III. MICROSCOPIC EXAMINATION
III. MICROSCOPIC EXAMINATION
A careful microscopic examination will often detect structures of great diagnostic importance in urine which seems perfectly clear, and from which only very slightsediment can be obtained with the centrifuge. Upon the other hand, cloudy urines with abundant sediment are often shown by the microscope to contain nothing of clinical significance.
Since the nature of the sediment soon changes, the urine must be examined while fresh, preferably within six hours after it is voided. The sediment is best obtained by means of the centrifuge. If a centrifuge is not available, the urine may be allowed to stand in a conical test-glass for six to twenty-four hours after adding some preservative (p. 48). The "torfuge" (Fig. 31) is said to be a very satisfactory substitute for the centrifuge, and is readily portable.
A small amount of the sediment should be transferred to a slide by means of a pipet. It is very important to do this properly. The best pipet is a small glass tube which has been drawn out at one end to a tip with rather small opening. The tube or glass containing the sediment is held on a level with the eye, the larger end of the pipet is closed with the index-finger, which must be dry, and thetip is carried down into the sediment. By carefully loosening the finger, but not entirely removing it, a small amount of the sediment is then allowed to run slowly into the pipet. Slightly rotating the pipet will aid in accomplishing this. After wiping off the urine which adheres to the outside, a drop from the pipet is placed upon a clean slide. A hair is then placed in the drop and a large cover-glass applied. Many workers use no cover. This offers a thicker layer and larger area of urine, the chance of finding scanty structures being proportionately increased. It has the disadvantage that any jarring of the room (as by persons walking about) sets the microscopic field into vibratory motion and makes it impossible to see anything clearly; and, since it does not allow of the use of high-power objectives, one cannot examine details as one often wishes to do. A large cover-glass with a hair beneath it avoids these disadvantages, and gives enough urine to find any structures which are present in sufficient number to have clinical significance, provided other points in the technic have been right. It is best, however, to examine several drops; and, when the sediment is abundant, drops from the upper and lower portions should be examined separately.
In examining urinary sediments microscopically no fault is so common, nor so fatal to good results, as improper illumination (seeFigs. 2 and 3), and none is so easily corrected. The light must be central and very subdued. The two-thirds objective should be used as a finder, while the one-sixth is reserved for examining details.
It is well to emphasize thatthe most common errors which result in failure to find important structures, when present, are lack of care in transferring the sediment to the slide, too strong illumination, and too great magnification.
In order to distinguish between similar structures it is often necessary to watch the effect upon them of certain reagents. This is especially true of the various unorganized sediments. They very frequently cannot be identified from their form alone. With the structures still in focus, a drop of the reagent may be placed at one edge of the cover-glass and drawn underneath it by the suction of a piece of blotting-paper touched to the opposite edge; or a small drop of the reagent and of the urine may be placed close together upon a slide and a cover gently lowered over them. As the two fluids mingle, the effect upon various structures may be seen.
Urinary sediments may be studied under three heads: A. Unorganized sediments. B. Organized sediments. C. Extraneous structures.
A. UNORGANIZEDSEDIMENTS
A. UNORGANIZEDSEDIMENTS
In general these have little diagnostic or prognostic significance. Most of them are substances normally present which have been precipitated from solution either because present in excessive amounts or, more frequently, because of some alteration in the urine (as in reaction, concentration, etc.) which may be purely physiologic, depending upon changes in diet or habits. Various substances are always precipitated during decomposition, which may take place either within or without the body. Unorganized sediments may be classified according to the reaction of the urine in which they are most likely to be found:
In acid urine: uric acid, amorphous urates, sodium urate, calcium oxalate, leucin and tyrosin, cystin, and fat-globules. Uric acid, the urates, and calcium oxalate arethe common deposits of acid urines; the others are less frequent, and depend less upon the reaction of the urine.
In alkaline urine: phosphates, calcium carbonate, and ammonium urate.
Other crystalline sediments (Fig. 32) which are rare and require no further mention are: calcium sulphate, cholesterin, hippuric acid, hematoidin, fatty acids, and indigo.
