Chapter II: The Urine (2)

A somewhat simpler application of this method, which is accurate enough for clinical purposes, is as follows: Take 8¾ c.c. (roughly, 9 c.c.) of Purdy's solution in a large test-tube, dilute with an equal volume of water, heat to boiling, and, while keeping the solution hot but not boiling, add the urine drop by drop from a medicine-dropper until the blue color is entirely gone. Toward the end add the drops very slowly, not more than 4 or 5 a minute. Divide 10 by the number of drops required to discharge the blue color; the quotient will be the percentage of glucose. If 20 drops were required, the percentage would be 10÷20 = 0.5 per cent. It is imperative that the drops be of such size that 20 of them will make 1 c.c. Test the dropper with urine, not water. If the drops are too large, draw out the tip of the dropper; if too small, file off the tip.

_Purdy's solution_ consists of pure crystalline copper sulphate, 4.752 gm.; potassium hydroxid, 23.5 gm.; ammonia (U.S.P.; sp. gr., 0.9), 350 c.c.; glycerin, 38 c.c.; distilled water, to make {81} 1000 c.c. Dissolve the copper sulphate and glycerin in 200 c.c. of the water by aid of gentle heat. In another 200 c.c. of water dissolve the potassium hydroxid. Mix the two solutions, and when cool, add the ammonia. Lastly, bring the whole up to 1000 c.c. with distilled water. This solution is of such strength that the copper in 35 c.c. will be reduced by exactly 0.02 gm. of glucose.

(2) Fehling's Method.--Take 10 c.c. Fehling's solution (made by mixing 5 c.c. each of the copper and alkaline solutions described on page 78) in a flask or beaker, add three or four volumes of water, boil, and add the urine very slowly from a buret until the solution is completely decolorized, heating but not boiling after each addition.

Fehling's solution is of such strength that the copper in 10 c.c. will be reduced by exactly 0.05 gm. of glucose. Therefore, the amount of urine required to decolorize the test solution contains just 0.05 gm. glucose, and the percentage is easily calculated.

(3) Fermentation Method.--This is convenient and satisfactory, its chief disadvantage being the time required. It {82} depends upon the fact that glucose is fermented by yeast with evolution of CO_{2}. The amount of gas evolved is an index of the amount of glucose. Einhorn's saccharimeter (Fig. 25) is the simplest apparatus.

The urine must be so diluted as to contain not more than 1 per cent. of glucose. A fragment of fresh yeast cake about the size of a split pea is mixed with a definite quantity of the urine measured in the tube which accompanies the apparatus. It should form an emulsion free from lumps or air-bubbles. The long arm of the apparatus is then filled with the mixture. At the end of fifteen to twenty-four hours fermentation will be complete, and the percentage of glucose can be read off upon the side of the tube. The result must then be multiplied by the degree of dilution. Since yeast itself sometimes gives off gas, a control test must be carried out with normal urine and the amount of gas evolved must be subtracted from that of the test.

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(2) Levulose, or fruit-sugar, is very rarely present in the urine except in association with glucose, and has about the same significance. Its name is derived from the fact that it rotates polarized light to the left. It behaves the same as glucose with all the ordinary tests, and can be distinguished only by polarization.

(3) Lactose, or milk-sugar, is sometimes present in the urine of nursing women and in that of women who have recently miscarried. It is of interest chiefly because it may be mistaken for glucose. It reduces copper, but does not ferment with yeast. In strong solution it can form crystals with phenylhydrazin, but is extremely unlikely to do so when the test is applied directly to the urine.

3. Acetone Bodies.--This is a group of closely related substances--acetone, diacetic acid, and beta-oxybutyric {83} acid. Acetone is derived from decomposition of diacetic acid, and this in turn from beta-oxybutyric acid by oxidation. The origin of beta-oxybutyric acid is not definitely known, but it is probable that its chief, if not its only, source is in some obscure metabolic disturbance with abnormal destruction of fats. The three substances generally appear in the urine in the order mentioned. When the disturbance is mild, acetone only appears; as it becomes more marked, diacetic acid is added, and finally beta-oxybutyric acid appears. The presence of beta-oxybutyric acid in the blood is probably the chief cause of the form of auto-intoxication known as "acid intoxication."

