Basic Examination of Urine PDF

CHAPTER 29 BASIC EXAMINATION OF URINE Roger S. Riley, Richard A. McPherson URINE FORMATION, 468 Basic (Routine) Urinalysis Procedure, Urinary Calculi, 505...

CHAPTER 29 BASIC EXAMINATION OF URINE Roger S. Riley, Richard A. McPherson URINE FORMATION, 468 Basic (Routine) Urinalysis Procedure, Urinary Calculi, 505 499 Laboratory Tests used to Investigate COMPONENTS OF BASIC (ROUTINE) URINALYSIS, 468 Automated Urinalysis, 500 Stone Formers, 507 Specimen Evaluation, 469 Automated Microscopy Analyzers, 503 Urinary Screening for Inherited Integrated, Fully Automated Metabolic Diseases, 507 Gross/Physical Examination, 469 Urinalysis Systems, 505 Additional Urine Testing Chemical Screening, 472 Modalities, 509 Examination of Urine Sediment, 486 SPECIAL TESTING AND MONITOR- ING TECHNIQUES, 505 SELECTED REFERENCES, 509 METHODS FOR URINALYSIS, 499 The purpose of this chapter is to highlight the pertinent information KEY POINTS that can be provided by the most common urine tests. Two main types  any different diseases can display abnormalities in the urine. There- M of urinalysis are currently performed. These include (1) the dipstick fore, examination of the urine is an important laboratory function. (reagent strip) urinalysis, which is commonly performed in screening laboratories, in physician offices, and as patient home testing; and (2)  asic urinalysis consists of gross examination of the urine as well B the basic (routine) urinalysis, which adds a microscopic examination of as a dipstick analysis for blood, white blood cells, sugar, and other urine sediment to the reagent strip urinalysis. These examinations utilize substances. The dipstick may be read manually or by an automated various laboratory disciplines, particularly chemistry and microscopy. In instrument. addition to these front-­line diagnostic procedures, new technologies—  microscopic analysis of urine may be necessary in many cases. This A including immunocytochemistry, molecular diagnostics, deoxyribonu- is done to detect cellular elements, casts, and crystals. Each of these cleic acid (DNA) ploidy, and cell cycle analysis—are constantly evolving items can be caused by several different disease states. to provide additional diagnostic and prognostic information. Urine  lthough microscopic examination of the urine is usually performed A microbiology studies, crucial to the diagnosis of infectious pathogens of manually, several automated instruments can perform this analysis. the urinary tract, are addressed elsewhere in this textbook. It is impor- tant to remember that each of these modalities has a certain clinical util-  ed blood cells within the urine can come from any point along the R ity. Table 29.1 lists the benefits of commonly ordered urine laboratory urinary tract. Dysmorphic red blood cells are often a sign of glomeru- examinations. lar disease.  he first voided morning urine, because it is the most concentrated, is T URINE FORMATION often the best specimen for analysis. Some procedures may require a 12-­or 24-­hour urine sample. In the normal adult, approximately 1200 mL of blood perfuses the kid-  pecific gravity and osmolality measurements reflect the concentrat- S neys each minute, which accounts for about 25% of the cardiac output. ing ability of the kidneys. After a period of dehydration, the osmolality The glomeruli (normally numbering at least 1 million per kidney) receive should be three to four times that of plasma. blood through afferent arterioles, and an ultrafiltrate of the plasma passes through each glomerulus into the Bowman space. From here, the filtrate  roteinuria greater than 4 g/day is seen in nephrotic syndrome. P is passed through the tubules and collecting ducts, where reabsorption or Although nephrotic syndrome is usually seen in primary renal disease, secretion of various substances and the concentration of urine can occur it is occasionally seen in a systemic disease that affects the kidneys. (Scott & Quaggin, 2015). Ultimately, the original glomerular filtrate vol-  etonuria can be seen in diabetic individuals. It can also be seen in K ume of about 180 L in 24 hours is reduced to about 1 to 2 L depending other states, such as febrile illness and cachexia. on the status of hydration. This urine formed in the kidneys passes from  he dipstick nitrite and leukocyte esterase tests are used to help diag- T the collecting ducts into the renal pelvis, ureters, bladder, and urethra to nose urinary tract infection. Positive results should be confirmed by be voided. microscopic analysis of the urine. The kidneys take part in several regulatory functions. Through glo- merular filtration and tubular secretion, numerous waste products—  rinary calculi are most commonly formed from calcium. Workup of U including nitrogenous products of protein catabolism, and both organic habitual stone formers should include analysis of both the urine and and inorganic acids and bases—are eliminated from the body. Fluid, elec- the stone. trolytes (including sodium, potassium, calcium, and magnesium), and acid-­ base status are regulated in homeostasis. Furthermore, the kidneys provide A significant amount of information can be obtained through the examina- important hormonal regulation with erythropoietin and renin production tion of urine. Careful examination enables the detection of disease pro- as well as vitamin D activation. Any derangement of these functions by cesses intrinsic to the urinary system, both functional (physiologic) and renal or systemic disease can be reflected as chemically or cytologically structural (anatomic), and sometimes unsuspected (Echeverry, Hortin, altered urine. & Rai, 2010; Haber, 1988; Takemura, Ishida, Inoue, Kobayashi, & Beck, 2000). The progression or regression of various lesions can be monitored COMPONENTS OF BASIC (ROUTINE) with only minimal distress to the patient. Furthermore, systemic disease URINALYSIS processes, such as endocrine or metabolic abnormalities, can be detected through the recognition of abnormal quantities of disease-­specific metabo- The basic (routine) urinalysis consists of four parts: specimen evalu- lites excreted in the urine. Laboratory urine tests will continue to play an ation, gross/physical examination, chemical screening, and sediment essential role in clinical medicine. examination. 468  TABLE 29.1 Benefits of Common Urine Laboratory Tests CLINICAL UTILITY Type of test Aims Screen Diagnosis Monitor Prognosis PART 3 Urine chemistry (reagent strip) Glucosuria +++ + + + Proteinuria Hematuria Leukocyturia Infection Wet urinalysis (routine) Diabetes ++++ ++ ++ + Proteinuria Hematuria Leukocyturia Infections Cylindruria Crystalluria Urine microbiology Infections ++ ++++ ++ + Urine cytology (conventional) Cancer + ++ + – Inflammation Viral infections Cytodiagnostic urinalysis Glomerular and renal tubular disorders + ++++ +++ ++ LUT disorders Nonbacterial infections Lithiasis Image cytometry and DNA analysis Urothelial cancer – ++ +++ +++ Flow cytometry Urothelial cancer – + +++ ++ From Schumann GB, Schumann JL, Marcussen N: Cytodiagnostic urinalysis of renal and lower urinary tract disorders, New York, 1995, Igaku-­Shoin Medical Publishers, with permission. LUT, Lower urinary tract; –, negative; +, low positive; ++++, strongly positive. SPECIMEN EVALUATION color to the urine is usually due to an ingested food or medication but may also be a clue to a specific disease state (Aycock & Kass, 2012). For color Before one proceeds with any examination, the urine specimen must be changes of urine in pediatric patients, see Cone (1968). Table 29.3 lists the evaluated in terms of its acceptability. Considerations include proper urine color changes associated with commonly used drugs. labeling, proper specimen for the requested examination, proper pre- Red Urine. The most common abnormal color for urine is red or red-­ servative, visible signs of contamination, and whether any transportation brown. When seen in females, menstrual flow contamination should be delays may have caused significant deterioration (Delanghe & Speeckaert, considered. Hematuria (presence of red blood cells [RBCs]), hemoglobin- 2016; Stankovic & DiLauri, 2008). Each laboratory should have written uria, and myoglobinuria may produce pink, red, or red-­brown coloration. and enforced guidelines for the acceptance or rejection of specimens. All three of these conditions