T connection 23 and is connected to the digital vacuum gauge 12.
In the preferred embodiment of this apparatus a solenoid valve 25 is connected to T connection 23 at the point illustrated by FIG. 2. The solenoid valve is used for a backing line (not illustrated) for dry nitrogen gas being connected to the turbomolecular pump 30. In the event that the vacuum or ultra-high vacuum system malfunctions and stops, the chamber is filled with inert nitrogen gas instead of moisture and hydrocarbon-containing air.
Turbomolecular pump 30 is used to create the ultra high vacuum of 1×10-8 mbar. to 1×10-10 mbar required to properly practice the process of this invention. The ultra high vacuum pump 30 can be any of a variety of commercially available vacuum pumping apparatus. The preferred embodiment is a turbomolecular pump and in particular a turbomolecular pump manufactured by Leybold-Heraeus (Model TMP-360). It is essential that the ultra high vacuum pump, whether it is a turbomolecular pump or not, yield a hydrocarbon free vacuum. As mentioned previously, the mechanical pump 20 is used to pump out gases which are transmitted through the ultra high vacuum pump 30 from sample chamber 90.
In the preferred embodiment of this invention a cooling fan 31 is used to cool the bearings of the turbomolecular pump or other ultra high vacuum pump 30. A heating bakeout jacket 32 heats the walls of the ultra high vacuum pump 30 while in operation to ensure that gases are desorbed from the inner surfaces of the ultra high vacuum pump. These gases and even liquids are converted from condensation on the inner surfaces of the turbomolecular pump to result in gases thus enhancing the vacuum created by the turbomolecular pump 30. Thermocouple 33 provides the connection to the energy source (not shown) for heating bakeout jacket 32.
Conflat® flange 40 is used to seal the turbomolecular pump to a first spool 50. Conflat® is a trademark of Varian Industries, Inc. and describes a brand of flange. The type of flange associated with "Conflat®" is well known to those skilled in the art and can generally be described as a first surface having a knife edge designed to penetrate a second abutting surface which is a soft metal. Although many state of the art sealing devices will function effectively to seal the members at the ultra high vacuums and temperatures desired, it has been found most preferable to use a 100 cf Conflat® flange which is a stainless steel flange with a copper O-ring seal. Of great importance in Conflat® flange seal 40 is the fact that it functions effectively at temperatures up to 150° C. during bakeout of the apparatus. This permits the effective formation of a seal with relatively standard sealing means. It would be virtually impossible to form the seal necessary if flange 40 were sealed with conventional, squeezable O-rings which are typically made from elastomeric material.
Spool 50 provides the conduit from the turbomolecular pump 30 to a gate valve 60. Spool piece 50 includes four Conflat® flanges. The first is the common Conflat® flange 40 with turbomolecular pump 30. The second is Conflat® flange 51. The third and fourth Conflat® flanges are identified by numerals 52 and 53. Conflat® flange 52 connects spool 50 to the residual gas analyzer 13 sensing head while Conflat® flange 53 provides the seal between the spool 50 and a Bayard-Alpert gauge
An electropneumatic, ultra high vacuum pendulum gate valve 60 comprises the main valve isolating the turbomolecular pump 30 from the sample chamber 90. A piston contained within piston housing 61 provides the mechanism for opening and closing gate valve 60. Solenoid valve 62 and nitrogen gas are used to actuate the opening and closing of gate valve 60.
A second spool piece 70 is illustrated in FIG. 2 but in more specific detail by FIG. 3. Reference should be made to FIG. 3. Second spool piece 70 provides feedthrough from the pendulum gate valve 60 to the sample chamber 90. Spool piece 70 has extensions 71, 72, 74 and 78 connected to the main portion of the spool piece housing. Flange 71 provides tubulation for electrical feedthrough to the control panel 10 from the sample chamber 90. Flange 72 is tubulation for the low pressure vacuum head. At the exterior end of tube 72 is located low vacuum gauge head 73. Low vacuum gauge head 73 is connected to digital vacuum gauge 12. An ultra high vacuum valve 75 to mechanical pump 21 is located at the end portion of extension 74 from spool piece 70. The valve 75 acts to control the preliminary or "rough" vacuum drawn on sample chamber 90. Conflat® flanges 76 and 77 are used to seal spool piece 70 to gate valve 60 and to ceramic insulator spool 80. The fourth extension from spool piece 70 is overpressure relief valve 78 shown by phantom line in FIG. 3.
Ceramic insulator spool 80 is inserted between spool piece 70 and sample chamber 90. Insulator spool 80 functions to prevent gross heat transfer from the elements of the apparatus above spool piece 70 to the cryogenic dewar 99 below (see FIG. 4). Without insulator spool 80 frost and ice frequently develop on the exterior of the ultra-high vacuum pump assembly 30 and other connected elements. Ceramic insulator spool 80 also permits more efficient utilization of the supercooling material, i.e. liquid nitrogen.
The sample chamber 90 is used to retain the sample holder 100. These components are illustrated in FIGS. 4 and 5. The sample chamber apparatus 90 includes a resin containing chamber 95 and a glass window 96 to provide visual access to the resin containing chamber 95. A gold sealed ultra high vacuum valve 97 and tubulation 98 to provide access for the resin into the sample chamber 90. Glass tube 91 is attached to sample chamber 90 via glass to metal adapter 92 and tubulation 88 which in turn is connected to a metal "T" flange 93. Calibrated leak valve 94 is used to flush or permeate the sample chamber 90 with dry nitrogen gas or other inert material. The tube 91 is used to include osmium tetroxide crystals for introduction of osmium vapors into the sample chamber 90 during staining operations. Support members 94 are used to maintain the relative spacing of tubulation 88 and 98 from the housing of sample chamber 90. Cryogenic dewar 99 is used to maintain the cryogenic cooling means, i.e., liquid nitrogen.
In the most preferred embodiment of the apparatus of this invention a device is provided for sensing and automatically controlling the level of supercoolant, i.e. liquid nitrogen, in cryogenic dewar 99. Inherent in the use of liquid nitrogen or other similar coolants is the boiling off of the coolant over a period of time. Thus the coolant level must be periodically replenished to maintain the desired level of cooling. This can be accomplished manually or a mechanism can be installed for automatically sensing and replenishing the coolant level.