(1) Acetone.--Minute traces, too small for the ordinary tests, may be present in the urine under normal conditions. Larger amounts are not uncommon in fevers, gastrointestinal disturbances, and certain nervous disorders. Occurrence of acetonuria in pregnancy suggests death of the fetus.

Acetonuria is practically always observed in acid intoxication, and, together with diaceturia, constitutes its most significant diagnostic sign. A similar or identical toxic condition, always accompanied by acetonuria and often fatal, is now being recognized as a not infrequent late effect of anesthesia, particularly of chloroform anesthesia. This postanesthetic toxemia is more likely to appear, and is more severe when the urine contains any notable amount of acetone before operation, which suggests the importance of routine examination for acetone in surgical cases.

Acetone is present in considerable amounts in many cases of diabetes mellitus, and is always present in severe {84} cases. Its amount is a better indication of the severity of the disease than is the amount of sugar. A progressive increase is a grave prognostic sign.

_Detection of Acetone_.--The urine may be tested directly, but it is best to distil it after adding a little phosphoric or hydrochloric acid to prevent foaming, and to test the first few cubic centimeters of distillate. A simple distilling apparatus is shown in Fig. 26. The test-tube may be attached to the delivery tube by means of a two-hole rubber cork as shown, the second hole serving as air vent, or, what is much less satisfactory, it may be tied in place with a string. Should the vapor not condense well, the test-tube may be immersed in a glass of cold water.

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(1) Gunning's Test.--To a few cubic centimeters of urine or distillate in a test-tube add a few drops of tincture of iodin {85} and of ammonia alternately until a heavy black cloud appears. This cloud will gradually clear up, and if acetone be present, iodoform, usually crystalline, will separate out. The iodoform can be recognized by its odor, especially upon heating (there is danger of explosion if the mixture be heated before the black cloud disappears), or by detection of the crystals microscopically. The latter, only, is safe, unless one has an unusually acute sense of smell. Iodoform crystals are yellowish, six-pointed stars or six-sided plates (Fig. 27).

This modification of Lieben's test is less sensitive than the original, but is sufficient for all clinical work; it has the advantage that alcohol does not cause confusion, and especially that the sediment of iodoform is practically always crystalline. When applied directly to the urine, phosphates are precipitated and may form star-shaped crystals which are very confusing to the inexperienced.

(2) Lange's Test.--This is a modification of the well-known Legal test. It is more sensitive and gives a sharper end-reaction. To a small quantity of urine add about one-twentieth its volume (1 drop for each 1 c.c.) of glacial acetic acid and a few drops of fresh concentrated aqueous solution of {86} sodium nitroprussid, and gently run a little ammonia upon its surface. If acetone be present, a purple ring will form within a few minutes at the junction of the two fluids.

(3) Trommer's Test.--This new test has proved very satisfactory in the hands of the writer. The urine need not be distilled. Alkalinize about 10 c.c. of the urine with 2 or 3 c.c. of 40 per cent. caustic soda solution, add 10 or 12 drops of 10 per cent. alcoholic solution of salicylous acid (salicyl aldehyd), heat the upper portion nearly to the boiling-point, and keep at this temperature five minutes or longer. In the presence of acetone a purplish-red color appears in the heated portion.

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(2) Diacetic acid occurs in the same conditions as acetone, but is less frequent and has more serious significance. In diabetes its presence is a grave symptom and often forewarns of approaching coma. It rarely or never occurs without acetone.

_Detection_.--The urine must be fresh.

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(1) Gerhardt's Test.--To a few cubic centimeters of the urine add solution of ferric chlorid (about 10 per cent.) drop by drop until the phosphates are precipitated; filter and add more of the ferric chlorid. If diacetic acid be present, the urine will assume a Bordeaux-red color which disappears upon boiling. A red or violet color which does not disappear upon boiling may be produced by other substances, as phenol, salicylates, and antipyrin.

(2) Lindemann's Test.--To about 10 c.c. of urine add 5 drops 30 per cent. acetic acid, 5 drops Lugol's solution, and 2 or 3 c.c. chloroform, and shake. The chloroform does not change color if diacetic acid be present, but becomes reddish-violet in its absence. This test is claimed by its advocates to be more sensitive and more reliable than Gerhardt's.

