Practical pathology

I. METHODS OF EXAMINATION.

Pathologic material is examined in the fresh state when it is desired to make a diagnosis in the shortest time possible, or when the processes of fixation and hardening produce such alterations in the morphology and chemic constitution of the cells that these features can be recognized only in the unfixed, fresh state. So far as the saving of time is concerned it is possible to take material removed during an operation, examine it in the fresh state, and return a diagnosis to the surgeon, while the patient is still on the table under the influence of the anæsthetic. It is not possible to do this with all tissues or with all pathologic conditions; but, when it can be done, the advantages of such a rapid diagnosis, in the saving of time, labor, expense and danger to the patient, are obvious. The best idea of the cell is also gained by its study in a fresh condition. Vital phenomena and certain morphologic features, as cilia, can be observed only in fresh material. Many of the chemic constituents of cells (glycogen, fat, mucin, pseudomucin, albumin-granules, cholesterin, etc.) are either lost, or are so changed by processes of fixation or hardening that they can no longer be recognized. The majority of specific chemic tests can be made in fresh tissues only. Moderate and slight degrees of fatty degeneration and cloudy swelling are easily recognized in the fresh state; in fixed and hardened preparations they may not be recognized at all. Particularly in the case of the heart-muscle is it necessary to make an examination of the fresh material when the diagnosis of these conditions is concerned. Further, a greater or less degree of shrinking is caused by many of the agents used in fixing and hardening, and this is avoided by the examination of the fresh material. In the case of pathologic fluids (sputum, urine, féces, etc.) an examination of the sediment in the fresh state should always be made as a matter of routine. The formed elements of these fluids are best determined by this means. In the case of tissues, a diagnosis made by means of scrapings, smears, teased bits of tissue, frozen sections, etc., should always be controlled by the examination of fixed and hardened material.

In the examination of fresh material the following methods are employed: Sedimentation, smears, scraping, crushing, teasing, maceration, sections, shaking or penciling, digestion, intravital staining, injection, the warm stage and “cultivation.”

1. Sedimentation. The formed elements of pathologic fluids (urine, sputum, pus, blood, exudates, transudates, cyst-contents, etc.) are examined by collecting the sediment of such fluids from the bottom of a sedimenting glass or bottle, by means of a capillary pipette controlled by the finger. While the sediment is passing up into the tube the pipette should be moved about the bottom of the vessel so as to get some of the sediment from all parts. When the fluid is rich in cellular elements sedimentation is not necessary; a drop of the fluid is placed upon the slide; if too thick it is diluted with physiologic salt-solution or serum. If poor in cellular elements the fluid must be centrifugalized by means of a water- or electric-centrifuge; and a drop of the sediment in the centrifuge tube is then removed by the pipette and placed upon the slide, and covered with a cover-glass. To facilitate the low-power examination of such sediments parallel streaks upon the slide may be made across its entire length, and examined without the use of cover-glasses. To apply the various reagents mentioned below it becomes necessary to use a cover-glass as directed.

2. Smears. A clean fresh cut is made into the organ or tissue, and a clean slide or cover-glass is drawn across the surface. Without permitting the smear to dry a drop of salt-solution or any desired reagent is put upon it, and it is then examined. This method is especially applicable to the study of the cells of the spleen, bone-marrow, lymphnodes, etc. Permanent balsam-mounts may be made of such smears by fixing with heat or alcohol and ether, staining, drying and mounting.

3. Scraping. A fresh cut is made into the organ or tissue, and the excess of blood absorbed by a pad of absorbent paper devoid of lint. A clean scalpel held at an angle of 45° is then drawn with some force back and forth over the cut-surface until its blade collects a sufficient amount of “tissue-juice” made up of the cells of the tissue. This is then put upon a slide, and covered with salt-solution or any desired reagent, and is then examined. This method is used especially for cellular infiltrations, soft tumors, and parenchymatous organs (spleen, lymphnodes, bone-marrow, liver, etc.), and for the inner wall of cysts (echinococcus-cysts, cysts lined with ciliated epithelium).