are easily detectable on reagent strip testing; A properly labeled specimen must have the patient’s full name and the however, further evaluation is necessary for absolute differentiation (see date and time of collection. Additional information may be required by the Blood, Hemoglobin, Hemosiderin, and Myoglobin in Urine section the institution, but these three essentials constitute minimum labeling later in the chapter). requirements. In the porphyrias, urine coloration is variable. It is usually red in con- The first voided morning urine, which is the most concentrated, is best genital erythropoietic porphyria and porphyria cutanea tarda; however, in for routine urinalysis. At times, a catheterized specimen or suprapubic col- lead porphyrinuria, the urine color is generally normal. In acute intermit- lected urine specimen is received. If a single specimen is submitted for tent hepatic porphyria, it is normal but darkens on standing. Red urine multiple measurements, bacteriologic examination should be done first also may be associated with the use of drugs and dyes in diagnostic tests— provided that the urine has been properly collected. With pediatric patients for example, phenolsulfonphthalein, which is sometimes used in assessing and persons in acute renal failure, only a small volume of urine may be renal function, will cause a red color in alkaline urine. Patients with an available for processing. In such cases, a notation should be made and the unstable hemoglobin may produce urine with red-­brown color that does measurements most pertinent to the diagnosis should be performed first. not give a positive indication of hemoglobin or bilirubin. The pigment is For quantitative measurements, timed (12-­or 24-­hour) urinary collection probably a dipyrrole or bilifuscin. An innocuous red urine associated with is preferred to random specimens. ingestion of beets is seen in genetically susceptible persons. Yellow-­Brown or Green-­Brown Urine. Yellow-­brown or green-­ GROSS/PHYSICAL EXAMINATION brown urine is generally associated with bile pigments, chiefly bilirubin. On shaking the urine specimen, a yellow foam may be seen, which dis- Appearance tinguishes bilirubin from a normal, dark, concentrated urine, which will Some of the more important changes in the gross appearance of urine are have white foam. In severe obstructive jaundice, the urine may be dark described in this section. A comprehensive list is provided in Table 29.2. green. Orange-­ Red or Orange-­ Brown Urine. Excreted urobilinogen is Color colorless but is converted in the presence of light and low pH to urobilin, The yellow color of urine is due largely to the pigment urochrome, excre- which is dark yellow to orange. Urobilin will not color the foam on shak- tion of which is generally proportional to the metabolic rate. It is increased ing; in this way, it may be confused with a concentrated normal urine. during fever, thyrotoxicosis, and starvation. Small quantities of urobilins Reagent strip testing would be confirmatory in this situation. and uroerythrin (pink pigment) also contribute to urine coloration. In nor- Dark Brown or Black Urine. Acid urine containing hemoglobin will mal individuals, both pale and dark yellow urine can be produced; these darken on standing because of the formation of methemoglobin. “Cola-­ differences are rough indicators of hydration and urine concentration. colored” urine may be seen with rhabdomyolysis (Keverline, 1998) and in Pale urine, typically of low specific gravity, is excreted following high fluid some patients taking l-­dopa. Rarer causes of dark-­brown urine are homo- intake; darker urine is seen when fluids are withheld. Note that pale urine gentisic acid (alkaptonuria) and melanin. Urine-­containing homogentisic of high specific gravity may be found in diabetes mellitus. An abnormal acid will darken more rapidly when alkaline. 469 extern.ir CHAPTER 29 BASIC EXAMINATION OF URINE TABLE 29.2 TABLE 29.3 Appearance and Color of Urine Urine Color Changes with Commonly Used Drugs* Drug Color Appearance Cause Remarks Alcohol, ethyl Pale, diuresis Colorless Very dilute urine Polyuria, diabetes insipidus Anthraquinone laxatives (senna, cascara) Reddish, alkaline; yellow-­ Cloudy Phosphates, carbonates Soluble in dilute acetic brown, acid acid Urates, uric acid Dissolve at 60° C and in Chlorzoxazone (Paraflex; muscle relaxant) Red alkali Deferoxamine mesylate (Desferal; chelates Red Leukocytes Insoluble in dilute acetic iron) acid Ethoxazene (Serenium; urinary analgesic) Orange, red Red blood cells Lyse in dilute acetic acid Fluorescein sodium (given IV) Yellow (“smoky”) Furazolidone (Furoxone, Tricofuron; an Brown Bacteria, yeasts Insoluble in dilute acetic antibacterial, antiprotozoal nitrofuran) acid Spermatozoa Insoluble in dilute acetic Indigo carmine dye (renal function, cys- Blue acid toscopy) Prostatic fluid Iron sorbitol (Jectofer; possibly other iron Brown on standing Mucin, mucous threads May be flocculent compounds forming iron sulfide in urine) Calculi, “gravel” Phosphates, oxalates Levodopa (l-­dopa; for parkinsonism) Red then brown, alkaline Clumps, pus, tissue Mepacrine (Atabrine; antimalarial, intesti- Yellow Fecal contamination Rectovesical fistula nal worms, Giardia) Radiographic dye In acid urine Methocarbamol (Robaxin; muscle relaxant) Green-­brown Milky Many neutrophils Insoluble in dilute acetic (pyuria) acid Methyldopa (Aldomet; antihypertensive) Darkens; if oxidizing agents Fat Nephrosis, crush injury— present, red to brown Lipiduria, opalescent soluble in ether Methylene blue (used to delineate fistulas) Blue, blue-­green Chyluria, milky Lymphatic obstruction— Metronidazole (Flagyl; for Trichomonas Darkening, reddish brown soluble in ether infection, amebiasis, Giardia) Emulsified paraffin Vaginal creams Nitrofurantoin (Furadantin; antibacterial) Brown-­yellow Yellow Acriflavine Green fluorescence Phenazopyridine (Pyridium; urinary an- Orange-­red, acid pH Yellow- Concentrated urine Dehydration, fever algesic), also compounded with sulfon- orange amides (e.g., Azo Gantrisin) Urobilin in excess No yellow foam Phenindione (Hedulin; anticoagulant. Im- Orange, alkaline; color Bilirubin Yellow foam if sufficient portant to distinguish from hematuria) disappears on acidifying bilirubin Phenol poisoning Brown; oxidized to qui- Yellow-green Bilirubin-biliverdin Yellow foam nones (green) Yellow-brown Bilirubin-biliverdin “Beer” brown, yellow foam Phenolphthalein (purgative) Red-­purple, alkaline pH } Red Hemoglobin Positive Phenolsulfonphthalein (also sulfobro- Pink-­red, alkaline pH Reagent strip Erythrocytes Positive mophthalein) for blood Myoglobin Positive Rifampin (Rifadin, Rimactane; tuberculosis Bright orange-­red Porphyrin May be colorless therapy) Fuscin, aniline dye Foods, candy Beets Yellow alkaline, genetic Riboflavin (multivitamins) Bright yellow Menstrual Clots, mucus Sulfasalazine (Azulfidine; for ulcerative Orange-­yellow, alkaline pH contamination colitis) Red-purple Porphyrins May be colorless *Other commonly used drugs have been noted to produce color change once or Red-brown Erythrocytes occasionally: amitriptyline (Elavil)—blue-­green; phenothiazines—red; triamterene Hemoglobin on (Dyrenium)—pale blue (blue fluorescence in acid urine). standing Methemoglobin Acid pH Myoglobin Muscle injury cause green or bluish-­green urine discoloration (Cotten & McCudden, Bilifuscin (dipyrrole) Result of unstable 2011; Meng et al., 2013). hemoglobin Purple Urine. Urine with a bright purple color has been reported in Brown-black Methemoglobin Blood, acid pH association with long-­term urinary catheterization with coexistent urinary Homogentisic acid On standing, alkaline; tract infection (Demelo-­Rodríguez et al., 2016). alkaptonuria Melanin On standing, rare Clarity (Character) Blue-green Indicans Small intestine infections Urine is normally clear; thus, the presence of particulate material in an Pseudomonas infections unspun specimen warrants further investigation. The differential diagnosis Chlorophyll Mouth deodorants for cloudy urine is broad and includes several nonpathologic entities. Tur- bidity may simply be due to the precipitation of crystals or nonpathologic salts referred to as amorphous. Phosphate, ammonium urate, and carbonate can precipitate in alkaline urine; these redissolve when acetic acid is added. Blue, Green, or Blue-­Green Urine. A blue, green, or