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{87} (3) Oxybutyric acid has much the same significance as diacetic acid, but is of more serious import. There is no satisfactory clinical test for it.

4. Bile.--Bile appears in the urine in all diseases which produce jaundice, often some days before the skin becomes yellow; and in many disorders of the liver not severe enough to cause jaundice. It also occurs in diseases with extensive and rapid destruction of red blood-corpuscles. Both bile-pigment and bile acids may be found. They generally occur together, but the pigment is not infrequently present alone. Bilirubin, only, occurs in freshly voided urine, the other pigments (biliverdin, bilifuscin, etc.) being produced from this by oxidation as the urine stands. The acids are almost never present without the pigments, and are, therefore, seldom tested for clinically.

_Detection of Bile-pigment_.--Bile-pigment gives the urine a greenish-yellow, yellow, or brown color, which upon shaking is imparted to the foam. Cells, casts, and other structures in the sediment may be stained brown or yellow. This, however, should not be accepted as proving the presence of bile without further tests.

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(1) Smith's Test.--Overlay the urine with tincture of iodin diluted with nine times its volume of alcohol. An emerald-green ring at the zone of contact shows the presence of bile-pigments. It is convenient to use a conical test-glass, one side of which is painted white.

(2) Gmelin's Test.--This consists in bringing slightly yellow nitric acid into contact with the urine. A play of colors, of which green and violet are most distinctive, denotes the presence of bile-pigment. Colorless nitric add will become yellow upon standing in the sunlight. The test may be applied {88} in various ways: by overlaying the acid with the urine; by bringing a drop of each together upon a porcelain plate; by filtering the urine through thick filter-paper, and touching the paper with a drop of the acid; and, probably best of all, by precipitating with lime-water, filtering, and touching the precipitate with a drop of the acid.

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_Detection of Bile Acids_.--Hay's test is simple, sensitive, and fairly reliable, and will, therefore, appeal to the practitioner. It depends upon the fact that bile acids lower surface tension. Other tests require isolation of the acids for any degree of accuracy.

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Hay's Test.--Upon the surface of the urine, which must not be warm, sprinkle a little finely powdered sulphur. If it sinks at once, bile acids are present to the amount of 0.01 per cent. or more; if only after gentle shaking, 0.0025 per cent. or more. If it remains floating, even after gentle shaking, bile acids are absent.

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5. Hemoglobin.--The presence in the urine of hemoglobin or pigments directly derived from it, accompanied by few, if any, red corpuscles, constitutes _hemoglobinuria_. It is a rare condition, and must be distinguished from _hematuria_, or _blood_ in the urine, which is common. In both conditions chemic tests will show hemoglobin, but in the latter the microscope will reveal the presence of red corpuscles. Urines which contain notable amounts of hemoglobin have a reddish or brown color, and may deposit a sediment of brown, granular pigment.

Hemoglobinuria occurs when there is such extensive destruction of red blood-cells within the body that the liver cannot transform all the hemoglobin set free into bile-pigment. The most important examples are seen in {89} poisoning, as by mushrooms and potassium chlorate, in malignant malaria (blackwater fever), and in the obscure condition known as "paroxysmal hemoglobinuria." This last is characterized by the appearance of large quantities of hemoglobin at intervals, usually following exposure to cold, the urine remaining free from hemoglobin between the attacks.

_Detection_.--Teichmann's test (p. 202) may be applied to the precipitate after boiling and filtering, but the guaiac test is more convenient in routine work.

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Guaiac Test.--Mix equal parts of "ozonized" turpentine and fresh tincture of guaiac which has been diluted with alcohol to a light sherry-wine color. In a test-tube or conical glass overlay the urine with this mixture. A bright blue ring will appear at the zone of contact within a few minutes if hemoglobin be present. The guaiac should be kept in an amber-colored bottle. Fresh turpentine can be "ozonized" by allowing it to stand a few days in an open vessel in the sunlight.

