Tissue preparation apparatus and method

An apparatus for mounting a tissue specimen on a chuck for sectioning in a cryostat including a base, a clamp for receiving a tissue preparation slide, a chuck holder secured to the base for holding the chuck in a predetermined orientation relative to the clamp, a support secured to the base and extending therefrom and terminating in a distal end, and, means for slideably securing said clamp to said support. The clamp is positionable between a first location in which the clamp is at a minimum distance from the chuck holder and a second location in which the clamp is at a maximum distance from the chuck holder. A method for using the apparatus is likewise disclosed.

TECHNICAL FIELD 
This invention relates to the preparation of tissue samples for sectioning, 
and specifically to preparation for tissue sectioning incidental to the 
Mohs tissue surgical technique. 
BACKGROUND ART 
The Mohs tissue surgical technique, which was developed by Frederic E. Mohs 
of Madison, Wis., is a method of removing skin tumors such as cutaneous 
malignancies and certain major carcinomas, and evaluating sections (very 
thin slices) of the tissue under a microscope. In order for Mohs surgery 
to be successful, high quality horizontally cut frozen tissue sections 
must be produced and microscopically reviewed to determine whether any 
residual tumor has spread beyond the tissue sample. 
The Mohs process begins with the excising of a tissue sample which includes 
the skin tumor. The tissue sample is then marked for orientation purposes, 
for example, by scoring with a scalpel and marking the sample immediately 
left or right of the score with ink, to allow the surgeon to determine 
where additional excisions must occur should the results of an inspection 
of a microscopic section of tissue sample indicate that the tumor has 
spread beyond the excised tissue sample. If residual tumor is indicated by 
the microscopic inspection, additional tissue is excised, and the 
procedure is repeated until there are no indications that the tumor has 
spread beyond the excised tissue samples. 
The surface of the excised tissue to be inspected is the curved, generally 
bowl-shaped surface that results from the passage of the scalpel below the 
surface of the skin. This bowl-shaped surface must be converted to a 
planar surface in order to be sliced by a device known in the art as a 
microtome. The microtome is typically located in a refrigerated unit, 
called a cryostat, which is capable of maintaining an internal temperature 
of -20 degrees Celsius or below. 
To enable this sectioning or slicing, the tissue must be mounted on a 
cryostat chuck with the flattened or planar surface exposed and 
perpendicular to the long axis of the cryostat chuck. The chuck and 
attached tissue sample are then placed into a chuck fixture in the 
cryostat where the tissue is cut into frozen sections having a thickness 
of only five to seven micrometers. Each section is then placed on a 
microscope slide and the section is stained by dipping the slide in 
solvents and various dye solutions. After the desired amount of staining 
is achieved, a clear glue-like substance is used to attach a thin layer of 
glass called a "cover slip". 
The dye causes cell walls, cell contents, and also extra-cellular material 
within the section, which would normally appear transparent, to be readily 
visible when viewed under a microscope for the presence of malignant cells 
and also for a host of inflammatory reaction to those malignant cells. If 
the surgeon determines that carcinoma cells are present in the section, 
further excision of tissue from the patient is necessary. 
As those skilled in the art will readily appreciate, if the first section 
does not include all of the formerly bowl-shaped surface, which may occur 
if the planar surface is not parallel to the path of relative movement 
between the cryostat knife and the tissue sample, then the surgeon must 
review subsequent deeper sections until a determination can be made that 
all of the formerly bowl-shaped surface has been evaluated. This can be a 
time consuming effort, since each section must be stained and 
microscopically examined and interpreted by the surgeon before a 
determination can be made as to whether further excision of tissue is 
necessary. Therefore, orientation of the mounted tissue sample so that the 
planar surface is parallel to the path of the cryostat knife is key to 
ensuring that the cutting time involved in sectioning the tissue sample 
will be a minimum, since that means that the first tissue section may be 
the only one that the surgeon needs to evaluate. 
The prior art discloses various methods and/or devices which attempt to 
solve this problem of flattening the bowl-shaped surface to obtain a 
perfect section (as defined herein below). The first method, referred to 
as the American Optical Heat Extractor, involves use of a copper jig to 
hold a cryostat chuck, and a solid metal cylinder which is movably 
attached to the jig. (This type of jig and metal cylinder was initially 
offered on cryostats manufactured by American Optical, and currently 
standard equipment on most cryostats regardless of the manufacturer.) The 
jig, chuck and cylinder are maintained at cryostat temperatures (-20 
degrees C.), and O.C.T. fluid (a clear, tissue mounting fluid such as this 
is sold under the brand name Tissue Tek II O.C.T. Compound, by Miles 
Laboratories, Inc.) is placed onto the tissue mounting surface of the 
chuck. (The O.C.T. has the general consistency and viscosity of egg 
whites, and freezes at a temperature below that at which the tissue 
samples freeze.) A tissue sample is then immediately placed onto the 
liquid O.C.T. with the bowl-shaped surface facing away from the tissue 
mounting surface of the chuck. The metal cylinder is then lowered onto the 
tissue sample, sandwiching the specimen between the chuck and the metal 
cylinder and extracting heat from both the tissue specimen and the O.C.T. 
After 30 seconds or so when both the tissue specimen and the O.C.T. are 
frozen, the cylinder is somehow jarred to free it from the tissue specimen 
and the O.C.T., leaving the tissue specimen and the O.C.T. frozen to the 
chuck. Once frozen, the O.C.T. acts as a glue by bonding the tissue to the 
chuck, and also surrounding and supporting the tissue sample so it can be 
subsequently sliced by the microtome within the cryostat. Unfortunately, 
the first tissue section produced using this method often fails to include 
the complete periphery of the tissue specimen, requiring the review of 
multiple sequential tissue sections to ensure that no tumor is present on 
the formerly bowl-shaped surface. 
A second method, referred to as the American Optical Tissue Presser, is a 
variation on the American Optical Heat Extractor, but includes a spring 
that partially supports the metal cylinder so that the full weight of the 
cylinder does not rest on the tissue specimen. Unfortunately, this method 
also often fails to produce first tissue sections which include the 
complete periphery of the tissue specimen. Accordingly, the review of 
multiple sequential tissue sections to ensure that no tumor is present on 
the formerly bowl-shaped surface is often required. 
