- Email: [email protected]
Office Laboratory Tests: Technic and Interpretation
OFFICE LABORATORY' TESTS Technic and Interpretation MALCOLM M. STANLEY, M.D .....
THE following laboratory tests were chosen for discussion because of their ready applicability to clinical problems frequently encountered by the practitioner, and because they can be done in the office, for the most part with a minimum of expensive or complicated equipment. URINE
I. Bile in the Urine.-Although commonly occurring in the urine in conjunction with bile salts and other components of bile, bilirubin is usually tested for because of its relative ease of detection. The presence of bilirubinuria indicates that there has been a "regurgitation"! of bile already formed by the liver into the blood stream from which it was excreted by the kidneys into the urine. This regurgitation may occur following necrosis of the liver cells as a result of infection ("infectious hepatitis"), the action of toxic substances such as arsenic ("toxic hepatitis"), or the effect of back pressure because of obstruction' of some portion of the biliary system ("obstructive jaundice"). In uncomplicated hemolytic icterus there is no bile in the urine. Hence, in an icteric patient this determination is of value in narrowing the possibilities as to the causative mechanism. Bilirubinuria in small amounts may be the earliest detectable sign in infectious hepatitis and should be searched for in obscure febrile illnesses of short duration with vague abdominal symptoms, particularly with a history of exposure, in order that the disease be diagnosed in its incigient (preicteric) stage. Routine tests at short intervals of the urines of industrial workers exposed to hepatotoxic agents have been of value in detecting those 'Yith early liver damage in order that they might be removed from the deleterious environment before serious harm had taken place. 2 TESTS
1. "FOAM" TEST A test tube half full of the urine is shaken vIgorously, holding the thumb over the open end. The color of the layer (,f foam which forms on top of the urine is compared against a white background with a normal From the Evans Memorial, Massachusetts Memorial Hospitals, and the Department of Medicine, Boston University School of Medicine, Boston, Massa·· chusetts . . Instructor in Medicine, Boston University School of Medicine; Fellow in Medicine, Evans Memorial, Massachusetts Memorial Hospitals. 1168
OFFICE LABORATORY TESTS
1169
urine treated in the same manner. If the foam is yellow the test is positive. It is to be emphasized that slight discoloration of the foam, indicating a small amount of bile, can be detected only by comparison with a normal control.
The foam test is a most sensitive method for detection of bile, and is the most easily performed of all. Its only disadvantage is that the presence of substances such as penicillin, streptomycin, phenolsulfonphthalein in acid solution, riboflavin, atabrine and possibly others (not, however, urochrome) will cause a false positive. Its interpretation is difficult when patients are receiving these drugs, so other confirmatory determinations must be made in those instances. 2. METHYU;NE BLUE TEST'-u
A 0.2 per cent aqueous solution of methylene blue chloride is added drop by drop (using a pipette which delivers 20 drops of the solution per cubic centimeter) to 5 cc. of a prebreakfast urine specimen. The reading (in drops) is taken as one less than the number needed to change the color from green to blue. If more than 8 drops are required a smaller measured amount of urine is diluted to 5 cc. with distilled water and the test repeated in order that a clearer end point may be obtained. If diluted, the number of drops is multiplied by the dilution factor. A reading greater than 4 drops has been regarded (by Gellis and Stokes, et al.) as a positive test for bilirubin in the urine. Readings of 2 to 4 drops inclusive have been regarded as suggestive and as an indication for additional studies. As Myers2 described the test, 2 drops of methylene blue solution were added to 10 cc. of urine. The solution remains dark blue if negative, but turns a brilliant green if positive for bile. He quantitated the test by progressively diluting the urine, keeping the total volume at 10 cc. by replacing increasing amounts of urine with distilled water, until a negative result was obtained.
Franke introduced the methylene blue test in 1931.3 This soon brought forth several communications in protest4 and approval,'" G just as did Myers' recent paper. 7- 11 The majority·of these authors state that a positive test results from the simple mixing of the two colors blue and yellow, and not from the oxidation of yellow bilirubin to green biliverdin with conversion of the methylene blue to the colorless leuko form as was Franke's original belief. The consensus and bulk of the evidence indicate that the blending of colors accounts for most of the green col or phenomenon. Because a positive test results from blending of the yellow and blue colors, false positives are also likely to occur when other pigments are present in the urine as in the foam test. Its most ardent advocates 7 • s admit its inferiority in sensitivity and specificity to the Harrison and diazo spot test. On the other hand, it is performed in a roughly quantitative manner and may be of value in following the course of patients with parenchymal liver disease such as infectious hepatitis, since the
1170
MALCOLM M. STANLEY
titer diminishes and the test becomes negative during convalescence when the icterus is still present and the serum bilirubin is still elevated. 3. DIAZO SPOT (GodfriedI2 ) Five cubic centimeters of 10 per cent barium chloride and 10 cc. of urine are mixed and the voluminous precipitate of insoluble barium salts is filtered. The wet filter is placed on a disk of dry filter paper so that the excess liquid is removed. To the precipitate on filter paper add 1 drop of Ehrlich's diazo reagent, 4 drops of 95 per cent alcohol, and 1 drop of phosphate buffer (6 per cent Na2HP04.12H20). A red to pink color indicates a positive reaction. If there are large amounts of bilirubin, the test works better when the urine is diluted.
(Ehrlich's Diazo Reagent: This is a mixture of two solutions, each of which keeps well, but which must be freshly mixed immediately prior to using. Solution 1: Sulphanilic acid, 5 gm.; concentrated hydrochloric acid, 50 cc.; distilled water to make 1000 cc. Solution 2: Sodium nitrite, 0.5 gm.; distilled water, 100 cc. Take 0.8 cc. of solution 2 and make up to 25 cc. with solution 1.) (Godfriedl2 13) Precipitate the bilirubin with barium chloride and filter as in the Diazo test (3 above). To the precipitate on filter paper 1-2 drops of Fouchet's reagent (25 per cent trichloroacetic acid containing 0.9 per cent ferric chloride) are added. If bilirubin is present, the characteristic blue to green color appears. 4. HARRISON SPOT
When the barium chloride and urine are mixed the barium ions and s.ulfate ions present in the urine form insoluble barium sulfate which precipitates. The bilirubin is adsorbed onto the barium sulfate particles, hence, is "concentrated." The blue to green color of a positive Harrison test is the result of oxidation of the adsorbed yellow bilirubin to biliverdin or bilicyanin. Both the Harrison and the diazo spot tests are "concentration" tests. Both the Harrison spot and diazo spot tests are very sensitive and quite specific. Their only drawback is that they are more complicated and require more time and equipment. According to Godfried,12 0.0020.008 mg. of bilirubin per 100 cc. of urine is detectable by these methods. 5. HARRISON MODIFICATION (Hawkinson, Watson. and Turner14 ) a. Preparation of Barium Impregnated Paper Strips.-Pieces of extra thick and retentive filter paper are allowed to remain briefly in a saturated aqueous solution of barium chloride. (Schleicher and Schull number 470 filter paper is recommended. Lighter grades are not satisfactory. We have found that ordinary unglazed heavy white blotting paper is quite adequate.) They are then dried in the air or, preferably, in a drying oven, after which they are cut into strips 4 inches long by 12 inch wide. b. Procedure.-One end of a barium chloride impregnated strip is placed in the urine sample to be tested, the strip being in an approximately vertical position, at least one half extending above the surface of
1171
OFFICE LABORATORY TESTS
the sample. After standing in the urine for from thirty seconds up to two minutes the strip is withdrawn and placed on a piece of dry paper such as a paper towel or any other absorbent paper. Inspection of the filter strip will usually reveal somewhat more color in that area which corresponded to the surface of the urine. Two to three drops of Fouchet's reagent (see above) are then dropped directly on this area. A positive test is denoted by the appearance of a green color, varying in intensity with the amount of bilirubin present. With smaller amounts the color is often detected as a faint green line running across the strip. The urine to be tested may be agitated by shaking, by bubbling air llrough the liquid, or by forcefully drawing it up and squirting it back from a syringe into a beaker several times so that there is a layer of foam on the surface. If the barium chloride impregnated strip is then inserted into the liquid through the layer of foam, and the test carried out otherwise as described above, we have noted that its sensitivity is markedly increased. In effect, this combines the principles of the foam test and the Harrison test. >
Hawkinson, Watson, and Turner consider that their modification is just as sensitive as the original Harrison test. This has been our experience with the procedure. The modification of the original Harrison technic is "especially suited for mass and serial usage" and would seem to have eliminated all the objections to the original method while retaining its advantages of sensitivity and specificity. It is easily and quickly performed and requires, besides the barium chloride impregnated strips, only one reagent which will keep indefinitely. Because of its extreme simplicity it would seem that the foam test is useful as a screening test which should be done on all urines as a part of the routine urinalysis. Confirmation of a positive foam test if necessary can be obtained with one of the more specific procedures. For this the modified Harrison test is recommended because of the ease and speed with which it can be performed. 11. Glycosuria.-The importance of testing the urine routinely for sugar is now too well recognized to require emphasis. The discovery of glycosuria during the course of a routine "check-up," particularly of the members of the family of a diabetic, is often the first evidence of diabetes. It may occur long before obvious symptoms make their appearance. Adherence to the custom of determining urine and blood sugars while the patient is in the fasting state when diabetes is suspected may, however, result in unnecessary delay in diagnosis. 16 In the mild diabetic the urine may be free from sugar and the blood sugar may be normal in the fasting state, particularly before breakfast after a ten to twelve hours' fast. Therefore, in patients who are suspected of having diabetes these tests should be performed on specimens taken two to four hours after a meal heavy in carbohydrate, or a glucose tolerance test should be done. Do not do SINGLE fasting blood and urine sugars when the object is to establish the diagnosis of diabetes in a doubtful case.