blue-­green Uric acid and urates cause a white, pink, or orange cloud in acid urine and discoloration of the urine is most commonly due to food dyes or addi- redissolve on warming to 60°C. tives, some foods, or medications, including amitriptyline, doxorubicin, Cloudy urine can be attributed to the presence of various cellular cimetidine, flutamide, indomethacin, methocarbamol, mitoxantrone, elements. Leukocytes may form a white cloud similar to that caused by phenylbutazone, phenergan, propofol, promethazine, triamterene, phosphates, but the cloud remains after acidification. Likewise, bacterial rinsapin, and sildenafil (Aycock & Kass, 2012; Brunzel, 2012; Strasinger growth may cause a uniform opalescence that is not removed by acidifica- & Di Lorenzo, 2014). Rare causes of blue or green urine include Pseu- tion or filtration. It has been suggested that turbidimetric assessment using domonas infection and some inherited diseases, such as Hartnup disease, a double-­beam turbidimeter may be useful for urine infection screening indicanemia, indicanuria, and familial hypercalcemia. In cancer patients (Livsey, 1995). Turbidity may also be due to RBCs, epithelial cells, sper- receiving chemotherapy, the methylene blue included in several dysuria matozoa, or prostatic fluid. Prostatic fluid normally contains a few leuko- medications, including Prosed DS, Trac Tabs, Urised, and Uroblue, may cytes and other formed elements. 470  Miscellaneous causes for cloudy urine include mucus from the lower Pathologic states that result in excess renal fluid loss/urine excretion urinary tract or genital tract, blood clots, menstrual discharge, and other can be divided into three groups. particulate material such as pieces of tissue, small calculi, clumps of pus, Defective Hormonal Regulation of Volume Homeostasis. Dia- and fecal material. Fecal material in urine may occur with a fistulous con- betes insipidus can be due to a deficiency (central/pituitary variety) of, or nection between the colon or rectum and bladder. Contamination with renal unresponsiveness (nephrogenic) to, antidiuretic hormone. In either powders or with antiseptics that become opaque with water (phenols) will situation, excessive thirst and water intake occur, together with marked PART 3 also cause a turbid urine. polyuria and nocturia. Up to 15 L of urine per day may be produced. Chyluria. This is a rare condition in which the urine contains Defective Renal Salt/Water Absorption. This can be due to the lymph. It is associated with obstruction to lymph flow and rupture of administration of diuretic agents or an abnormality of the renal tubules, lymphatic vessels into the renal pelvis, ureters, bladder, or urethra (Gut- resulting in sodium wasting or impairment of the countercurrent mecha- tilla et al., 2017). Although parasitic infection with Wuchereria bancrofti nism. In progressive chronic renal failure, functioning renal tissue is dimin- (filariasis) is the prevailing cause (Cortvriend et al., 1998), abdominal ished and the ability to concentrate urine is gradually lost. To excrete the lymph node enlargement, tumors, scoliosis surgery, and pregnancy have daily renal water and solute load, an increase in urine volume per residual also been associated with chyluria. Even with filariasis, this condition nephron results and the urine eventually becomes isoosmotic with the is rare. plasma ultrafiltrate. The appearance of the urine varies with the amount of lymph present, Osmotic Diuresis. In diabetes mellitus with hyperglycemia, an exces- ranging from clear to opalescent or milky. Clots may form and, if sufficient sive amount of glucose is excreted, causing a solute diuresis. lymph is present, the urine may layer with the chylomicrons on top and fibrin and cells beneath. Chylomicrons may not be apparent microscopi- Decreases in Urine Volume cally unless they have coalesced as microglobules. This fatty material can Oliguria is the excretion of less than 500 mL of urine per 24 hours, and be extracted from urine using an equal volume of ether or chloroform. anuria is the near-­complete suppression of urine formation. Water depri- Urine phosphates, in contradistinction, will not clear with this method. vation will cause a decrease in urine volume even before signs of dehy- Pseudochyluria occurs with the use of paraffin-­based vaginal creams for the dration appear. Oliguria can be rather abrupt in onset, as can acute renal treatment of Candida infection. failure, or it may be due to a chronic progressive renal disease. In either Lipiduria. Fat globules appear in the urine most often with nephrotic case, retention of nitrogenous waste products (azotemia) can occur (see syndrome; these consist of neutral fats (triglycerides) and cholesterol Chapter 15). The causes of acute renal failure are classically categorized (Streather et al., 1993). Lipiduria can also be present in patients who have as follows. sustained skeletal trauma with fractures to major long bones or the pelvis. Prerenal. Loss of intravascular volume may result from hemorrhage Presumably, the source of lipid is exposed fatty marrow. Keep in mind that or from dehydration associated with prolonged diarrhea, vomiting, excess in addition to these endogenous lipids, oily contaminants such as paraffin sweating, or severe burns. So-­called third spacing is the shifting of intravas- may float on the urine surface. Microscopic examination of the urine may cular fluids to extracellular spaces. Additionally, conditions such as conges- be required to classify fatty materials as Oil Red O–positive droplets or tive heart failure, sepsis, anaphylaxis, or renal artery embolic occlusion may cholesterol esters with polarization. result in a decrease in renal blood flow. Postrenal. Bilateral hydronephrosis, resulting from high-­grade or Odor long-­standing obstruction of the urinary tract, may be associated with Urine normally will have a faint, aromatic odor of undetermined source. a marked decrease in urine flow and even anuria. This can occur with Specimens with extensive bacterial overgrowth can be recognized by an prostatic hyperplasia and carcinoma. Bilateral ureteral obstruction due ammoniacal, fetid odor. Additionally, ingestion of asparagus or thymol to stones, clots, and sloughed tissue, and urethral obstruction due to produces distinctive odors in urine. stricture or valves, are other forms of obstruction. The anuria associated Characteristic urine odors associated with amino acid disorders include with sulfonamide therapy and dehydration is due to obstruction caused the following: by the precipitation of crystals in the renal tubules when the urinary pH is acidic. Cystinuria Rotten eggs Renal Parenchymal Disease. This should be considered after other Hawkinsinuria Swimming pool prerenal and postrenal causes of oliguria have been ruled out. The list of Ketoacidosis Sweet, fruity conditions is extensive and includes various vascular disorders, glomeru- Isovaleric acidemia and glutaric acidemia Sweaty feet lonephritis, interstitial nephritis, and ATN. A common cause of ATN is renal ischemia due to heart failure or hypotension. Numerous nephro- Maple syrup urine disease (MSUD) Maple syrup toxic agents may produce ATN, including several antibiotics, mercury, Methionine malabsorption Cabbage, hops cadmium, carbon tetrachloride, and glycerol. Other causes include hemo- Phenylketonuria Mousy, musty globinuria and myoglobinuria, associated with hemolysis and muscle dam- Trimethylaminuria Rotting fish age, respectively, as well as excessive quantities of intratubular proteins or Tyrosinemia Rancid crystals. Chronic renal failure, a progressive and irreversible loss of renal func- Lack of odor in urine from patients with acute renal failure suggests acute tion, results from several disease entities. These include hypertensive and tubular necrosis (ATN) rather than prerenal failure. diabetes-­associated nephrosclerosis, chronic glomerulonephritis, polycys- tic kidney disease, and other urologic disorders. Urinary specific gravity Urine Volume is low and proteinuria, casts, and renal cells may be evident. Pyelonephri- Under ordinary conditions, the main determinant of urine volume is water tis or interstitial nephritis will cause predominantly tubular dysfunction intake. The average adult produces from 600 to 2000 mL of urine per day, with polyuria early in the disease, but later oliguria of chronic