The sample holder 100, as shown in FIGS. 5 through 8, is used to retain the actual tissue samples. Typically the cryogenic bath environment 99 is liquid nitrogen contained by a dewar. The essential characteristic is that the tissue temperature must not exceed -140° C. The thermoconductivity of the cryogenic energy from the cryogenic bath environment 99 to the sample holder 100 is inherent in the structure. Reference here is made specifically to FIG. 4. Sample holder 100 is characterized by construction from materials that are thermally conductive. It is essential that sample holder 100 be thermally conductive.
In the most preferred embodiment of this invention radiant heating means 125 are provided to permit a source of radiant energy to the tissue samples. Typically the radiant heating means are controlled by rheostats or thermostats. Temperature indicating means such as identified as component 14 of control panel 10 are typically used so that the temperature of the environment and tissue samples can be specifically controlled. In the preferred embodiment of this invention the radiant heating means and temperature indicating means are all operated by a microprocessor of a computer within precise defined ranges.
Other forms of energy are equally useful with the apparatus of this invention. More particularly, electromagnetic energy sources such as microwaves, radio waves, acoustic sound waves, visual light waves and ultraviolet or near ultraviolet waves may be used. Magnetic flux is also useful, especially in combination with any of the above enumerated energy forms. Combinations of the above may be used depending on the application and sample to which the apparatus is placed. Infrared radiation should be avoided. Sample characteristics are of paramount importance in determining the energy source which is ultimately selected.
In actual operation a tissue sample is vitrified to liquid nitrogen temperatures, i.e., less than -140° C. The tissue is then transferred from a storage dewar to the thermally conductive sample holder 100 under liquid nitrogen temperatures with prechilled forceps.
The sample holder 100 is placed into the precooled sample chamber 90. Thermocouple wire 102 extending from sample holder 100 is then connected to mating wire 104 extending down from spool piece 70 (see FIG. 3). Likewise, heater wire 103 is then connected to mating flow through wire 105 extending from spool piece 70. The specimen chamber 90 is then connected to the spool 70 via Conflat® flange 77. This connection must be accomplished in the liquid nitrogen bath. Mechanical pump 21 is then activated and the spool 70 and sample chamber 90 are evacuated (rough pumped) to approximately 1×10-3 mbar. The valves connecting the mechanical pump to sample chamber 90 is then closed and the main valve 60 between the turbomolecular pump 30 and sample chamber 90 is opened. At this time the drying process begins.
The system is allowed to thermally equilibrate while being constantly monitored by the instrumentation in control panel 10. The turbomolecular pump draws a vacuum of approximately 1×10-8 mbar. to 1×10-10 mbar. The samples themselves are monitored by the residual gas analyzer 13 which includes a quadrapole mass spectrometer.
When the tissue is in equilibrium as indicated by no change in temperature for one to five and preferably two to four hours, the temperature controller is raised from -150° C. to about -70° C. Preferably the temperature is raised at a rate of 1° C. per hour to 3° C. per hour or more. In the most preferred embodiment the temperature is raised at a rate of from 1° C. per hour to 10° C. per hour. When the residual gas analyzer 13 shows no increase in water vapor after an increase in temperature the tissue is determined to be dry (typically at -85° C. to -70° C.). The temperature is then increased to 25° C. When the tissue has reached 25° C. the liquid nitrogen level in the dewar is allowed to drop and the outside walls of the sample chamber 90 are warmed to room temperature.
Optionally osmium vapor may then be introduced through glass to metal adapter 92. Subsequently the osmium vapors are removed by recrystallization in a liquid nitrogen trap. Also, the resin material is added from resin chamber 95 through tubulation 98. The tissue may then be removed to polymerize the resin.
FIG. 1 is a schematic illustration of the process for use of the equipment of this invention. The portion of FIG. 1 which is included within a dotted line is not asserted to be new or novel, only the apparatus which is used to implement these steps is new and novel. As can be readily understood from the foregoing description and the flow chart of FIG. 1, the essence of this invention amounts to vitrification, molecular distillation, sublimation, dehydration and tissue equilibration. This is a process and result which has not been heretofore thought possible. By use of the apparatus of this invention, it is possible to achieve medical goals heretofore thought to be impossible.
Although the preferred embodiment of the apparatus of this invention has been described hereinabove in some detail, it should be appreciated that a variety of embodiments will be readily available to a person designing cryopreparation apparatus for a specific end use. The description of the apparatus of this invention is not intended to be limiting on this invention, but is merely illustrative of the preferred embodiment of this invention. Other apparatus and components which incorporate modifications or changes to that which has been described herein are equally included within this application.
It is essential to the proper functioning of the apparatus of this invention that the sample holder 100 be sized and designed to be fittably received by sample chamber 90 and to maintain one or more tissue samples in the proper condition of vitrification during equilibration, sublimation and dehydration. The sample holder 100, which has shown specific utility in the apparatus of this invention, is illustrated more specifically in FIGS. 4, 5, 6, 7 and 8.
Referring now specifically to FIG. 5, the sample holder 100 consists of a solid block of thermally conductive material 110, preferably copper, silver, gold, sapphire, or diamond, and combinations or alloys of copper, silver, gold, sapphire, or diamond. In one preferred embodiment an alloy of silver and copper plated with gold is used. In another preferred embodiment, a solid sapphire block or sapphire coated metallic block are used. In still another preferred embodiment, a metallic block is diamond coated. These illustrations of preferred embodiments are not intended to be limiting on the invention. Any combination of materials that are functionally effective, i.e., thermally conductive, at the temperatures and pressures encountered in the subject process and apparatus can be used.
A plurality of wells 111 have been created in one surface of metal block 110. In the most preferred embodiment, fifty wells 111 are arranged in a circular configuration as shown in more detail in FIG. 8. The specific arrangement of wells 111 in solid block 110 is not critical, although functional limitations facilitating heating and water removal must be observed.
A central aperture 126 is also found in metal block 110. Radiant heat means 125 is insertable into aperture 126. The wells 111 create tissue reservoirs. The vitrified tissue samples are individually inserted into tissue reservoirs 112 with prechilled forceps as previously disclosed.