1.In Acid Urine.—(1)Uric-acid Crystals.—These crystals are the red grains—"gravel" or "red sand"—which are often seen adhering to the sides and bottom of a vessel containing urine. Microscopically, they are yellow or reddish-brown crystals, which differ greatly in size and shape. The most characteristic forms (Plate III and Fig. 33) are "whetstones"; roset-like clusters of prisms and whetstones; and rhombic plates, which have usually a paler color than the other forms and are sometimes colorless. Recognition of the crystals depends less upon their shape than upon their color, the reaction of the urine, and the facts that they are soluble in caustic soda solution and insoluble in hydrochloric or acetic acid.When ammonia is added, they dissolve and crystals of ammonium urate appear.
A deposit of uric-acid crystals has no significance unless it occurs before or very soon after the urine is voided. Every urine, if kept acid, will in time deposit its uric acid. Factors which favor an early deposit are high acidity, diminished urinary pigments, and excessive excretion of uric acid. The chief clinical interest of the crystals lies in their tendency to form calculi, owing to the readiness with which they collect about any solid object. Their presence in the freshly voided urine in clusters of crystals suggests stone in the kidney or bladder, especially if blood is also present (seeFig. 62).
(2)Amorphous Urates.—These are chiefly urates ofsodium and potassium which are thrown out of solution as a yellow or red "brick-dust" deposit. In pale urines this sediment is almost white. It disappears upon heating. A deposit of amorphous urates is very common in concentrated and strongly acid urines, especially in cold weather, and has no clinical significance. Under the microscope it appears as fine yellowish granules, often so abundant as to obscure all other structures (Plate III). In such cases the urine should be warmed before examining. Amorphous urates are readily soluble in caustic soda solutions. When treated with hydrochloric or acetic acid they slowly dissolve and rhombic crystals of uric acid appear.
Rarely, sodium urate occurs in crystalline form—slender prisms, arranged in fan- or sheaf-like structures (Fig. 32).
(3)Calcium Oxalate.—Characteristic of calcium oxalate are colorless, glistening, octahedral crystals, giving the appearance of small squares crossed by two intersecting diagonal lines—the so-called "envelop crystals" (Fig. 47). They vary greatly in size, being sometimes so small as to seem mere points of light with medium-power objectives. Unusual forms, which, however, seldom occur except in conjunction with the octahedra, are colorless dumb-bells, spheres, and variations of the octahedra (Fig. 34). The spheres might be mistaken for globules of fat or red blood-corpuscles. Crystals of calcium oxalate are insoluble in acetic acid or caustic soda. They are dissolved by strong hydrochloric acid, and recrystallize as octahedra upon addition of ammonia. They are sometimes encountered in alkaline urine.
The crystals are commonly found in the urine after ingestion of vegetables rich in oxalic acid, as tomatoes,spinach, asparagus, and rhubarb. They have no definite significance pathologically. They often appear in digestive disturbances, in neurasthenia, and when the oxidizing power of the system is diminished. Like uric acid, their chief clinical interest lies in their tendency to form calculi, and their presence in fresh urine, together with evidences of renal or cystic irritation, should be viewed with suspicion, particularly if they are clumped in small masses.
(4)Leucin and Tyrosin.—-Crystals are deposited only when the substances are present in considerable amount. When present in smaller amount, they will usually be deposited if a drop of the urine be slowly evaporated upon a slide. They generally appear together, and are of rare occurrence, usually indicating severe fatty destruction of the liver, such as occurs in acute yellow atrophy and phosphorus-poisoning.
The crystals cannot be identified from their morphology alone, since other substances, notably calcium phosphate (Fig. 38) and ammonium urate, may take similar or identical forms.
Leucin crystals(Fig. 35) are slightly yellow, oily-looking spheres, many of them with radial and concentricstriations. Some may be merged together in clusters. They are not soluble in hydrochloric acid nor in ether.
Tyrosincrystallizes in very fine colorless needles, usually arranged in sheaves, with a marked constriction at the middle (Fig. 35). It is soluble in ammonia and hydrochloric acid, but not in acetic acid.