4. Crushing. A small bit of the tissue is cut out with the scissors or scalpel, and placed upon the slide. A cover-glass is then placed upon it and pressed down so firmly that the bit of tissue is spread out in a thin film or layer beneath the cover-glass. Reagents are introduced beneath the cover-glass, as desired. (See below.) This method is used in the examination of the lung, kidneys and brain for fatty embolism, and of the brain and spinal-cord for “fat-granule” cells, pigment, calcified ganglion-cells, Negri bodies, etc. It is also frequently used in the bacteriologic examination of tissues (crushing of tubercles, etc.).

5. Teasing. A small bit of the tissue is cut out and placed upon the slide or in a staining-dish and covered with physiologic salt-solution. It is then teased with fine needles until divided into its ultimate elements. Hard tumors (mature connective-tissue tumors, fibrosarcomata, etc.), muscle (examination for trichina), nerve-trunks, etc., are best examined in this way.

6. Maceration. In the case of some tissues the ultimate histologic elements are so firmly held together that they cannot be separated without the aid of a macerating- or dissociation-fluid. (See below.) Digestion is also used for the same end. The tissue should be as fresh as possible and cut into small bits, which are placed in the maceration-fluid in watch-glasses or staining-dishes for twenty-four hours or longer. The macerated bits are then teased until the finest elements are separated. In the case of very minute elements the teasing may be carried on under a hand-lens or the stereoscopic binocular microscope. During the process of maceration all parts of the macerating tissue must be kept covered with the macerating-fluid, or the uncovered portions will become hardened.

7. Section-cutting. Sections of fresh tissue may be made with the curved shears, simple razor, double-bladed razor and the freezing-microtome.

a. Curved Shears. The tissue is put upon a stretch, and from the surface a thin, flat section is cut out with the scissors. With care a fairly thin section may be obtained in this way. It may be examined by pressing it upon a slide beneath a cover-glass, or it may be treated as a frozen section.

b. Simple Razor. As a part of their required laboratory training medical students should learn how to make working sections with a simple razor. Such a technical knowledge is sure to be of practical use at some time or other. With a little practice sections sufficiently thin for ordinary diagnostic work can be cut. If the piece of tissue is large it may be held in the hand; but when small or soft it may be placed in a matrix of hardened liver, pith, potato, apple, firm lard or butter, paraffin, etc., and cut at the same time with the latter. Both blade and tissue should be wet with physiologic salt-solution. The blade, which must be very sharp, should be drawn through the tissue by a shoulder movement, with the wrist-joint fixed. As the sections are cut they are floated off of the razor-blade into physiologic salt-solution, and thence treated as desired.

c. Double-bladed Knife. This consists of two parallel blades arranged so that the space between them can be changed by means of screws. For firm material the blades should be close together; for soft material farther apart. The blades should be dipped into physiologic salt-solution to fill up the space between the blades. The instrument is then drawn through the tissue, and the section between the blades floated out in salt-solution. Some workers use this double-bladed knife to make a perpendicular cut into the tissue, then turning it to either side to cut the lower edge of the section and removing the blades with the section between them. Both the single- and the double-bladed razors require practice for successful section-cutting. Further, it must be borne in mind that sections of fresh tissue obtained by these methods are unsuited for complicated staining methods and can be used only for the simplest staining processes. Nevertheless, in a fairly large proportion of pathologic conditions it is possible to secure a diagnosis by these methods.