1172
MALCOLM M. STANLEY
1.
QUANTITATIVE ESTIMATIONS OF URINARY GLUCOSE
The necessity for basing the determination of adequacy of control of diabetics on the quantitative estimation of sugar spilled in the urine during the twenty-four-hour period, rather than on qualitative tests on periodic specimens, has recently been reiteratedY Especially when controlled on protamine zinc insulin alone are diabetics liable to spill small amounts of sugar after meals. When the total for twenty-four hours is measured, the amount is often found to be insignificant. It has been our goal to control patients in the clinic so that 5 gm. of glucose or less are lost in the urine per day. A.
MODIFIED BENEDICT METHOD!8
To 2.5 cc. of reagent in a pyrex test tube add approximately 1 gm. of anhydrous sodium carbonate. Heat slowly to boiling over a low flame. Add the urine slowly, drop by drop, from a pipette graduated in tenths of a cubic centimeter, while the solution is kept boiling. The end point is complete absence of blue color. The white precipitate of cuprous thiocyanate by contrast makes the end point easier to determine. If x is the number of cc. of urine required for reduction, the formula for computing the percentage of glucose is as follows: 0.005 - - X 100 = percentage of sugar x
If there is a high concentration of glucose, it may be advantageous to dilute the urine 1: 10, in which case the formula is: 0.005 - - - X 1000 = percentage of sugar (in original urine) x
The reagent (2.5 cc. are reduced by 5 mg. glucose) is composed of the following (only the copper sulfate need be weighed accurately): Copper sulfate (pure crystallized) ................ Sodium carbonate (crystallized) .................. or Anhydrous sodium carbonate ................ Sodium or potassium citrate, C.P .................. Potassium thiocyanate, C.P ....................... Potassium ferrocyanide solution (5 per cent) ....... Distilled water, to make .........................
18.0 . . 200.0 . 100.0 . 200.0 . 125.0 . 5.0 . 1000.0
gm. gm. gm. gm. gm. cc. cc.
With the aid of heat dissolve the carbonate, citrate and thiocyanate in about 700 cc. of water and filter. Dissolve the copper sulfate in 100 cc. of water and pour slowly into the other fluid, stirring constantly. Add the ferrocyanide solution, cool and dilute to 1000 cc. This reagent may also be purchased already prepared from many pharmaceutical firms.
OFFICE LABORATORY TESTS
2.
1173
METHODS BY WHICH DIABETIC PATmNTS THEMSELVES CAN DETECT GLYCOSURIA
Every well-trained diabetic should learn how to test his own urine for sugar and ketones, and should perform at least the former test every day. There are several different outfits which are available for these procedures. A.
BENEDICT QUALITATIVE TEST's
Eight drops of urine are added to 5 cc. (approximately 1 teaspoonful) of Benedict's qualitative solution and thoroughly mixed by shaking. The test tube is placed in boiling water for five minutes. The amount of sugar is estimated by the degree of reduction, from green to red.
The reagent contains copper sulfate, sodium or potassium citrate and sodium carbonate. The reaction is the familiar one of reduction by glucose of the cupric salt to cuprous hydrate (yellow) or cuprous oxide (red). A pint of solution (enough for approximately 100 tests) costs from 59 cents to $1.00. Two test tubes and a medicine dropper cost an additional 25 cents. Each test, therefore, costs less than 1 cent. This procedure is quite satisfactory when performed by the patient or physician according to directions. It is the least expensive. However, the variations in results are amazing when the reagent, the urine, or both are not measured, or the boiling is inadequate. The test requires several minutes and the equipment is bulky and difficult to carry on one's person. An external source of heat is necessary. Because of these inconveniences, particularly during vacations when traveling or visiting, patients often neglect this procedure entirely. When dietary indiscretions common to such periods occur, derangement of control may be serious without the patient's knowledge. B.'L"CLINITEST" TABLET METHon '9
This set is packed in a small plastic case (8 by 5 by 3.5 cm.) which fits into the pocket or purse and which contains all the necessary materials (dropper, test tube and bottle of 36 reagent tablets). The top of the case is designed for collection of the urine specimen. The retail price of the kit is $1.75 (includes 36 tablets). Additional tablets are 60 cents for 36. Each test costs approximately 1.7 cents, disregarding the initial cost of the kit. Twenty-five one hundredths milliliter (5 drops) of urine is added to 0.5 ml. (10 drops) of water in the small test tube and one reagent tablet is dropped into this solution. A boiling reaction occurs immediately and if glucose is present a colored precipitate of cuprous oxide will form. The degree of reduction is estimated as in the Benedict qualitative test. A color scale for comparison is part of the kit.
1174
MALCOLM M. STANLEY
The components of the reagent tablet are essentially the same as in the Benedict solution except for the presence of sodium hydroxide, citric acid and sodium bicarbonate instead of sodium carbonate and sodium citrate. However, upon being dissolved the tablet produces sodium citrate, carbon dioxide and a very small quantity of sodium bicarbonate. In addition, the heat resulting from the ionization of the solid caustic and the neutralization between sodium hydroxide and citric acid is sufficient for the reduction of the cupric ions in the presence of glucose. Recently, testing of several thousand diabetic urines by trained laboratory technicians, using both the Benedict qualitative and the Clinitest methods, has shown agreement in 84 per cent of the specimens. 19 As the test can be done in less than a minute and requires no external source of heat, it is of great value to patients who are traveling, staying at hotels, and the like. This set combines the virtues of simplicity, convenience, portability and inexpensiveness and is the best outfit available for the purpose. C.
"SHEFTEL" TABLET METHOD""
21
This urine sugar test case is a somewhat elaborate outfit in a 12 by 11 by 3.7 cm. plastic box. The box opens so that the top forms a rack which holds: (1) the test tube in position for heating, (2) two vials, one containing 60 copper sulfate reagent tablets and the other 40 methenamine tablets which are burned for a source of heat, (3) a graduated dropper-pipette, and (4) a combination glass spoon and rubber test tube cleaner. A color scale and durable book of instructions are also provided. The retail price is $3.75, including the above reagent and fuel tablets. Each test costs 2% cents, disregarding the initial cost of the outfit. To 1.5 cc. of water in the test tube are added 0.25 cc. of urine and a copper suIfate reagent tablet. A methenamine tablet is placed on the metal well just below the bottom of the test tube, and lighted. The fuel tablet bums for 2% minutes, boiling the solution and carrying the reduction reaction to completion.
The reagent tablet employs the same chemicals that are used for the Benedict qualitative test, namely copper sulfate, sodium or potassium citrate, and sodium carbonate, but with the addition of acacia and creatinine. The big disadvantage is that an external source of heat is required. Both the initial cost and the cost of each test are relatively high. The case is slightly large to be carried conveniently in the pocket. A separate container must be provided for the urine to be tested. On the other hand, the procedure may be done precisely and in a roughly quantitative manner. The case is sturdily constructed and quite durable.