renal failure with night urine generally not in excess of 400 mL. In pregnancy, the usual supervenes. diurnal variation may be reversed. Young children, compared with adults, Specific Gravity and Osmolality may excrete about three to four times as much urine per kilogram of body weight. Measurement of urine output during timed intervals may be valu- The volume of excreted urine and the concentrations of its solutes are able in clinical diagnosis. varied by the kidney to maintain the homeostasis of body fluid and elec- trolytes. Specific gravity and osmolality measurements reflect the relative Increases in Urine Volume degree of concentration or dilution of a urine specimen. This, in turn, Production of more than 2000 mL of urine in 24 hours is termed polyuria; aids in evaluating the concentrating and diluting abilities of the kidneys. nocturnal polyuria (nocturia) is excretion of more than 500 mL of urine at Both of these indices, as well as urine color, have been found to be reli- night with a specific gravity of less than 1.018. In general, high volumes of able indicators of hydration status (Armstrong et al., 1998; McKenzie urine tend to result in a low specific gravity. et al., 2017). Excessive intake of water (polydipsia) will result in polyuria, as will con- The specific gravity of a specimen indicates the relative proportions sumption of certain drugs with a diuretic effect, such as caffeine, alcohol, of dissolved solid components to total volume of the specimen; in other thiazides, and other diuretics. Intravenous solutions may increase the urine words, it reflects the density of the specimen. Osmolality, on the other output. Increased salt intake and high-­protein diets will require more water hand, indicates the number of particles of solute per unit of solution. Larger for excretion. Nocturia is common in elderly individuals, with a prevalence particles, such as proteins and sugars, tend to elevate the specific gravity of 80% to 90% by 80 years of age in both sexes (Weiss & Blaivas, 2000). more than smaller electrolytes. In critical circumstances, the measurement 471 extern.ir  of osmolality of urine (and plasma) is preferred to the measurement of Urinometer. This is a hydrometer that is adapted to directly measure CHAPTER 29 BASIC EXAMINATION OF URINE specific gravity (Imran et al., 2010; Voinescu et al., 2002). the specific gravity of urine at room temperature. It should be checked each day by measuring the specific gravity of distilled water. If the uri- Specific Gravity nometer does not give a reading of 1.000, an appropriate correction must Urea (20%), sodium chloride (25%), sulfate, and phosphate contribute be applied to all readings taken with that urinometer. The accuracy of a most of the specific gravity of normal urine. Normal adults with adequate urinometer may be further checked with solutions of known specific grav- fluid intake will produce urine of specific gravity 1.016 to 1.022 over a 24-­ ity. An automated urinometer using a capacitive sensor has been described hour period. However, normal kidneys have the ability to produce urine (Eklund et al., 2015; Slettengren et al., 2019). with a specific gravity that ranges from 1.003 to 1.035. If a random speci- Because temperature influences the specific gravity, urine samples men of urine has a specific gravity of 1.023 or more, the concentrating abil- should be allowed to come to room temperature before a reading is made ity can be considered normal. Minimum specific gravity after a standard or a correction of 0.001 should be made for each 3°C above or below the water load should be less than 1.007. calibration temperature indicated on the urinometer. Corrections must Urines of low specific gravity are called hyposthenuric, with the specific also be made for protein or glucose present; subtract 0.003 for every 1 g/ gravity less than 1.007. In diabetes insipidus, loss of concentrating abil- dL of protein and 0.004 for every 1 g/dL of glucose. ity (as described earlier) results in production of large volumes of urine Procedure. The urinometer vessel is filled three-­fourths full with with a specific gravity as low as 1.001 (specific gravity of water is 1.000). urine (minimum volume required is about 15 mL). The urinometer is Prolonged excretion of urine with low specific gravity can also be seen inserted with a spinning motion to make sure that it is floating freely. with various renal abnormalities, including pyelonephritis and glomeru- (When reading the urinometer, be sure that it is not touching the sides lonephritis. High specific gravity can be seen after excess water loss/dehy- or the bottom of the cylinder. Avoid surface bubbles, which obscure the dration, adrenal insufficiency, hepatic disease, or congestive heart failure. meniscus.) Read the bottom of the meniscus. When little or no variability is noted between several specimens from a Falling Drop Method. This is a direct method for measuring patient and the specific gravity is fixed at about 1.010, this is known as isos- specific gravity. It is more accurate than the refractometer and is thenuria. This finding is indicative of severe renal damage with disruption more precise than the urinometer. This method utilizes a specially of both concentrating and diluting abilities. designed column filled with water-­i mmiscible oil. A measured drop of Methods. Several methods are available to measure specific gravity— urine is introduced into the column; as this drop falls, it encounters reagent strip, refractometer, urinometer, and the falling drop method. two beams of light. Breaking the first beam starts a timer and break- Reagent Strip. This is an indirect method for measuring specific ing the second turns it off. The falling time is measured electroni- gravity. The reagent area has three main ingredients present: polyelec- cally and is expressed as a specific gravity (Marable, 1963). Despite trolyte, indicator substance, and buffer. The principle of this method is its accuracy and use of small specimen volume, this method is not based on the pKa change of pretreated polyelectrolytes in relation to the widely practiced. ionic concentration of the urine. When the ionic concentration is high, the pKa (acid dissociation constant) is decreased, as is the pH. The indica- Osmolality tor substance then changes color relative to ionic concentration, which is The normal adult with a normal fluid intake will produce urine of translated to specific gravity values. The results obtained by this method about 500 to 850 mOsm/kg water. The normal kidney is able to pro- should be used with caution, since it is not affected by high amounts of duce a urine osmolality in the range of 800 to 1400 mOsm/kg water glucose, protein, or radiographic contrast material, all of which tend to in dehydration, and a minimal osmolality of 40 to 80 mOsm/kg water elevate the specific gravity readings obtained from refractometers and uri- during water diuresis. After a period of dehydration, the osmolality of nometers, described in the following sections. The reagent strip assay for the urine should be three to four times that of the plasma (e.g., with a urine pH must be performed carefully to avoid runover from adjacent test plasma osmolality of 285 mOsm/kg water, the urine osmolality should areas, which can cause false readings. be at least 855 mOsm/kg water). The influence of age and gender on Refractometer (see Chapter 4). This is also an indirect method. urine osmolality is poorly documented; however, in one study, males The refractive index of a solution is related to the content of dissolved sol- of all ages had higher urine osmolality and total osmole excretion than ids present. The index is the ratio of the velocity of light in air to the veloc- females (Perinpam, 2016). ity of light in a solution. It varies directly with the proportion of particles Methods. The freezing-­ point depression method is commonly in solution and, therefore, with the specific gravity. employed. A solution containing 1 osmol or 1000 mOsm/kg water The optical analog handheld clinical refractometer is a device that depresses the freezing point 1.86°C below the freezing point of water. For requires only a few drops of urine (unlike the 15 mL of urine necessary methods, see Chapter 4. with the urinometer). Although the refractometer measures the refractive index of a solution, the scale used is valid only for urine