(5)Cystin crystalsare colorless, highly refractive, rather thick, hexagonal plates with well-defined edges. They lie either singly or superimposed to form more or less irregular clusters (Fig. 36). Uric acid sometimestakes this form and must be excluded. Cystin is soluble in hydrochloric acid, insoluble in acetic; it is readily soluble in ammonia and recrystallizes upon addition of acetic acid.
Cystin crystals are very rare, and when found, point to cystin calculus.
(6)Fat-globules.—Fat appears in the urine as highly refractive globules of various sizes, frequently very small. These globules are easily recognized from the fact that they are stained black by osmic acid and red by Sudan III. The stain may be applied upon the slide, as already described (p. 103). Osmic acid should be used in 1 per cent. aqueous solution; Sudan III in saturated solution in 70 per cent. alcohol.
Fat in the urine is usually a contamination from unclean vessels, oiled catheters, etc. A very small amount may be present after ingestion of large quantities of cod-liver oil or other fats. In fatty degeneration of the kidney, as in phosphorus-poisoning and chronic parenchymatous nephritis, fat-globules are commonly seen, both free in the urine and embedded in cells and tube-casts.
Inchyluria, or admixture of chyle with the urine as a result of rupture of a lymph-vessel, minute droplets of fat are so numerous as to give the urine a milky appearance. Chyluria occurs most frequently as a symptom of infection byFilaria sanguinis hominis.
2.In Alkaline Urine.—(1)Phosphates.—While most common in alkaline urine, phosphates are sometimes deposited in neutral or feebly acid urines. The usual forms are: (a) Ammoniomagnesium phosphate crystals; (b) acid calcium phosphate crystals; and (c) amorphous phosphates. All are readily soluble in acetic acid.
(a)Ammoniomagnesium Phosphate Crystals.—They arethe common "triple phosphate" crystals, which are generally easily recognized (Figs. 37 and63, andPlate IV). They are colorless, except when bile-stained. Their usual form is some modification of the prism, with oblique ends. Most typical are the well-known "coffin-lid" and "hip-roof" forms. The long axis of the hip-roof crystal is often so shortened that it resembles the envelop crystal of calcium oxalate. It does not, however, have the same luster; this, and its solubility in acetic acid, will always prevent confusion.
When rapidly deposited, as by artificial precipitation, triple phosphate often takes feathery, star- or leaf-like forms. These gradually develop into the more common prisms. X-forms may be produced by partial solution of prisms.
(b)Acid Calcium Phosphate Crystals.—In feebly add, neutral, or feebly alkaline urines acid calcium phosphate, wrongly called "neutral calcium phosphate," is not infrequently deposited in the form of colorless prismsarranged in stars and rosets (Fig. 38, 1). The individual prisms are usually slender, with one beveled, wedge-like end, but are sometimes needle-like. They may sometimes take forms resembling tyrosin (Fig. 38, 2), calcium sulphate, or hippuric acid, but are readily distinguished by their solubility in acetic acid.
Calcium phosphate often forms large, thin, irregular, usually granular, colorless plates, which are easily recognized (Fig. 38, 3).
(c)Amorphous Phosphates.—The earthy phosphates are thrown out of solution in most alkaline and many neutral urines as a white, amorphous sediment, which may be mistaken for pus macroscopically. Under the microscope the sediment is seen to consist of numerous colorless granules, distinguished from amorphous urates by their color, their solubility in acetic acid, and the reaction of the urine.
The various phosphatic deposits frequently occur together. They are sometimes due to excessive excretion of phosphoric acid, but usually merely indicate that the urine has become, or is becoming, alkaline.
(2)Calcium carbonatemay sometimes be mingled with the phosphate deposits, usually as amorphous granules, or, more rarely, as colorless spheres and dumb-bells, Fig. 39, which are soluble in acetic acid with gas-formation.
(3)Ammonium Urate Crystals.—This is the only urate deposited in alkaline urine. It forms opaque yellow crystals, usually in the form of spheres (Plate IV, andFig. 63), which are often covered with fine or coarse spicules, "thorn-apple crystals." Sometimes dumb-bells, compact sheaves of fine needles, and irregular rhizome forms are seen (Fig. 40). Upon addition of acetic acid they dissolve, and rhombic plates of uric acid appear.
These crystals occur only when ammonia is present in excess. They are generally found along with the phosphates in decomposing urine and have no clinical significance.