This test is very sensitive, and a negative result proves the absence of hemoglobin. Positive results are not conclusive, because numerous other substances--few of them likely to be found in the urine--may produce the blue color. That most likely to cause confusion is pus, but the blue color produced by it disappears upon heating. The thin film of copper often left in a test-tube after testing for sugar may give the reaction, as may also the fumes from an open bottle of bromin.

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6. Diazo Substances.--Certain unknown substances sometimes present in the urine give a characteristic color reaction--the "diazo reaction" of Ehrlich--when treated with diazo-benzol-sulphonic acid and ammonia. This reaction has much clinical value provided its limitations {90} be recognized. It is at best an empirical test and must be interpreted in the light of clinical symptoms. Although it has been met with in a considerable number of diseases, its usefulness is practically limited to typhoid fever, tuberculosis, and measles.

(1) Typhoid Fever.--Practically all cases give a positive reaction, which varies in intensity with the severity of the disease. It is so constantly present that it may be said to be "negatively pathognomonic": if negative at a stage of the disease when it should be positive, typhoid is almost certainly absent. Upon the other hand, a reaction when the urine is highly diluted (1:50 or more) has much positive diagnostic value, since this dilution prevents the reaction in most conditions which might be mistaken for typhoid; but it should be noted that mild cases of typhoid may not give it at this dilution. Ordinarily the diazo appears a little earlier than the Widal reaction--about the fourth or fifth day--but it may be delayed. In contrast to the Widal, it begins to fade about the end of the second week, and soon thereafter entirely disappears. An early disappearance is a favorable sign. It reappears during a relapse, and thus helps to distinguish between a relapse and a complication, in which it does not reappear.

(2) Tuberculosis.--The diazo reaction has been obtained in many forms of the disease. It has little or no diagnostic value. Its continued presence in pulmonary tuberculosis is, however, a grave prognostic sign, even when the physical signs are slight. After it once appears it generally persists more or less intermittently until death, the average length of life after its appearance being about six months. The reaction is often temporarily present in {91} mild cases during febrile complications, and has then no significance.

(3) Measles.--A positive reaction is frequently obtained in measles, and may help to distinguish this disease from German measles, in which it does not occur.

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Technic.--Although the test is really a very simple one, careful attention to technic is imperative. Many of the early workers were very lax in this regard. Faulty technic and failure to record the stage of the disease in which the tests were made have probably been responsible for the bulk of the conflicting results reported.

Certain drugs often given in tuberculosis and typhoid interfere with or prevent the reaction. The chief are creosote, tannic acid and its compounds, opium and its alkaloids, salol, phenol, and the iodids. The reagents are:

(1) Saturated solution sulphanilic acid in 5 per cent. hydrochloric acid.

(2) 0.5 per cent. aqueous solution sodium nitrite.

(3) Strong ammonia.

Mix forty parts of (1) and one part of (2). In a test-tube take equal parts of this mixture and the urine, and pour 1 or 2 c.c. of the ammonia upon its surface. If the reaction be positive, a garnet ring will form at the junction of the two fluids; and upon shaking, a distinct pink color will be imparted to the foam. The color of the foam is the essential feature. If desired, the mixture may be well shaken before the ammonia is added: the pink color will then instantly appear in that portion of the foam which the ammonia has reached, and can be readily seen. The color varies from eosin-pink to deep crimson, depending upon the intensity of the reaction. A doubtful reaction should be considered negative.

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7. Pancreatic Reaction.--Cammidge has shown that in cases of pancreatitis a substance capable of forming {92} crystals with phenylhydrazin can be developed by boiling the urine with a mineral acid, and has offered the following test as an aid in diagnosis of this obscure condition. The nature both of this substance and the antecedent substance from which it is derived is not known. As originally proposed, the test was complicated and probably not trustworthy, but with his improved and simplified technic, Cammidge has had very promising results. In 200 consecutive examinations in which the diagnosis was confirmed, _postmortem_ or at operation, 67 cases of pancreatitis (65 chronic, 2 acute) gave positive reactions; 4 cases of cancer of the pancreas were positive, 12 negative; 4 cases in which no pancreatitis was found were positive, 113 were negative. Normal urines do not give the reaction. The difficulty and importance of diagnosis in pancreatitis warrant inclusion of the method here even though its true value cannot be definitely assigned. While the test is somewhat tedious, all the manipulations are simple and require no apparatus but flasks, test-tubes, and funnels.

