Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a process and apparatus for
histological analysis. In particular, it relates to a
method of preparing a sample for analysis by means of
freeze imprinting.
The usual histological analysis is based on the use
of a freezing cryotome. In medical practice, the surgeon
takes a section of the tissue from the diseased part of
the patient and gives this section to the pathologist who
first freezes the section of the tissue and then cuts it
into slices, applies color and carries out in this manner
the microscopic diagnosis.
-~ Another method, which is very inaccurate, consists of
pressing the tissue sample onto a slide whereb~ a very
small amoun~ of the tissue material is left on the slide.
Unfortunately, this tissue is frequently in poor condition~
It is known, for Instance from DOS 2,704,300 that
a possible techni~ue is to provide a small cylinder of
anatomic material which, mounted in a suitable apparatus,
is sliced with a blade such as to expose a fresh surface
of the cylinder. Immediately after the cut, the surface
; is "fixed" by immersion of the cylinder in a refrigerating
liquid, such as liquid nitrogen. The sample obtained in
this manner is not only very thick, but the test surface
suffers the usual damages caused by the friction produced
by the blade.
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Other apparatuses are also known for cooling anatomic
samples, such as British Patent 1,023,597, or to cool
a sample on a microscopic slide, such as French Patent
1,403,270, or to cool the slide body of a microscope as
in U.S. Patent 3,580,658.
The apparatus of the present invention has very sub-
stantial advantages over the previously known methods and,
most importantly, it makes possible histological analysis
of a monolayer.
The microscopic reproduction of a normal or a patho-
logical organ is the most trustworthy and accurate method
of analysis and the thinner is the layer of cells being
examined under the microscope, the more trustworthy and
accurate are the results. The ideal condition is to
examine a monocellular layer which reproduces the structure
exactly. In this manner it is possible to see in the
positive picture the cells of the parenchyma, i.e., the
epithelial and other tissues which perform the special
functions of the organ and in the negative picture vessels
and connective tissue, and stroma which will form some
pockets. These pockets may also be interpreted
quantitatively.
In accordance with the concept of the present inven-
tion, a thermal shock is created between the section
of the organ tissue and the slide on which it is being
supported in such manner that as a result of the thermal
shock, the cells are induced to exfoliate to form a
monolayer on the slide. This is achieved while keeping
intact the submicroscopic arrangement, and particularly
the enzymatic liposomial arrangement.
Thus the present invention in its broadest aspect
relates to an apparatus for histological analysis which
comprises a refrigerating device adapted to spray a
cryogenic liquid on the surface of a microscope slide
whereby the surface of the slide is chilled to at least
-4C, at least one cooling fan which increases the
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evaporation of the cryogenic liquid; and an automatic
press plate adapted to apply a measurable and graduated
pressure on an organ tissue section placed on the chilled
` slide, whereby a ~onolayer of the tissue exfoliates onto
the slide.
In order to better illustrate the present invention,
reference is made to the sensation of cold which one
receives when one touches with bare hands a frozen surface,
and particularly a very cold metal surface. Thus, it is
well known that there is a sensation of instantaneous
adhesiveness between the skin and the metal surface, these
surfaces being at very different temperatures. In reality,
if the metal surface is sufficiently cold, the skin when
removed from the surface leaves thereon a thin cellular
layer.
With the apparatus of this invention, the press plate
may also be temperature controlled, e.g. chilled to a
desired temperature. It is also possible to automatically
advance the slide whereby several organ sample depositions
may be made on the same slide. According to another pre-
ferred feature, the slide may be vibrated by means of a
vibrator to assist in the exfoliation of the sample.
Certain preferred embodiments of the invention will now
be illustrated by the attached drawing which schematically
illustrates the apparatus of the invention.
~; As shown, the device includes a cylinder 1 containing a
cryogenic liquid 2, e.g. ethyl chloride. Above the liquid
in the cylinder 1 is a gas phase 3 pressurized above the
level of the cryogenic liquid 2.