OFFICE LABORATORY TESTS
1175
D. "GALATEST" POWDER METHOD"
This "outfit" is composed of two tubes of dry reagent powder (one for testing for sugar and the other for acetone) and a medicine dropper contained in a flimsy unpainted wooden box 10.7 by 6 by 3 cm. (1) The white "Galatest" powder (for sugar detection) is a mixture of bismuth oxychloride, sodium hydroxide and sodium silicate. (2) The "Denco" powder (for acetone detection) is a mixture of sodium carbonate, ammonium sulfate and sodium nitroprusside. The cost of the set is $2.25, or $1.00 for each of the tubes of powder. Each tube contains material for approximately 100 tests, so the cost is about 1 cent each. Deposit on a piece of plain white paper a little of the "Galatest" powder (covering an area abcut the size of the little finger nail). Use dropper and deposit 1 small drop of urine on the powder. The presence of sugar is indicated by a change in the color of the powder ranging from gra~-green to black, depending upon the amount.
In this instance the glucose reduces the bismuth oxychloride to black bismuth oxide in the cold. It is easily portable, and the procedure is very simple to perform: Again, however, a separate container must be provided for collection of the urine. The colors are not as sharply defined as in the more familiar copper reduction tests, and a maximum of 1 per cent glucose in undiluted urine can be detected. (To detect acetone the above procedure is followed, using instead "Denco" powder and 2 to 3 drops of urine. A positive test is indicated by color changes ranging from light lavender to dark purple, and requires about one minute for completion. If no acetone is present, the powder will take on a grayish-yellow color.)
STOOL Every patient who enters the hospital or who undergoes a physical examination as a periodic routine procedure or for other reasons should have a stool examination, for occult blood at least. Occult blood in the stools is confirmatory evidence in many cases of malignancy of the stomach or large bowel or active peptic ulcer. A persistently positive test may provide the reason for an obscure anemia or diatrhea. An adequate specimen can be obtained on the gloved 6nger at the time the digital rectal examination is made. Only rarely must this procedure be postponed because of an insufficient amount of feces in the rectum. Unfortunately, examination of the stool, although it may often be diagnostic, is the most frequently neglected laboratory test. CLINICAL TESTS FOR OCCULT BLOOD
1. TECHNIC FOR GUAIAC TEST
Place a small amount of material (feces, gastric contents) in a depression on a white spot plate (tile) and mix with several drops of glacial
1176
MALCOLM M. STANLEY
acetic acid. Add 2 or 3 drops of gum guaiac solution and mix again. Add 2 or 3 drops of 3 per cent hydrogen peroxide. Development of a green or blue calor is a positive reaction. The rapidity with which the calor develops and the depth of calor are some indications of the amount of hemoglobin present. REAGENTS (should be kept in dark bottles, as they are decomposed by light).1. Gum guaiac: 1 gm. of gum guaiac in 60 cc. of 95 per cent alcohol or in 60 cc. glacial acetic acid. 2. Hydrogen peroxide: 3 per cent aqueous solution. Keep refrigerated if possible. (In case of a negative result, it is well to perform the test on a small amount of blood if there is any doubt as to the potency of the reagents, particularly the hydrogen peroxide.)
2. TECHNIC FOR BENZIDINE TEST
A saturated solution of "benzidine base for blood detection" in glacial acetic acid is used inste;o.d of the gum guaiac solution. The test is otherwise performed in the same way as the guaiac on the stool.
Blood and certain other substances contain the enzyme peroxidase which catalyzes the oxidation of the base compound (guaiacol or benzidine base) to a green or blue colored compleX'. An excess of hydrogen peroxide rapidly destroys this colored complex and also oxidizes and destroys hemoglobin. Therefore, hydrogen peroxide must not be added in excess and must be added after the guaiac or benzidine solution. Factors Influencing the Tests and Their Interpretation.-Feces contain substances which inhibit the peroxidase reaction. Bramkamp23 found that when hemoglobin was mixed with the feces, five times as much was needed to produce a positive benzidine reaction as was necessary when it was in watery solution. A concentration of 0.04 per cent blood freshly mixed with the stool will cause a positive benzidine test. The ingestion of 1.5 to 2 gm. of hemoglobin (9.4 to 12.5 cc. of blood containing 16 gm. of hemoglobin per 100 cc.) over a day's time resulted in positive tests in only 35 to 48.5 per cent of the normal subjects, however. 24 Pancreatic digestion is probably largely responsible for this variable loss of ability to reduce peroxides which occurs on passage of blood through the intestines,23 although other factors such as intestinal motility may influence it. 24 Thus, when blood is taken by mouth, ninety times as much may be required to produce a positive test as when the blood is freshly mixed with the stool. Hence when the site of bleeding is in the esophagus, stomach or duodenum, relatively large amounts of blood must be lost before it can be detected by the usual tests. In contrast to this, as little as 0.04 per cent blood theoretically will produce a positive reaction when the source of bleeding is in the large bowel. . The benzidine reaction will often be positive on the stools of normal persons if their diets contain meat. The guaiac test is somewhat less sensitive, hence it is not necessary to have the patient on a meat-free
OFFICE LABORATORY TESTS
1177
diet, particularly if only a blue or strong blue-green color is regarded as positive, and not the lighter shades of green. It is for this reason that we routinely employ the guaiac test, realizing, however, that it is not as sensitive as the benzidine test, and that it will not detect small hemorrhages. In testing for hemoglobin in the urine, the extreme sensitivity of the benzidine test is a virtue and makes it preferable to the guaiac test in this instance. False Positives.-A number of other substances will produce the green or blue color reaction besides hemoglobin. The common ones are milk, pus, ferrous sulfate, bromides, iodides, and copper compounds (such as the copper precipitate left on the walls of test tubes after Benedict's test for sugar). Usually administration of ferrous iron by mouth, although causing the stool to appear "tarry," will not produce a strongly positive guaiac reaction. This is because the unabsorbed iron is either found "in firm organic combination"26 after its passage through the gut, in which nonionized form it will not catalyze the reaction, or it is oxidized to the ferric state soon after defecation, when exposed to air. In rare instances, however, iron therapy will cause a false positive guaiac test, apparently because some of the ferrous form appears in the feces unchanged. We have seen at least one case in which there was good evidence that ferrous sulfate caused a false positive guaiac test on the stool. From consideration of the above facts it may be seen that a negative test for fecal occult blood does not rule out small amounts of bleeding, particularly if it occurs high in the gastrointestinal tract; neither does a positive test necessarily indicate that the stool contains blood. Despite these limitations the procedure is an extremely valuable one and should be performed routinely in the office and clinic as well as in the hospital. ERYTHROCYTE SEDIMENTATION RATE
In the presence of inflammation and tissue destruction of any sort, there are various nonspecific responses among the more important of which is an increased production of plasma fibrinogen in the liver. 27 The rate of sedimentation of the red cells has been observed to parallel these increases in blood Rbrinogen. 28 Thus, it is elevated in acute and chronic infectious diseases, malignant neoplasms, leukemia and lymphoma, acute and chronic nephritis in the active stage, coronary thrombosis and thromboses in general. It is also elevated in normal and abnormal pregnancy, and during the menstrual flow. It is usually depressed in severe cardiac decompensation, presumably because of interference with hepatic function. It may also be decreased in various forms of severe liver disease, especially in the chronic stages, such as cirrhosis of the liver. It is not altered by fever itself such as produced by the hypertherm, but it is often elevated in the presence of fever, in
1178
MALCOLM M. STANLEY
which cases it is presumed that both the fever and the elevated erythrocyte sedimentation rate represent responses to an underlying inflammatory process. The erythrocyte sedimentation rate is often advantageously used as an aid in determining the progress and treatment of certain chronic infections, such as rheumatic fever, tuberculosis and arthritis. It should be emphasized that it is entirely a nonspecific test,29. 30 and hence is of no diagnostic value beyond that of a simple indication of the presence, and presumably the amount, of inflammatory reaction. Two of the several technics available appear to be quite satisfactory.28 One, the Westergren method,31 is quite simple to perform and produces reliable results in most instances. In the presence of severe anemia, however, it may be inaccurate. In such instances the RourkeErnstene32 method may be preferable, although for routine use the increased time and effort required for its performance makes it less satisfactory than the Westergren method. TECHNIC OF THE WESTERGREN METHOD
Draw venous blood without stasis. Add exactly 3 cc. of blood to 0.75 cc. of 3.8 per cent sodium citrate solution in a calibrated tube. It is better to use a tube such as the ordinary calibrated centrifuge tube rather than depending upon the calibrations of the syringe to measure the desired quantity. Mix the blood and draw into Westergren tube to the height of 200 mm. (to the zero mark). Determine the distance of fall of the erythrocyte column (the distance between the meniscus and the line of demarcation between the red cell layer and plasma) at the end of exactly one hour.