B. ORGANIZEDSEDIMENTS
B. ORGANIZEDSEDIMENTS
The principal organized structures in urinary sediments are: tube-casts; epithelial cells; pus-corpuscles; red blood-corpuscles; spermatozoa; bacteria; and animal parasites. They are much more important than the unorganized sediments just considered.
1.Tube-casts.—These interesting structures are albuminous molds of the uriniferous tubules. Their presence in the urine probably always indicates some pathologic change in the kidney, although this change may be very slight or transitory. Large numbers may be present in temporary irritations and congestions.They do not in themselves, therefore, imply organic disease of the kidney.They probably occur only in urine which contains, or has recently contained, albumin.
While it is not possible to draw a sharp dividing-line between the different varieties, casts may be classified as: (1) Hyaline casts; (2) waxy casts; (3) granular casts; (4) fatty casts; (5) casts containing organized structures—(a) epithelial casts; (b) blood-casts; (c) pus-casts; (d) bacterial casts. As will be seen, practically all varieties are modifications of the hyaline.
The significance of the different varieties is more readily understood if one considers their mode of formation. Albuminous material, the source and nature of which are not definitely known, probably enters the lumen of a uriniferous tubule in a fluid or plastic state. It there hardens into a mold, which, when washed out by theurine, retains the shape of the tubule, and contains within its substance whatever structures and débris were lying free within the tubule or were loosely attached to its wall. If the tubule be small and have its usual lining of epithelium, the cast will be narrow; if it be large or entirely denuded of epithelium, the cast will be broad. A cast, therefore, indicates the condition of the tubule in which it is formed.
The search for casts must be carefully made. The urine must be fresh, since hyaline casts soon dissolve when it becomes alkaline. It should be thoroughly centrifugalized. When the sediment is abundant, casts, being light structures, will be found near the top. In cystitis, where casts may be entirely hidden by the pus, the bladder should be irrigated to remove as much of the pus as possible and the next urine examined. In order to prevent solution of the casts the urine, if alkaline, must be rendered acid by previous administration of boric acid or other drugs.
(1)Hyaline Casts.—Typically, these are colorless, homogeneous, semitransparent, cylindric structures, with parallel sides and usually rounded ends. Not infrequently they are more opaque or show a few granules or an occasional cell, either adhering to them or contained within their substance. Generally they are straight or curved; less commonly, convoluted. Their length and breadth vary greatly: they are sometimes so long as to extend across several fields of a medium-power objective, but are usually much shorter; in breadth, they vary from one to seven or eight times the diameter of a red blood-corpuscle. (See Figs.2, 41, 42, and46.)
Hyaline casts are the least significant of all the casts,and occur in many slight and transitory conditions. Small numbers are common following ether anesthesia, in fevers, after excessive exercise, and in congestions and irritations of the kidney. They are always present, and are usually stained yellow when the urine contains much bile. While they are found in all organic diseases of the kidney, theyare most important in chronic interstitial nephritis. Here they are seldom abundant, but their constant presence is the most reliable urinary sign of the disease. Small areas of chronic interstitial change are probably responsible for the few hyaline casts so frequently found in the urine of elderly persons.
Very broad hyaline casts commonly indicate complete desquamation of the tubular epithelium, such as occurs in the late stages of nephritis.
(2)Waxy Casts.—Like hyaline casts, these are homogeneous when typical, but frequently contain a few granules or an occasional cell. They are much more opaque than the hyaline variety, and are usually shorter and broader, with irregular, broken ends, and sometimes appear to be segmented. They are grayish or colorless, and have a dull waxy look, as if cut from paraffin (Figs. 43 and61). They are sometimes composed of material which gives the amyloid reactions. Waxy casts are found in most advanced cases of nephritis, where they are an unfavorable sign.
Casts which resemble waxy casts but have a distinctlyyellow color, as if cut from beeswax (so-called "fibrinous casts"), are often seen in acute nephritis. They have less serious significance than the true waxy variety.
(3)Granular Casts.—These are merely hyaline casts in which numerous granules are embedded (Figs. 42, 44,46, and61).