After insertion, the tissue samples are then covered with reservoir cover 113. Reservoir cover 113 includes a wire mesh section 114 and a side wall 115. The configuration of reservoir cover 113 is shown with more specificity in FIG. 6. Wire mesh section 114 is attached to side wall 115 by special vacuum adhesives. Solder is not appropriate because of the out-gas properties of most solders. The finer the mesh of 114 the more effective the desired gas transfer. Reservoir cover 113 also functions to protect the tissue samples from the effects of sudden changes in pressure such as when the gate valve 60 is opened or closed.
Teflon® spacers 120 are intermittently spaced around the exterior surface of solid metal block 110 to provide the proper spacing from the wall or other chilled surface of sample chamber 90. A Teflon® sleeve 119 is threaded into central aperture 126 to protect connecting wires 102 and 103. Although the sample holder 100 is shown in a size and configuration which is appropriate for cryopreparation of tissue samples for analysis, it should be understood that a person of ordinary skill in the art can equally prepare a sample holder to accommodate larger tissue masses or other forms of tissue.
In the most preferred embodiment of the sample holder 100 of this invention, the configuration is as shown in FIGS. 5, 7 and 8. Specifically, the radiant heating means 125 and the thermocouple connection 122 are both enclosed in aperture 126. The heater 125 and thermocouple connection 122 are insulated from one another, but are both enclosed within central aperture 126.
Solid block 110 also includes vertical lip 127 protruding vertically from the circumferential surface 128 of solid block 110. Vertical lip 127 permits the input of tissue fixation chemicals, i.e., resin and osmium, from above the ceramic insulators. The advantage enjoyed by such vertical infiltration is the ability to resin infiltrate at any temperature. In the past with other sample holders, it has been necessary to infiltrate at room temperatures. However, with vertical lip 127, the infiltrating chemicals can be maintained at or near the surface of the tissue reservoirs as illustrated in Figure 7. Preventing spillover of the chemicals enables the resin impregnation at a variety of temperatures. This is a distinct commercial advantage in the practice of this invention.
In actual practice, the reservoir covers 113, when inserted into tissue reservoirs 112, define a plane 129. In order to function effectively, it is preferred that vertical lip 127 extend below the plane 129. Vertical lip 12 functions to trap and redirect infiltration chemicals and maintains the chemicals on or near the surface of block 110 where the wells 111 are located.
In actual practice, individual tissue samples are placed in reservoirs 111 and reservoir cover 113 is inserted over the top of the tissue sample. As illustrated by FIG. 5, the covers 113 extend slightly above the surface of sample holder 100 to provide means for grasping the covers 113 during insertion or removal. In the most preferred embodiment of this invention slits 116 are provided in side wall 115 to give some flexibility to the covers 113, again to assist insertion and removal. The apertured surface 114 of covers 113 permits dehydration and sublimation without forming unnecessary moisture on the walls of reservoirs 112. It should also be understood that the preferred material for use in forming thermally conductive block 110 is sapphire although other materials, i.e., copper, silver, gold, diamond and alloys or combinations of the same, have been shown to be equally viable. The characterizing function of thermally conductive block 110 is the ability to transmit ultra low temperatures to the tissue samples and to maintain performance characteristics under the ultra high vacuum and ultra low temperature conditions of the cryopreparation apparatus and process of this invention.
Radiant heating means 125 are illustrated in FIG. 5 and provide a source of radiant heat to the tissue samples. Radiant heating means 125 are controlled by control panel 10. Control panel 10 permits infinite variability to the radiant heating means. In particular, temperature reader/recorder 14 and chart recorder 15 maintain information and control over the temperature of tissue reservoirs 112 and the tissue samples contained therein. As has been specifically pointed out hereinabove, control of sample temperature and the environmental temperature surrounding the tissue samples is absolutely essential to the effective functionality of the apparatus of this invention.
The most preferred embodiment of the sample holder 100 is illustrated in FIGS. 7 and 8. Included in the most preferred embodiment is radiant heating means 125 which are shown in aperture 126. The most preferred form of radiant heating means 125 is a 220 volt/100 watt cartridge heater. The heating system is made more efficient by coating the interior, polished (spectral) surface of side wall 115 of reservoir cover 113 with a material which permits the efficient transfer of radiation energy to the specimen. Thus, in the preferred embodiment, the walls 115 are treated with potassium sulfide to turn the interior surface walls black and thus provide the mechanism for controlled radiant heating of the tissue sample. In some embodiments the interior surfaces of wells 111 are likewise spectral.
Thus, the radiant heating means, i.e., cartridge heater 125, is controlled by temperature reader/recorder 14. The heating mechanism is selectively activated manually or preferably by a programmable computer or microprocessor to maintain the desired temperature or temperature rate of change. Upon heating, the thermally conductive block 110 conducts heat energy to the tissue reservoirs 112 and the heat energy is absorbed by the spectral coating on reservoir cover 113 and/or side wall 115. The spectral coating then acts as the source of radiant heat to the tissue samples.
Although the preferred embodiment of the specimen holder of this invention has been described hereinabove in some detail, it should be appreciated that a variety of embodiments will be readily available to a person designing an apparatus for a specific end use. The description of the preferred sample holder of this invention is not intended to be limiting on this invention, but is merely illustrative of the preferred embodiment of this invention. Other specimen holders which incorporate modifications or changes to that which has been described hereinabove are equally included within this application.