d. Freezing Microtome. Much more satisfactory sections can be obtained by freezing the fresh tissue and cutting it upon the freezing microtome. Various types of these instruments are in the market. The freezing is accomplished by the use of ether, ethyl-chloride or fluid carbonic acid gas. The ether and ethyl-chloride freezing-microtomes consist essentially of a metal plate or hollow box on which the tissue rests and against the under-surface of which a spray of ether or ethyl-chloride is forced by means of a rubber bulb connected with a supply of the freezing agent in a bottle. Such freezing attachments can be attached to any microtome, but special instruments as Jung’s “student’s freezing microtome,” Cathcart’s, Bausch and Lomb’s or Becker’s ether-freezing microtome can be recommended for this purpose. (See Fig. 52.) The tissue to be frozen must be of small size and not more than 3-4 mm. thick. It is placed upon the freezing plate in a drop of water, white of egg or thick gum-arabic, and pressed firmly against the plate. The spray must not be too constant or strong, but should be given with regular pauses of about a second to allow the ether to evaporate. The tissue must be firmly frozen, but not so hard that it crumbles. It is usually difficult to freeze the upper part of the tissue hard enough to give good sections, and this half-frozen tissue must be trimmed off with the knife until good sections are secured. As the sections are cut they are removed from the knife-blade with the finger and put into physiologic salt-solution that has been recently boiled to drive off the air, so that, in thawing, there may be no formation of air-bubbles in the tissue to cause artefacts. If put into strong alcohol diffusion-currents may damage the tissue; a succession of graded solutions should, therefore, be used if the sections are to be fixed and hardened. The further treatment of frozen sections is given below. The relative slowness of the method of freezing, its greater cost, the necessity of the frequent replacement of the rubber bulbs and tubing, and the greater amount of trouble required by the use of ether or ethyl-chloride are disadvantages that can be avoided by the use of the carbonic acid freezing microtome. Where much work is done by the freezing method the use of the latter is advised.

The carbonic acid outfit consists of a microtome arranged so that it can be connected with a cylinder of compressed carbonic acid gas, as shown in the Aschoff-Becker or Bardeen models. (Figs 53 and 54.) The cylinders or drums containing a charge of 15-30 lbs. of the liquid carbonic-acid are furnished by the trade at reasonable rates. It is not necessary to buy the drums, as they are replaced by full ones as needed. They are provided with valves and can be fastened in an upright position or laid flat upon a table, in any way convenient for the attachment to the microtome. Connection is made between the valve of the cylinder and the object-holder of the microtome by means of strong rubber tubing which should be securely wired at both ends, or by a flexible metallic tube. The latter is preferable, as the rubber-tubing often bursts, or is so stretched by the pressure of the gas that it must be frequently replaced. Object-holders with flexible metallic tube attachment for use in an ordinary microtome are supplied by Bausch and Lomb and other firms. My personal experience makes me prefer the Bardeen freezing microtome (see Fig. 54) to all others. It is cheap and can be easily attached to the valve of the gas-cylinder, which is fastened horizontally to the table-top, as it has a screw-thread fitted to the uniform thread of the drum-valve. The object-holder can be raised or lowered between the glass-tracks on which the knife runs, so that sections of a definite thickness can be obtained. The most satisfactory knife is that of the type of a plane-bit set in a wooden handle. (See Fig. 55.) It must be well honed and stropped. The tissue, which should not be more than 5 mm. thick, is placed upon the object-holder in a drop of water, albumin fixative, or saturated solution of gum-arabic, and pressed firmly against the plate as the gas is turned on slowly and evenly. Freezing is usually accomplished in one-half to one minute. The best results are obtained by turning the gas on for about 15-20 seconds, then turning it off for several seconds; the tissue will continue to freeze; if not hard enough the gas is turned on again for 15 seconds and then turned off. If necessary this may be repeated until the tissue is properly frozen. The interruption of the gas-flow prevents over-freezing and thereby lessens the amount of change in the cells. When sufficiently frozen the gas is then turned off; the knife, with bevel-edge down, is held in the right hand with its handle between the palm and the ball of the thumb with the back of the hand uppermost; and the edge of the knife set at an angle of 45° to the tracks, along which it is rapidly pushed back and forth, shaving the sections from the frozen tissue as the latter is pushed up above the level of the tracks by means of the micrometer screw turned with the left hand. The screw should have been adjusted at the proper height before freezing, so that no time is lost in getting the object-holder up to the height for cutting. By holding the elbow and fore-arm closely against the body and pushing from the elbow with wrist-joint fixed the shaving of sections can be accomplished very quickly, so that with one freezing several hundred sections can be obtained. The microtome-screw should not be turned by the left hand until the knife on its return has cleared the tissue. A little practice in co-ordinating movements is necessary for expert work. The sections should be allowed to collect upon the knife-blade until a large number have been cut; they are then swept off the blade by the finger into cold freshly-boiled physiologic salt-solution. Sections of tissue that have been previously fixed can be put into 60 per cent alcohol, where the sections will unroll and straighten out perfectly flat. When the method of freezing by alternately turning on and off the gas, as given above, is followed, and the amount of freezing in the interval noted, there is no danger of over-freezing, and the breaking and crumbling of sections, with the production of marked artefacts, is avoided. When over-freezing has occurred the block may be partly thawed out by the finger. But over-freezing, as well as repeated freezing and thawing, may cause so much damage to fresh tissue that a diagnosis cannot be obtained from the frozen sections. After thawing out in the physiologic salt-solution the sections of fresh tissue obtained by freezing are treated according to the methods given in the next section of this chapter.