Finely granular castscontain many fine granules, are usually shorter, broader, and more opaque than the hyaline variety, and are more conspicuous. Their color is grayish or pale yellow.
Coarsely granular castscontain larger granules and are darker in color than the finely granular, being often dark brown owing to presence of altered blood-pigment. They are usually shorter and more irregular in outline, and more frequently have irregularly broken ends.
(4)Fatty Casts.—Small droplets of fat may at times be seen in any variety of cast. Those in which the droplets are numerous are called fatty casts (Figs. 43 and 44). The fat-globules are not difficult to recognize. Staining with osmic acid or Sudan (p. 109) will remove any doubt as to their nature.
The granules and fat-droplets seen in casts are products of epithelial degeneration. Granular and fatty casts, therefore, always indicate partial or complete disintegration of the renal epithelium. The finely granular variety is the least significant, and is found when the epithelium is only moderately affected. Coarsely granular, and especially fatty casts, indicate a serious parenchymatous nephritis.
(5)Casts Containing Organized Structures.—Cells and other structures are frequently seen adherent to a cast or embedded within it. (SeeFigs. 41 and 42). When numerous, they give name to the cast.
(a)Epithelial castscontain epithelial cells from the renal tubules. They always imply desquamation of epithelium, which rarely occurs except in parenchymatous inflammations (Figs. 60 and 61).
(b)Blood-castscontain red blood-corpuscles, usually much degenerated (Figs. 45 and60). They always indicate hemorrhage into the tubules, which is mostcommon in acute nephritis or an acute exacerbation of a chronic nephritis.
(c)Pus-casts(seeFig. 62), composed almost wholly of pus-corpuscles, are uncommon, and point to a chronic suppurative process in the kidney.
(d) Truebacterial castsare rare. They indicate a septic condition in the kidney. Bacteria may permeate a cast after the urine is voided.
The structures most likely to be mistaken for casts are:
(1)Mucous Threads.—Mucus frequently appears in the form of long strands which slightly resemble hyaline casts (Fig. 46). They are, however, more ribbon-like, have less well-defined edges, and usually show faint longitudinal striations. Their ends taper to a point or are split or curled upon themselves, and are never evenly rounded, as is commonly the case with hyaline casts.
(2)Cylindroids.—This name is sometimes given to themucous threads just described, but is more properly applied to certain peculiar structures more nearly allied to casts. They resemble hyaline casts in structure, but differ in being broader at one end and tapering to a slender tail, which is often twisted or curled upon itself (Fig. 46). They frequently occur in the urine along with hyaline casts, and have no definite pathologic significance.
(3)Masses of amorphous uratesorphosphatesorvery small crystals(Fig. 47), which accidentally take a cylindric form, or shreds of mucus covered with granules, closely resemble granular casts. Application of gentle heat or appropriate chemicals will serve to differentiate them. When urine contains both mucus and granules, large numbers of these "pseudo-casts," all lying in the same direction, can be produced by slightly moving the cover-glass from side to side. It is possible—as in urate infarcts of infants—for urates to be molded into cylindric bodies within the renal tubules.
(4)Hairsandfibersof wool, cotton, etc. These could be mistaken for casts only by beginners. One can easilybecome familiar with their appearance by suspending them in water and examining with the microscope (Fig. 57).
(5)Hyphæofmoldsare not infrequently mistaken for hyaline casts. Their higher degree of refraction, their jointed or branching structure, and the accompanying spores will differentiate them (Fig. 58).
2.Epithelial Cells.—A few cells from various parts of the urinary tract occur in every urine. A marked increase indicates some pathologic condition at the site of their origin. It is sometimes, but by no means always, possible to locate their source from their form. Any epithelial cell may be so granular from degenerative changes that the nucleus is obscured. They are usually divided into three groups:
(1)Small, round,orpolyhedral cellsare about the size of pus-corpuscles, or a little larger, with a single round nucleus. Such cells may come from the deeper layers of any part of the urinary tract. They are uncommon in normal urine. When they are dark in color, very granular, and contain a comparatively large nucleus, they probably come from the renal tubules, but their origin in the kidney is not proved unless they are found embedded in casts. Renal cells are abundant in parenchymatous nephritis, especially the acute form. They are nearly always fatty—most markedly so in chronic parenchymatous nephritis, where theirsubstance is sometimes wholly replaced by fat-droplets ("compound granule cells") (see Figs.44, 48,60, and61).