Cited Patent | Filing date | Publication date | Applicant | Title |
US3679450 * | 16 Mar 1970 | 25 Jul 1972 | Roy Nutt | Biopsy method |
US3745290 * | 1 Mar 1972 | 10 Jul 1973 | Gen Electric | Inductively heatable utensils or vessels for heating,serving and storing food |
US3942519 * | 12 Aug 1974 | 9 Mar 1976 | Ultrasonic Systems, Inc. | Method of ultrasonic cryogenic cataract removal |
US4055904 * | 2 Apr 1976 | 1 Nov 1977 | Tousimis Research Corporation | Automatic critical point drying apparatus |
US4056855 * | 7 Apr 1976 | 8 Nov 1977 | Charles Kelman | Intraocular lens and method of implanting same |
US4063068 * | 12 Aug 1976 | 13 Dec 1977 | Minnesota Mining And Manufacturing Company | Food heating and cooking receptacle |
US4100158 * | 2 Jul 1976 | 11 Jul 1978 | Johnson, Matthey & Co., Limited | Osmium compounds |
US4120991 * | 10 Dec 1976 | 17 Oct 1978 | Technicon Instruments Corporation | Process for mounting tissue sections with an U.V. light curable mounting medium |
US4197658 * | 12 May 1978 | 15 Apr 1980 | Fts Systems, Inc. | Tissue freeze dryer |
US4232453 * | 25 Sep 1978 | 11 Nov 1980 | C. Reichert Optische Werke, Ag | Device for freeze drying and synthetic resin impregnation when necessary of small biological objects for electron microscopic examination |
US4248821 * | 25 Jul 1979 | 3 Feb 1981 | Dellen Adrian F Van | Method and device for embedding a specimen for microscopic examination |
US4266111 * | 11 Jan 1979 | 5 May 1981 | Wentgate Engineers (1976) Limited | Apparatus for transferring work through a region of reduced pressure for the performance of an operation thereon |
US4269713 * | 31 Aug 1979 | 26 May 1981 | Kuraray Co., Ltd. | Ethylene-vinyl alcohol copolymer membrane and a method for producing the same |
US4278623 * | 11 Jan 1977 | 14 Jul 1981 | Union Carbide Corporation | Ultra-fine fibers and method for making same |
US4278701 * | 8 Nov 1979 | 14 Jul 1981 | Hagens Gunther Von | Animal and vegetal tissues permanently preserved by synthetic resin impregnation |
US4331591 * | 1 Oct 1979 | 25 May 1982 | Ciba-Geigy Corporation | Chemical process for the production of α-aminophosphonic acids and peptide derivatives |
US4336691 * | 13 Dec 1979 | 29 Jun 1982 | The Board Of Trustees Of The Leland Stanford Junior University | Cryojet rapid freezing apparatus |
US4337240 * | 7 Mar 1979 | 29 Jun 1982 | New England Nuclear Corporation | Denatured albumin complexes for radioscintigraphic imaging and evaluation of reticuloendothelial systems |
US4347671 * | 3 Apr 1980 | 7 Sep 1982 | Kernforschungsanlage Juich, Gesellschaft mit beschrankter Haftung | Vacuum-drying method and apparatus |
US4379003 * | 30 Jul 1980 | 5 Apr 1983 | Bell Telephone Laboratories, Incorporated | Magnetic devices by selective reduction of oxides |
US4449305 * | 17 Jun 1982 | 22 May 1984 | Societe D'utilisation Scientifique Et Industrielle Du Froid Usifroid | Freeze-drying apparatus |
US4510169 * | 23 Aug 1983 | 9 Apr 1985 | The Board Of Regents, The University Of Texas | Method and apparatus for cryopreparing biological tissue for ultrastructural analysis |
US4567847 * | 30 Nov 1984 | 4 Feb 1986 | Board Of Regents, The University Of Texas System | Apparatus and method for cryopreparing biological tissue for ultrastructural analysis |
US4619257 * | 30 Nov 1984 | 28 Oct 1986 | Board Of Regents, The University Of Texas System | Apparatus and method for cryopreparing corneal tissue for surgical procedures |
EP0136014A2 * | 8 Aug 1984 | 3 Apr 1985 | Board Of Regents The University Of Texas System | Method and apparatus for cryopreparing biological tissue for ultrastructural analysis |
EP0184417A2 * | 2 Dec 1985 | 11 Jun 1986 | Board Of Regents The University Of Texas System | Apparatus and method for cryopreparing biological tissue for ultrastructural analysis |
Reference | ||
1 | * | G. Smolin and R. Throft, The Cornea, Scientific Foundations and Clinical Practice, 437 464 (1983). |
2 | G. Smolin and R. Throft, The Cornea, Scientific Foundations and Clinical Practice, 437-464 (1983). | |
3 | * | J. Linner, S. Livesey, D. Harrison, A. Steiner, A New Technique for Removal of Amorphous Phase Tissue Without Ice Crystal Damage: A Preparative Method for Ultrastructural Analysis and Immunoelectron Microscopy, Journal of Histochemistry and Cytochemistry, 34, 1123 1135 (1986). |
4 | J. Linner, S. Livesey, D. Harrison, A. Steiner, A New Technique for Removal of Amorphous Phase Tissue Without Ice Crystal Damage: A Preparative Method for Ultrastructural Analysis and Immunoelectron Microscopy, Journal of Histochemistry and Cytochemistry, 34, 1123-1135 (1986). | |
5 | * | Sleytr, U. B. and Robards, A. W., Understanding the Artefact Problem in Freeze Fracture Replication: A Review, Journal of Microscopy, 126, 101 122 (1982). |
6 | Sleytr, U. B. and Robards, A. W., Understanding the Artefact Problem in Freeze-Fracture Replication: A Review, Journal of Microscopy, 126, 101-122 (1982). | |
7 | * | T. Casey and D. Mayer, Corneal Grafting, Principles Practice, 81 86, 271 280 (1984). |
8 | T. Casey and D. Mayer, Corneal Grafting, Principles Practice, 81-86, 271-280 (1984). | |
9 | * | Terracio, L. and Schwabe, K. G., Freezing and Drying of Biological Tissues for Election Microscopy, Journal of Histochemistry and Cytochemistry, 29, 1021 1028 (1981). |
10 | Terracio, L. and Schwabe, K. G., Freezing and Drying of Biological Tissues for Election Microscopy, Journal of Histochemistry and Cytochemistry, 29, 1021-1028 (1981). |
Citing Patent | Filing date | Publication date | Applicant | Title |
