U.S. patent application number 10/486491 was filed with the patent office on 2004-12-09 for process and apparatus for preparing and presenting a tissue sample for histological study.
Invention is credited to Petit, Michael G..
Application Number | 20040248237 10/486491 |
Document ID | / |
Family ID | 25458211 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248237 |
Kind Code |
A1 |
Petit, Michael G. |
December 9, 2004 |
Process and apparatus for preparing and presenting a tissue sample
for histological study
Abstract
An apparatus and method for preserving cytological specimens for
histological or histopathological use. A cytological sample from a
biological tissue such as a tumor or a tumor cell line is
dehydrated, disbursed and evenly distributed throughout a volume of
molten material. The molten material is then drawn into a tubular
member such as a pipette and allowed to solidify. Upon
solidification, the cylindrical specimen is partially or completely
extrudedfrom the tube for further processing, including fixation,
dehydration and molten paraffin infiltration and embedding as
required for presentation to a sectioning device such as a
microtome. Thin circular slices of the cylindrical specimen are
removed from the cylindrical specimen and transferred to a slide,
fixed and stained as desired. The device and method enable the
production of a slide bearing a spatially homogeneous distribution
of cytological material for microscopic examination.
Inventors: |
Petit, Michael G.;
(Tulipanhaven, DK) |
Correspondence
Address: |
Michael G. Petit
PO Box 91929
Santa Barbara
CA
93190-1929
US
|
Family ID: |
25458211 |
Appl. No.: |
10/486491 |
Filed: |
February 9, 2004 |
PCT Filed: |
August 12, 2002 |
PCT NO: |
PCT/DK02/00532 |
Current U.S.
Class: |
435/40.52 |
Current CPC
Class: |
G01N 1/06 20130101; G01N
1/36 20130101; G01N 1/30 20130101; Y10T 436/2525 20150115 |
Class at
Publication: |
435/040.52 |
International
Class: |
G01N 033/48; G01N
001/30 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 13, 2001 |
US |
09929642 |
Claims
1. A method for preparing a portion of a tissue comprising cells
for microscopic examination comprising the steps of: (a) placing
the portion of tissue into a volume of a fluid embedding medium,
then (b) disrupting said portion until said cells comprising said
portion are distributed substantially homogeneously throughout said
volume of said fluid embedding medium; then (c) introducing a
portion of said volume of said fluid embedding medium into an axial
bore in a tubular member; then (d) solidifying said fluid embedding
material within said axial bore to form a cylindrical plug; then
(e) extruding a portion of said cylindrical plug from said axial
bore and separating a portion of said cylindrical plug to form a
separated portion; then (f) mounting said separated portion of said
cylindrical plug on a rigid support substrate.
2. The method of claim 1 wherein said tissue is a cell line.
3. The method of claim 1 or 2 wherein the introducing is with the
same and/or different said fluid embedding mediums into at least
two bore holes; and extruding of at least two formed plugs to form
a new plug containing said at least two plugs.
4. A method for preparing a portion of a tissue comprising cells
for microscopic examination comprising the steps of: (a) placing
the portion of tissue into a volume of a fluid embedding medium,
then (b) disrupting said portion until said cells comprising said
portion are distributed substantially homogeneously throughout said
volume of said fluid embedding medium; then (c) introducing a
portion of said volume of said fluid embedding medium into an axial
bore in a tubular member; then (d) solidifying said fluid embedding
material within said axial bore to form a cylindrical plug; then
(e) separating a portion of said tubular member containing a
portion of said cylindrical plug from said tubular member to form a
separated portion; then (f) mounting said separated portion of said
tubular member and cylindrical plug on a rigid support
substrate.
5. A method for preparing a portion of a tissue comprising cells
for microscopic examination comprising the steps of: (a) placing
the portion of tissue into a volume of a fluid embedding medium,
then (b) disrupting said portion until said cells comprising said
portion are distributed substantially homogeneously throughout said
volume of said fluid embedding medium; then (c) introducing a
portion of said volume of said fluid embedding medium into a
plurality of axial bores in an extrusion device; then (d)
solidifying said fluid embedding material within said axial bores
to form a plurality of cylindrical plugs; then (e) extruding a
portion of said cylindrical plugs from said axial bores and
separating a portion of said cylindrical plugs to form a plurality
of separated portions; then (f) mounting said plurality of
separated portion of said cylindrical plugs on a rigid support
substrate.
6. The method of claim 5 wherein said tissue is a cell line.
Description
1. FIELD OF THE INVENTION
[0001] The present invention relates to a method and apparatus for
embedding and presenting tissue samples for histological
examination and, more particularly, a method for preparing a tissue
sample for microscopic examination.
2. PRIOR ART
[0002] A procedure for preparing tissue samples for microscopic
examination by embedding the tissue in paraffin and slicing the
paraffin-embedded tissue into thin layers with a microtome for
mounting on a slide is well known in the art. Preparatory to
embedding, the tissue is pretreated in various solutions selected
in accordance with the particular examination being conducted.