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Technic.--Careful attention to detail is imperative. An ordinary routine examination is first made. Albumin and sugar, if present, must be removed: the former, by acidifying with acetic acid, boiling, and filtering; the latter, by fermentation with yeast after the first step of the method proper. An alkaline urine should be made slightly acid with hydrochloric acid.

(1) Forty cubic centimeters of the urine, which has been rendered perfectly clear by repeated filtration through the same filter-paper, are placed in a small flask, treated with 1 c.c. concentrated hydrochloric acid and gently boiled on a sand-bath for ten minutes, a funnel with long stem being placed in the neck of the flask to act as a condenser (Fig. 28). After {93} boiling, the urine is cooled in a stream of cold water and brought to its original bulk with distilled water; 8 gm. of lead carbonate are then added to neutralize the acid. The fluid is allowed to stand a few minutes and then filtered through well-moistened fine-grained filter-paper until perfectly clear.

(2) The filtrate is shaken up with 8 gm. powdered tribasic lead acetate and filtered. The excess of lead is then removed by passing hydrogen sulphid gas through the fluid or by shaking well with 4 gm. finely powdered sodium sulphate, heating to boiling, cooling to as low a temperature as possible in a stream of water, and filtering as before until perfectly clear.

(3) Ten cubic centimeters of the filtrate are then made up to 17 c.c. with distilled water, and added to a mixture of 0.8 gm. phenylhydrazin hydrochlorate, 2 gm. powdered sodium acetate, and 1 c.c. 50 per cent. acetic acid in a small flask {94} with funnel condenser. This is boiled on a sand-bath for ten minutes, and filtered while hot through filter-paper moistened with hot water into a test-tube with a 15 c.c. mark. Should the filtrate not reach this mark, make up to 15 c.c. with hot distilled water. Allow to cool slowly.

(4) In well-marked cases of pancreatitis a yellow precipitate appears within a few hours; in milder cases, it may not appear for twelve hours. The microscope shows this sediment to consist of "long, light yellow, flexible, hair-like crystals arranged in sheaves, which, when irrigated with 33 per cent. sulphuric acid, melt away and disappear in ten to fifteen seconds after the acid first touches them" (Fig. 29).

(5) To exclude traces of glucose which might be overlooked in the preliminary examination a control test should be carried out in the same manner with omission of step (1).

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{95} 8. Drugs.--The effect of various drugs upon the color of the urine has been mentioned (p. 50). Most poisons are eliminated in the urine, but their detection is more properly discussed in works upon toxicology. A few drugs which are of interest to the practitioner, and which can be detected by comparatively simple methods, are mentioned here.

Acetanilid and Phenacetin.--The urine is evaporated by gentle heat to about half its volume, boiled for a few minutes with about one-fifth its volume of strong hydrochloric acid, and shaken out with ether. The ether is evaporated, the residue dissolved in water, and the following test applied: To about 10 c.c. are added a few cubic centimeters of 3 per cent. phenol, followed by a weak solution of chromium trioxid (chromic acid) drop by drop. The fluid assumes a red color, which changes to blue when ammonia is added. If the urine is very pale, extraction with ether may be omitted.

Antipyrin.--This drug gives a dark-red color when a few drops of 10 per cent. ferric chlorid are added to the urine. The color does not disappear upon boiling, which excludes diacetic acid.

Arsenic.--_Reinsch's Test_.--Add to the urine in a test-tube or small flask about one-seventh its volume of hydrochloric acid, introduce a piece of bright copper-foil about one-eighth-inch square, and boil for several minutes. If arsenic be present, a dark-gray film is deposited upon the copper. The test is more delicate if the urine be concentrated by slow evaporation. This test is well known and is widely used, but is not so reliable as the following:

_Gutzeit's Test_.--In a large test-tube place a little {96} arsenic-free zinc, and add 5 to 10 c.c. pure dilute hydrochloric acid and a few drops of iodin solution (Gram's solution will answer), then add 5 to 10 c.c. of the urine. At once cover the mouth of the tube with a filter-paper cap moistened with saturated aqueous solution of silver nitrate (1:1). If arsenic be present, the paper quickly becomes lemon-yellow, owing to formation of a compound of silver arsenid and silver nitrate, and turns black when touched with a drop of water. To make sure that the reagents are arsenic-free, the paper cap may be applied for a few minutes before the urine is added.