and cannot be used to indicate the specific gravity of salt or sugar solutions. This should be CHEMICAL SCREENING kept in mind if salt solutions are to be used for calibration. Special graphs Reagent strips are the primary method used for the chemical examination or tables are required to convert refractive index scale numbers to solute of urine. Although easily used, they represent multiple complex, state-­of-­ concentrations in aqueous solutions if this should be required (Ameri- the-­art chemical reactions (Pugia, 2000). Box 29.1 lists recommendations can Optical Catalog Number 10403). The specific gravity reading on the for both storage and use of reagent strips. Although reading of the strips refractometer is generally slightly lower than a urinometer reading on the has traditionally been done manually, automated instruments, such as the same urine specimen by about 0.002. Digital refractometers are now avail- Bayer Atlas (Siemens Medical Solutions Diagnostics, Tarrytown, NY) and able for human clinical applications. Beckman Coulter Diagnostics iChemVELOCITY (Beckman Coulter, Procedure. A temperature-­ c ompensated hand model is widely Inc., Brea, CA), are now available that will aspirate a precise amount of used. The instrument is temperature compensated between 60°F and urine, deposit it on the dipstick, and read the chemical reactions on the 100°F (15°C to 38°C). It is damaged by heat above 150°F (66°C) and reagent strip by reflectance (Lyon et al., 2003; Penders et al., 2002; Wu, by immersion of the eyepiece and focusing ring in water. It should 2010). These systems provide excellent reproducibility of results and are read zero with distilled water; the zero reading can be reset if neces- not prone to some of the inconsistencies that occur when human hands try sary by breaking the seal over the setscrew, turning it with a small to time the reactions and when human eyes attempt to discriminate differ- screwdriver, and resealing. Check calibration daily. Copper sulfate ent shades of color reactions. solutions can be adjusted to monitor a high specific gravity level as an It should be noted that reagent strip methods are changed periodically, additional check. sensitivities and color reactions altered, and new measurements added. To make a specific gravity determination of urine, clean the surfaces Manufacturers supply tables of common interfering substances, which of the cover and prism with a drop of distilled water and a damp cloth, should be consulted. Interference with ascorbic acid and drugs produc- and allow them to dry. Close the cover. Hold horizontally and apply a ing colored urine, such as phenazopyridine (Pyridium) and other azo com- drop of urine at the notched bottom of the cover so that it flows over pounds as well as methylthioninium chloride (methylene blue), may be the prism surface by capillary action. Point the instrument toward a encountered. light source at an angle that gives optimal contrast. Rotate the eyepiece The chemical measures most commonly found on reagent strips will until the scale is in focus. Read directly on the specific gravity scale the be discussed first, with less commonly measured chemical parameters fol- sharp dividing line between light and dark contrast. The entire pro- lowing. A discussion on the clinical application of each analyte will precede cedure should be repeated with a second drop of urine from the same reagent strip and other methods. Confirmatory methods will be included sample. when available and necessary. 472  BOX 29.1 The urine tends to become less acid following a meal (the so-­called alkaline Recommendations for Reagent Strips tide). This was long believed to be a urinary compensation for gastric acid secretion; however, recent studies do not support this view (Johnson et al., Storage 1995). Sodium bicarbonate, potassium citrate, and acetazolamide may be Protect from moisture and excessive heat. used to induce alkaline urine in the treatment of some calculi, particularly Store in cool, dry area but not in a refrigerator. those composed of uric acid, cystine, or calcium oxalate. These agents may PART 3 Check for discoloration with each use; discoloration may indicate loss of also be used in some urinary tract infections (the antibiotics neomycin, reactivity. kanamycin, and streptomycin are more active in alkaline urine), in sulfon- Do not use discolored strips or tablets. amide therapy, and in the treatment of salicylate poisoning. Keep container tightly stoppered. The capacity to exchange hydrogen ion for cation and the formation Check manufacturer’s directions with each new lot number for changes of ammonia are decreased when tubular function is impaired. In classic in procedure. renal tubular acidosis, glomerular filtration is normal, but the distal tubu- Testing lar ability to form ammonia and exchange hydrogen ions for cations is Test urine as soon as possible after receipt. defective. Systemic acidosis results. The urine is relatively alkaline, and Remove only enough strips for immediate use; recap tightly. the pH cannot be lowered below 6 to 6.5, even with administration of an Test a well-­mixed, unspun urine sample. acid-­loading substance. Additionally, titratable acidity and the concentra- Urine samples must be at room temperature before testing. tion of ammonium are decreased (Singh et al., 1995). In proximal renal Do not touch the test area with fingers. tubular acidosis, bicarbonate wasting occurs. This can also be seen in Fan- Do not use reagent strips in the presence of volatile acids or alkaline fumes. coni syndrome. Dip reagent strip into urine briefly—no longer than 1 second. In metabolic alkalosis, an alkaline urine with higher levels of urinary Drain excess urine off—run edge of strip along rim of tube, or blot edge bicarbonate is produced, and ammonia production is decreased. The kid- on absorbent paper. ney may produce urine with a pH as high as 7.8. In respiratory alkalosis, Do not allow reagents to run together. an alkaline urine is produced that is associated with increased excretion of Do not lay reagent strip directly on workbench surface. bicarbonate. Follow exact timing recommendations for each chemical test. Hold reagent strip close to the color chart and read under good lighting. Methods Know sources of error, sensitivity, and specificity of each test on the Reagent Strip. Indicators methyl red and bromothymol blue give a reagent strip. range of orange, green, and blue colors as the pH rises, permitting estima- Think! Make correlations between patient history and individual test, and tion of pH values to within half a unit within the range of 5 to 9. It should then follow through. be read immediately, but time is not critical. Care should be taken not to have excessively wet strips where acid buffer from the protein patch runs into the pH patch, causing it to become orange. Urine pH Measurement of urine pH and acidity must always be made on freshly The kidneys and lungs normally work in concert to maintain acid-­base voided specimens. If precise measurements are required, the container equilibrium. The lung excretes carbon dioxide, whereas the renal con- should be filled to minimize the amount of dead space, and the urine cov- tribution is that of reclaiming and generating bicarbonate and secreting ered tightly. The container should be kept cold, preferably on ice, but not ammonium ions. The proximal renal tubule is responsible for the bulk of frozen (Cook et al., 2007). On standing, the pH tends to rise because of the bicarbonate reabsorption/generation, and the distal tubule provides the loss of carbon dioxide and because bacterial growth produces ammonia remaining function. from urea. The tubular cells exchange hydrogen ions for sodium of the glomeru- pH Electrode. Although the estimate of the pH obtainable by indica- lar filtrate. The metabolic activity of the body produces nonvolatile acids, tor strip is usually sufficient, more accurate measurement with a pH meter principally sulfuric, phosphoric, and hydrochloric acids, but also small and glass electrode may be indicated in some clinical circumstances, such amounts of pyruvic, lactic, and citric acids and ketone bodies. These are as the diagnosis and treatment of patients with disturbances of acid-­base excreted by the glomerulus as salts (sodium, potassium, calcium, and balance or monitoring urine alkalinization in patients receiving