Method of preparing tissue

Patented Jan. 22, 1946 ¢3

2,393,580

UNITED STATES PATENT OFFICE

2,393,580

METHOD OF PREPARING TISSUE

Edwin C. Weiskopf, New York, N. Y.

No Drawing. Application March 31, 1942,

Serial No. 436,967

Claims.

This invention relates to a method of preparing tissue for microscopic examination thereof.

The preparation of tissue to enable the microscopic examination thereof involves a plurality of treatments of the tissue prior to the cutting of the sections from the tissue specimens for the 'staining and mounting of the sections on the microscope slides. More particularly, in the preparation of the tissue it is necessary to immerse the issue successively in a plurality of liquid agents for certain lengths of time, first to fix the tissue, then to wash the same for removing the fixative, then to dehydrate the tissue, usually by immersion of the tissue successively in a plurality of alcohols or other dehydration agents, then to immerse the tissue in a clearing agent, and thereafter to infiltrate the tissue with an infiltration agent such as, for example, paraffin, celloidin, etc. After the tissue is thus treated, it is cut into sections of the desired thickness; then the paraffin or other infiltration medium is removed from said sections, either by a solvent or by heat, after which the sections are stained and mounted on the slides.

Due to the numerous treatments to which the tissue is subjected, as briefly described above, and due to the relatively long time required for each treatment by the several agents, respectively, in accordance with the methods heretofore practiced by pathologists and others concerned with the microscopic examination of tissue, the preparation of tissue, especially to and through the stage of preparation at which the tissue is infiltrated with paraffin or other suitable agent, required a great deal of time. For example, prior to the present invention, when it was desired to utilize Zenker’s solution as the fixative the time required for this fixation treatment alone was about two days.

I have discovered a method of treating the tissue whereby greatly to reduce the time required for preparing the tissue for microscopic examination. More particularly, I have discovered that if the tissue is immersed in the liquid agent and is slowly moved in said liquid while immersed therein, the time required for treating the tissue at each step in the course of its preparation for microscopic examination is greatly reduced, and better sections are obtained. It will be obvious of course to pathologists and others skilled in the art that the reduction of the time required for the preparation of tissue is a highly important advantage, because, among other things, it is possible to prepare slides in a relatively short time for use in cases where the (CI. 117—3) prompt examination of the tissue is an important factor and because more tissue specimens . can be prepared in a given length of time with the same implements and apparatus than was here5 tofore possible. The advantage last mentioned is especially significant in hospital or other laboratories in which a large number of tissue specimens must be prepared and examined.

Referring now more specifically to the method 10 of the present invention, the tissue which is to be prepared for microscopic examination can be in any of the customary sizes, for example, such sizes as are usual in performing autopsies, currettings, and biopsies. The tissue of the desired, 15 necessary, or available size, when treated with a liquid for fixing the tissue, is immersed in any suitable liquid according to the preference of the pathologist, for example,, in any of the following fixatives: Formalin, Bouin, Zenker’s, Halle and 20 Vande-Grift. While immersed in the fixation liquid, the tissue is kept in motion preferably byrotating the tissue specimen slowly in said liquid. The tissue is positioned in the liquid so that it is completely immersed therein and so that its sur25 face of maximum area is in vertical position, or approximately in vertical position, within the receptacle containing said liquid. The container in which the tissue specimen is carried in said vertical position is rotated about a vertical axis ³⁰ within the liquid agent in the receptacle therefor at a slow speed, the preferred speed being about two revolutions per minute. The speed of rotation may be varied from this preferred speed, but should be sufficiently low to avoid substan35 tial movement of the body of liquid while the tissue is rotated therein. In general, a suitable range of speed is from one revolution to five revolutions per minute, but the present method is not to be limited precisely to speeds within this ⁴θ range, it being understood, however, that for proper results the speed of rotation of the tissue in the liquids should be kept sufficiently low to prevent substantial movement of the liquid in which the tissue is immersed.