The normal values for the erythrocyte sedimentation rate by this method are 1 to 7 mm. per hour for men and 4 to 11 mm. per hour for women and children. The values above 15 mm. per hour are abnormal. Values of from 12 to 15 mm. per hour are of questionable significance. TECHNIC FOR THE ROUHKE-EHNSTENE METHOD
Draw venous blood without stasis. Place 5 cc. in a bottle with dry oxalate mixture (4 mg. of potassium oxalate and 6 mg. of ammonium oxalate-HelIer and PauI 33 ). Fill the Wintrobe tube to "10" mark (100 mm.). The level of the red cell layer is read each five minutes for one hour. Uncorrected Sedimentation Rate.-Add the millimeters of sedimentation in the two fastest five minute periods and divide the sum by ten. This gives the most rapid rate of fall in millimeters per minute. H ematocrit (percentage of packed cells) .-Centrifuge the tube for one hour at 3,000 R.P.M. and read the percentage erythrocytes. Per cent ce11s =
height of packed erythrocytes . X 100 total height of column of cells and plasma
"Corrected Sedimentation Rate."-Dcrive the "Corrected Sedimentation Rate" from the uncorrected sedimentation rate and the hematocrit using the reference chart of Rourke and Ernstene. 32
OFFICE LABORATORY TESTS
H'~
'pecial Precautions for Performing Sedimentation Rate by Either chnic.-l. Always use the proper anticoagulant for the technic permed. ~. Perform the test as soon as possible after taking the sample. If )Od stands more than two hours, the sedimentation rate is slowed :l the test is valueless. 3. Be certain that the tube is exactly vertical. An inclination of only )er cent may accelerate settling velocity by 30 per cent. t Wide fluctuations of temperature influence the rate of sedimenion. 5. Any tendency of the blood sample to clot and any gross hemolysis ralidates the result. Determination of the erythrocyte sedimentation rate, particularly the estergren type, is such a simple procedure that it can readily be done the office. The necessary apparatus, racks and tubes are quite inexnsive. REFERENCES
· Rich, A. R.: The Pathogenesis of the Forms of Jaundice. Bull. Johns Hopkins Hosp., 47:338-377, 1930. · Myers, C. P.: Use of Methylene Blue in Testing for Bilirubin in the Urine. J. Indust. Hyg. & Toxicol., 27:52-55 (Feb.) 1945. · Franke, K.: Methylenblau, ein einfachcs sehr empnndliches Reagens zum Nachweis von Bilirubin. 1Ied. Klin., 27:94-96 (Jan. 16) 1931. · Roch, M.: Ueber Methylenblau als Reagens zum Nachweis von Bilirubin. Med. Klin.,27:589-590 (April 17) 1931. · Seide, J. and Zink, K.: Ueber die Verwendbarkeit der Methylenblauprobe zum Nachweis von Bilirubin. Deutsche Med. Wchnschr., 57:1744-1745 (Oct. 9) 1931. · Fellinger, K. and Menkes, K.: Ueber quantitative Bilirubinbestimmungen im Ham mit der Methylenblaumethode. Wein. klin. Wchnschr., 46:133-134 (Feb. 3) 1933. · Gellis, S. S. and Stokes, J.: The Methylene Blue Test in Infectious (Epidemic) Hepatitis. J.A.M.A., 128:782-783 (July 14) 1945. · Gellis, S., Neefe, J., Reinhold, J. and Stokes, J.: Methylene Blue Test for Bilirubin in the Urine. Correspondence, J.A.M.A., 128:826 (July 14) 1945. I. Editorial: Methylene Blue Test for Urinary 'Bilirubin. J.A.M.A., 127:1058 (April 21) 1945. '. Watson, J., Meads, M. and Castle, W.: Tests for Urinary Bilirubin. Corrcspondence, J.A.M.A., 128:308 (May 26) 1945. Figge, F. H. J.: The Green Color of a Methylene Blue-Bilirubin Mixture. Correspondence, J.A.M.A., 128:613-614 (June 23) 1945. Godfried, E. G.: Clinical Tests for Bilirubin in Urine. Biochem. J., 28:20562060, 1934. :. Harrison, G.: Chemical Methods in Clinical Medicine. London, J. and A. Churchill, 1937. :. Hawkinson, V., Watson, C. J. and Turner, R. H.: A Modincation of Harrison's Test for Bilirubin in the Urine Especially Suited for Mass and Serial Usage. J.A.M.A., 129:514-515 (Oct. 13) 1945. ;. Foord, A. and Baisinger, C.: Comparison of Tests for Bilirubin in Urine. Am. J. Clin. Path., 10:238-244, 1940. !.
1180
MALCOLM M. STANLEY
16. Joslin, E. P., Root, H. F., White, P. and Marble, A.: The Treatment of Di· abetes Mellitus, 7th Ed. Philadelphia, Lea & Febiger, 1940, p. 189. 17. John, H. J.: Does the Fractional Daily Urine Examination on a Diabetic Pa· tient Furnish Us the Ideal Means of Control? South. M. J., 39:60-63 (Jan.) 1946. 18. Benedict, S. R.: The Detection and Estimation of Glucose in Urine. J.A.M.A., 57: 1193-1194 (Oct. 7) 1911. 19. Kasper, J. and Jeffrey, I.: A Simplified Benedict Test for Glycosuria. Am. J. Clin. Path., Technical Section, 8:117-120, 1944. 20. Sheftel, A. G.: A Combined Qualitative and Quantitative Test for Sugar in the Urine. M. J. & Rec., 126:663-664, 1927. 21. Rhodehamel, R. H., Rose, C. L. and Chen, K. K.: A Rapid Method of Esti· mating Urine Sugar. M. Rec., 145:324-325, 1937. 22. Stanley, P.: The Detection of Sugar in Urine. Am. J. M. Technol., 6:263, 1940. 23. Bramkamp, R. G.: The Benzidine Reaction: Some Observations Relating to Its Clinical Application. J. Lab. & Clin. Med., 14:1087-1091, 1929. 24. Kirschen, M., Sorter, H. and Necheles, H.: Occult Blood: With a Note on the Use of Carmine for the Marking of Stools. Am. J. Digest. Dis. & Nutrition, 9:154-156 (May) 1942. 25. Kiefer, E. D.: Detection of Occult Blood in Feces. Am. J. Surg., 25:530-535, 1934. 26. Goodman, L. and Gilman, A.: The Pharmacological Basis of Therapeutics. New York, The Macmillan Company, 1941, p. 1109. 27. Ham, T. H. and Curtis, F. C.: Plasma Fibrinogen Response in Man. Medicine, 17:413-445, 1938. 28. Ham, T. H. and Curtis, F. C.: Sedimentation Rate of Erythrocytes. Medicine, 17:447-517, 1938. 29. Cutler, J.: The Graphic Presentation of the Blood Sedimentation Rate. A Study in Pulmonary Tuberculosis. Am. J. M. Sc., 171:882-901, 1926. 30. Cutler, J. W.: The Practical Application of the Blood Sedimentation Test in General Medicine. Am. J. M. Sc., 183:643-657, 1932. 31. Westergren, A.: The Technique of the Red Cell Sedimentation Reaction. Am. Rev. Tuberc., 14:94-101, 1926. 32. Rourke, M. D. and Ernstene, A. C.: A Method for Correcting the Erythrocyte Sedimentation Rate for Variations in the Cell Volume Percentage of Blood. J. Clin. Investigation, 8:545-559, 1930. 33. Heller, V. G. and Paul, H.: Changes in Cell Volume Produced by Varying Concentrations of Different Anticoagulants. J. Lab. & Clin. Med., 19:777786, 1934.
