NEW STAINS IN TISSUE DIAGNOSIS

Transcription

1 NEW STAINS IN TISSUE DIAGNOSIS CHARLES F. GESCHICKTER, M.D. (Prom the Surgical Pathological Lahoratory of the Johns Hopkins Hospital ami Univel'sily, Baltimore, Maryland) The use of stains has long been essential to the study of the histologic characteristics of cancer as well as of normal and other diseased tissues. The practical need, however, for a rapid recognition of cancer tissue for diagnosis in the operating room or by the pathologist elsewhere, in cooperation with the practitioner, has led to the attempt to find some dye which will act as a specific indicator for the presence of malignancy. In attempting to use histological stains in cancer diagnosis, the technic used in the preparation of tissue for microscopic work must be the fundamental starting point. No matter what method is used in bringing the stain into contact with the cell, the fact remains that in studying the problem experimentally there must be some means for the experimenter to observe and record how the dye acts in the presence of the tumor cells. There are available, of course, numerous histologic methods, as frozen sections, stained smears on slides, hanging drop preparations of cells suspended in fluid, and the cutting of hardened and embedded tissues for microscopic examination, but no single method employed at the present time entirely meets the problem. The reasons for this are obvious. If tissue is employed that has been fixed and hardened and embedded in paraffin or celloidin, many of its original characteristics may be lost or changed by the innumerable steps in the technic, which involve the death of the cell, the combination of the cell protein with the fixing reagents, and the change in the acid-base reaction brought about by embedding and other processes. The time element necessary for such fixed section technic is also too long to make it useful in the operating room at biopsy. If, on the other hand, frozen sections are used, we are immediately restricted in the type of dyes and dye solvents which may be applied to the tissue. It thus becomes necessary, in attempting the development of a new rapid method for tissue diagnosis, to employ new solvents for 2169

2 2170 CHARLES F. GESCHICKTER staining, by means of which acid and basic dyes may be mixed in any desired proportion and at the same time alcoholic solutions and other solvents injurious to tissue be avoided. Such a method involves pre-treating sections of fresh tissue in certain buffer solutions, so that pictures may be obtained in fresh tissue which are identical, or directly comparable, with those obtained on fixed tissue. Finally, a technic for dehydrating and clearing frozen sections is required, which will permit mounting in balsam without marked shrinkage or loss of color, so that the staining reaction may be preserved for study and recorded. The staining technic which is presented here is not offered as a specific stain for cancer diagnosis but represents an attempt to obtain a more satisfactory histologic procedure for the study of various dyestuffs when employed in the presence of freshly excised cancer tissue. It represents an endeavor to correlate what is seen in the fixed section with what is found in tissue immediately after its removal from the body, and with the processes which are going on while that tissue still remains a vital part of its surroundings. This technic makes possible the use of acid-base staining reactions on fixed tissues and the use of the same reactions with fresh tissue, and in this manner not only throws much light upon the living biological properties of the tumor, but also permits an evaluation of the subsequent changes brought about by the technic itself. A buffer solution is used as a prestaining bath for the purpose of counteracting lactic acid formation in fresh tissue and bringing the section before staining, regardless of how fixed, as near as possible to the neutrality point. This buffer solution is a mixture of potassium acid phosphate and sodium hydroxide with a reaction of ph 7.0. From this buffer solution the cut sections are passed into a staining mixture which is dissolved in ethylene glycol, diluted by a small addition of ethyl alcohol (80 parts ethylene glycol to 20 parts of 95 per cent alcohol). This solvent not only dissolves stains which will precipitate in water, or even in alcohol, but at the same time has sufficient viscosity to prevent shrinkage and injury to the fresh tissue. Its action on cells in a viable state is relatively non-toxic and thus permits a great variability in the dyestuffs used. When these non-aqueous stains have once penetrated the tissues, they do not decolorize readily, and the sections are, therefore, more easily handled when dehydration and clearing must be employed. At present the dyes used have been various derivatives

3 PLATE I PHOTOMICROGRAPH OF BENIGN AND MALIGNANT AREAS IN A SECTION TAKEN FROM CARCINOMA m' THE RECTUM The section at the time of photographing was ten months old and the tissue was fresh, without fixation, at the time of staining. Preservation and dehydration were carried out as described in the text. Stained by thionin-eosinate mixture.