It must be emphasized here that the process of freezing is an active one, and alters the relation of cell-structures. With many fresh tissues the changes resulting from the freezing are so great that no diagnosis can be made. It seems necessary here to warn against the routine employment of the rapid method of freezing and staining fresh tissues in the diagnosis of material obtained by surgical operation. It has become a fad with some surgeons to make a pathologic diagnosis by the freezing method while the patient is on the table. Consequently, as the result of diagnoses made by the rapid freezing and staining method, many mistakes are made, even by supposed experts in this line. Particularly in the diagnosis of sarcoma is it easy to make mistakes because of the altered aspect of the cells caused by freezing. Normal lymphnodes, tonsils and inflammatory infiltrations may look like spindle-cell sarcoma in the sections prepared by the rapid freezing and staining method; and the exact nature of many other pathologic conditions cannot be accurately determined from such sections. On the other hand, a certain number of pathologic conditions can always be recognized in sections obtained in this way, and this fact justifies the employment of the method when properly controlled. In all cases in which the pathologic condition is not clearly evident in sections obtained by the rapid freezing and staining method no diagnosis should be given. In such cases the tissue should be fixed and then cut upon the freezing-microtome, or imbedded in celloidin or paraffin and then cut. Even when the patient is upon the table the tissue removed can be put into a 10 per cent formol solution for a few minutes and then frozen directly in gum without washing out the formalin. The longer the time that can be used for this short preliminary fixation the better the sections will be and the less the production of artefacts by the freezing. The process of fixation can be hastened by warming the fixing-fluid. I advise this short fixation before freezing in all cases of operative diagnostic work when the diagnosis is wanted as soon as possible. For all other work with the freezing microtome, when the question of time is not so important, fixation with formol for 12-24 hours should be carried out. This combination of formol-fixation and the freezing method permits the early diagnosis of autopsy and operation material, makes possible the demonstration of fat and other substances altered or dissolved out by the imbedding methods, and is a convenient way of selecting tissues, requiring more complicated staining processes. (See also Page 239.) The further treatment of sections of fresh tissue obtained by freezing will be found in the second section of this chapter.

8. Penciling or Shaking. For the demonstration of the stroma or reticulum either fresh or fixed sections may be placed upon a slide in an abundance of fluid and gently penciled with a fine, blunt camel’s hair brush until the fluid becomes cloudy. The cloudy fluid is washed away and replaced by fresh as long as cells are given off. The same results may be obtained by shaking the sections in a test-tube until the cells are shaken out of the stroma. The removal of the cells from the section is shown by its greater transparency. One of the practical applications of these methods in diagnostic work is the differentiation between alveolar round-cell sarcomata and carcinomata.