Atropin will cause dilatation of the pupil when a few drops of the urine are placed in the eye of a cat or rabbit.

Bromids can be detected by acidifying about 10 c.c. of the urine with dilute sulphuric acid, adding a few drops of fuming nitric acid and a few cubic centimeters of chloroform, and shaking. In the presence of bromin the chloroform, which settles to the bottom, assumes a yellow color.

Iodin--from ingestion of iodids or absorption from iodoform dressings--is tested for in the same way as the bromids, the chloroform assuming a pink to reddish-violet color. To detect traces, a large quantity of urine should be rendered alkaline with sodium carbonate and greatly concentrated by evaporation before testing.

Lead.--No simple method is sufficiently sensitive to detect the traces of lead which occur in the urine in chronic poisoning. Of the more sensitive methods, that of Arthur Lederer is probably best suited to the practitioner:

It is essential that all apparatus used be lead-free. Five hundred cubic centimeters of the urine are acidified {97} with 70 c.c. pure sulphuric acid, and heated in a beaker or porcelain dish. About 20 to 25 gm. of potassium persulphate are added a little at a time. This should decolorize the urine, leaving it only slightly yellow. If it darkens upon heating, a few more crystals of potassium persulphate are added, the burner being first removed to prevent boiling over; if it becomes cloudy, a small amount of sulphuric acid is added. It is then boiled until it has evaporated to 250 c.c. or less. After cooling, an equal volume of alcohol is added, and the mixture allowed to stand in a cool place for four or five hours, during which time all the lead will be precipitated as insoluble sulphate.

The mixture is then filtered through a small, close-grained filter-paper (preferably an ashless, quantitative filter-paper), and any sediment remaining in the beaker or dish is carefully washed out with alcohol and filtered. A test-tube is placed underneath the funnel; a hole is punched through the tip of the filter with a small glass rod, and all the precipitate (which may be so slight as to be scarcely visible) washed down into the test-tube with a jet of distilled water from a wash-bottle, using as little water as possible. Ten cubic centimeters will usually suffice. This fluid is then heated, adding crystals of sodium acetate until it becomes perfectly clear. It now contains all the lead of the 500 c.c. urine in the form of lead acetate. It is allowed to cool, and hydrogen sulphid gas is passed through it for about five minutes. The slightest yellowish-brown discoloration indicates the presence of lead. A very slight discoloration can be best seen when looked at from above. For comparison, the gas may be passed through a test-tube containing an equal amount of distilled water. The quantity of lead can be {98} determined by comparing the discoloration with that produced by passing the gas through lead acetate (sugar of lead) solutions of known strength. One part of lead acetate crystals contains 0.54 part of lead. Hydrogen sulphid is easily prepared in the simple apparatus shown in Fig. 30. A small quantity of iron sulphid is placed in the test-tube; a little dilute hydrochloric acid is added; the cork is replaced; and the delivery tube is inserted to the bottom of fluid to be tested.

Mercury.--Traces can be detected in the urine for a considerable time after the use of mercury compounds by ingestion or inunction.

About a liter of urine is acidified with 10 c.c. hydrochloric acid, and a small piece of copper-foil or gauze is introduced. This is gently heated for an hour, and allowed to stand for twenty-four hours. The metal is then removed, and washed successively with very dilute sodium hydroxid solution, alcohol, and ether. When dry, {99} it is placed in a long, slender test-tube, and the lower portion of the tube is heated to redness. If mercury be present, it will volatilize and condense in the upper portion of the tube as small, shining globules which can be seen with a hand-magnifier or low power of the microscope. If, now, a crystal of iodin be dropped into the tube and gently heated, the mercury upon the side of the tube is changed first to the yellow iodid and later to the red iodid which are recognized by their color.