(2)Irregular cellsare considerably larger than the preceding. They are round, pear-shaped, or spindle-shaped, or may have tail-like processes, and are hence named large round, pyriform, spindle, or caudate cells respectively. Each contains a round or oval distinct nucleus. Their usual source is the deeper layers of the urinary tract, especially of the bladder. Caudate forms come most commonly from the pelvis of the kidney (see Figs. 49,b, 50,62, and63).
(3)Squamousorpavement cellsare large flat cells, each with a small, distinct, round or oval nucleus (Fig. 49,a). They are derived from the superficial layers of the ureters, bladder, urethra, or vagina. Those from the bladder are generally rounded, while those from the vagina are larger, thinner, and more angular. Great numbers of these vaginal cells, together with pus-corpuscles, may be present when leukorrhea exists.
3.Pus-corpuscles.—A very few leukocytes are present in normal urine. They are more abundant when mucus is present. An excess of leukocytes, mainly of the polymorphonuclear variety, with albumin, constitutespyuria—pus in the urine.
When at all abundant, pus forms a white sediment resembling amorphous phosphates macroscopically. Under the microscope the corpuscles appear as very granular cells, about twice the diameter of a red blood-corpuscle (Figs. 51 and63). In freshly voided urine many exhibit ameboid motion, assuming irregular outlines. Each contains one irregular nucleus or several small, rounded nuclei. The nuclei are obscured or entirely hidden by the granules, but may be brought clearly into view by running a little acetic acid under the cover-glass. This enables one to easily distinguish pus-corpuscles from small round epithelial cells, which resemble them in size, but have a single, rather large, round nucleus.
Pyuria indicates suppuration in some part of the urinarytract—urethritis, cystitis, pyelitis, etc.—or may be due to contamination from the vagina, in which case many vaginal epithelial cells will also be present. In general, the source of the pus can be determined only by the accompanying structures (epithelia, casts) or by the clinical signs.
A fairly accurate idea of the quantity of pus from day to day may be had by shaking the urine thoroughly and counting the number of corpuscles per cubic millimeter upon the Thoma-Zeiss blood-counting slide.
4.Red Blood-corpuscles.—Urine which contains blood is always albuminous. Very small amounts do not alter its macroscopic appearance. Larger amounts alter it considerably. Blood from the kidneys is generally intimately mixed with the urine and gives it a hazy reddish or brown color. When from the lower urinary tract, it is not so intimately mixed, and settles more quickly to the bottom, the color is brighter, and small clots are often present.
Red blood-corpuscles are not usually difficult to recognize with the microscope. When very fresh, they have a normal appearance, being yellowish discs of uniform size (normal blood). When they have been in the urine anyconsiderable time, their hemoglobin may be dissolved out, and they then appear as faint colorless circles or "shadow cells" (abnormal blood), and are more difficult to see (Fig. 52; see also Figs.45and60). They are apt to be swollen in dilute and crenated in concentrated urines. The microscopic findings may be corroborated by chemic tests for hemoglobin, although the microscope may show a few red corpuscles when the chemic tests are negative.
When not due to contamination from menstrual discharge, blood in the urine, orhematuria, is always pathologic. Blood comes from the kidney tubules in severe hyperemia, in some forms of nephritis, and in renal tuberculosis and malignant disease. The finding of blood-casts is the only certain means of diagnosing the kidney as its source. Blood comes from the pelvis of the kidney in renal calculus (Fig. 62), and is then usually intermittent, small in amount, and accompanied by a little pus and perhaps crystals of the substance forming the stone. Considerable hemorrhages from the bladder may occur in vesical calculus, tuberculosis, and newgrowths. Small amounts of blood generally accompany acute cystitis.
5.Spermatozoaare generally present in the urine of men after nocturnal emissions, after epileptic convulsions, and in spermatorrhea. They may be found in the urine of both sexes following coitus. They are easily recognizedfrom their characteristic structure (Fig. 53). The one-sixth objective should be used, with subdued light and careful focusing.