high-­dose ammonium salts) and, together with ammonia produced by the proximal methotrexate therapy or undergoing treatment for nephrolithiasis (Kwong tubules, can then go on to trap secreted hydrogen ions for elimination in et al., 2013; Wockenfus et al., 2013). the urine (see Chapter 15). Because the pH meter may tend to drift, it must be standardized with three buffers of known pH immediately before use. After standardiza- Normal pH tion, spray the electrodes with distilled water, clean, and dry with a tissue. The average adult on a normal diet excretes about 50 to 100 mEq of hydro- Immerse the electrode in the urine sample and report the pH of urine at gen ions in 24 hours to produce urine of about pH 6. In healthy individuals, the temperature of measurement. urine pH may vary from 4.6 to 8. Titratable Acidity of Urine. The pH of the urine is largely depen- dent on the amounts of monobasic and dibasic phosphate present. Titrat- Acid Urine able acidity is measured by titrating an aliquot of 24-­hour urine (collected Acid urine may be produced by a diet high in meat protein and with some on ice) with 0.1 N NaOH, with pH 7.4 as an end point. Measurement may fruits, such as cranberries. During the mild respiratory acidosis of sleep, a be used, together with urinary ammonia determination, in patients with more acid urine may be formed. Also, therapeutic acidification of the urine chronic acidosis of obscure origin. Normal titratable acidity is in the range by various pharmacologic agents—including ammonium chloride, methio- of 200 to 500 mL 0.1 N NaOH (or 6 mL 0.1 N NaOH per kg of body nine, and methenamine mandelate—is used in the treatment of some cal- weight) or 20 to 40 mEq/24 hours. This procedure is described in previous culi. This would include phosphate and calcium carbonate stones, which editions of this textbook. tend to develop in alkaline urine. In acid-­base disturbances, the pH of the urine reflects attempts at Protein in Urine compensation by the kidneys. Patients with metabolic or respiratory Normally, up to 150 mg of protein is excreted in the urine daily, with acidosis should produce acid urine with increased titratable acidity and the average urine protein concentration varying from 2 to 10 mg/dL ammonium ion concentration. In diabetic ketoacidosis, large quantities depending on urine volume. Anderson has demonstrated more than of hydrogen ions are excreted, much as ammonium ion. In potassium 200 urinary proteins, derived from both plasma and the urinary tract depletion, such as in hypokalemic alkalosis of prolonged vomiting or in (Anderson et al., 1979). About one-­third is albumin, and the remaining hypercorticism, or with prolonged use of diuretics, paradoxical aciduria plasma proteins include small globulins, such as α-­, β-­, and γ-­globulins. with slightly acid urine may occur in the presence of a metabolic alkalo- Plasma proteins with a molecular weight less than 50,000 to 60,000 pass sis. Low urine pH is associated with metabolic syndrome, chronic kidney through the glomerular basement membrane and are normally reab- disease, and an increased incidence of nephrolithiasis (Nakanishi et al., sorbed by proximal tubular cells. Albumin (molecular weight 69,000) 2012). is apparently filtered, but only in very small amounts. Retinol binding, β2-­microglobulin, immunoglobulin (Ig) light chains, and lysozyme are Alkaline Urine excreted in small amounts. Tamm-­Horsfall glycoprotein (uromucoid), Alkaline urine may be induced by a diet high in certain fruits and vegeta- secreted by distal tubular cells and cells of the ascending loop of Henle, bles, especially citrus fruits (Welch, Mulligan, Bingham, & Khaw, 2008). constitutes one-­third or more of the total normal protein loss (Devuyst 473 extern.ir  et al., 2017). IgA in secretions of the urinary tract, enzymes, and proteins Proteinuria in Older Adults CHAPTER 29 BASIC EXAMINATION OF URINE from tubular epithelial cells, other desquamated cells, and leukocytes The incidence of significant proteinuria found on urinalysis in the older also contribute to urine protein. adult population is substantially increased when compared with patients Detection of an abnormal amount of protein in urine is an important younger than 60 years of age (Verma et al., 2012). It has been estimated indicator of renal disease because protein has a very low maximal tubular that the older adult population in general has a threefold to fourfold rate of reabsorption; increased filtration of protein quickly saturates the greater incidence of glomerulonephritis, and approximately one-­quarter reabsorptive mechanism. Screening methods are routinely used to differ- of those affected have a minimal change–like disorder that may respond to entiate normal protein excretion from abnormal and, therefore, should not steroid therapy. Occult malignancies in this population may also give rise detect less than about 8 to 10 mg/dL in a normal adult with a normal to membranous glomerulonephritis, with resultant proteinuria (Threatte, rate of urine flow. The reagent strip method is sensitive to albumin while 1986). acid precipitation methods detect all proteins and, therefore, will indicate the presence of globulins as well as albumin. It should be noted that a Proteinuria Quantification very dilute random urine specimen may have a falsely low protein value. More useful information for the diagnosis of kidney disease and for fol- Because a positive result for protein is significant, it should be confirmed lowing the response to treatment is obtained by quantitatively analyz- by a second method and on repeated specimens. Depending on the history ing the amount of protein excreted over a 24-­hour period. It should and examination, confirmatory measurements for elevated protein should be noted that the accuracy of measurements of any quantitative urine be accompanied by the evaluation of renal function, examination of the determination depends on the adequacy and completeness of the timed urine sediment, and urine culture. urine collection. Erroneous results are often related to collection prob- Functional proteinuria is usually less than 0.5 g/day and can be seen lems. Repeat measurements may be needed to decide whether the pro- in various situations in which dehydration contributes to the level of teinuria is intermittent or persistent. Due to the difficulty in collecting protein measured in urine. With strenuous exercise, a mixture of high-­ timed urine specimens, a random untimed (i.e., “spot”) urine specimen and low-­molecular-­weight proteins appears in the urine, and many casts, for the determination of the urine albumin-­to-­creatinine ratio (ACR) or both hyaline and granular, can be seen. Functional proteinuria may also total urinary protein-­to-­creatinine ratio (PCR) is recommended as an accompany congestive heart failure, cold exposure, and fever. In any acceptable alternative specimen by the Laboratory Working Group of event, the proteinuria resolves with appropriate treatment or rest within the National Kidney Disease Education Program and other authorities 2 to 3 days. (Group KDIGOKCW, 2013; Rodby, 2016). To determine the PCR, the Intermittent, transient proteinuria can occasionally be seen in patients urine protein (mg/dL) reading from the dipstick is divided by the urine with a normal history, normal physical examination findings, and other- creatinine (mg/mL) (Ginsberg et al., 1983). The ratio is normally less wise normal renal function. Except for the occasional proteinuria, urinaly- than 0.2 g protein/g creatinine in adults and children over 2 years of age, sis is also normal. These patients are typically followed every 6 months to and less than 0.5 g protein/g creatinine in children under 2 years of age. check for hypertension or other abnormalities, and the overall prognosis is Since the creatinine excretion rate is proportional to the patient’s age, sex, good. A transient proteinuria may also occur in normal pregnancy, but any and weight and can be predicted from the serum creatinine (Cockcroft & proteinuria in pregnancy is an important finding and requires investiga- Gault, 1976), an estimated 24-­hour protein excretion rate can be obtained tion. Persistent proteinuria of 1 to 2 g/day in an asymptomatic person, or by multiplying the PCR by the creatinine excretion rate. The result is when accompanied by hematuria, has a poorer prognosis than intermittent