US4964280 * | 17 Aug 1989 | 23 Oct 1990 | Board Of Regents | Method and apparatus for cryopreparing biological tissue |
US5154007 * | 13 Mar 1990 | 13 Oct 1992 | Board Of Regents University Of Texas System | Method and apparatus for cryopreparing biological tissue |
US5431952 * | 28 Feb 1994 | 11 Jul 1995 | Board Of Trustees Operating Michigan State University | Method for preservation of biological tissue |
US6652583 | 9 Apr 2001 | 25 Nov 2003 | Rhode Island Hospital | Cardiac valve replacement |
US7665226 * | 11 Apr 2005 | 23 Feb 2010 | Kitakyushu Foundation For The Advancement Of Industry, Science & Technology | Method for drying under reduced pressure using microwaves |
US7832216 * | 16 Mar 2007 | 16 Nov 2010 | Siemens Plc | Apparatus for cooling |
US7966746 * | 24 Apr 2007 | 28 Jun 2011 | Medical Instill Technologies, LLC | Needle penetrable and laser resealable lyophilization method |
US8056256 * | 17 Sep 2008 | 15 Nov 2011 | Slack Associates, Inc. | Method for reconditioning FCR APG-68 tactical radar units |
US8171652 | 27 Jun 2011 | 8 May 2012 | Medical Instill Technologies, Inc. | Penetrable and resealable lyophilization method |
US8272411 | 3 Aug 2009 | 25 Sep 2012 | Medical Instill Technologies, Inc. | Lyophilization method and device |
US8701307 | 29 Nov 2012 | 22 Apr 2014 | Howard C. Slack | Method for cleaning and reconditioning FCR APG-68 tactical radar units |
US8904664 | 17 Jan 2013 | 9 Dec 2014 | Mimedx Group, Inc. | Dehydration device and methods for drying biological materials |
US9180145 | 9 Oct 2013 | 10 Nov 2015 | Mimedx Group, Inc. | Compositions and methods for recruiting and localizing stem cells |
US9222728 | 24 Apr 2007 | 29 Dec 2015 | Medinstill Development Llc | Penetrable and resealable lyophilization device |
US9463206 | 18 Dec 2012 | 11 Oct 2016 | Mimedx Group, Inc. | Cross-linked dehydrated placental tissue grafts and methods for making and using the same |
US9555062 | 4 Nov 2014 | 31 Jan 2017 | Mimedx Group, Inc. | Dehydration device and methods for drying biological materials |
US20040106991 * | 24 Nov 2003 | 3 Jun 2004 | Hopkins Richard A. | Cardiac valve replacement |
US20040241043 * | 19 Mar 2004 | 2 Dec 2004 | Stephan Sattler | Automatic analyzer |
US20060070392 * | 5 Oct 2004 | 6 Apr 2006 | Washington University | Apparatus for freezing a biological sample |
US20070214821 * | 16 Mar 2007 | 20 Sep 2007 | Siemens Magnet Technology Ltd. | Apparatus for cooling |
US20070271811 * | 11 Apr 2005 | 29 Nov 2007 | Takaharu Tsuruta | Method And Apparatus For Drying Under Reduced Pressure Using Microwaves |
US20080028632 * | 24 Apr 2007 | 7 Feb 2008 | Daniel Py | Needle penetrable and laser resealable lyophilization device and related method |
US20100064541 * | 17 Sep 2008 | 18 Mar 2010 | Slack Howard C | Method for reconditioning fcr apg-68 tactical radar units |
USRE33567 * | 21 Oct 1988 | 9 Apr 1991 | Micromeritics Instrument Corporation | Temperature controlling apparatus for use with pore volume and surface area analyzers |
CN101105358B | 19 Mar 2007 | 3 Nov 2010 | 英国西门子公司 | Cooling apparatus |
DE10312197A1 * | 19 Mar 2003 | 30 Sep 2004 | Roche Diagnostics Gmbh | Probenbehandlungsgerät, insbesondere automatisches Analysegerät |
EP2384702A1 * | 7 May 2010 | 9 Nov 2011 | Asociación Centro de Investigación Cooperativa en Biociencias - CIC bioGUNE | Device for storing cryo-grid storage boxes |
WO2009065585A2 | 20 Nov 2008 | 28 May 2009 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Ultra-rapid freezing device and method |
WO2009065585A3 * | 20 Nov 2008 | 11 Mar 2010 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Ultra-rapid freezing device and method |
WO2011138424A1 | 6 May 2011 | 10 Nov 2011 | ASOCIACIÓN CENTRO DE INVESTIGACIÓN COOPERATIVA EN BIOCIENCIAS - CIC bioGUNE | Device for storing cryo-grid storage boxes |
WO2014028328A1 * | 9 Aug 2013 | 20 Feb 2014 | Mimedx Group Inc. | Dehydration device and methods for drying biological materials |
U.S. Classification | 62/264, 34/92, 118/50.1, 62/51.1, 422/131, 435/2 |
International Classification | G01N1/42 |
Cooperative Classification | G01N1/42 |
European Classification | G01N1/42 |
Date | Code | Event | Description |
13 Apr 1987 | AS | Assignment | Owner name: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:LINNER, JOHN G.;LIVESEY, STEPHEN A.;REEL/FRAME:004693/0859 Effective date: 19870413 |
30 Sep 1991 | FPAY | Fee payment | Year of fee payment: 4 |
2 Jan 1996 | REMI | Maintenance fee reminder mailed | |
26 May 1996 | LAPS | Lapse for failure to pay maintenance fees | |
6 Aug 1996 | FP | Expired due to failure to pay maintenance fee | Effective date: 19960529 |
21 Jan 2000 | AS | Assignment | Owner name: TRANSAMERICA BUSINESS CREDIT CORPORATION, ILLINOIS Free format text: SECURITY AGREEMENT;ASSIGNOR:LIFECELL CORPORATION;REEL/FRAME:010539/0026 Effective date: 19991206 |
1 May 2008 | AS | Assignment | Owner name: LIFECELL CORPORATION, NEW JERSEY Free format text: RELEASE BY SECURED PARTY;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION (SUCCESSOR-IN-INTEREST TO TRANSAMERICA BUSINESS CREDIT CORPORATION);REEL/FRAME:020876/0934 Effective date: 20080423 |


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You may not have heard of it (yet), but it could be the future of world motorsport
Is it a bird? Is it a plane? Well, sort of on that last one actually, or at least that's where the DeltaWing Le Mans prototype gets its name from. Think of Concorde (you probably already were) and you're somewhere near.