A third method, referred to as the Bard Parker scalpel handle method, 
involves freezing the tissue specimen to the chuck while using the flat 
handle of a metal scalpel to flatten the bowl shaped surface while the 
temperature drops. The surgeon moves the scalpel handle back and forth 
across the tissue sample and "eyeballs" the relative flatness of the 
bowl-shaped surface. The scalpel is removed before it has a chance to 
stick to the freezing O.C.T. and tissue sample. This line-of-sight method 
becomes less exact when the edges of the tissue sample curl under or sink 
lower than the back-and-forth path of the scalpel handle. As a result, 
this method also often fails to produce first tissue sections which 
include the complete periphery of the tissue specimen. 
A fourth method, referred to as the glass slide method, is the same as the 
Bard Parker scalpel handle method, except that a glass microscope slide is 
substituted for the scalpel handle. Alternately, the tissue specimen may 
be frozen to the glass slide, a drop of O.C.T. placed on the tissue, and 
then the slide is inverted and frozen to the chuck using the line-of-sight 
method. A fifth method, referred to as the forceps method, is the same as 
the Bard Parker scalpel handle method, except that a forceps handle is 
substituted for the scalpel handle. Both the fourth and fifth methods 
suffer from the same reliance on the "eyeball" method of the Bard Parker 
method, and accordingly, each method also often fails to produce first 
tissue sections which include the complete periphery of the tissue 
specimen. 
A sixth method, referred to as the Miami Special, involves a specially 
designed pair of pliers having a chuck holder attached to one jaw and a 
flat metal plate attached to the other jaw. The bowl-shaped surface of the 
tissue specimen is frozen to the flat metal plate, and then a tissue chuck 
with O.C.T. on the tissue mounting surface thereof is placed into the 
chuck holder with the tissue mounting surface of the chuck facing the 
tissue specimen. The jaws are then closed, sandwiching the tissue specimen 
and O.C.T. between the tissue mounting surface of the chuck and the flat 
metal plate. A coolant is then used to freeze the O.C.T., usually by 
immersing the end of the pliers holding the tissue sample in liquid 
nitrogen. While the Miami Special represents a significant improvement 
over the "eyeball" methods discussed above, the flat metal plate is only 
parallel to the tissue mounting surface of the chuck at one position of 
the jaws, and therefore the Miami Special almost always yields a 
flattened, formerly bowl-shaped surface that is at a slant relative to the 
tissue mounting surface of the chuck. Accordingly, the Miami Special also 
often fails to produce first tissue sections which include the complete 
periphery of the tissue specimen. 
A seventh method involves use of a cryostat chuck, a polished metal disk, 
and a two-part metal jig. The bowl-shaped surface of the tissue sample is 
flattened by cooling the metal disk to -20 degrees Celsius and rolling the 
bowl-shaped surface against the metal disk. The tissue freezes to the 
metal disk which prevents return of the original bowl-shape, and the disk 
and tissue are placed in a cryostat to prevent thawing of the tissue. 
While the metal disk and attached tissue are maintained at a subfreezing 
temperature, a warm cryostat chuck is covered with O.C.T. fluid, and 
placed into a fixed portion of a jig located in the cryostat. When 
solidification of the O.C.T begins, the metal disk is placed in a mobile 
portion of the jig, and brought into apposition with the partially 
solidified O.C.T. compound by sliding the mobile portion of the jig onto 
the fixed portion of the jig, and allowing all components to stabilize at 
-20 degrees Celsius. The mobile jig is then removed, and the metal disk is 
"popped" off leaving the tissue sample on the cryostat chuck. An alternate 
version of this method involves the use of a nitrogen cooled, polished 
metal disk to eliminate the need to work within the confines of the 
cryostat when flattening the tissue sample. 
An eighth method, involves a Cryomold, something akin to a clear, thin 
plastic envelope in the shape of an ice cube tray for a single cube. A 
thin layer of O.C.T. is added to the inside bottom of the Cryomold, which 
is then placed against the bowl-shaped surface of the tissue sample to be 
examined. The Cryomold is placed on the freezing bar within the cryostat, 
and, working within the confines of the Cryostat, the surgeon flattens the 
tissue sample with forceps as the O.C.T. and tissue sample freeze. 
Additional O.C.T. is then added to fill the Cryomold. The tissue chuck 
then is placed on the gelatinous surface, and the entire arrangement, 
including the tissue sample, is allowed to freeze in the cryostat. After 
freezing is complete, the plastic envelope is peeled away and the tissue 
sample is ready for sectioning. 
One problem with the Cryomold is that, because the Cryomold is flexible, it 
must be remain on a hard, flat surface (such as the freezing bar in the 
cryostat) until the tissue sample has been flattened and frozen to the 
Cryomold with the O.C.T., and therefore actual freezing of the tissue 
sample to the bottom of the Cryomold cannot be directly observed. Since 
the O.C.T. on the bottom freezes uniformly, when the O.C.T. freezes at the 
positions where the peripheral edge is being held to the bottom, it is 
also freezing at those positions where the periphery is not being held to 
the bottom, so that when the surgeon seeks to freeze these other positions 
of the edge to the bottom, the O.C.T. has solidified and cannot be 
squeezed out, thereby supporting the edge off the bottom at these 
positions. When additional O.C.T. (at room temperature) is added to the 
Cryomold, the frozen tissue can thaw and curl at the peripheral edge, and 
due to the relatively large volume of O.C.T. which is required to fill the 
Cryomold, the freezing of the O.C.T. to the tissue chuck takes 
considerably longer than many other methods known in the art. If the 
tissue sample floats or curls into undesirable positions before complete 
freezing of the tissue sample and O.C.T. occurs, the tissue sample and 
O.C.T. must be thawed and the embedding process repeated until the tissue 
sample is frozen to the bottom of the Cryomold. Once frozen, the surgeon 
may raise the Cryomold from the freezing bar and view the bowl-shaped 
surface of the tissue sample to determine whether the entire periphery has 
been frozen to the inside bottom of the Cryomold. If the surgeon 
determines that the entire periphery of the bowl-shaped surface is not 
frozen to the bottom of the Cryomold, the tissue sample and O.C.T. must be 
thawed and the embedding process repeated until the entire periphery is 
visible. Since the Cryomold method uses O.C.T., which is clear (at room 
temperature, white when frozen), to bond the bowl-shaped surface of the 
tissue sample to the bottom of the Cryomold, it may not be readily 
apparent whether the entire periphery is located is a single plane as 
desired, or whether pockets of O.C.T. have lifted portions of the 
bowl-shaped surface off the bottom of the Cryomold. As a result of the 
foregoing, the surgeon may need to remove in excess of 300 microns of 
tissue before obtaining a perfect section. 