Typically, prior to paraffin embedding, the tissue sample is fixed,
dehydrated, cleared, infiltrated with molten paraffin and,
depending on the test, stained.
[0003] In order to impregnate the tissue sample, the sample is
dehydrated, most commonly with isopropyl alcohol, and then immersed
in liquid paraffin. The step of paraffin impregnation normally
takes place at ambient pressure and at a temperature slightly
higher than the melting point of the embedding material. In the
event that the embedding material is paraffins, the melting point
lies approximately between 50 degrees C. and 58 degrees C. The
replacement of the isopropyl alcohol contained in the tissue of the
tissue samples with paraffin is effected by dissolving the
isopropyl alcohol in paraffin such that the concentration of the
paraffin increases in the tissue sample. When the paraffin
solidifies, thin sections can be sliced from the tissue samples
embedded in paraffin block, mounted on a slide and examined under a
microscope.
[0004] Berger, in U.S. Pat. No. 5,089,288, discloses a method of
impregnating a tissue sample with paraffin in which a tissue
sample, which has been fixed with isopropyl alcohol, is maintained
under vacuum in a treatment vessel and the molten paraffin and
tissue sample are simultaneously subjected to ultrasonic vibration
effective to remove the isopropyl alcohol from the tissue sample
and to impregnate the tissue sample with the paraffin.
[0005] McCormick, in U.S. Pat. No. 5,665,398, discloses a system
for providing an embedded tissue specimen subsequent to fluid
treatment of the specimen and preparatory to histological
examination. The system includes the combination of a cassette for
use in the preparation of tissue specimens for histological
examination and an embedding mold having a first cavity for
receiving the treated specimen and a second cavity for receiving
the cassette. The system includes means for dispensing a
predetermined amount of molten wax into the embedding mold.
[0006] U.S. Pat. No. 5,080,869 to McCormick describes a cassette
for efficiently processing tissue samples. The cassette is
stackable and can be used for preparing a plurality of specimens.
The cassette generally includes a plurality of apertures disposed
in the walls of the cassette for passage of processing fluids in a
direction both orthogonal and parallel to the plane of the bottom
wall of the cassette. The cassette also includes a sloping
extension of the front wall of the cassette for ease in placing
indicia on the cassette for identification of the sample. Further
examples of systems and methods useful for preparing
paraffin-impregnated tissue samples for histological examination
are disclosed in U.S. Pat. No. 5,843 to Kerrod, et al., and U.S.
Pat. No. 4,569,647 to McCormick.
[0007] A problem with the prior art methods of embedding and
sectioning tissue is the clumping of cells such that cells of
interest may not be present in a particular field of view. It is,
therefore, desirable to have a method for preparing tissue for
sectioning such that cells comprising the tissue are more or less
evenly distributed on the section mounted on a slide thereby
improving the probability of a particular cell type of interest
being present in a particular section and field of view.
SUMMARY
[0008] It is a first object of the invention to provide a method
for processing a tissue sample for mounting on a slide. It is a
further object of the invention to provide a method for preparing a
tissue for sectioning wherein the cells comprising the tissue are
substantially evenly distributed throughout the tissue preparation
and a tissue section derived therefrom.
[0009] It is yet a further object of the invention to provide an
apparatus and method for using the apparatus operable for preparing
and presenting a tissue for sectioning. It is an overall object of
the invention to provide a method and apparatus for preparing a
tissue sample for examination wherein the probability of a cell of
interest being disposed within a field of view of the examiner is
enhanced when compared with prior art methods of tissue sample
preparation.
[0010] The features of the invention believed to be novel are set
forth with particularity in the appended claims. However the
invention itself, both as to organization and method of operation,
together with further objects and advantages thereof may be best be
understood by reference to the following description taken in
conjunction with the accompanying drawings in which:
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic diagram illustrating the sequence of
steps used for tissue sample preparation in accordance with the
present invention.
[0012] FIG. 2 is a longitudinal cross-sectional view of a preferred
embodiment of a pipette operable for forming a cylindrical tissue
specimen suitable for sectioning.
[0013] FIG. 3 and FIG. 4 are schematic diagrams illustrating the
sequences of steps of a preferred embodiment using a number of bore
holes.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0014] The method for preparing a tissue sampling for storage and
sectioning is illustrated diagrammatically in FIG. 1. A tissue
sample 10 is prepared for embedding in a solid or semisolid support
such as, for example, agarose or paraffins, by immersing the tissue
in a suitable fluid medium, or a series of fluid mediums 11, to
dehydrate and/or clear the tissue. The dehydrated tissue is then
transferred into a volume of molten paraffins 12 and the tissue,
which may comprise a cell line, fragmented and disbursed throughout
the volume of melt by cell dispersing means 13 such as a sonicator
or mechanical agitator.