9. Digestion. For the demonstration of stroma, parasites, etc., the tissues may be digested with gastric or pancreatic ferments until the required elements are freed. A freshly prepared pepsin in 0.2 per cent HCl in the incubator for 3-5 hours will digest fibrin in fresh clots. Sections of fixed tissues may be imbedded in paraffin, cut, and digested on the slide with Grübler’s pancreatin according to the method of Flint.

10. Intravital and Supravital Staining. Various methods have been advised for the intravital staining of cell-granules. Intravenous or intraperitoneal injections of methylene blue, alum carmine, neutral red and other stains will produce intracellular granule-staining in various organs of experimental animals. In the study of low forms of animal life staining solutions may be injected, or the animal or its parts may be examined in staining fluids. Human material can be examined by this method immediately after being removed from the body by operation or within 1-2 hours after death. (For details of these methods see article on “Färbungen, intravitale,” Encyklopädie der mikroskopischen Technik.)

11. Injection. Injections for the demonstration of blood-vessels, lymphatics, ducts of glands, etc., are rarely used in pathologic work. The organs to be injected must be fresh, warm from the body, if possible. The vessels should be washed out by a freshly filtered 8 per cent sodium nitrate or sodium sulphate solution, followed by physiologic salt-solution. A cannula is introduced into a main vessel, tightly secured, and then connected with a syringe or a gravity injection-apparatus giving a constant pressure. The injection-mass is then injected under a low pressure. In the case of injections into lymphatic vessels the cannula should be introduced into the largest lymphatics at the periphery of the blood-vessels where larger lymph-vessels are more easily found. Injections are made with either cold or warm solutions; the latter are preferable but require that the organ to be injected be warmed by immersing it in water at a temperature of 40°C. The injection fluid must be of the same temperature. After the warm injection is given the organ is put into ice-cold 10 per cent formol solution until fixed and then after-hardened, imbedded and stained as desired. After the use of cold injections the tissues are fixed in 10 per cent formol, alcohol, or any other desired solution, and treated according to the end sought. Nuclear and diffuse stains contrasting with the color of the injection mass should be used. The process of injection requires great care; the pressure must be carefully regulated to prevent extravasations, and the injection-fluid must be free from air-bubbles. The injection is continued until the organ appears diffusely stained. Blood-vessels are fixed in their natural blood-injection by such agents as formol and chromic acid, so that stains acting upon the red blood cells cause the veins and capillaries to appear as if they had been injected.

The following injection masses are advised:—

12. Warm Stage. For the study of vital phenomena in the living cell Ross’s electrical warmer is recommended. It can be slipped on and off the slide without changing the focus, and is managed without any difficulty. It keeps the centre of the slide at a temperature of 37°C. Reagents can be applied as desired. Deetjen’s agar may be used as a medium for the preservation of living cells. (See Methods of Blood-Examination.) Various forms of warm and moist chambers used in experimental embryological work can also be utilized in experimental pathology.

13. Tissue-cultivation. The embryologic methods of growing tissues in lymph and blood-plasma as developed by Harrison, Burrows and Carrel have been applied in pathologic work to the experimental study of repair and regeneration, grafting, transplantation and tumor-transplantation. (For methods see Harrison, Journal of Exper. Zoology, 1910; Burrows, Jour. of Amer. Med. Assoc., 1910; Carrel, Jour. of Amer. Med. Assoc., 1910.)

II. REAGENTS USED IN THE EXAMINATION OF FRESH TISSUES.

In the examination of fresh tissue it is often desirable to use certain reagents for the purpose of making chemical tests or to bring out some structures more prominently than others. To introduce these reagents beneath the cover-glass in such a way as to get the desired effect without disturbing given fields requires some practice with a very simple technical method. The preparation is first examined in salt-solution, and the cover-glass adjusted so that it has a slight rim of fluid about its edge, but not enough to make it float. The reagent to be applied is dropped with a glass-dropper at one side of the cover-glass, while at the other the salt-solution is removed slowly by a piece of absorbent paper. The changes produced in the tissue-elements during the progress of the reagent can be observed under low or high powers. Care must be taken to change the fluids so slowly that isolated cells will not be washed away.