Morphin.--Add sufficient ammonia to the urine to render it distinctly ammoniacal, and shake thoroughly with a considerable quantity of pure acetic ether. Separate the ether and evaporate to dryness. To a little of the residue in a watch-glass or porcelain dish add a few drops of formaldehyd-sulphuric acid, which has been freshly prepared by adding one drop of formalin to 1 c.c. pure concentrated sulphuric acid. If morphin be present, this will produce a purple-red color, which changes to violet, blue-violet, and finally nearly pure blue.

Phenol.--As has been stated, the urine following phenol poisoning turns olive-green and then brownish-black upon standing. Tests are of value in recognizing poisoning from ingestion and in detecting absorption from carbolized dressings.

The urine is acidulated with hydrochloric acid and distilled. To the first few cubic centimeters of distillate is added 10 per cent. solution of ferric chlorid drop by drop. The presence of phenol causes a deep amethyst-blue color, as in Uffelmann's test for lactic acid.

Phenolphthalein, which is now being used as a cathartic under the name of _purgen_, gives a bright pink color when the urine is rendered alkaline with caustic soda.

{100} Quinin.--A considerable quantity of the urine is rendered alkaline with ammonia and extracted with ether; the ether is evaporated, and a portion of the residue dissolved in about twenty drops of dilute alcohol. The alcoholic solution is acidulated with dilute sulphuric acid, a drop of an alcoholic solution of iodin (tincture of iodin diluted about ten times) is added, and the mixture is warmed. Upon cooling, an iodin compound of quinin (herapathite) will separate out in the form of a microcrystalline sediment of green plates.

The remainder of the residue may be dissolved in a little dilute sulphuric acid. This solution will show a characteristic blue fluorescence when quinin is present.

Resinous drugs cause a white precipitate like that of albumin when strong nitric acid is added to the urine. This is dissolved by alcohol.

Salicylates, salol, and similar drugs give a bluish-violet color, which disappears upon heating, upon addition of a few drops of 10 per cent. ferric chlorid solution. When the quantity of salicylates is small, the urine may be acidified with hydrochloric acid and extracted with ether, the ether evaporated, and the test applied to an aqueous solution of the residue.

Tannin and its compounds appear in the urine as gallic acid, and the urine becomes greenish-black (inky, if much gallic acid be present) when treated with a solution of ferric chlorid.

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 slight {101} sediment 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 the {102} tip 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 (see Figs. 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 that _the 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_.

{103} 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. UNORGANIZED SEDIMENTS

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 are {104} the 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. {105} 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 (see Fig. 62).

(2) Amorphous Urates.--These are chiefly urates of {106} sodium 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, {107} 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 concentric {108} striations. Some may be merged together in clusters. They are not soluble in hydrochloric acid nor in ether.

_Tyrosin_ crystallizes 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 crystals are 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 sometimes {109} takes 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.

In _chyluria_, 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 by _Filaria 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 are {110} the common "triple phosphate" crystals, which are generally easily recognized (Figs. 37 and 63, and Plate 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 prisms {111} arranged 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.

{112} (2) Calcium carbonate may 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, and Fig. 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.

{113} 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. ORGANIZED SEDIMENTS

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 the {114} urine, 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, and 46.)

Hyaline casts are the least significant of all the casts, {115} 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, they {116} are 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 and 61). 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 distinctly {117} yellow 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, and 61).

_Finely granular casts_ contain 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 casts_ contain 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.

{118} 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. (See Figs. 41 and 42). When numerous, they give name to the cast.

(_a_) _Epithelial casts_ contain 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-casts_ contain red blood-corpuscles, usually much degenerated (Figs. 45 and 60). They always indicate hemorrhage into the tubules, which is most {119} common in acute nephritis or an acute exacerbation of a chronic nephritis.

(_c_) _Pus-casts_ (see Fig. 62), composed almost wholly of pus-corpuscles, are uncommon, and point to a chronic suppurative process in the kidney.

(_d_) True _bacterial casts_ are 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 the {120} mucous 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 urates or phosphates or very 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) Hairs and fibers of wool, cotton, etc. These could be mistaken for casts only by beginners. One can easily {121} become familiar with their appearance by suspending them in water and examining with the microscope (Fig. 57).

(5) Hyphæ of molds are 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: