U.S. patent number 3,870,789 [Application Number 05/311,349] was granted by the patent office on 1975-03-11 for enhancement of morphologic detail in tissue.
Invention is credited to Kurt W. Mikat.
United States Patent |
3,870,789 |
Mikat |
March 11, 1975 |
ENHANCEMENT OF MORPHOLOGIC DETAIL IN TISSUE
Abstract
Tissue is prepared for microscpic examination by subjecting the
tissue to fixation during centrifugation to achieve a significant
cell flattening with resultant dispersion of cell contents and
improved cytological detail while maintaining the substantial
integrity of morphologic pattern of cell entities. A preferred
centrifugation apparatus comprises a tissue retaining clip
positioned on a centrifuge drum with provision for spraying a
programmed sequence of tissue treating substances into said
drum.
Inventors: |
Mikat; Kurt W. (Grand Blanc,
MI) |
Family
ID: |
23206511 |
Appl.
No.: |
05/311,349 |
Filed: |
December 1, 1972 |
Current U.S.
Class: |
435/40.52;
422/72 |
Current CPC
Class: |
G01N
1/30 (20130101); G01N 1/31 (20130101); G01N
2001/305 (20130101) |
Current International
Class: |
G01N
1/31 (20060101); G01N 1/30 (20060101); G01n
001/00 (); G01n 001/30 (); G02b 021/34 () |
Field of
Search: |
;424/3 ;117/3,101
;118/44,52,56,326,500 ;233/26 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Watson, J. Lab & Clin. Med., Vol. 68, Sept. 1966, pp. 494-501.
.
Martinazzi, AJCP, Vol. 56, Dec. 1971, pp. 719-722..
|
Primary Examiner: Meyers; Albert T.
Assistant Examiner: Fagelson; A. P.
Attorney, Agent or Firm: Hauke, Gifford, Patalidis &
Dumont
Claims
I claim:
1. A method for preparing a tissue for microscopic examination said
tissue having intercellular connective matter, said method
comprising the steps of subjecting said tissue to a centrifugal
force of sufficient magnitude and duration to flatten the cells of
said tissue and simultaneously treating said tissue with a tissue
fixative to prevent, following such centrifugation, said flattened
cells from reverting significantly toward their original shape.
2. The method of claim 1 wherein said duration of centrifugation is
sufficient to substantially double the cross sectional area of a
cell nucleus.
3. The method of claim 1 wherein said magnitude of centrifugal
force and said duration of centrifugation are sufficient to effect
a flattening of cells with some dispersion of cell contents but
insufficient to destroy morphologic cell patterns.
4. The method of claim 1 wherein said centrifugation step is
carried out over a period of about 16 hours.
5. The method of claim 1 wherein said tissue is enveloped in a
material which is permeable to tissue processing fluids.
6. The method of claim 1 wherein said tissue fixative is
formalin.
7. A method for preparing a tissue for microscopic examination,
said tissue having intercellular connective matter, said method
comprising the steps of
a. enveloping said tissue in a material which is permeable to a
selected tissue fixative;
b. subjecting said tissue to a centrifugal force sufficient to
effect a flattening of tissue cells with some dispersion of cell
contents but insufficient to destroy morphologic cell patterns;
c. subjecting said tissue, while it is subject to said centrifugal
force, to the action of said fixative for a period of time
sufficient to effect a fixation sufficient to prevent, following
such centrifugation, said flattened cells from reverting
significantly toward their original shape.
Description
BACKGROUND OF THE INVENTION
The invention deals with the preparation of human or other tissue
for microscopic examination. It has been the usual practice to
immerse a sample of tissue successively in a number of tissue
processing fluids to fix the tissue, to wash out the fixative, to
dehydrate the tissue, to infiltrate the tissue with paraffin, and
the like prior to cutting the tissue into sections of desired
thickness. The sections are then mounted on slides and stained.
Attempts have been made to enhance cellular detail in such tissue
specimens. Some enhancement has been ascribed to a surface tension
effect, a flattening by surface tension tending to disperse the
cellular components in the plane of the slide surface allowing more
discernible cellular detail than if the cell were in its original
and generally more spherical shape. Similarly, it has been observed
that brief periods of centrifugation of smears; for example, 3 to
30 minutes; result in enhancement of cytological detail with the
apparent enlargement of the cells as flattening is increased.
Similarly, attempts have been made to attain improved results by
stretching cells or by applying relatively large pressures to
cells.
It is therefore an object of this invention to provide an improved
method and apparatus for the enhancement of cellular detail in
tissue prepared for microscopic examination.
SUMMARY OF THE INVENTION
The present invention provides for the fixation of tissue under
centrifugation to achieve flattening and enhanced morphologic
detail. The tissue is simultaneously subjected to centrifugation
and fixation for a period of time sufficient to achieve the desired
flattening of the cells and the desired fixation to prevent the
cells from returning to their more natural shapes. By way of
example, a period of 16 hours is a suitable period of time.
A preferred centrifuge of the invention comprises a means,
preferably a clip and a permeable envelope, for securing a tissue
sample to the wall of a centrifuge drum and comprises a means for
spraying a fixative or other tissue processing fluid, or a number
of fluids sequentially, into the centrifuge drum in a manner to
effect the fixing of the tissue while the tissue is being subjected
to centrifugation.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic cross sectional view illustrating an
embodiment of the invention;
FIG. 2 is an enlarged diagrammatic cross sectional view of the
tissue cube shown in FIG. 1;
FIG. 3 is a diagrammatic cross sectional view illustrating the
appearance of the tissue cube of FIG. 2 following
centrifugation;
FIG. 4 is a diagrammatic sectional view taken along the line 4--4
of FIG. 3;
FIG. 5 is a diagrammatic cross sectional view of an embodiment of
the invention illustrating a preferred manner of centrifugation and
fixation of tissue; and
FIG. 6 is an enlarged diagrammatic cross sectional view of the
holding clip of FIG. 5 together with a tissue specimen and fluid
permeable envelope.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring more specifically to FIG. 1, a filter paper disc or
sponge disc 10, is placed in the bottom of a centrifuge cup 12, the
disc 10 being wetted with tissue fixing solution such as 10 percent
formalin. A disc of smooth paper 14 is placed over disc 10 and a
sample of tissue 16 is placed in the center of disc 14. The tissue
16 is covered with a smooth paper disc 18.
Referring more particularly to FIGS. 2, 3, and 4; the tissue cube
16 is shown with embedded spheres, such as sphere 20, which
illustrate various cell entities one may desire to view with
improved visability. FIG. 2 represents the initial fresh tissue,
FIG. 3 the flattened tissue following centrifugation, and FIG. 4 a
section as it would appear on the slide.
Referring more particularly to FIG. 5 a centrifuge, indicated
generally as 22, is provided with a housing 24, an access door 26
and a motor 28. The motor 28 drives a shaft 30 which rotates within
journal elements 32, 34.
Mounted on shaft 30 for rotation therewith are a processing fluid
conduit 36 provided with a plurality of spray nozzles, such as
nozzle 38, and a cylindrical centrifuge drum 40 provided on the
interior of its outer wall with clips, such as clip 42. Drum 40 is
perforated as at 43 and is provided with a bottom drainage port
44.
A cylindrical liner 46 provided with a bottom drainage port 48 is
positioned intermediate the drum 40 and housing 24.
A pump 50 selectively draws processing fluid from storage bottles;
such as bottles 52, 54, 56; via tubes 58, 60, 62 respectively and
via valve 64. Conduit 66 receives said fluid from pump 50 and
delivers it to rotating processing fluid conduit 36. Valve 64 is
joined to drainage port 48 by tube 65 for the recycling of
processing fluid via pump 50 or for discharge via tube 67. A pump
68 pumps a temperature control liquid from a sump pit, indicated
generally as 70, through conduit 72 to coil 74 surrounding the
liner 46. The coil 74 terminates at opening 76 to return
temperature control fluid to sump pit 70.
The clip 42 secures a fluid permeable envelope 78 containing a
tissue sample 16 to the centrifuge drum 40 (FIG. 6).
DESCRIPTION OF OPERATION
By way of example flat-bottomed, 1 inch diameter centrifuge cups 12
were prepared in the following manner (FIG. 1). A filter paper disc
10 was placed in the bottom of the cup and covered with 1 ml. of 10
percent formalin. A disc of smooth paper 14 slightly smaller than
the formalin-soaked disc was placed over the disc 10. Samples of
various fresh human and rat tissues including kidney, testis,
brain, spleen, liver and striated muscle were cut into cubes 16
measuring approximately 5 mm. per side. Each tissue cube 16 was
then placed in the center of a paper disc 14 in a cup 12 and
covered by a smooth paper disc 18 to concentrate the formalin fumes
for fixation.
Using a free-swinging centrifuge head (not shown) on a size 2
International Centrifuge (International Equipment Company, Boston,
Mass.) the tissue cubes 16 were centrifuged at 1,000 g (2,200
rev/min) for 16 hr.. They were then removed and processed through
paraffin embedding in the usual manner for microscopic evaluation,
cutting sections at 2-3 mm thickness (FIG. 4).
Two sets of controls were prepared: One consisted of corresponding
tissue cubes fixed in centrifuge cups prepared as for the
experimental tissues but without centrifugation. The other set of
tissue cubes were fixed and processed as routine surgical specimens
by well known prior art methods.
The fixation of tissue under centrifugation, as described, resulted
in a pronounced flattening of tissue cubes 16 into disc or
"pancake" forms (FIG. 3). The greatest flattening occurred with the
longer periods of centrifugation (16 hr) and with tissues having a
loose connective tissue framework as in testis, brain and liver.
The more densely fibrous tissues had more resistance to flattening
but still showed enhanced detail. The integrity of morphologic
patterns of cell entities, that is histological patterns and
cellular relationships, were retained in all cases but spread over
a large area (FIG. 4).
Ocular micrometer measurements confirmed enlargement of all
cellular types, some with doubling of the average diameter. Nuclear
details were generally clearer. A few aspects of tissue detail
became less clear such as delicate basement membranes, presumably
because these elements flattened out in the plane of the slide
surface rather than being clearly observed parallel to the path of
light. The controls fixed in centrifuge cups appeared similar to
those processed as routine surgical specimens with no significant
differences in morphology.
The tissue blocks 10 were also rotated 90.degree. and were
sectioned across the cells. Definite flattening with apparent
elongation of all cells was apparent (FIG. 3).
In a preferred apparatus (FIGS. 5 and 6) for carrying out the
simultaneous centrifugation and fixation of tissue, a tissue sample
16 is placed in an envelope 78 which is attached to centrifuge drum
40 by a clip 42 (FIG. 6). The centrifuge drum 40 is then rotated at
about 1,000 g, for example, for about 16 hours. The envelope 78 is
made of a material which is permeable to the selected fixative or
other processing fluid. It, of course, must resist disintegration
by fluid action. Generally a porous paper, a loosely woven fabric,
or mesh plastic is suitable.
During the centrifugation processing, fluids are drawn sequentially
from as many receptacles (e.g., 52, 54, 56) as needed, (for
fixatives, wash solutions, dehydration agents, etc.), the sequence
and duration of use being controlled by the programmed valve 64.
Such programming systems are now well known in the art and their
description here would serve no purpose. The processing fluid being
pumped by pump 50 at any given time passes through tube 66 which
drains into rotating fluid channel 36 surrounding shaft 30. The
rotating fluid is projected outwardly through the nozzles 38 to wet
the sample 16 and/or to subject the sample 16 to fumes from the
projected processing fluid. The processing fluid passes through the
openings 43, spins out over the rim of centrifuge drum 40 or drains
from the centrifuge drum 40 through port 44 and falls into the
bottom of cylindrical liner 46. The processing fluid then drains
through port 48 into tube 65 for recyling via valve 64 and pump 50
or, alternatively, for discharge via valve 64 and discharge tube
67.
The temperature of the interior of the centrifuge is controlled by
a temperature control fluid which is pumped by pump 68 from sump
pit 70 through tube 72 to coil 74. The temperature control fluid
returns to the sump pit 70 via discharge tube 76. A temperature
control device (not shown) for controlling the temperature of the
temperature control device is positioned in sump pit 70.
Although 16 hours of centrifugation and treatment of tissue has
been mentioned above as a suitable length of time in which to
secure the advantages of the present invention, it is not critical.
As indicated one may wish to use a longer period of time in the
case of structures particularly resistant to flattening or more
slowly responsive to a desirable processing procedure. Similarly,
and in the interest of economy, one may find a shorter period of
time satisfactory for the purpose at hand. It may be noted,
however, that time is here being reckoned in hours whereas in prior
art methods the attainment of some improved morphologic detail in
smears by centrifugation alone (that is, without simultaneous
fixation), time has been reckoned in minutes (3-30 minutes, for
example). Similarly, although a centrifugal force of 1,000 g. is
suitable, this may be varied to fit specific needs of the
investigator in the enhancement of selected detail in a selected
tissue.
With further reference to smears, and, for example, to blood smears
prepared by spreading the film with the two slide method, cells in
the relatively thinner film areas appear relatively larger with
their contents more diffuse. In flattened tissue sections prepared
in accordance with the present invention the advantage of the
relatively larger appearing cells in smears is attained but with a
preservation of morphologic cell patterns; that is, the
preservation of the spatial and geometric relationship of cell
entities in essentially the same pattern as in the unflattened cell
with, of course, the exception that such entities are likely to be
relatively more dispersed.
Preparation of tissue sections in accordance with the present
invention also results in increased cell nuclear diameters, often
twice that of control cells -- a result which is greater than that
attained by the mechanical stretching of cells. Where the observed
cell is in mitosis the chromosones are more dispersed allowing
better evaluation, including counting and classification to a
certain extent.
The invention has found use in testicular biopsies for infertility
studies where cellular clarity has previously been disappointing,
in definitive cellular identification of tumors where lack of
cellular differentiation had previously made specific diagnosis
difficult, and in tissue chromosonal analysis.
It will be apparent to one skilled in the art to which the
invention pertains that various changes and modifications may be
made therein without departing from the spirit of the invention or
the scope of the appended claims.
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