usually normalized for body surface area and expressed as the 24-­hour (transient) or postural proteinuria. protein excretion in g/day per 1.73 m2 body surface area. The ACR is rec- Recent interest has focused on the importance of proteinuria in deter- ommended by some authorities as an alternative to the PCR in untimed mining risk for adverse outcomes in chronic kidney disease (CKD) (Krstic urine specimens, with an ACR greater than 30 mg albumin/g creatinine et al., 2016; Levey et al., 2003; Sarnak & Astor, 2011). Current guidelines considered abnormal (Lamb et al., 2009). for classifying stages of CKD are based on estimated glomerular filtration Heavy Proteinuria (>4 g/day). Heavy protein loss is characteristi- rate; however, heavy proteinuria was found to be independently associ- cally seen with nephrotic syndrome. Classically, a low serum albumin level, ated with a twofold or greater increase in all-­cause mortality, myocardial generalized edema, and increased serum lipids (cholesterol, triglycerides, infarction, and progression to renal failure (Hemmelgarn et al., 2010). A and phosphatides) accompany this disorder. Lipoproteins, low-­density and systematic review and meta-­analysis of 26 cohort studies involving 169,949 very-­low-­density, are increased in serum, whereas high-­density lipopro- individuals showed a strong and continuous association between protein- tein, a smaller molecule, has been demonstrated in the urine (de Men- uria and risk for coronary artery disease, leading those authors to suggest doza et al., 1976). It has been suggested that loss of lipoprotein lipase in the routine incorporation of testing for proteinuria into assessment of car- urine contributes to the rise in serum lipid levels. γ-­Globulin is also lost diovascular risk (Perkovic et al., 2008). in the urine, which may contribute to susceptibility to bacterial infections Hereditary proteinuria syndromes are rare and have heterogeneous commonly found in nephrotic patients. When lipid is lost in urine, many forms, ranging from congenital nephrotic syndrome with severe pro- granular casts, fatty casts, and fat-­filled renal tubular epithelial cells (oval teinuria to focal segmental glomerulosclerosis with moderate proteinuria fat bodies) are found in the sediment. Cholesterol ester droplets may be (Benoit et al., 2010; Caridi et al., 2010). Progression to end-­stage renal demonstrable by polarization. disease is a common outcome. Specific diagnosis is possible with genetic Nephrotic syndrome is principally associated with glomerular dys- testing for mutations in the genes for various structural proteins of the function/damage due to (1) primary renal diseases, including idiopathic glomerulus (Joshi et al., 2013; Machuca et al., 2009; Tryggvason et al., disease, and (2) systemic diseases with renal involvement (Certikova-­ 2006). Chabova & Tesar, 2013). Transient or mechanical causes include severe Postural Proteinuria congestive heart failure, constrictive pericarditis, and renal vein throm- bosis. The last can be a consequence of nephrotic syndrome because of Postural (orthostatic) proteinuria occurs in 3% to 5% of apparently healthy losses of anticlotting factors in urine and elevation of serum fibrinogen. young adults. In this condition, proteinuria is found during the day but not In children, a common cause of nephrotic syndrome is minimal change at night, when a recumbent position is assumed. Persistent proteinuria may disease (also known as nil lesion), a steroid-­responsive glomerular disorder. develop in some of these healthy subjects at a later date; renal biopsies have Acute, rapidly progressive, and chronic types of glomerulonephritis are shown abnormalities of the glomerulus or obstruction of the left renal vein causes of heavy proteinuria and may be accompanied by urinary eryth- in a few cases (Milani et al., 2010; Robinson et al., 1961). Proteinuria is rocytes or erythrocyte casts. Diabetes mellitus and lupus erythematosus apparently related to an exaggerated lordotic position and may result from are systemic diseases that frequently cause glomerular injury and heavy renal congestion or ischemia. The total daily excretion of protein rarely proteinuria. Urine sediment may be “telescoped,” that is, may display exceeds 1 g and, in most instances, no other evidence of renal disease is all types of cells and casts in lupus nephritis or with a hypersensitivity apparent. reaction (Schreiner, 1955). Malaria, malignant hypertension, toxemia of To evaluate the possibility of postural proteinuria, the patient is pregnancy, heavy metals (gold, mercury), drugs (penicillamine), neoplasia instructed to empty the bladder upon going to bed in the evening. Imme- in general, amyloidosis, sickle cell disease, renal transplant rejection, and, diately upon arising in the morning, the patient voids and saves this speci- rarely, primary antiphospholipid syndrome (Levy et al., 1998) are addi- men. After 2 hours of standing and walking about, the patient voids again tional causes of heavy proteinuria. and saves the specimen. The two urine specimens are assessed for protein; Moderate Proteinuria (1.0–4.0 g/day). Moderate protein- if the first is negative and the second positive, the patient may have pos- uria may be found in the vast majority of renal diseases, including those tural proteinuria. Frequent examination of the patient should be made to mentioned previously, as well as nephrosclerosis, multiple myeloma, reevaluate this condition. and toxic nephropathies. Also included are degenerative, malignant, and 474 inflammatory conditions of the lower urinary tract, including irritative Jones proteinuria is associated with multiple myeloma, macroglobulin- conditions such as the presence of calculi. emia, primary (AL) amyloidosis, and malignant lymphomas. The incidence Minimal Proteinuria (3–4 g/day). Loss or reduction of the fixed negative charge these lower urine albumin levels ranging from 20 to 200 mg/L (or an on the glomerular basement membrane allows albumin to permeate into approximate rate of excretion of 20 to 200 μg/min) are an indicator the Bowman space in large quantities, more than can be reabsorbed by the of early and possibly reversible glomerular damage (Mogensen, 1984; proximal tubular cells. When serum albumin is lost in urine, other proteins Viberti et al., 1982). In diabetic patients, microalbuminuria is associ- of similar size or charge are also lost (e.g., antithrombin, transferrin, preal- ated with a fourfold to sixfold increase in cardiovascular mortality and bumin, α1-­acid glycoprotein, α1-­antitrypsin). Because tubular function may is an independent risk factor for renal mortality (Bakris, 1996; Roshan still be normal, very small plasma proteins are largely reabsorbed. Large & Stanton, 2013; Zelmanovitz et al., 1998). It is also more prevalent in proteins, in contradistinction, are not seen in urine while the glomeru- hypertensive subjects and may be an indicator of subclinical renal and lus is still selective (e.g., α2-­macroglobulin, β-­lipoprotein). As larger pro- extrarenal organ damage (Gerber et al., 1998; Viazzi et al., 2016). Vari- teins appear, the proteinuria is less selective, indicating greater damage ous analytic methods have been introduced, including immunologic test to the glomerulus (e.g., with membranous nephropathy and proliferative systems and dye-­binding chemical test strips, both of which are discussed glomerulonephritis). in the next section. Mechanisms of proteinuria in diabetic kidney disease with specific attention to glomerular damage were recently reviewed, with special rec- Methods ognition that CKD is actually a multifactorial, complex disease involving Several screening and quantitative methods are available for the analysis a combination of both glomerular and tubulointerstitial scarring (Baines of protein in urine (Hay-­Lombardie et al., 2018; Viswanathan & Upad- & Brunskill, 2011; Erkan, 2013; Garg & Rabelink, 2011; Han et al., 2017; hyay, 2011). Because a positive screening test may have serious implica- Magee et al., 2017). tions, it is important to be able to confirm results by a second, different Tubular Pattern. This is associated with loss of a small amount of method. Common screening tests include the qualitative/semiquantita- urinary protein that would otherwise be largely reabsorbed. These pro- tive colorimetric reagent strip test and