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5 PLATE II PHOTOMICROGRAPH OF A PARAFFIN SECTION FROM A EWING'S SARCOMA OF BONE Stained by thionin-eosinate mixture.

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7 NEW STAINS IN TISSUE DIAGNOSIS 2171 of thionin, chemically compounded with eosin. These dyes are relatively amphoteric, the blue or basic portion giving a nuclear stain and the pink or acid portion staining the cytoplasm. The novel element in their use depends, of course, upon the solvent used, since many of these compounds are insoluble in water. The staining solution found most satisfactory to date consists of: A. Thionin-eosinate, 1 per cent, two parts. B. Eosin yellowish (alcohol soluble only), 1 per cent, two parts. C. Azure A, 1 per cent, one part. Each of these powdered dyes is first dissolved in alcohol ethylene glycol solution, and the resulting solutions are mixed in the proportions indicated. The mixture should be allowed to stand without filtering for twenty-four hours, with intermittent shaking, in order that any precipitate formed may pass again into solution. All the dyestuffs used are obtainable commercially, and were supplied by the National Aniline and Chemical Company, 40 Rector Street, New York City, with the exception of the thionineosinate, which was prepared by Mr. W. C. Holmes in the Color Laboratories of the Department of Agriculture, Washington, D.C. This dye compound is prepared by the precipitation of two molecular weights of thionin hydrochloride and one molecular weight of the sodium salt of eosin in aqueous solution. After staining, the sections are washed and then dehydrated, cleared, and mounted. The dehydration and clearing of sections which have had no previous fixation is a difficult matter, since the solutions customarily employed either shrink unfixed tissue too markedly or decolorize the usual synthetic dyes. It was necessary, therefore, to employ organic solvents hitherto unused in staining technics, which were neither injurious to the tissue nor to the staining agents. The dehydrating reagent must be miscible with water, whereas the clearing reagent must be miscible with balsam or the various mounting gums. Diethylene glycol monobutyl ether has been found satisfactory as a dehydrating reagent and normal butyl phthalate has proved valuable as a clearing reagent. These two compounds make it possible for the section to be transferred, after staining, to the slide, and a permanent cover slip mounted in gum damar immediately applied. A rapid and relatively permanent section is thus obtained. These sections have the advantage of the frozen section in rapidity and, to a large degree, the per-

8 2172 CHARLES F. GESCHICKTER manency and differentiation of structure seen in a section of fixed and embedded material. More important still, the use of the method makes possible the application of a single staining technic interchangeably to fresh frozen sections, formalin-fixed frozen sections, and tissues embedded in paraffin or celloidin. Thus staining properties of cancer tissue which can be demonstrated by careful studies on fixed material can be applied to the fresh material which is obtained for immediate section in the operating room. This is proving a very valuable aid in the study of cancer material and also a great saving of time and money in preparing tissue for diagnosis. TECHNIC A. The sections before staining should be allowed to remain in the buffer for about thirty seconds. If the tissue has been fixed for many months in formalin, or other preservative, it should be left in the buffer solution for about one hour. B. From the buffer solution the sections are stained by immersing in the dye for from forty-five seconds to one minute. Fixed sections of tissue preserved for some months may require two to three minutes. C. After removal from the stain, the tissues are washed in tap water and are ready for dehydrating and mounting. D. Dehydrating is accomplished by passing the washed sections into di-ethylene glycol monobutyl ether and floating the sections in this fluid for twenty to thirty seconds. E. Sections are then passed into normal butyl phthalate, from which the sections are floated on to a glass slide, blotted, and mounted in a drop of gum damar.