The DeltaWing project has been knocking around for a while, and though having secured a place at this year's Le Mans 24 Hours under the Automobile Club de L'Ouest's 'Project 56' experimental technology entry, it hasn't had an engine to power it. Until now.
Nissan has now revealed it will be backing the DeltaWing project as an official partner with the tripod-like racer getting a 300hp, 1.6-litre turbocharged four-cylinder motor loosely related to that of the top-spec Juke 1.6 DIG-T. Not the sonorous V12 or intricate experimental petrol-electric hybrid powertrain you might have been hoping for given the car's Batman's-trackday-toy looks. But the stealthy racer is really quite an innovative, well thought out and truly relevant project, based on existing technology in order to keep costs (relatively) low and maximise efficiency.
And there's that word most of us wouldn't associate with the glamour and excitement of motor racing - efficiency. But while most driving enthusiasts see it as a byword for dull and drab motoring, with power reined in to improve fuel economy efficiency in racing actually means more speed.
The whole concept has been based on a strategy of half the weight and half the drag of a conventional LMP car, and in doing so has allowed the DeltaWing's engineers to halve the tyres' effective contact patch at the front, therefore halving the friction and drag thanks to that cigar-like front profile. Importantly, that means fuel consumption is also cut by 50 per cent.
According to DeltaWing's Chief Designer Ben Bowlby, "to halve the drag the DeltaWing doesn't employ any wings, relying solely on downforce created by ground effect and the car's body." That means it'll be extremely slippery down the 3.7-mile long Mulsanne Straight and the reason why it can achieve speeds of a modern prototype with under half the power. But more importantly, it'll be affected less by turbulent air in traffic - a massive factor with 56 cars on track, even if the Le Mans lap is eight and a half miles round.
On the pace
Bowlby is bullish about the programme's prospects, citing, "the ACO has given us a target lap time of 3:40 around Le Mans [that's in between LMP1 and LMP2 pace] with an intended top speed of 300km/h." The latter of which DeltaWing has already exceeded in testing.
For those of you thinking pie-in-the-sky stuff and recalling examples such as Aston Martin's ambitious but ultimately doomed attempt at Le Mans last year (the DW is actually loosely based on last year's Aston LMP1 chassis), you might be surprised.
With backing from Dan Gurney's All American Racing outfit, American Le Mans Series founder and owner Don Panoz, two-time ALMS champions Highcroft Racing and now the might of Nissan in the engine department, DeltaWing carries some serious clout.
So how does it drive?
According to Marino Franchitti, now confirmed to drive the car at Le Mans, "it's like a normal racing car - it has its own idiosyncrasies but it's easy to drive. You have to apex a corner with the rear wheels as opposed to the fronts in a normal car, due to the rear being the widest point, but it handles really well and is easy to position. Everything is very progressive and the aerodynamic balance is very consistent and controllable - it's just like a normal car when it breaks away."
Under that slim nose are two specially developed tyres from Michelin just four inches in width. And that's all the rubber DeltaWing has to play with up front. Yet despite the comparative lack of rubber the team is boasting of corner speeds similar to more conventional prototypes.
That's because the car's weight distribution is biased rearwards to the tune of 73:27, putting all the mass behind the centre of gravity and offering excellent stability, especially in a straight line and under braking. The car's brake balance is weighted towards the rear too, making use of the significantly larger 12.5-inch wide rear tyres. That rearward weight distribution should mean less weight transfer under braking (well over 50 per cent of the braking force is behind the centre of gravity), as well as more traction under power, equating to a less wayward tail than you might otherwise imagine.
Going the distance
"With only 40 litres of fuel to carry and much less mass we're hoping to go further on a stint than the regular prototypes," reckons Franchitti. And we can well believe him. From the time the Scot has already spent behind the wheel of the DeltaWing, he outlined that triple-stinting tyres at Le Mans won't be an issue, and that the car's inherent, almost benign balance - despite its outlandish looks - means degradation in the rubber won't upset the chassis or bite the driver.
It's a futuristic looking piece of kit, and there are some proper innovations in the design - not least the clever "half everything" principle and its manifestation in the metal (or should that be carbon fibre). The developments in efficiency through reduced drag, lower mass and a small turbocharged engine are relevant to the future of road cars as well.
According to Nissan Europe General Manager, Darren Cox, "DeltaWing is about innovation and relevance to road cars. The developments in efficiency will challenge the way people think about motorsport and benefit road car progress in a sustainable way through real-world technology - downsizing and improving aerodynamic efficiency as two examples."
That's all well and good for the future, but in the here and now the DeltaWing has a job to do.
"The car is the first real wholesale change since putting engines in the back of Formula 1 cars or the development of wings - fans are drawn to it and it's so important in engaging the next generation of sportscar followers. It's great to be involved in something so exciting," believes Franchitti.
It won't be competing for the podium proper, unfortunately, as it's in an experimental class of one. The team has been warned by the ACO to not get in the way of anybody's race, and it's well aware of that. Given the claims it's making though, Franchitti and Co. might not have to be checking their mirrors all that frequently.

subset: commoncrawl


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High Caliber Hunting Review
Price: $2.99 (currently on sale for $0.99)
Version: 1.0.0
App Reviewed on: iPhone 4
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Hunting games are just one of those staples of the iOS platform that have been around since the inception of game development on the device. Though it is easy to assume that the primary consumer of said titles might have a noticeable twang to their voice or a close resemblance to a spouse, several developers (including Activision) have already catered to this unorthodox audience. Electronic Arts has decided that they can no longer allow this lucrative business to remain untouched either, recently releasing High Caliber Hunting as their answer to the popular Deer Hunter brand. Is this another shameless regurgitation, championed by the EA of old, or are we looking at the birth of something new and special?
If you are not much for reading long pages of contextual dialog, you are in luck. High Caliber Hunting lacks heavily in the plotline department. All you need to know is that you are wearing camo, hiding in a bush somewhere, and looking to plant Bambi in a pre-mature grave. The closest you will get to progressing through the game is selecting the different hunting regions that you can unlock through gameplay, consisting of broad sweeping locations such as Texas, Alaska, Canada, and Iowa.