The cylinder 1 connects via valve 4 and connector tube
5 to a nozzle 6 which is arranged to direct a spray or jet
of cryogenic liquid onto the surface of a microscope slide
9. Fans 7 and 8 blow air, assisted by deflectors 7' and
8', on the lower surface of slide 9 to accelerate the evap-
oration of the cryogenic liquid. This aids in speeding
the cooling of the slide.
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The slide is supported by guides 10 and 11 which permit
lateral movement. The anatomic section to be exfoliated
is placed on the slide 9 and is compressed on it by means
of the press plate 12, which is connected to a toothed
shaft 13, moved up and down by a toothed wheel 14 forming
part of a pinion gear arrangement. With this arrangement,
the press plate 12 may exert a graduated pressure on the
section bein~ analyzed. The anatomic section is then
pressed onto the slide 9 after the latter has reached a
fixed temperature. This usually occurs 3 to 4 minutes
after the valve 4 has been opened and the cooling fans 7
and 8 have been operational.
FinaIly, the guides 10 and 11 and the slide 9 are
subjected to vibration by means of a vibrator 15 and this
subjects the anatomic section to a mechanical shock, which
in conjunction with the thermal shock facilitates the
exfoliation.
The apparatus may also be provided with a device for
automatically advancing the slide 9 so that several
depositions at different pressures may be made on the same
slide and information may be obtained with respect to the
degree of separation of the cells after the second, third,
fourth, etc. pressing. In this manner, it is possible to
reconstruct, layer by layer, the properties of the tissue.
Such results could not be achieved by MeanS of conventional
histological analysis. The degree of cellular adhesion to
the chilled slide is also an indication of the density of
the neoplasiae.
It will also be understood that various modifications
of the present invention are possible within the scope and
spirit of the present claims. For instance, the fans 7
and 8 and vibrating device 15 may be electrically activated
by a battery or by an electric current. The cryogenic
liquid may be varied depending on the desired results and
there may be used a gaseous material which can remain
liquid at very low temperatures, such as li~uid nitrogen.
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The pressurized gas may of the same material as the cryo-
genic liquid and the rate of discharge of the liquid when
the valve 4 is open may be accelerated by warming the cyl-
inder particularly in the region of the pressurized gas 3.
The apparatus of the present invention has the very
important advantage of very small dimensions and may have a
size of at the most in the range of 30 to 40 cm. This is a
feature which makes it extremely easy to handle and easy to
transport.
The cooling time varies from one bioptic section to
another and also depends upon the thickness of the slide,
but in any event, the period of time is very short, ranging
to a few tenths of a second. At the end of the cooling
period, the section is removed with tweezers, the slide
is removed from the guide and the slide is then ready for
instantaneous application of color.
According to another embodiment, the slide may be
precolored, in which case the system requires no more than
3 to 5 minutes total time Erom when the apparatus begins
to be operated up to the time when the slide is ready for
the diagnostic analysis.
From experiments which have been carried out with the
apparatus of the present invention it has been determined
that:
1. information can be obtained on the structure of the
parenchima being examined which undergoes exfoliation
in the form of a monolayer on the slide;
2. the properties of the parenchima can be determined
with respect to the density of the cells which have
undergone desquamation;
3. information is available as to the presence in the
negative of vascular structures,
4. it is also possible to determine the density of the
cells in successive pressings as an index of the
excess of cellular layers with respect to the
possibility of the stroma being bound~
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Finally, the device of the present invention makes it
possible to achieve a very substantial detailed diagnostic
study of the cells with respect to nucleus, nucleoli,
mitosis, the presence of mast cells and their quantitiz-
ation ratio between cells and cells; in case of tumors, the
relationship between neoplastic cells and normal cells;
relationship between tissues which are immuno-reactive and
normal tissues or pathological tissues as in the case of
infiltration in the liver of inflammatory substances or
phlogistic reactions in areas of tumors to be examined
in detail.