In a ninth method, a variation of the American Optical Heat Extractor 
referred to as the cork method, a frozen tissue "well" is prepared by 
making a ring of O.C.T. compound around a rubber stopper on a glass slide 
at -20 degrees Celsius. Upon freezing of the O.C.T., the stopper is 
removed, and the bottom of the well is warmed with a fingertip and the 
excess O.C.T. is removed with a cotton swab. The excised tissue is placed 
into the well with the bowl-shaped surface facing the slide and allowed to 
freeze inside the cryostat at -20 degrees Celsius while a metal probe is 
used to press the bowl-shaped surface against the glass slide during the 
freezing process. After the tissue is completely frozen, the well is 
filled with additional O.C.T. and a metal heat sink is applied for 
approximately 3 minutes to speed the freezing process and help flatten the 
tissue. The frozen tissue sample is then gently pushed off the slide after 
warming the undersurface of the slide with the fingertips. The tissue 
sample is then inverted and mounted onto a metal chuck with additional 
O.C.T. and the heat extractor at -20 degrees Celsius for approximately 1 
minute, and when broken away is ready for sectioning. When mounting the 
tissue sample to the grooved surface of the tissue chuck, the surface to 
be cut is visually aligned during freezing, again with the goal, often not 
attained, of mounting the flattened, formerly bowl-shaped surface of the 
specimen so that it is parallel to the grooved mounting surface of the 
tissue chuck. 
A tenth method, referred to as the Motley method, uses a cylindrical chuck 
holder within a sleeve which is vertically oriented and slideably 
positioned thereabout. The chuck holder includes a pipe for delivering 
liquid nitrogen into the sleeve (from a source which is controlled by a 
foot-actuated valve), and vent holes for allowing the gaseous nitrogen to 
escape from within the sleeve. The top of the sleeve defines a plane which 
is parallel to the plane in which the tissue chuck is held by the chuck 
holder. A microscope slide is placed on the top of the sleeve so as to 
form a bridge, and the bowl-shaped surface of the tissue sample is pressed 
into contact with the slide with forceps while liquid nitrogen is sprayed 
on the opposite side of the slide via the pipe, thus freezing the tissue 
sample to the slide. The slide and sleeve are lifted away from the chuck 
holder, and a tissue chuck having O.C.T. thereon (at room temperature) is 
then placed in the chuck holder. The slide is then inverted (so that the 
tissue sample is now frozen to the lower surface of the slide) and the 
sleeve and slide are then placed back over the chuck holder and, using 
both hands to support the sleeve and hold the slide to the top thereof, 
the surgeon slides the sleeve down over the chuck holder until the tissue 
sample rests in the O.C.T. on the tissue chuck. The foot pedal is then 
actuated to spray liquid nitrogen against the underside of the chuck until 
the O.C.T. freezes. The surgeon's finger is then used to warm the slide 
until the tissue separates therefrom. 
One drawback to the Motley method is that as the tissue sample is being 
pressed down onto the top side of the microscope slide, liquid nitrogen is 
sprayed against the bottom side, and so completeness of attachment of the 
tissue sample peripheral edge to the slide cannot be determined until the 
after the tissue sample is completely frozen and the slide can be flipped 
over and viewed, by which time frozen condensation will likely frost the 
slide, making inspection difficult. If inspection does reveal incomplete 
attachment, the tissue sample must be melted and the attachment process 
repeated. Slide breakage may occur due to the relatively large diameter of 
the sleeve and the force required to press some tissue samples flat 
against the slide, and because there is no seal between the microscope 
slide and the sleeve, escaping nitrogen gas blows out the top of the 
sleeve towards the surgeon and may splatter O.C.T. in the direction of the 
surgeon. Additionally, since the frozen tissue sample begins to warm as 
soon as the nitrogen spray ceases, time is of the essence in lowering the 
sleeve below the chuck holder, placing the chuck with O.C.T. thereon into 
the chuck holder, raising the sleeve, placing the slide with frozen tissue 
on top of the sleeve and lowering the sleeve until the tissue sample rests 
in the O.C.T. If this process takes too long, the tissue will melt away 
from the slide, and the chuck (with dripping O.C.T.) must be removed from 
the chuck holder and the process of freezing the tissue sample to the 
slide must be repeated. As the device is used, excess O.C.T. is likely to 
find its way between the chuck holder and the sleeve making raising and 
lowering of the sleeve more difficult. If the O.C.T. freezes to the sleeve 
and chuck holder, it may be impossible to remove the sleeve prior to 
removing the frozen chuck and tissue sample, making their removal 
difficult. If the O.C.T. freezes to the chuck and chuck holder, it may be 
impossible to remove the chuck from the chuck holder. 
An eleventh method, referred to as the cooled embedding head, eliminates 
the need to operate within the confines of the cryostat by utilizing an 
embedding head having a polished, planar metal surface which is cooled by 
CO.sub.2 to sub-freezing temperatures. The bowl-shaped surface of the 
tissue sample is flattened by manipulating the tissue to adhere to the 
cold metal of the polished surface so the once bowl-shaped surface is 
flattened down onto the head. With the tissue sample frozen to the 
embedding head, O.C.T. fluid is poured over the frozen tissue and, due to 
the temperature of the embedding head, the O.C.T. immediately begins to 
freeze. A tissue chuck received within a spring loaded tissue chuck holder 
and having a grooved mounting surface at room temperature is lowered by a 
system of levers, so that the grooved surface of the tissue chuck is 
brought into contact with the O.C.T. as is freezes. An additional nozzle 
through which CO.sub.2 gas can be sprayed is directed at the tissue chuck 
to facilitate rapid cooling of the tissue chuck and freezing of the O.C.T. 
to the tissue chuck. When the O.C.T. solidifies, the plane of the tissue 
chuck is parallel to that of the polished, planar metal surface of the 
embedding head and the tissue which is adhering to it. If the O.C.T. 
sufficiently adheres to the grooved surface of the tissue chuck, then the 
attached tissue sample embedded in the O.C.T. is forcibly separated from 
the polished, planar metal surface of the embedding head. The chuck with 
frozen tissue is then placed in the cryostat for tissue sectioning. 