THE following laboratory tests were chosen for discussion because of their ready applicability to clinical problems frequently encountered by the practitioner, and because they can be done in the office, for the most part with a minimum of expensive or complicated equipment. URINE
I. Bile in the Urine.-Although commonly occurring in the urine in conjunction with bile salts and other components of bile, bilirubin is usually tested for because of its relative ease of detection. The presence of bilirubinuria indicates that there has been a "regurgitation"! of bile already formed by the liver into the blood stream from which it was excreted by the kidneys into the urine. This regurgitation may occur following necrosis of the liver cells as a result of infection ("infectious hepatitis"), the action of toxic substances such as arsenic ("toxic hepatitis"), or the effect of back pressure because of obstruction' of some portion of the biliary system ("obstructive jaundice"). In uncomplicated hemolytic icterus there is no bile in the urine. Hence, in an icteric patient this determination is of value in narrowing the possibilities as to the causative mechanism. Bilirubinuria in small amounts may be the earliest detectable sign in infectious hepatitis and should be searched for in obscure febrile illnesses of short duration with vague abdominal symptoms, particularly with a history of exposure, in order that the disease be diagnosed in its incigient (preicteric) stage. Routine tests at short intervals of the urines of industrial workers exposed to hepatotoxic agents have been of value in detecting those 'Yith early liver damage in order that they might be removed from the deleterious environment before serious harm had taken place. 2 TESTS
1. "FOAM" TEST A test tube half full of the urine is shaken vIgorously, holding the thumb over the open end. The color of the layer (,f foam which forms on top of the urine is compared against a white background with a normal From the Evans Memorial, Massachusetts Memorial Hospitals, and the Department of Medicine, Boston University School of Medicine, Boston, Massa·· chusetts . . Instructor in Medicine, Boston University School of Medicine; Fellow in Medicine, Evans Memorial, Massachusetts Memorial Hospitals. 1168
OFFICE LABORATORY TESTS
1169
urine treated in the same manner. If the foam is yellow the test is positive. It is to be emphasized that slight discoloration of the foam, indicating a small amount of bile, can be detected only by comparison with a normal control.
The foam test is a most sensitive method for detection of bile, and is the most easily performed of all. Its only disadvantage is that the presence of substances such as penicillin, streptomycin, phenolsulfonphthalein in acid solution, riboflavin, atabrine and possibly others (not, however, urochrome) will cause a false positive. Its interpretation is difficult when patients are receiving these drugs, so other confirmatory determinations must be made in those instances. 2. METHYU;NE BLUE TEST'-u
A 0.2 per cent aqueous solution of methylene blue chloride is added drop by drop (using a pipette which delivers 20 drops of the solution per cubic centimeter) to 5 cc. of a prebreakfast urine specimen. The reading (in drops) is taken as one less than the number needed to change the color from green to blue. If more than 8 drops are required a smaller measured amount of urine is diluted to 5 cc. with distilled water and the test repeated in order that a clearer end point may be obtained. If diluted, the number of drops is multiplied by the dilution factor. A reading greater than 4 drops has been regarded (by Gellis and Stokes, et al.) as a positive test for bilirubin in the urine. Readings of 2 to 4 drops inclusive have been regarded as suggestive and as an indication for additional studies. As Myers2 described the test, 2 drops of methylene blue solution were added to 10 cc. of urine. The solution remains dark blue if negative, but turns a brilliant green if positive for bile. He quantitated the test by progressively diluting the urine, keeping the total volume at 10 cc. by replacing increasing amounts of urine with distilled water, until a negative result was obtained.
Franke introduced the methylene blue test in 1931.3 This soon brought forth several communications in protest4 and approval,'" G just as did Myers' recent paper. 7- 11 The majority·of these authors state that a positive test results from the simple mixing of the two colors blue and yellow, and not from the oxidation of yellow bilirubin to green biliverdin with conversion of the methylene blue to the colorless leuko form as was Franke's original belief. The consensus and bulk of the evidence indicate that the blending of colors accounts for most of the green col or phenomenon. Because a positive test results from blending of the yellow and blue colors, false positives are also likely to occur when other pigments are present in the urine as in the foam test. Its most ardent advocates 7 • s admit its inferiority in sensitivity and specificity to the Harrison and diazo spot test. On the other hand, it is performed in a roughly quantitative manner and may be of value in following the course of patients with parenchymal liver disease such as infectious hepatitis, since the
1170
MALCOLM M. STANLEY
titer diminishes and the test becomes negative during convalescence when the icterus is still present and the serum bilirubin is still elevated. 3. DIAZO SPOT (GodfriedI2 ) Five cubic centimeters of 10 per cent barium chloride and 10 cc. of urine are mixed and the voluminous precipitate of insoluble barium salts is filtered. The wet filter is placed on a disk of dry filter paper so that the excess liquid is removed. To the precipitate on filter paper add 1 drop of Ehrlich's diazo reagent, 4 drops of 95 per cent alcohol, and 1 drop of phosphate buffer (6 per cent Na2HP04.12H20). A red to pink color indicates a positive reaction. If there are large amounts of bilirubin, the test works better when the urine is diluted.
(Ehrlich's Diazo Reagent: This is a mixture of two solutions, each of which keeps well, but which must be freshly mixed immediately prior to using. Solution 1: Sulphanilic acid, 5 gm.; concentrated hydrochloric acid, 50 cc.; distilled water to make 1000 cc. Solution 2: Sodium nitrite, 0.5 gm.; distilled water, 100 cc. Take 0.8 cc. of solution 2 and make up to 25 cc. with solution 1.) (Godfriedl2 13) Precipitate the bilirubin with barium chloride and filter as in the Diazo test (3 above). To the precipitate on filter paper 1-2 drops of Fouchet's reagent (25 per cent trichloroacetic acid containing 0.9 per cent ferric chloride) are added. If bilirubin is present, the characteristic blue to green color appears. 4. HARRISON SPOT
When the barium chloride and urine are mixed the barium ions and s.ulfate ions present in the urine form insoluble barium sulfate which precipitates. The bilirubin is adsorbed onto the barium sulfate particles, hence, is "concentrated." The blue to green color of a positive Harrison test is the result of oxidation of the adsorbed yellow bilirubin to biliverdin or bilicyanin. Both the Harrison and the diazo spot tests are "concentration" tests. Both the Harrison spot and diazo spot tests are very sensitive and quite specific. Their only drawback is that they are more complicated and require more time and equipment. According to Godfried,12 0.0020.008 mg. of bilirubin per 100 cc. of urine is detectable by these methods. 5. HARRISON MODIFICATION (Hawkinson, Watson. and Turner14 ) a. Preparation of Barium Impregnated Paper Strips.-Pieces of extra thick and retentive filter paper are allowed to remain briefly in a saturated aqueous solution of barium chloride. (Schleicher and Schull number 470 filter paper is recommended. Lighter grades are not satisfactory. We have found that ordinary unglazed heavy white blotting paper is quite adequate.) They are then dried in the air or, preferably, in a drying oven, after which they are cut into strips 4 inches long by 12 inch wide. b. Procedure.-One end of a barium chloride impregnated strip is placed in the urine sample to be tested, the strip being in an approximately vertical position, at least one half extending above the surface of
1171
OFFICE LABORATORY TESTS
the sample. After standing in the urine for from thirty seconds up to two minutes the strip is withdrawn and placed on a piece of dry paper such as a paper towel or any other absorbent paper. Inspection of the filter strip will usually reveal somewhat more color in that area which corresponded to the surface of the urine. Two to three drops of Fouchet's reagent (see above) are then dropped directly on this area. A positive test is denoted by the appearance of a green color, varying in intensity with the amount of bilirubin present. With smaller amounts the color is often detected as a faint green line running across the strip. The urine to be tested may be agitated by shaking, by bubbling air llrough the liquid, or by forcefully drawing it up and squirting it back from a syringe into a beaker several times so that there is a layer of foam on the surface. If the barium chloride impregnated strip is then inserted into the liquid through the layer of foam, and the test carried out otherwise as described above, we have noted that its sensitivity is markedly increased. In effect, this combines the principles of the foam test and the Harrison test. >
Hawkinson, Watson, and Turner consider that their modification is just as sensitive as the original Harrison test. This has been our experience with the procedure. The modification of the original Harrison technic is "especially suited for mass and serial usage" and would seem to have eliminated all the objections to the original method while retaining its advantages of sensitivity and specificity. It is easily and quickly performed and requires, besides the barium chloride impregnated strips, only one reagent which will keep indefinitely. Because of its extreme simplicity it would seem that the foam test is useful as a screening test which should be done on all urines as a part of the routine urinalysis. Confirmation of a positive foam test if necessary can be obtained with one of the more specific procedures. For this the modified Harrison test is recommended because of the ease and speed with which it can be performed. 11. Glycosuria.-The importance of testing the urine routinely for sugar is now too well recognized to require emphasis. The discovery of glycosuria during the course of a routine "check-up," particularly of the members of the family of a diabetic, is often the first evidence of diabetes. It may occur long before obvious symptoms make their appearance. Adherence to the custom of determining urine and blood sugars while the patient is in the fasting state when diabetes is suspected may, however, result in unnecessary delay in diagnosis. 16 In the mild diabetic the urine may be free from sugar and the blood sugar may be normal in the fasting state, particularly before breakfast after a ten to twelve hours' fast. Therefore, in patients who are suspected of having diabetes these tests should be performed on specimens taken two to four hours after a meal heavy in carbohydrate, or a glucose tolerance test should be done. Do not do SINGLE fasting blood and urine sugars when the object is to establish the diagnosis of diabetes in a doubtful case.