Where the game lacks in the linear structure department, it makes up for in solid variety of environments. Players will find themselves traversing through the plains of Texas, the wintery wonderland of "The Great White North," or whatever the hell Iowa has to offer (cornfields, perhaps?). Accordingly, the wildlife also shifts dramatically depending upon your setting, but that will never change their behaviors of walking from left to right across the screen, looking for some unseen foliage to consume.
The biggest issue with High Caliber Hunting is the one dimensional gameplay. Everything is see Thumper, shoot Thumper, with little to change up the formula. This is perfectly sufficient to support the title through several short play sessions, but eventually you find yourself craving more. Whether it be more weapons, more mobility, or even more game types, everything feels more piecemeal than what you might expect to find in a full product. That is why it should come as no surprise that from the launch day, that additional downloadable content has been available. Sadly, this isn't just a side effect of the publisher, any more, it is becoming the norm.
It would be a crime to deprive hunting fans of a game like High Caliber Hunting, but if you are on the fence, you may be better served placing your deer blind elsewhere and waiting for another target to trot along.


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Imatinib Mesylate in Treating Patients With Myelofibrosis
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Condition | Intervention | Phase |
Chronic Myelomonocytic Leukemia Essential Thrombocythemia Polycythemia Vera Primary Myelofibrosis |
Drug: imatinib mesylate Other: laboratory biomarker analysis |
Phase 2 |
Study Type: | Interventional |
Study Design: | Intervention Model: Single Group Assignment Masking: Open Label Primary Purpose: Treatment |
Official Title: | A Phase II Study Of Gleevec (Imatinib Mesylate Formerly Known as STI-571) In Patients With Myelofibrosis |
- Clinical responses in terms of improvement in anemia and splenomegaly as previously published for myelofibrosis [ Time Frame: Up to 12 months ]
- Frequency of adverse events graded according to the National Cancer Institute Common Toxicity Criteria (NCI CTC) v2.0 [ Time Frame: Up to 12 months ]Exact 95% confidence intervals generated using the binomial distribution.
- Progression-free survival [ Time Frame: Up to 12 months ]Analyzed using the Kaplan-Meier method.
- Bone marrow response [ Time Frame: Up to 12 months ]
Enrollment: | 18 |
Study Start Date: | April 2002 |
Primary Completion Date: | August 2007 (Final data collection date for primary outcome measure) |
Arms | Assigned Interventions |
Experimental: Treatment (imatinib mesylate)
Patients receive oral imatinib mesylate once or twice daily on days 1-28. Courses repeat every 28 days for 12 months in the absence of disease progression or unacceptable toxicity.
Drug: imatinib mesylate
Given orally
Other Names:
Other: laboratory biomarker analysis
Correlative studies
Detailed Description:
I. To determine the response rate (complete and partial) to STI-571 in patients with myelofibrosis.
II. To determine the safety of STI-571 in patients with myelofibrosis.
I. To determine the effects of STI-571 on bone marrow morphology (including effects on marrow fibrosis, osteosclerosis and cellularity) in patients with myelofibrosis.
II. To assess the effects of STI-571 on surrogate biologic endpoints including PDGFR expression (by immunohistochemistry), PDGFR signaling, and circulating progenitor (CD34 positive) cells.
III. To determine the effects of STI-571 on bone marrow cytogenetics in patients with an abnormal karyotype.
OUTLINE: This is a multicenter study. Patients are stratified according to Dupriez risk score (low vs intermediate vs high).
Patients receive oral imatinib mesylate once or twice daily on days 1-28. Courses repeat every 28 days for 12 months in the absence of disease progression or unacceptable toxicity.
Ages Eligible for Study: | 18 Years and older (Adult, Senior) |
Sexes Eligible for Study: | All |
Accepts Healthy Volunteers: | No |
Inclusion Criteria:
Patients must have histologic confirmation of one of the following diseases-
- Myeloid metaplasia with myelofibrosis (this includes all subtypes- chronic idiopathic myelofibrosis or agnogenic myeloid metaplasia, post thrombocythemic and post polycythemic myelofibrosis) or
- Chronic myelomonocytic leukemia (CMMOL) with t(5;12)(q31-33;p12) or TEL-PDGFRβ rearrangement; patients with CMMOL and the t(5;7)(q31-33;q11.2) or other chromosomal translocations resulting in activation of PDGFR will also be eligible
- Patients must have anemia (hemoglobin < 11 g/dL) or palpable splenomegaly (measured in cm from costal margin- to eligible); patients with palpable splenomegaly must have spleen size documented ultrasonographically as well; they must also meet standard diagnostic criteria for MMM* or CMMOL; patients with MMM must have thrombocytopenia (platelet count < 100 x 10^9/L) to be eligible; they must be Philadelphia chromosome or (BCR/ABL) rearrangement negative
- Patients with CMMOL must also have the t(5;12)(q31-33;p12) or TEL-PDGFRβ rearrangement to be eligible
The Italian diagnostic criteria for MMM
Necessary criteria
- Diffuse bone marrow fibrosis
- Absence of the Philadelphia chromosome or BCR-ABL rearrangement in peripheral blood cells
Optional criteria
- Splenomegaly of any grade
- Anisopoikilocytosis with tear drop erythrocytes
- Presence of circulating immature myeloid cells
- Presence of circulating erythroblasts
- Presence of clusters of megakaryoblasts and anomalous megakaryocytes in bone marrow sections
- Myeloid metaplasia
Diagnosis of MMM is acceptable if the following combinations are present
- The two necessary criteria plus any other two optional criteria when splenomegaly is present OR
- The two necessary criteria plus any other four optional criteria when splenomegaly is absent
- Patients may have had prior chemotherapy or radiation therapy including splenic irradiation; prior therapy with erythropoietin, GCSF or androgenic steroids is also permitted; there is no limit to number of prior regimens received; at least 4 weeks must have elapsed since prior chemo, radiation or other therapy
- ECOG performance status =< 2 (Karnofsky >= 60%)
- Total bilirubin < 1.5 X institutional upper limit of normal
- AST(SGOT)/ALT(SGPT) <2.5 X institutional upper limit of normal unless due to disease
- Serum creatinine < 2 X institutional upper limit of normal
- Patients must not be pregnant or nursing because STI-571 at the recommended therapeutic dose may be harmful to the developing fetus or newborn; for this reason women of child-bearing potential and men must agree to use an effective contraceptive method; women of reproductive potential must have a negative pregnancy test within 7 days prior to registration; since interactions with oral contraceptives cannot be excluded at present, male and female patients of reproductive potential must agree to employ an effective barrier method of birth control throughout the study and for up to 3 months following discontinuation of study drug
- Ability to understand and the willingness to sign a written informed consent document
World Health Organization (WHO) diagnostic criteria for CMMOL:
- Persistent peripheral blood monocytosis > 1 x 10^9/L
- Absence of the Philadelphia chromosome or BCR/ABL fusion gene
- Fewer than 20% blasts in the blood or bone marrow
Dysplasia in one or more myeloid lineages; if myelodysplasia is absent or minimal, the diagnosis of CMML may still be made if the other criteria (1-3) are met and:
- An acquired clonal cytogenetic abnormality is present in the marrow cells
- The monocytosis has been persistent for at least 3 months
- Other causes for monocytosis have been excluded
Exclusion Criteria:
- Patients who have had chemotherapy or radiotherapy within 4 weeks (6 weeks for nitrosoureas or mitomycin C) prior to entering the study or those who have not recovered from adverse events due to agents administered more than 4 weeks earlier
- Patients may not be receiving any other investigational agents
- History of allergic reactions attributed to compounds of similar chemical or biologic composition to STI-571
- Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements
- Pregnant women are excluded from this study because STI-571 is an agent with the potential for teratogenic or abortifacient effects; because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with STI-571, STI-571 should not be administered patients who are breastfeeding
- HIV-positive patients receiving combination anti-retroviral therapy are excluded from the study because of possible pharmacokinetic interactions with STI-571; appropriate studies will be undertaken in patients receiving combination anti-retroviral therapy when indicated
- Because warfarin is metabolized through the CYP450 system, and since gastrointestinal bleeding may occur with STI-571, no therapeutic anticoagulation with warfarin will be permitted in patients participating in this study; as an alternative, therapeutic anticoagulation may be accomplished using low-molecular weight heparin (e.g. Lovenox) or heparin
Please refer to this study by its ClinicalTrials.gov identifier: NCT00039416
United States, Illinois | |
University of Chicago | |
Chicago, Illinois, United States, 60637 |
Principal Investigator: | Olatoyosi Odenike | University of Chicago |
Responsible Party: | National Cancer Institute (NCI) |
ClinicalTrials.gov Identifier: | NCT00039416 History of Changes |
Other Study ID Numbers: |
NCI-2012-02895 NCI-2012-02895 ( Registry Identifier: CTRP (Clinical Trial Reporting Program) ) CDR0000069381 11498A ( Other Identifier: University of Chicago ) 5669 ( Other Identifier: CTEP ) N01CM62201 ( US NIH Grant/Contract Award Number ) P30CA014599 ( US NIH Grant/Contract Award Number ) |
Study First Received: | June 6, 2002 |
Last Updated: | February 24, 2014 |
Additional relevant MeSH terms:
Primary Myelofibrosis Polycythemia Polycythemia Vera Thrombocytosis Thrombocythemia, Essential Leukemia, Myelomonocytic, Chronic Myeloproliferative Disorders Bone Marrow Diseases Hematologic Diseases Blood Platelet Disorders Blood Coagulation Disorders |
Hemorrhagic Disorders Leukemia, Myeloid Leukemia Neoplasms by Histologic Type Neoplasms Myelodysplastic-Myeloproliferative Diseases Imatinib Mesylate Antineoplastic Agents Protein Kinase Inhibitors Enzyme Inhibitors Molecular Mechanisms of Pharmacological Action |
ClinicalTrials.gov processed this record on March 27, 2017


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With Senator Grassley's 99 county tour stopping through this week, I would like to take the opportunity to advocate for policies to benefit our state with one of the most powerful people in the Senate.
The election is next Tuesday, November 4th, and the importance of paying close attention to who is asking for your vote cannot be understated.
Joe, I enjoyed your editorial "Abortion shouldn't be only Pro-life Cause" (Joe Foreman column Feb. 13) and I'm going to prove your point by focusing on just one thing.


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Tuesday, March 13, 2012
Tomato & Vodka Spaghetti
Whenever I make a big Italian meal for a special occasion this Tomato and Vodka Spaghetti always features as the second course. It is super easy to make but so delicious and will go down a treat with everyone. I just made this for my Mum's Birthday meal and it was a hit. I like to save a tablespoon of the vodka to add to the end so that you can still taste it but if you prefer you can just add it all at the same time, before you add the cream. And of course if you prefer you can miss the vodka out and still have a delicious sauce.
The recipe states 500g of spaghetti so as part of large meal that would feed 8-10 people, but for a main course 5-6 depending how hungry you all are!
2 tbsp olive oil
3 fat cloves of garlic, crushed
700ml jar (approx.) passata
1 tsp sugar (optional)
Salt & pepper
4 tbsp vodka
125ml cream
1 tbsp butter
Extra virgin olive oil
500g spaghetti
Heat the olive oil in a frying pan then gently cook the garlic until softened but not coloured. Add the passata then add a little water to the jar and swirl it round to get all of the tomatoes. Add the tomato and water to the pan then add the sugar, salt and pepper. Leave to simmer gently for 20-30 minutes then taste to check the seasoning.
While the sauce is simmering cook the spaghetti in a large pan of boiling, salted water according to the instructions on the packet. When the pasta is ready reserve some of the cooking water, then drain the spaghetti.
Add 3 tbsp of the vodka and leave to simmer for another minute or 2 then add the cream to the sauce and continue to gently cook for a further couple of minutes. Turn off the heat and add the remaining tablespoon of vodka to the sauce. Add the drained spaghetti and toss it through the sauce along with some of the cooking water. Add the tablespoon of butter and a good drizzle of olive oil and stir until the spaghetti is evenly coated and you have a silky smooth sauce. Serve at once with a little extra freshly ground black pepper and olive oil.
11 comments:
- AnonymousMarch 14, 2012 at 9:32 AM
Ah, memories of New Year in Melrose. My assistant chef could have been more efficient. Mind you she will probably claim the same!! It tasted great though despite the chaos.
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