One of the disadvantages of this latter method is that the surgeon has no 
way to determine whether the tissue sample is properly flattened against 
the embedding head until after the tissue sample is separated therefrom. 
Therefore, if for any reason the tissue sample failed to completely 
flatten against the embedding head (e.g. a crease is formed in the 
bowl-shaped surface during flattening of the sample, an air bubble is 
trapped between the embedding head and the tissue sample during the 
process of attaching the tissue sample to the embedding head, etc.), 
tissue sections cut from the tissue sample will not include the entire 
surface of the formerly bowl-shaped surface. If this situation goes 
undetected, the tissue section may not include cancerous material which 
was otherwise detectable. If the surgeon is somehow able to detect a 
crease or bubble in the tissue sample after freezing the tissue sample to 
the embedding head, the tissue sample must be thawed, rinsed and refrozen 
to the embedding head. However, such excessive thawing and refreezing of 
the tissue sample causes cell lysis, (breakage of the cell walls in the 
tissue sample and leakage of cell contents) which significantly changes 
the tendency of cells to absorb stain during the staining process 
described above, and gives cells a deflated and less defined architecture. 
This varied stain absorption and shrinkage of cells can make 
interpretation of the finished slides more difficult and error prone. 
Another disadvantage of this latter process is that once the O.C.T. is 
placed on top of the frozen tissue sample and embedding head, it 
immediately begins to cool, which leads to both condensation of humidity 
on the exposed surfaces of the O.C.T. and a dramatic increase in the 
viscosity of the O.C.T. As those skilled in the art will readily 
appreciate, the condensation becomes a frost which creates an interface 
between the tissue chuck and the O.C.T., and the increased viscosity 
reduces the tendency of the O.C.T. to flow into the voids of the textured 
surface of the tissue chuck, both of which may result in inadequate 
bonding of the O.C.T. to the tissue chuck and subsequent detachment of the 
tissue sample from the tissue chuck when the surgeon attempts to forcibly 
break away the frozen tissue sample and O.C.T. from the embedding head. 
Quickly lowering the tissue chuck onto the O.C.T. immediately after 
placing the O.C.T. on the embedding head can alleviate some of the effects 
of condensation and increased viscosity, but it may not allow adequate 
time for air present in the voids of the textured surface of the tissue 
chuck to escape, thereby preventing the O.C.T. from flowing into the voids 
and producing inadequate bonding of the O.C.T. to the chuck and the 
attendant problem described above. Likewise, placing an excess amount of 
O.C.T. on the embedding head and tissue sample while keeping the heat 
transfer rate of the embedding head constant will allow the surgeon a 
little more time for the O.C.T. to flow into voids of the textured surface 
of the tissue chuck (due to the sheer volume of the O.C.T.), but if the 
excess O.C.T. flows down the sides of the embedding head and bonds 
thereto, problems associated with separation of the tissue sample and 
O.C.T. from the embedding head may be aggravated when this O.C.T. freezes 
in the form of icicles. So dealing with the problems of attaching the 
chuck to the tissue and rapidly freezing the O.C.T. on the embedding head 
can cause additional problems when the time comes to remove the chuck, 
tissue sample, and O.C.T. from the embedding head. Thus, timing and the 
skill of the operator (whether a surgeon or a technician) becomes 
critically important to the tenth method. 
Although problems associated with detachment of the tissue sample from the 
tissue chuck (when the surgeon attempts to forcibly break away the frozen 
tissue sample and O.C.T. from the embedding head) can be addressed by 
wiping a film of oil (such as petroleum jelly) on the embedding head prior 
to flattening the bowl-shaped surface thereto, this obviously makes it 
more difficult to get the tissue to adhere to the embedding head in the 
first place since the purpose of the oil is to reduce the tendency of the 
tissue sample to adhere to the embedding head. In addition, it adds one 
more step to the tissue sample preparation, since the embedding head must 
be re-oiled for each tissue sample. (Of course, if the embedding head 
becomes nicked or scratched during the course of normal use, this 
separation problem will be further aggravated.) 
Most importantly, any method of preparing tissue samples which requires 
forcibly separating the flattened, formerly bowl-shaped surface of the 
tissue sample from the object to which it is adhered has the inherent risk 
that, when the tissue sample is separated therefrom, the very cancer cells 
which the surgeon is searching for may remain adhered to that object, and 
therefore not appear on the tissue slices produced in the cryostat. The 
nature of cancer cells increases the likelihood for the occurrence of this 
problem, because cancer cells are delicate and friable, and have no 
significant structural support as compared to healthy skin tissue. 
Furthermore, any method which relies on warming of such object to release 
the formerly bowl-shaped surface of the tissue sample therefrom introduces 
the problems associated with cell lysis described above. 
An eleventh method is disclosed in U.S. Pat. No. 4,752,347 issued to Rada 
on Jun. 21, 1988, which is hereby incorporated by reference. Rada 
discloses a method and apparatus in which a tissue sample is placed onto a 
polished disk platform and covered with a flexible plastic membrane, such 
as polyethylene plastic sheet material. A vacuum source is activated, 
which evacuates air from between the membrane and the platform, drawing 
the bowl-shaped surface of the tissue sample toward the platform. Liquid 
nitrogen is then used to freeze the tissue sample to the platform, and 
once the tissue sample is frozen to the platform, the membrane is peeled 
away from the platform and the tissue sample. In one embodiment, O.C.T. is 
applied to the platform on which the tissue sample is located and to a 
corrugated platform such as a tissue chuck. After the O.C.T. has partially 
solidified, the platforms are mated together and the O.C.T. is allowed to 
solidify. Then the platforms are forcibly separated, or heated if need be, 
to remove the tissue sample from the platform to which it was originally 
frozen and leave it frozen to the corrugated platform. Unfortunately, 
since the invention disclosed in Rada relies on heat or force to free the 
tissue sample from the platform to which it was originally mounted, it 
suffers from the same problems associated therewith and described above. 