1172
MALCOLM M. STANLEY
1.
QUANTITATIVE ESTIMATIONS OF URINARY GLUCOSE
The necessity for basing the determination of adequacy of control of diabetics on the quantitative estimation of sugar spilled in the urine during the twenty-four-hour period, rather than on qualitative tests on periodic specimens, has recently been reiteratedY Especially when controlled on protamine zinc insulin alone are diabetics liable to spill small amounts of sugar after meals. When the total for twenty-four hours is measured, the amount is often found to be insignificant. It has been our goal to control patients in the clinic so that 5 gm. of glucose or less are lost in the urine per day. A.
MODIFIED BENEDICT METHOD!8
To 2.5 cc. of reagent in a pyrex test tube add approximately 1 gm. of anhydrous sodium carbonate. Heat slowly to boiling over a low flame. Add the urine slowly, drop by drop, from a pipette graduated in tenths of a cubic centimeter, while the solution is kept boiling. The end point is complete absence of blue color. The white precipitate of cuprous thiocyanate by contrast makes the end point easier to determine. If x is the number of cc. of urine required for reduction, the formula for computing the percentage of glucose is as follows: 0.005 - - X 100 = percentage of sugar x
If there is a high concentration of glucose, it may be advantageous to dilute the urine 1: 10, in which case the formula is: 0.005 - - - X 1000 = percentage of sugar (in original urine) x
The reagent (2.5 cc. are reduced by 5 mg. glucose) is composed of the following (only the copper sulfate need be weighed accurately): Copper sulfate (pure crystallized) ................ Sodium carbonate (crystallized) .................. or Anhydrous sodium carbonate ................ Sodium or potassium citrate, C.P .................. Potassium thiocyanate, C.P ....................... Potassium ferrocyanide solution (5 per cent) ....... Distilled water, to make .........................
18.0 . . 200.0 . 100.0 . 200.0 . 125.0 . 5.0 . 1000.0
gm. gm. gm. gm. gm. cc. cc.
With the aid of heat dissolve the carbonate, citrate and thiocyanate in about 700 cc. of water and filter. Dissolve the copper sulfate in 100 cc. of water and pour slowly into the other fluid, stirring constantly. Add the ferrocyanide solution, cool and dilute to 1000 cc. This reagent may also be purchased already prepared from many pharmaceutical firms.
OFFICE LABORATORY TESTS
2.
1173
METHODS BY WHICH DIABETIC PATmNTS THEMSELVES CAN DETECT GLYCOSURIA
Every well-trained diabetic should learn how to test his own urine for sugar and ketones, and should perform at least the former test every day. There are several different outfits which are available for these procedures. A.
BENEDICT QUALITATIVE TEST's
Eight drops of urine are added to 5 cc. (approximately 1 teaspoonful) of Benedict's qualitative solution and thoroughly mixed by shaking. The test tube is placed in boiling water for five minutes. The amount of sugar is estimated by the degree of reduction, from green to red.
The reagent contains copper sulfate, sodium or potassium citrate and sodium carbonate. The reaction is the familiar one of reduction by glucose of the cupric salt to cuprous hydrate (yellow) or cuprous oxide (red). A pint of solution (enough for approximately 100 tests) costs from 59 cents to $1.00. Two test tubes and a medicine dropper cost an additional 25 cents. Each test, therefore, costs less than 1 cent. This procedure is quite satisfactory when performed by the patient or physician according to directions. It is the least expensive. However, the variations in results are amazing when the reagent, the urine, or both are not measured, or the boiling is inadequate. The test requires several minutes and the equipment is bulky and difficult to carry on one's person. An external source of heat is necessary. Because of these inconveniences, particularly during vacations when traveling or visiting, patients often neglect this procedure entirely. When dietary indiscretions common to such periods occur, derangement of control may be serious without the patient's knowledge. B.'L"CLINITEST" TABLET METHon '9
This set is packed in a small plastic case (8 by 5 by 3.5 cm.) which fits into the pocket or purse and which contains all the necessary materials (dropper, test tube and bottle of 36 reagent tablets). The top of the case is designed for collection of the urine specimen. The retail price of the kit is $1.75 (includes 36 tablets). Additional tablets are 60 cents for 36. Each test costs approximately 1.7 cents, disregarding the initial cost of the kit. Twenty-five one hundredths milliliter (5 drops) of urine is added to 0.5 ml. (10 drops) of water in the small test tube and one reagent tablet is dropped into this solution. A boiling reaction occurs immediately and if glucose is present a colored precipitate of cuprous oxide will form. The degree of reduction is estimated as in the Benedict qualitative test. A color scale for comparison is part of the kit.
1174
MALCOLM M. STANLEY
The components of the reagent tablet are essentially the same as in the Benedict solution except for the presence of sodium hydroxide, citric acid and sodium bicarbonate instead of sodium carbonate and sodium citrate. However, upon being dissolved the tablet produces sodium citrate, carbon dioxide and a very small quantity of sodium bicarbonate. In addition, the heat resulting from the ionization of the solid caustic and the neutralization between sodium hydroxide and citric acid is sufficient for the reduction of the cupric ions in the presence of glucose. Recently, testing of several thousand diabetic urines by trained laboratory technicians, using both the Benedict qualitative and the Clinitest methods, has shown agreement in 84 per cent of the specimens. 19 As the test can be done in less than a minute and requires no external source of heat, it is of great value to patients who are traveling, staying at hotels, and the like. This set combines the virtues of simplicity, convenience, portability and inexpensiveness and is the best outfit available for the purpose. C.
"SHEFTEL" TABLET METHOD""
21
This urine sugar test case is a somewhat elaborate outfit in a 12 by 11 by 3.7 cm. plastic box. The box opens so that the top forms a rack which holds: (1) the test tube in position for heating, (2) two vials, one containing 60 copper sulfate reagent tablets and the other 40 methenamine tablets which are burned for a source of heat, (3) a graduated dropper-pipette, and (4) a combination glass spoon and rubber test tube cleaner. A color scale and durable book of instructions are also provided. The retail price is $3.75, including the above reagent and fuel tablets. Each test costs 2% cents, disregarding the initial cost of the outfit. To 1.5 cc. of water in the test tube are added 0.25 cc. of urine and a copper suIfate reagent tablet. A methenamine tablet is placed on the metal well just below the bottom of the test tube, and lighted. The fuel tablet bums for 2% minutes, boiling the solution and carrying the reduction reaction to completion.
The reagent tablet employs the same chemicals that are used for the Benedict qualitative test, namely copper sulfate, sodium or potassium citrate, and sodium carbonate, but with the addition of acacia and creatinine. The big disadvantage is that an external source of heat is required. Both the initial cost and the cost of each test are relatively high. The case is slightly large to be carried conveniently in the pocket. A separate container must be provided for the urine to be tested. On the other hand, the procedure may be done precisely and in a roughly quantitative manner. The case is sturdily constructed and quite durable.