⁴³ The procedure described above in reference to the fixation of the tissue is repeated in each of the subsequent stages of preparing the tissue, that is in the washing of the tissue following the fixation thereof, in the dehydration of the tissue 59 by the immersion thereof successively in alcohols, dioxane, acetone or other dehydrators, in the clearing of the tissue during the immersion thereof in chloroform, benzine, xylol, cedarwood oil, aniline, etc., and in the infiltration of the de55 hydrated tissue during the immersion thereof in

2,383,680 liquid paraffin, celloidin, etc. The method of the present invention is greatly facilitated by the use of the automatic immersion apparatus described in my application Ser. No. 402,787, filed July 17, 1941.

The length of time during which the tissue Is immersed in the several liquid agents can be varied and is ordinarily varied in accordance with the particular methods followed by the different pathologists. It will be understood that pathologists, who are highly skilled experts, follow their own individual preferences in respect to methods of preparing tissue. In general, it may be stated that when the tissue is moved in the liquid as described above, in accordance with the present invention, the time of immersion can be reduced by at least 50%, and in the case of fixation by Zenker’s solution which ordinarily required about two days time, the fixation can be accomplished in accordance with the present invention in from 15 minutes to four hours depending upon the nature of the tissue, the size of the specimen, and the preference of the pathologist under whose direction the tissue is prepared. Thus it will be understood that while the time required for preparing the tissue in accordance with the present method is much less than was heretofore necessary the precise length of time of immersion of the specimen in the various liquid agents will depend upon the wishes of the various pathologists or other skilled artisans under whose direction the tissue is prepared.

After the tissue is prepared as described above, it is cut into sections as heretofore, following which the sections are treated in order to remove the paraffin, preliminary to the staining of said sections. If desired the sections can be turned in the paraffin removing liquid and in the staining liquid substantially in the same way as described above in the treatment of the tissue up through the stage of the infiltration thereof.

The following are examples of the technique of two eminent pathologists for preparing tissue in accordance with the method of the present invention:

Example I

1. Trim specimens of human tissue to thickness of about 2 mm. to 3 mm.

2. Place specimens in Zenker’s fixation fluid and fix for five hours. (Small specimens such as endometrium and skin biopsies will fix in even shorter time.)

3. Remove specimens from Zenker’s fluid and wash in water for about two hours in receptacle in which water is continually drained off and replaced by fresh water. (A siphon operated washer is used for this purpose.)

After the specimens are washed, they are immersed in the following’ liquids successively for the lengths of time indicated:

4. Alcohol, 95% for one hour.

5. Alcohol, 95% for one hour.

6. Dioxane I (with calcium chlorid) for one hour.

7. Dioxane II (with calcium chlorid) for one hour.

8. Dioxane III for one hour.

9. Dioxane IV for one hour.

10. After the tissue is treated in dioxane IV, they are infiltrated with paraffin by immersing the tissue in a paraffin bath for one hour, after which the tissue is removed from said bath and immersed in a fresh paraffin bath for two hours.

Example II

1. Fixative—VandeGrift’s—two to ten hours depending on size of specimens of tissue (human) ; trim specimens and immerse in fixative for eight hours.

2. Acetone for thirty minutes.

3. Acetone for thirty minutes.

4. Acetone for thirty minutes.

5. Acetone for thirty minutes.

6. Chloroform 85% and paraffin 15% for two hours.

7. Paraffin for two hours.

8. Paraffin for two hours.

It will be understood that during the immersion of the tissue in each of the liquids, including the paraffin baths, as set forth in the above examples of technique, the tissue specimens are continuously turned, in accordance with the present invention, as described and that this turning of the tissue and the transfer thereof from one liquid to another and the washing of the tissue take place automatically in the automatic immersion and tissue-turning apparatus described in my above mentioned application. After the tissue is embedded in paraffin it is cut into sections, deparafflnized, stained, and mounted on the slides.

While I have described the preferred mode of practicing my invention, it will be understood that the latter is susceptible to various modifications which will occur to those skilled in the art. Therefore, I do not wish to be limited to the invention as herein specifically described except as may be required by the scope of the appended claims.