As those skilled in the art will readily appreciate, in order to obtain a 
perfect section (i.e. a tissue slice which includes the entire flattened, 
formerly bowl-shaped surface, including the epidermal periphery thereof) 
the plane in which the flattened, formerly bowl-shaped surface lies must 
be substantially parallel to the plane in which the cryostat knife moves 
relative to the tissue sample. For example, to obtain a perfect section 
having a thickness of only 5 micrometers from a tissue sample having a 
flattened, formerly bowl-shaped surface measuring 1 centimeter in 
diameter, the acute cutting angle between the flattened, formerly 
bowl-shaped surface and the plane in which the cryostat knife moves 
relative to the tissue sample must be less than 30 thousandths of a degree 
(i.e. the arctangent of 5.times.10.sup.-6 /1.times.10.sup.-2). For tissue 
samples having a larger diameter, the angle must be even less. The 
relatively low percentages of perfect sections produced by the prior art 
indicate that none consistently provides a cutting angle within the 
acceptable tolerance. 
Adjustable chuck fixtures are available within most cryostats to assist 
orientation of the planar surface in those situations where initially the 
planar surface is not parallel to the path of relative movement between 
the cryostat knife and the tissue sample. However, adjustable fixtures are 
expensive, and adjustment of the fixture can be dangerous due to the close 
proximity of the cryostat knife. Further, the fixture must still be 
adjusted to be within the cutting angle tolerance described above, and 
adjusting the fixture to the correct orientation is an iterative process 
that can consume a considerable amount of time. Adjusting the fixture to 
an angle for a specific tissue sample means that the next tissue sample 
will likely require adjustment of the fixture as well. If done 
incorrectly, this may require evaluation of many subsequent slices in 
order to view all of the formerly bowl-shaped surface. 
Cryostats are generally designed such that when the chuck is placed within 
a chuck fixture within the cryostat, the tissue mounting surface of the 
chuck is parallel to the path of relative movement between the cryostat 
knife and the chuck. Therefore, as long as the planar surface is parallel 
to the path of relative movement between the cryostat knife and the tissue 
sample, the first slice should be the only section that need be evaluated. 
Unfortunately, despite the various methods and devices disclosed in the 
prior art to assist in obtaining a perfect tissue section, the problem 
persists. 
What is needed is a quick, inspectable means and method of mounting a 
tissue sample to a cryostat chuck such that the planar, formerly 
bowl-shaped surface is consistently parallel to the tissue mounting 
surface of the chuck, does not require forcible removal of the tissue 
sample from an object or warming of the object to obtain separation of the 
tissue sample therefrom, and which does not require the timing or level of 
operator skill required by the prior art. 
SUMMARY OF THE INVENTION 
It is therefore an object of the present invention to provide an improved 
apparatus for preparing tissue samples for sectioning by a cryostat. 
Another object of the present invention is to provide an apparatus which 
precisely orients tissue samples for optimum sectioning. 
Another object of the present invention is to provide for visual inspection 
of the flattened bowl-shaped surface of the tissue sample prior to contact 
with the O.C.T. compound. 
Another object of the present invention is to provide an apparatus which 
facilitates manipulation of the tissue sample for optimum sectioning. 
Another object of the present invention is to provide an apparatus which is 
time and cost effective, so as to reduce the overall surgical time and 
expense necessary to effect the total excision of a malignancy. 
Another object of the present invention is to provide an apparatus which is 
relatively simple to use, economical to manufacture, and particularly well 
adapted for the proposed usage thereof. 
Another object of the present invention to provide an improved method for 
preparing tissue samples for sectioning by a cryostat. 
According to the present invention, an apparatus for mounting a tissue 
specimen on a chuck for sectioning in a cryostat is disclosed, which 
apparatus comprises a base, a clamp for receiving a glass tissue 
preparation slide, a chuck holder secured to the base for holding the 
chuck in a predetermined orientation relative to the clamp, a support 
secured to the base and extending therefrom and terminating in a distal 
end, and, means for slideably securing said clamp to said support. The 
clamp is positionable between a first location in which the clamp is at a 
minimum distance from the chuck holder and a second location in which the 
clamp is at a maximum distance from the chuck holder. Additionally, the 
present invention discloses a method for mounting a tissue specimen on a 
tissue mounting surface of a tissue chuck for sectioning in a cryostat or 
the like comprising providing a chuck holder for holding the tissue 
mounting surface of the chuck essentially parallel to a primary reference 
plane. The primary reference plane is defined by primary arms of a clamp 
that is slideably moveable with respect to the chuck holder without 
changing the relative orientation of the chuck holder to the primary 
reference plane. The chuck is secured into the chuck holder such that the 
tissue mounting surface of the chuck is substantially parallel to the 
primary reference plane, and a puddle of tissue mounting fluid is placed 
on the tissue mounting surface of the chuck. A surface of the tissue 
specimen to be sectioned is then frozen to one face of a glass tissue 
preparation slide, and the glass tissue preparation slide is received 
within the clamp such that the one face is parallel to the primary 
reference plane. The clamp is then slid towards the chuck holder until the 
tissue sample is immersed in the tissue mounting fluid, and coolant is 
then sprayed on the slide, freezing the tissue mounting fluid to the 
tissue mounting surface of the chuck, the one face of the slide, and the 
tissue sample. The slide is then removed from the tissue sample and frozen 
tissue mounting fluid. 
The foregoing and other features and advantages of the present invention 
will become more apparent from the following description and accompanying 
drawings.

BEST MODE FOR CARRYING OUT THE INVENTION 
The apparatus 10 of the present invention as shown in FIG. 1 includes a 
base 12 having a planar base surface 14, to which is secured a chuck 
holder 16 and a hollow clamp support 18. The support 18 extends from the 
base surface 14 and terminates in a distal end 20. The present invention 
further includes a clamp 22 for receiving a tissue preparation slide 24 of 
the type known in the art and typically made of glass. The tissue mounting 
slide has two faces 25,27 which are parallel to each other, and an edge 29 
which defines the perimeter of the two faces 25,27. 
The clamp 22 is fixedly secured to a conveyor block 26, preferably by bolts 
28 which extend through holes (not shown) in the clamp 22 that are 
slightly larger than that diameter which would be necessary to simply 
accommodate the shaft of the bolt 28 extending therethrough. This slightly 
larger diameter allows for minor adjustments in the relative orientation 
between the clamp 22, and both the conveyor block 26 and the chuck holder 
16. A rail 30 that is perpendicular to the base surface 14 is fixedly 
secured to the support 18 by bolts, screws or other manner known in the 
art, and the conveyor block 26 is movably secured to the rail 30. As shown 
in FIG. 2, in the preferred embodiment of the present invention the 
conveyor block 26 has two internal loop paths 32, and the rail 30 has two 
longitudinally extending rail grooves 34 on opposite sides of the rail 30. 
(Although only one of the loop paths 32 and one of the rail grooves 34 is 
shown in FIG. 2, it is to be understood that the conveyor block 26 and 
rail 30 are symmetric about the length of the rail 30.) Each of the loop 
paths 32 is located adjacent one of the rail grooves 34, and a portion of 
each loop path 32 is parallel to, and opens into, the groove 34 adjacent 
thereto. 
The open portion of each loop path 32 constitutes a loop groove 36 which 
has the same dimensions, and opposes the rail groove 34 adjacent thereto. 
Ball bearings 38 having diameters only slightly less than the minimum 
width 40 of each loop path 32 are located therein, and the quantity of 
ball bearings 38 is such that the portion of each loop path 32 which is 
open to the rail groove 34 adjacent thereto is substantially filled with 
ball bearings 38 along the length thereof. Thus, as the conveyor block 26 
moves along the rail 30, each ball bearing 38 rolling in each loop path 32 
rolls into the open portion thereof and into the adjacent rail groove 34, 
rolls in the rail groove 34 along the length of the loop groove 36, and 
then rolls back into the closed portion of the loop path 32. 
Each loop path 32 contains a sufficient quantity of ball bearings 38 such 
that the portion of each loop path 32 between the rail groove 34 and the 
loop groove 36 always has ball bearings 38 extending substantially along 
the entire length thereof, thereby interlocking the conveyor block 26 and 
the rail 30. Additionally, the gap 42 between each rail groove 34 and the 
adjacent loop groove 36 opposed thereto is sized such that the gap 42 is 
essentially equal to the diameter of the ball bearings 38. As those 
skilled in the art will readily appreciate, such a design allows the 
conveyor block 26 to move freely along the rail 30, but provides no 
degrees of freedom of rotation of the conveyor block 26 with respect the 
rail 30. 
Thus the conveyor block 26 is slideably secured to the support 18 by the 
rail 30 and constrained thereby to travel along a linear path 44 that is 
perpendicular to the base surface 14, while the conveyor block 26 and the 
ball bearings 38 therein cooperate with the rail grooves 34 to prevent 
rotation of the block 26 relative to the rail 30. At the end 46 of the 
rail 30 opposite the base 12, a stop 48 is provided to prevent the 
conveyor block 26 from sliding off that end 46. The clamp 22 is thus 
positionable between a first location 50, as shown in FIG. 3, in which the 
clamp 22 is at a minimum distance from the chuck holder 16, (and may in 
fact be in contact therewith), and a second location 52 in which the 
conveyor block 26 contacts the stop 48 and the clamp 22 is at a maximum 
distance from the chuck holder 16, as shown in FIGS. 1 and 4. 
In the preferred embodiment of the present invention, a pulley 54 having an 
annular channel 56 therein is rotatably mounted in the support 18 adjacent 
the distal end 20. A cable 58 received within the channel 56 has a first 
end connected to the conveyor block 26, and a second end connected to a 
counterweight 60 that is suspended within the hollow support 18. 
Consequently, the counterweight 60 provides a predetermined force which 
acts on the clamp 22, through cable 58 and the conveyor block 26, to 
provide a predetermined force acting on the clamp 22 which tends to move 
the clamp 22 from the first location 50 to the second location. In the 
preferred embodiment of the present invention, the counterweight 60 is 
substantially equal to the combined weight of the conveyor block 26, the 
clamp 22, the bolts 28 that secure the clamp 22 to the conveyor block 26, 
and a typical glass microscope slide 24 with a tissue sample 62 secured 
thereto. Thus, once the slide 24 is released from the clamp 22, as 
described herein below, the weight of the counterweight 60 exceeds the 
combined weight of the conveyor block 26, the clamp 22, the bolts 28. 
Referring again to FIG. 1, the clamp 22 of the present invention includes 
two primary arms 64 in spaced relation to each other and extending away 
from the conveyor block 26. These primary arms 64 are fixed relative to 
the conveyor block 26, and preferably are integral with the portion of the 
clamp 22 which is bolted to the conveyor block 26. Referring to FIG. 3, a 
wall 37 extends between the primary arms 64, as does a hood 39 which 
extends away from the wall 37 and is integral with the primary arms 64. 
The wall 37 has a terminal edge 41 which is integral with a land 43. A 
slide stop 35 extends from the land 43 adjacent to each of the primary 
arms 64 to aid in proper positioning of the microscope slide 24, as 
described below. The wall 37 is preferably offset from each slide stop 35 
by 1/8 to 1/4 of an inch, so that when a slide 24 is positioned within the 
clamp 22 against the slide stops 35, the slide 24, primary arms 64, wall 
37, and hood 39 form a swirl pocket 45 immediately adjacent the slide 24. 
Referring back to FIG. 1, the clamp 22 further includes a hinge 66 below 
the primary arms 64, and two secondary arms 68, in spaced relation to each 
other, are secured to the hinge 66. Thus, the hinge 66 provides for 
rotation of the secondary arms 68 relative to the conveyor block 26, such 
that the secondary arms 68 are rotatable between a first position 70 
proximate the primary arms 64, as shown in FIG. 1, and a second position 
72 distant therefrom at which the arms may be parallel to the rail 30, as 
shown in FIG. 4. 
The clamp 22 has a locking mechanism 74 therein for locking the secondary 
arms 68 in the first position 70 (proximate the primary arms 64) for the 
purpose of clamping a microscope slide 24 between the primary and 
secondary arms 64,68. The locking mechanism 74, shown in cross-section in 
FIGS. 2 and 5, comprises a dovetail slot 76 in the clamp 22 extending away 
from the block 26, and a dovetail hinge support 78 slideably received in 
the dovetail slot 76. A positioning handle 80 is provided to facilitate 
selective positioning of the dovetail hinge support 78. As shown in FIG. 