OFFICE LABORATORY TESTS
1175
D. "GALATEST" POWDER METHOD"
This "outfit" is composed of two tubes of dry reagent powder (one for testing for sugar and the other for acetone) and a medicine dropper contained in a flimsy unpainted wooden box 10.7 by 6 by 3 cm. (1) The white "Galatest" powder (for sugar detection) is a mixture of bismuth oxychloride, sodium hydroxide and sodium silicate. (2) The "Denco" powder (for acetone detection) is a mixture of sodium carbonate, ammonium sulfate and sodium nitroprusside. The cost of the set is $2.25, or $1.00 for each of the tubes of powder. Each tube contains material for approximately 100 tests, so the cost is about 1 cent each. Deposit on a piece of plain white paper a little of the "Galatest" powder (covering an area abcut the size of the little finger nail). Use dropper and deposit 1 small drop of urine on the powder. The presence of sugar is indicated by a change in the color of the powder ranging from gra~-green to black, depending upon the amount.
In this instance the glucose reduces the bismuth oxychloride to black bismuth oxide in the cold. It is easily portable, and the procedure is very simple to perform: Again, however, a separate container must be provided for collection of the urine. The colors are not as sharply defined as in the more familiar copper reduction tests, and a maximum of 1 per cent glucose in undiluted urine can be detected. (To detect acetone the above procedure is followed, using instead "Denco" powder and 2 to 3 drops of urine. A positive test is indicated by color changes ranging from light lavender to dark purple, and requires about one minute for completion. If no acetone is present, the powder will take on a grayish-yellow color.)
STOOL Every patient who enters the hospital or who undergoes a physical examination as a periodic routine procedure or for other reasons should have a stool examination, for occult blood at least. Occult blood in the stools is confirmatory evidence in many cases of malignancy of the stomach or large bowel or active peptic ulcer. A persistently positive test may provide the reason for an obscure anemia or diatrhea. An adequate specimen can be obtained on the gloved 6nger at the time the digital rectal examination is made. Only rarely must this procedure be postponed because of an insufficient amount of feces in the rectum. Unfortunately, examination of the stool, although it may often be diagnostic, is the most frequently neglected laboratory test. CLINICAL TESTS FOR OCCULT BLOOD
1. TECHNIC FOR GUAIAC TEST
Place a small amount of material (feces, gastric contents) in a depression on a white spot plate (tile) and mix with several drops of glacial
1176
MALCOLM M. STANLEY
acetic acid. Add 2 or 3 drops of gum guaiac solution and mix again. Add 2 or 3 drops of 3 per cent hydrogen peroxide. Development of a green or blue calor is a positive reaction. The rapidity with which the calor develops and the depth of calor are some indications of the amount of hemoglobin present. REAGENTS (should be kept in dark bottles, as they are decomposed by light).1. Gum guaiac: 1 gm. of gum guaiac in 60 cc. of 95 per cent alcohol or in 60 cc. glacial acetic acid. 2. Hydrogen peroxide: 3 per cent aqueous solution. Keep refrigerated if possible. (In case of a negative result, it is well to perform the test on a small amount of blood if there is any doubt as to the potency of the reagents, particularly the hydrogen peroxide.)
2. TECHNIC FOR BENZIDINE TEST
A saturated solution of "benzidine base for blood detection" in glacial acetic acid is used inste;o.d of the gum guaiac solution. The test is otherwise performed in the same way as the guaiac on the stool.
Blood and certain other substances contain the enzyme peroxidase which catalyzes the oxidation of the base compound (guaiacol or benzidine base) to a green or blue colored compleX'. An excess of hydrogen peroxide rapidly destroys this colored complex and also oxidizes and destroys hemoglobin. Therefore, hydrogen peroxide must not be added in excess and must be added after the guaiac or benzidine solution. Factors Influencing the Tests and Their Interpretation.-Feces contain substances which inhibit the peroxidase reaction. Bramkamp23 found that when hemoglobin was mixed with the feces, five times as much was needed to produce a positive benzidine reaction as was necessary when it was in watery solution. A concentration of 0.04 per cent blood freshly mixed with the stool will cause a positive benzidine test. The ingestion of 1.5 to 2 gm. of hemoglobin (9.4 to 12.5 cc. of blood containing 16 gm. of hemoglobin per 100 cc.) over a day's time resulted in positive tests in only 35 to 48.5 per cent of the normal subjects, however. 24 Pancreatic digestion is probably largely responsible for this variable loss of ability to reduce peroxides which occurs on passage of blood through the intestines,23 although other factors such as intestinal motility may influence it. 24 Thus, when blood is taken by mouth, ninety times as much may be required to produce a positive test as when the blood is freshly mixed with the stool. Hence when the site of bleeding is in the esophagus, stomach or duodenum, relatively large amounts of blood must be lost before it can be detected by the usual tests. In contrast to this, as little as 0.04 per cent blood theoretically will produce a positive reaction when the source of bleeding is in the large bowel. . The benzidine reaction will often be positive on the stools of normal persons if their diets contain meat. The guaiac test is somewhat less sensitive, hence it is not necessary to have the patient on a meat-free
OFFICE LABORATORY TESTS
1177
diet, particularly if only a blue or strong blue-green color is regarded as positive, and not the lighter shades of green. It is for this reason that we routinely employ the guaiac test, realizing, however, that it is not as sensitive as the benzidine test, and that it will not detect small hemorrhages. In testing for hemoglobin in the urine, the extreme sensitivity of the benzidine test is a virtue and makes it preferable to the guaiac test in this instance. False Positives.-A number of other substances will produce the green or blue color reaction besides hemoglobin. The common ones are milk, pus, ferrous sulfate, bromides, iodides, and copper compounds (such as the copper precipitate left on the walls of test tubes after Benedict's test for sugar). Usually administration of ferrous iron by mouth, although causing the stool to appear "tarry," will not produce a strongly positive guaiac reaction. This is because the unabsorbed iron is either found "in firm organic combination"26 after its passage through the gut, in which nonionized form it will not catalyze the reaction, or it is oxidized to the ferric state soon after defecation, when exposed to air. In rare instances, however, iron therapy will cause a false positive guaiac test, apparently because some of the ferrous form appears in the feces unchanged. We have seen at least one case in which there was good evidence that ferrous sulfate caused a false positive guaiac test on the stool. From consideration of the above facts it may be seen that a negative test for fecal occult blood does not rule out small amounts of bleeding, particularly if it occurs high in the gastrointestinal tract; neither does a positive test necessarily indicate that the stool contains blood. Despite these limitations the procedure is an extremely valuable one and should be performed routinely in the office and clinic as well as in the hospital. ERYTHROCYTE SEDIMENTATION RATE
In the presence of inflammation and tissue destruction of any sort, there are various nonspecific responses among the more important of which is an increased production of plasma fibrinogen in the liver. 27 The rate of sedimentation of the red cells has been observed to parallel these increases in blood Rbrinogen. 28 Thus, it is elevated in acute and chronic infectious diseases, malignant neoplasms, leukemia and lymphoma, acute and chronic nephritis in the active stage, coronary thrombosis and thromboses in general. It is also elevated in normal and abnormal pregnancy, and during the menstrual flow. It is usually depressed in severe cardiac decompensation, presumably because of interference with hepatic function. It may also be decreased in various forms of severe liver disease, especially in the chronic stages, such as cirrhosis of the liver. It is not altered by fever itself such as produced by the hypertherm, but it is often elevated in the presence of fever, in
1178
MALCOLM M. STANLEY
which cases it is presumed that both the fever and the elevated erythrocyte sedimentation rate represent responses to an underlying inflammatory process. The erythrocyte sedimentation rate is often advantageously used as an aid in determining the progress and treatment of certain chronic infections, such as rheumatic fever, tuberculosis and arthritis. It should be emphasized that it is entirely a nonspecific test,29. 30 and hence is of no diagnostic value beyond that of a simple indication of the presence, and presumably the amount, of inflammatory reaction. Two of the several technics available appear to be quite satisfactory.28 One, the Westergren method,31 is quite simple to perform and produces reliable results in most instances. In the presence of severe anemia, however, it may be inaccurate. In such instances the RourkeErnstene32 method may be preferable, although for routine use the increased time and effort required for its performance makes it less satisfactory than the Westergren method. TECHNIC OF THE WESTERGREN METHOD
Draw venous blood without stasis. Add exactly 3 cc. of blood to 0.75 cc. of 3.8 per cent sodium citrate solution in a calibrated tube. It is better to use a tube such as the ordinary calibrated centrifuge tube rather than depending upon the calibrations of the syringe to measure the desired quantity. Mix the blood and draw into Westergren tube to the height of 200 mm. (to the zero mark). Determine the distance of fall of the erythrocyte column (the distance between the meniscus and the line of demarcation between the red cell layer and plasma) at the end of exactly one hour.