precipitation-­based testing (Table teins most often are of low molecular weight (e.g., α1-­microglobulin, 29.4). N-­acetyl-­β-­D-­glucosaminidase [NAG], β-­ globulins such as β2-­ Accurate results are obtained with reagent strips only when albu- microglobulin, light-­ chain immunoglobulins, cystatin C, and lyso- min is increased (White et al., 2011). Because of the lack of sensitivity zyme), usually without a clear predilection for albumin-­sized molecules. of the reagent strip to globulins, it may be necessary to use an acid By radioimmunoassay, β2-­microglobulin excretion has been measured precipitation method for screening purposes. This will depend on the in microgram amounts in urine as an indication of tubular damage; its patient population and the diseases being screened. Reagent strips do normal excretion is about 100 μg/day. A tubular pattern proteinuria have the advantage of avoiding false-­positive reactions with organic occurs with renal tubular diseases such as Fanconi syndrome, cystino- iodides, such as those used for radiographic contrast, and tolbutamides sis, Wilson disease, and pyelonephritis, and with renal transplantation or other drugs. rejection. The amount of proteinuria is typically lower than that seen Most other qualitative screening methods rely on protein precipita- with glomerular disease, at about 1 to 2 g/day. Tubular proteinuria may tion (e.g., with heat and acetic acid, with nitric acid, with sulfosalicylic acid be missed by the reagent strip test because of the absence or very low [SSA] and trichloroacetic acid). These methods will precipitate globulins amounts of albumin, but it may be detected by an acid precipitation as well as albumin. In practice, negative reagent strips with positive SSA method (Skoberne et al., 2017). In addition, urinary tubular indexes methods in urine specimens are attributable to radiographic dye, to peni- comparing tubular proteins to albumin, particularly the ratios of α-­1-­m cillins, and, rarely, to an isolated increase in globulins. Sulfosalicylic and to albumin and NAG to albumin, reportedly have a high sensitivity and trichloroacetic acids are used to precipitate protein in the cold and are used specificity in the differentiation between primary tubulointerstitial dis- as a convenient screening method. The sensitivity may be as low as 0.25 eases and primary glomerular diseases (Skoberne et al., 2017). There mg/dL depending on the technique used. is also interest in the utilization of specific molecules released into the With the intense interest in utilizing proteinuria as a risk strati- urine during tubular injury as monitors of renal insufficiency in patients fier for both diabetic and nondiabetic nephropathy, as well as other with diabetic nephropathy. The biomarkers under study include neutrophil-­gelatinase–associated lipocalin (NGAL), kidney injury mol- TABLE 29.4 ecule 1 (KIM-­1), liver–fatty-­acid–binding protein (L-­FABP), calprotec- tin, inflammatory cytokines, and others (Azimi, 2017; Kim et al., 2018; Screening Test for Detection of Proteinuria Lowenstein & Grantham, 2016; Tramonti & Kanwar, 2013; Tummala- Urine Constituents or palli et al., 2016). Condition Reagent Strip Acid Precipitation Overflow Proteinuria. Overflow proteinuria is due to the overflow of excess levels of a protein in the circulation, and can be seen with hemo- Highly buffered alkaline May cause FP May cause FN globin, myoglobin, or immunoglobulin loss into the urine. These pro- urine teins are not initially associated with glomerular or tubular diseases but Drug metabolites No effect May cause FP may themselves cause renal damage. Myoglobin may cause ATN (see the Radiocontrast media No effect May cause FP Blood, Hemoglobin, Hemosiderin, and Myoglobin in Urine section, and Turbidity No effect May cause FP case 4 in Chapter 9). Hemoglobin in low amounts is not thought to be toxic Quaternary ammonium May cause FP No effect unless hypovolemia is present. groups or chlorhexidine Bence Jones Proteinuria. Bence Jones protein is the light chain from a monoclonal immunoglobulin that is filtered in the kidney. Bence FN, False negative; FP, false positive. 475 extern.ir  conditions such as preeclampsia and coronary artery disease, recom- blank is used. For a comparison of biuret methods with the SSA turbidity CHAPTER 29 BASIC EXAMINATION OF URINE mendations for measurement have centered on methods for urine albu- method, see Lizana and colleagues (Lizana et al., 1977). min quantification rather than total protein (Benz-­de Bretagne et al., Several dye-­binding colorimetric methods are available to quantitate 2018; Lamb et al., 2009; Lamb et al., 2013). Urine albumin measure- urine protein. These include Coomassie blue, Ponceau S, and benzetho- ments are considered much more standardized and reliable than total nium chloride turbidity methods (McElderry et al., 1982). Pyrogallol Red-­ protein at low concentrations, where assessment of risk for progres- Molybdate will also react with protein to form a blueish-­purple complex sion in chronic kidney disease is important for diagnosis and planning that absorbs at 600 nm. therapy. Methods used to quantitate urinary protein have not been satisfactory. Reagent Strip. This method takes advantage of the protein error of Participants in the College of American Pathologists proficiency testing pH indicators. Because proteins carry a charge at physiologic pH, their surveys will be aware that the mean values reported vary twofold between presence will elicit a pH change. The reagent strip is impregnated with methods, with the SSA method producing high values. Precision is poor, tetrabromophenol blue buffered to an acid pH of 3, or tetrachlorophenol-­ with the SSA turbidimetric method showing the poorest coefficient of tetrabromosulfophthalein. In the absence of protein, the strip is yellow; 30 variation. The TCA-­biuret, Coomassie blue, and TCA turbidity methods to 60 seconds following urine application, variable shades of green develop show closer agreement and about half the coefficient of variation of the depending on the type and concentration of protein present. Results may SSA method. Problems arise from nonstandardized methods. With turbid- be read in a “plus” system as negative, trace, and 1+ to 4+. Most methods ity methods, these include different acid concentrations and timing, along will detect 5 to 20 mg of albumin per deciliter. with variation in the protein standard. As stated previously, reagent strips tend to be more sensitive to Microalbuminuria Determination Methods. Very small albumin than to globulins, Bence Jones protein, or mucoprotein. amounts of proteins, such as albumin and β2-­microglobulin, are “Trace” results may be seen with physiologic normal excretion of pro- measured by immunologic means using antibodies to the proteins, tein in concentrated urine specimens from healthy individuals. High nephelometric methods, immunoassay, protein strip electrophoresis, salt levels will lower results. Exceptionally alkaline and/or highly buff- high-­performance liquid chromatography (HPLC), or other means ered urine samples may give positive results in the absence of signifi- (Benz-­de Bretagne et al., 2018). The Micral II test strip (Boehringer cant proteinuria (e.g., with a patient on alkaline medication or with Mannheim, Indianapolis, IN) is an immunologic test system that bacterial contamination). False-­positive results can occur with highly gives an almost immediate, reliable semiquantitative determination pigmented urine, quaternary ammonium compounds, amidoamines of low urine albumin concentrations (Kutter, 1998). Oxytetracycline in fabric softeners, chlorhexidine, and excessive leaching of the acid may interfere with this method, causing higher readings. There is buffer of the test strip by excessive wetting. The method is unaf- no interference with pH. A newer method, Clinitek microalbumin fected by urine turbidity, radiographic media, and most drugs or their (Bayer Diagnostics, Tarrytown, NY), is a highly sensitive dye-­binding metabolites. method (Graziani et al., 2009; Guy et al., 2009). It has the further Sulfosalicylic Acid Method—Qualitative. This method advantage of an additional test pad for simultaneous measurement depends on formation of a precipitate for determination of the presence of creatinine concentration. This method is not absolutely specific of protein. for albumin, for the dy