[0015] Alternatively, the paraffin/cell slurry is extruded through
a warm motion less mixer tube section before entering a cold tube
section. The resulting endless plug is homogeneous. Motion less
mixers are commercially available tubes or pipes, which force the
flowing material to mix while passing various internal obstacles.
The tubes often have series of flow dividers placed in different
angles in the tube. The tube can be part of a continuous sonicator
allowing for large scale homogenizing of the hot and flowing
paraffin/cell melt.
[0016] After the cells comprising the tissue are disbursed
throughout the volume of melt 14, a pipette 15 having an axial bore
is employed to draw the volume of melt into the bore. The volume of
melt containing the disbursed tissue is allowed to cool below its
melting point within the axial bore. Upon hardening, the
cylindrical plug thus formed within the axial bore may be totally
or partially extruded from the pipette for storage and/or
sectioning. The pipette may also be used to store the tissue. The
extruded material may also be further processed as required for
other examinations of the tissue such as electron microscopy or
fluorescence spectrometry.
[0017] The pipette may be placed in a receiver port 20 in a
microtome 21 and a thin disc 22 of the plug 14 extruded from the
bore as shown in FIG. 2. A microtome blade 23 is advanced to
separate the extruded disc from the plug. The disc 22 is then
transferred to a support such as a microscope slide 24 for staining
and examination. The diameter of the axial bore, and hence, the
diameter of the plug and disc, may be "pin-like", having a diameter
of .about.0.1 mm. or "wafer-like", with a diameter of .about.1.5
cm. or even greater. The pipette may be any, preferably rigid,
tubular member. Preferred materials are glass and rigid or semi
rigid elastomers such as polyethylene, polypropylene or
polystyrene. The means for drawing the melt into the axial bore of
the tube include vacuum aspiration, centrifugation and capillary
action. A preferred means of vacuum aspiration is a plunger 25
slidably disposed within the axial bore of the tubular member.
While traditional paraffins may be used in accordance with the
present method, a preferred embedding medium is a hydrophilic gel
such as agarose.
[0018] In a variation of the method for preparing a portion of a
tissue comprising cells for microscopic examination presented
above, the tubular member comprises a polymeric material that
remains a rigid at the melt temperature of the embedding medium and
is easily separable from itself as, for example, by being sliced by
a knife blade. The cylindrical plug of embedded tissue within the
axial bore of the polymeric tubular member need not be extruded
from the bore prior to mounting. In accordance with the alternate
method, a portion of tissue is placed into a volume of a fluid
embedding medium, then the cells comprising the tissue are
disrupted until the cells comprising the portion are distributed
substantially homogeneously throughout the volume of molten fluid
embedding medium. A portion of the volume of the fluid embedding
medium is drawn into the axial bore of the polymeric tubular member
and permitted to solidify within the axial bore to form a
cylindrical plug. After the cylindrical plug is formed, a portion
of the tubular member (containing a portion of the cylindrical
plug) is then transversely sliced or otherwise separated from the
remainder of the tubular member to form a separated portion. The
separated portion, which includes a thin disc of embedded cells
circumferentially bounded by and contained within a ring comprising
a portion of the polymeric tubular member, is then mounted on a
rigid support substrate.
[0019] It is of advantage to make preparations which contain
multiple and different tissues or cells.
[0020] In another aspect of the invention, a heated or cold metal
block (31) with a number of bore holes (32) connected to movable
stoppers (33) in one end can each be filled with the same or
different tissues or cells in a suitable liquid (34). By applying
sonic waves or other mechanical movements (35) to the entire block,
the cells are dispersed evenly in each borehole.
[0021] The metal block is allowed to cool and the liquids solidify
in each hole. Applying pressure or pushing (41) the plugs upward or
downward partly into a container (42) attached to the metal block
the plugs will stand free and in parallel in the container
(43).
[0022] The container (41) is filled with paraffin with preferable a
lower melting point and allowed to solidify by cooling covering the
plugs (44).
[0023] The resulting paraffin or gel is cut (45) or sectioned to
give a thin slap of paraffin or gel containing slides from each of
the plugs (46). The thin slice is mounted on slides (47) and
further processed as required for examination of tissues in
microscopes.
[0024] In accordance with the process of the present invention, the
size of the sections can be controlled and the cells comprising the
tissue are substantially homogeneously distributed within the plug
and, therefore, a section removed therefrom. While particular
embodiments of the present invention have been illustrated and
described, it would be obvious to those skilled in the art that
various other changes and modifications can be made without
departing from the spirit and scope of the invention. For example,
any fluid embedding material that can be solidified after being
transferred to the axial bore of the tube such as upon standing,
addition of a catalyst to the fluid or UV irradiation may be used
in accordance with the present method. It is therefore intended to
cover in the appended claims all such changes and modifications
that are within the scope of this invention.
* * * * *