2, by sliding the dovetail hinge support 78 away from the support 18, the 
dovetail hinge support 78 is positionable relative to the hinge 66 so as 
to prevent rotation of the secondary arms 68 away from the primary arms 
64, thus locking the secondary arms 68 in place. Conversely, as shown in 
FIG. 5, by sliding the dovetail hinge support 78 toward the support 18, 
the dovetail hinge support 78 is positionable relative to the hinge 66 so 
as to allow rotation of the secondary arms 68 away from the primary arms 
64. As shown in FIG. 6, an adjustable bearing cap 200 is attached to one 
end of the dovetail hinge support 78. The bearing cap 200 is secured to 
the dovetail hinge support 78 by two small screws 202 which are threaded 
into the cap 200 but are not threaded into the dovetail hinge support 78. 
Sandwiched between the head 204 of each screw 202 and the dovetail hinge 
support 78 is an "O-ring" 206 made of neoprene or a similar material to 
allow the cap 200 to be tilted slightly with respect to the dovetail hinge 
support 78. A third screw 208, which is preferably an allen head screw, is 
threaded into the dovetail hinge support 78 but does not extend into the 
bearing cap 200. Instead, the third screw 208 bears on the underside 210 
of the bearing cap 200, such that advancing the third screw 208 raises the 
leading edge 212 of the bearing cap 200. This adjustable feature of the 
bearing cap 200 allows for increasing or decreasing interference between 
the bearing cap 200 and the hinge 66 through adjustment of the relative 
position of the bearing cap 200 to the hinge 66 which compensates for any 
wear which might occur due to rubbing of the bearing cap 200 against the 
hinge 66. 
Each of the secondary arms 68 preferably includes an "O-ring" 82 made of 
neoprene or a similar material to act both as a cushion between the 
secondary arms 68 and the microscope slide 24, and to provide a frictional 
force to hold the slide 24 securely in place when the clamp 22 is in the 
locked position, as shown in FIG. 1. 
The chuck holder 16 serves the purpose of holding a cryostat chuck 84 in a 
predetermined orientation relative to the clamp 22, such that as shown in 
FIG. 4, the mounting surface 86 of the chuck 84 is essentially parallel to 
a primary reference plane 88 described in greater detail below. It is to 
be understood that the mounting surface 86 of the chuck 84 is textured or 
grooved to maximize the adherence of the tissue sample 62 to the mounting 
surface 86, and that therefore the mounting surface 86 is not actually 
planar. Accordingly, the term "essentially parallel to the primary 
reference plane 88" as used herein means that the mounting surface 86, 
excluding such texturing, lies within a plane that is substantially 
parallel to the primary reference plane 88. As those skilled in the art 
will readily appreciate, the presence of the "O-rings" 82 ensure that when 
a slide 24 is secured in the clamp 22, the slide 24 will be parallel to 
the primary reference plane 88 even if the secondary arms 68 are not 
exactly parallel to the primary arms 64. 
Referring now to FIG. 7, the chuck holder 16 preferably is a solid cylinder 
90 of rigid material having a coefficient of heat transfer less than most 
metals. A shaft hole 100, which has a diameter sized to receive the shaft 
102 of the chuck 84, extends from the upper surface 94 of the chuck holder 
16 along the centerline 96 thereof, which is parallel to the rail 30. The 
diameter of the upper surface 94 is preferably smaller than the diameter 
of the tissue mounting surface 86 of the chuck 84 to facilitate removal of 
the chuck 84 from the chuck holder 16. The chuck 84 typically includes a 
lip 300 made of a material such as neoprene to allow for easier, and more 
comfortable, handling of the chuck 84 when it has been cooled to 
sub-freezing temperatures. An orifice 118 in the chuck holder 16, as shown 
in FIG. 1, intersects the shaft hole 100 to provide access to the end 120 
of the shaft 102 within the chuck holder 16 for the purpose of 
facilitating removal of the chuck 84 from the chuck holder 16 by pressing 
upwards on the end 120 of the shaft 102. This orifice 118 is appropriately 
sized so as to permit insertion of a thumb or finger. 
Referring again to FIG. 4, rotation of the secondary arms 68 defines two 
secondary reference planes 122,124, and the chuck holder 16 is located 
between the secondary reference planes 122,124. Thus, rotation of the 
secondary arms 68 is not subject to interference with the chuck holder 16. 
Each of the primary arms 64 has a lower surface 126 facing the base 12, 
and together the lower surfaces 126, the terminal edge 41, and the land 
43, as shown in FIG. 3, all lie in the same plane and define the primary 
reference plane 88. The primary reference plane 88 is perpendicular to the 
linear path 44 along which the conveyor block 26 is constrained to travel. 
As those skilled in the art will readily appreciate, since the microscope 
slide has two faces 25,27 which are parallel to each other, and one face 
27 of the slide lies flush against the lower surfaces 126 of the primary 
arms 64 when the slide 24 is received within the clamp 22, both faces 
25,27 are parallel to the primary reference plane 88 when the slide 24 is 
received within the clamp 22. Therefore, the clamp 22 is positionable 
between the first location 50 and a second location 52, and both faces 
25,27 of the tissue mounting slide 24 are substantially parallel to the 
tissue mounting surface 86 of the chuck 84 at the first location 50, the 
second location 52, and all locations therebetween. Additionally, the 
primary arms 64 of the clamp 22 are slideably moveable with respect to the 
chuck holder 16 without changing the relative orientation of the primary 
arms 64 to the chuck holder 16. 
Preferably the primary arms 64, as shown in FIG. 4, are centered over the 
chuck holder 16 and the spacing 128 between the primary arms 64 is less 
than the diameter of the mounting surface 86 of the chuck 84 in the chuck 
holder 16, so that movement of the clamp 22 towards the chuck holder 16 
necessarily ceases when the primary arms 64 contact the mounting surface 
86 of the chuck 84 when no slide 24 is present in the clamp 22, and when a 
slide 24 is present in the clamp 22, the interaction of the primary arms 
64 and the mounting surface 86 of the chuck 84 does not produce a 
significant bending moment in the slide 24 and thereby cause breakage of 
the slide 24. 