The normal values for the erythrocyte sedimentation rate by this method are 1 to 7 mm. per hour for men and 4 to 11 mm. per hour for women and children. The values above 15 mm. per hour are abnormal. Values of from 12 to 15 mm. per hour are of questionable significance. TECHNIC FOR THE ROUHKE-EHNSTENE METHOD
Draw venous blood without stasis. Place 5 cc. in a bottle with dry oxalate mixture (4 mg. of potassium oxalate and 6 mg. of ammonium oxalate-HelIer and PauI 33 ). Fill the Wintrobe tube to "10" mark (100 mm.). The level of the red cell layer is read each five minutes for one hour. Uncorrected Sedimentation Rate.-Add the millimeters of sedimentation in the two fastest five minute periods and divide the sum by ten. This gives the most rapid rate of fall in millimeters per minute. H ematocrit (percentage of packed cells) .-Centrifuge the tube for one hour at 3,000 R.P.M. and read the percentage erythrocytes. Per cent ce11s =
height of packed erythrocytes . X 100 total height of column of cells and plasma
"Corrected Sedimentation Rate."-Dcrive the "Corrected Sedimentation Rate" from the uncorrected sedimentation rate and the hematocrit using the reference chart of Rourke and Ernstene. 32
OFFICE LABORATORY TESTS
H'~
'pecial Precautions for Performing Sedimentation Rate by Either chnic.-l. Always use the proper anticoagulant for the technic permed. ~. Perform the test as soon as possible after taking the sample. If )Od stands more than two hours, the sedimentation rate is slowed :l the test is valueless. 3. Be certain that the tube is exactly vertical. An inclination of only )er cent may accelerate settling velocity by 30 per cent. t Wide fluctuations of temperature influence the rate of sedimenion. 5. Any tendency of the blood sample to clot and any gross hemolysis ralidates the result. Determination of the erythrocyte sedimentation rate, particularly the estergren type, is such a simple procedure that it can readily be done the office. The necessary apparatus, racks and tubes are quite inexnsive. REFERENCES
· Rich, A. R.: The Pathogenesis of the Forms of Jaundice. Bull. Johns Hopkins Hosp., 47:338-377, 1930. · Myers, C. P.: Use of Methylene Blue in Testing for Bilirubin in the Urine. J. Indust. Hyg. & Toxicol., 27:52-55 (Feb.) 1945. · Franke, K.: Methylenblau, ein einfachcs sehr empnndliches Reagens zum Nachweis von Bilirubin. 1Ied. Klin., 27:94-96 (Jan. 16) 1931. · Roch, M.: Ueber Methylenblau als Reagens zum Nachweis von Bilirubin. Med. Klin.,27:589-590 (April 17) 1931. · Seide, J. and Zink, K.: Ueber die Verwendbarkeit der Methylenblauprobe zum Nachweis von Bilirubin. Deutsche Med. Wchnschr., 57:1744-1745 (Oct. 9) 1931. · Fellinger, K. and Menkes, K.: Ueber quantitative Bilirubinbestimmungen im Ham mit der Methylenblaumethode. Wein. klin. Wchnschr., 46:133-134 (Feb. 3) 1933. · Gellis, S. S. and Stokes, J.: The Methylene Blue Test in Infectious (Epidemic) Hepatitis. J.A.M.A., 128:782-783 (July 14) 1945. · Gellis, S., Neefe, J., Reinhold, J. and Stokes, J.: Methylene Blue Test for Bilirubin in the Urine. Correspondence, J.A.M.A., 128:826 (July 14) 1945. I. Editorial: Methylene Blue Test for Urinary 'Bilirubin. J.A.M.A., 127:1058 (April 21) 1945. '. Watson, J., Meads, M. and Castle, W.: Tests for Urinary Bilirubin. Corrcspondence, J.A.M.A., 128:308 (May 26) 1945. Figge, F. H. J.: The Green Color of a Methylene Blue-Bilirubin Mixture. Correspondence, J.A.M.A., 128:613-614 (June 23) 1945. Godfried, E. G.: Clinical Tests for Bilirubin in Urine. Biochem. J., 28:20562060, 1934. :. Harrison, G.: Chemical Methods in Clinical Medicine. London, J. and A. Churchill, 1937. :. Hawkinson, V., Watson, C. J. and Turner, R. H.: A Modincation of Harrison's Test for Bilirubin in the Urine Especially Suited for Mass and Serial Usage. J.A.M.A., 129:514-515 (Oct. 13) 1945. ;. Foord, A. and Baisinger, C.: Comparison of Tests for Bilirubin in Urine. Am. J. Clin. Path., 10:238-244, 1940. !.
1180
MALCOLM M. STANLEY
16. Joslin, E. P., Root, H. F., White, P. and Marble, A.: The Treatment of Di· abetes Mellitus, 7th Ed. Philadelphia, Lea & Febiger, 1940, p. 189. 17. John, H. J.: Does the Fractional Daily Urine Examination on a Diabetic Pa· tient Furnish Us the Ideal Means of Control? South. M. J., 39:60-63 (Jan.) 1946. 18. Benedict, S. R.: The Detection and Estimation of Glucose in Urine. J.A.M.A., 57: 1193-1194 (Oct. 7) 1911. 19. Kasper, J. and Jeffrey, I.: A Simplified Benedict Test for Glycosuria. Am. J. Clin. Path., Technical Section, 8:117-120, 1944. 20. Sheftel, A. G.: A Combined Qualitative and Quantitative Test for Sugar in the Urine. M. J. & Rec., 126:663-664, 1927. 21. Rhodehamel, R. H., Rose, C. L. and Chen, K. K.: A Rapid Method of Esti· mating Urine Sugar. M. Rec., 145:324-325, 1937. 22. Stanley, P.: The Detection of Sugar in Urine. Am. J. M. Technol., 6:263, 1940. 23. Bramkamp, R. G.: The Benzidine Reaction: Some Observations Relating to Its Clinical Application. J. Lab. & Clin. Med., 14:1087-1091, 1929. 24. Kirschen, M., Sorter, H. and Necheles, H.: Occult Blood: With a Note on the Use of Carmine for the Marking of Stools. Am. J. Digest. Dis. & Nutrition, 9:154-156 (May) 1942. 25. Kiefer, E. D.: Detection of Occult Blood in Feces. Am. J. Surg., 25:530-535, 1934. 26. Goodman, L. and Gilman, A.: The Pharmacological Basis of Therapeutics. New York, The Macmillan Company, 1941, p. 1109. 27. Ham, T. H. and Curtis, F. C.: Plasma Fibrinogen Response in Man. Medicine, 17:413-445, 1938. 28. Ham, T. H. and Curtis, F. C.: Sedimentation Rate of Erythrocytes. Medicine, 17:447-517, 1938. 29. Cutler, J.: The Graphic Presentation of the Blood Sedimentation Rate. A Study in Pulmonary Tuberculosis. Am. J. M. Sc., 171:882-901, 1926. 30. Cutler, J. W.: The Practical Application of the Blood Sedimentation Test in General Medicine. Am. J. M. Sc., 183:643-657, 1932. 31. Westergren, A.: The Technique of the Red Cell Sedimentation Reaction. Am. Rev. Tuberc., 14:94-101, 1926. 32. Rourke, M. D. and Ernstene, A. C.: A Method for Correcting the Erythrocyte Sedimentation Rate for Variations in the Cell Volume Percentage of Blood. J. Clin. Investigation, 8:545-559, 1930. 33. Heller, V. G. and Paul, H.: Changes in Cell Volume Produced by Varying Concentrations of Different Anticoagulants. J. Lab. & Clin. Med., 19:777786, 1934.