In operating the apparatus of the present invention, the clamp 22 is raised 
away from the chuck holder 16 and a chuck 84 is placed therein. The 
surgeon excises the skin tumor from the patient using the Mohs technique 
described above, producing a tissue sample 62 having a bowl-shaped surface 
130. As shown in FIG. 8, the bowl-shaped surface 130 is pressed onto one 
face of a glass microscope slide 24 while a coolant such as liquid 
nitrogen is sprayed on the opposite face of the slide 24. As the 
bowl-shaped surface 130 is pressed against the nitrogen chilled slide 24, 
the bowl-shaped surface 130 freezes to the slide 24. By judiciously 
working around the periphery of the tissue sample 62 while pressing the 
sample 62 against the slide 24 (and intermittently spraying the opposite 
side of the slide 24 with nitrogen to maintain the slide 24 below freezing 
temperature), the entire bowl-shaped surface 130 can be frozen to the 
slide 24, thus flattening the surface which had been bowl-shaped. Because 
the surgeon can directly view the bowl-shaped surface of the sample 62 as 
the surgeon is freezing the sample 62 to the slide, the surgeon can ensure 
that the bowl-shaped surface does not become creased as it is pressed 
against the slide 24 and that no air bubbles become trapped between the 
sample 62 and the slide 24. 
A small puddle of O.C.T. or similar tissue mounting fluid is deposited at 
the center of the mounting surface 86 of the chuck 84. With the surface of 
the slide 24 on which the tissue sample 62 is mounted facing the chuck 84 
as shown in FIG. 1, the microscope slide 24 is then positioned against the 
lower surfaces 126 of the primary arms 64 and slid towards the rail 30 
until the slide 24 contacts each of the slide stops 35. Using the 
positioning handle 80, the surgeon raises the secondary arms 68 into 
contact with the slide 24 by extending the dovetail support 78 from the 
dovetail slot 76 until the dovetail support 78 moves bearing cap 200 so 
that bearing cap 200 contacts and rotates the hinge 66 into the position 
at which the secondary arms 68 swing up and contact the slide 24, 
sandwiching the slide 24 between the primary and secondary arms 64,68. If 
necessary, the slide 24 is adjusted to center the tissue sample 62 over 
the puddle of O.C.T., and the dovetail support 78 is extended slightly 
further to support the hinge 66 and prevent the secondary arms 68 from 
rotating away from the primary arms 64. 
As those skilled in the art will readily appreciate, since the slide 24 is 
flat, and the mounting surface 86 of the chuck 84 is parallel to the 
primary reference plane 88 defined by the lower surfaces 126 of the 
primary arms 64, clamping the slide 24 firmly against the lower surfaces 
126 of the primary arms 64 necessarily positions the lower face 25 of the 
slide 24 in a plane that is substantially parallel to the plane in which 
the tissue mounting surface 86 of the chuck 84 is located. During the 
process that follows, liquid nitrogen may be sprayed onto the upper 
surface of the slide 24 (into the swirl pocket 45 between the primary arms 
64) as needed to keep the tissue sample 62 frozen to the slide 24. 
As shown in FIG. 3, the slide 24 is lowered into contact with the O.C.T. 
fluid on the mounting surface 86 of the chuck 84, so that the tissue 
sample 62 is immersed in the O.C.T. Coolant such as liquid nitrogen is 
then sprayed into the swirl pocket 45 until the O.C.T. freezes. As those 
skilled in the art will readily appreciate, since the slide 24 is being 
held firmly against the land 43 and the lower surface 126 of each of the 
primary arms 64, as long as the coolant is sprayed directly into the 
pocket 45, no splattering of the O.C.T. will occur as a result of the 
coolant spray, since the slide 24 shields the O.C.T. from the blast of the 
coolant spray. 
Once the tissue sample 62 has been frozen to the mounting surface 86 of the 
chuck 84, the secondary arms 68 are rotated downward to clear the slide 24 
as shown in FIG. 4, by retracting the dovetail support 78 into the 
dovetail slot 76 until the bearing cap 200 of the dovetail support 78 no 
longer contacts the hinge 66. The entire clamp 22 is then moved up and 
away from the slide 24 by a gentle upward tap on the clamp pin 132. The 
chuck 84, with attached tissue sample 62 and slide 24, can then be removed 
from the chuck holder 16 by reaching into the second orifice 118 of the 
chuck holder 16 with a finger and pressing upward on the end 120 of the 
chuck shaft 102. Further spraying of the slide 24 with liquid nitrogen 
causes the slide 24 to release from the sample 62 due to the relative 
differences in the coefficients of thermal expansion between the glass 
slide 24 and the water-based tissue sample 62. Thus, the tissue sample 62 
is freed from the slide 24 without thawing the sample 62 or forcibly 
removing it therefrom, thereby avoiding the problems discussed above 
associated with these methods of releasing the sample 62 from the object 
to which it is attached. The resulting tissue sample 62 is frozen to the 
chuck 84 such that the formerly bowl-shaped surface is now parallel to the 
mounting surface 86 of the chuck 84. The chuck 84 can then be placed in a 
cryostat, and the tissue sample 62 sliced parallel to the mounting surface 
86, sectioning the entire formerly bowl-shaped surface, including the 
peripheral edge thereof, with a single slice of the cryostat knife. 
As those skilled in the art will readily appreciate, the first or second 
slice of tissue will produce a section of the tissue sample 62 that, 
through examination under a microscope, will indicate whether the tumor 
has spread beyond the tissue sample 62. Accordingly, the surgeon can 
quickly determine whether additional tissue must be removed to excise all 
of the tumor. 
The present invention provides a quick, inspectable means and method of 
mounting a tissue sample to a cryostat chuck such that the planar, 
formerly bowl-shaped surface is consistently parallel to the tissue 
mounting surface of the chuck. Additionally, the present invention does 
not require the application of force or heat to the tissue sample to 
obtain removal of the tissue sample from the object to which it has been 
frozen. As a result, the present invention does not require the timing or 
level of operator skill required by the prior art to obtain consistently 
perfect tissue sections. 
Although this invention has been shown and described with respect to 
detailed embodiments thereof, it will be understood by those skilled in 
the art that various changes in form and detail thereof may be made 
without departing from the spirit and scope of the claimed invention.