ANALYSIS OF URINE AND OTHER BODY FLUIDS MACROSCOPIC AND CHEMICAL EXAMINATION OF URINE OUTLINE • Introduction • Physical Examination of Urine o Urine color ▪ Abnormal Urine ▪ Laboratory Correlation of Urine o Clarity o Specific gravity • Chemical Examination of Urine o Protein o Glucose o Ketones o Blood o Bilirubin o Urobilinogen o Nitrite o Leukocyte • Interpretation of the Urinalysis • INTRODUCTION • Urine analysis is the oldest clinical laboratory test that is still being performed today. In old times, only the physical characteristics of urine were tested - color, clarity, odor, and taste. Although as of recent, taste is no longer being tested due to the availability of chemical tests that would serve the same purpose as evaluating taste. • Apart from evaluating the physical characteristics of urine samples, chemical and microscopic testing is now part of the urinalysis. • Chemical testing traditionally involved only testing for pH, specific gravity, glucose, and protein (4-parameter testing) but has now been expanded to include testing for hemoglobin, bilirubin, urobilinogen, ketones, leukocyte esterase, and nitrite (10-parameter). Recently, ascorbic acid has been added (11-parameter) because of the interferences that may be caused by the presence of ascorbic acid in urine. PHYSICAL EXAMINATION OF URINE COLOR • varies from almost colorless to black • due to: o normal metabolic functions o physical activity, o ingested materials o pathologic conditions • Normal urine color : pale yellow, yellow, and dark yellow • Pigments o Urochrome - product of endogenous metabolism o Uroerythrin o Urobilin ABNORMAL URINE • Dark Yellow/ Amber/ Orange o May not always signify a normal conc. Urine o Can be caused by bilirubin o + Bilirubin: yellow foam appears when specimen is shaken; may also contain hepatitis virus o Medication of phenazopyridine • Brown/Black o (-) Blood o May contain homogentisic acid or melanoma o Medication: levodopa, methyldopa, phenol derivatives, and metronidazole (Flagyl) • Red/Pink/ Brown o Presence of Blood o Hemoglobin o Myoglobin o Menstrual contamination (should also be reported) o Medications: rifampin, phenolphthalein, phenindione, and phenothiazines o Tea colored - caused by infection of S. pyogenes • Blue/Green o Pathogenic cause: UTI caused by Psuedomonas spp. o Intestinal infections resulting in increased urinary indicant o Ingestion of breath deodorizers (Clorets) = GREEN o Medication: methocarbamol (Robaxin), methylene blue, and amitriptyline (Elavil) = BLUE o Bacterial infection caused by Klebsiella or Providencia spp = PURPLE • Describe urine color with color only, do not describe using objects LABORATORY CORRELATION OF URINE COLOR (BOOK) Color Cause Clinical/Laboratory Correlations Colorless Recent fluid consumption Commonly observed with random specimens Pale yellow Polyuria or diabetes insipidus Increased 24-hour volume and low specific gravity Diabetes mellitus Elevated specific gravity and positive glucose test resul Dilute random specimen Recent fluid consumption Dark Yellow Concentrated specimen May be normal after strenuous exercise or in first morning specimen B complex vitamins Dehydration Fever or burns Bilirubin Yellow foam when shaken and positive chemical test results for bilirubin Acriflavine Negative bile test results and possible green fluorescence Nitrofurantoin Drug commonly administered for urinary tract infections Orange-yellow Phenazopyridine (Pyridium) Drug commonly administered for urinary tract infections Phenindione Anticoagulant, orange in alkaline urine, colorless in acid urine Yellow-green Bilirubin oxidized to biliverdin Colored foam in acidic urine and false-negative chemical test results for bilirubin Green Pseudomonas infection Positive urine culture Blue-green Amitriptyline Antidepressant Methocarbamol (Robaxin) Muscle relaxant, may be green-brown Clorets None Indican Bacterial infections, intestinal disorders Methylene blue Fistulas Phenol When oxidized Pink Red RBCs Cloudy urine with positive chemical test results for blood and RBCs visible microscopically Hemoglobin Clear urine with positive chemical test results for
Color Cause Clinical/Laboratory Correlations blood; intravascular hemolysis Myoglobin Clear urine with positive chemical test results for blood; muscle damage Beets Alkaline urine of genetically susceptible persons Rifampin Tuberculosis medication Menstrual contamination Cloudy specimen with RBCs, mucus, and clots Port wine Porphyrins Negative test for blood, may require additional testing Red-brown RBCs oxidized to methemoglobin Seen in acidic urine after standing; positive chemical test result for blood Myoglobin Brown Homogentisic acid (alkaptonuria) Seen in alkaline urine after standing; specific tests are available Black Malignant melanoma Urine darkens on standing and reacts with nitroprusside and ferric chloride Melanin or melanogen Phenol derivatives Interfere with copper reduction tests Argyrol (antiseptic) Color disappears with ferric chloride Methyldopa or levodopa Antihypertensive Metronidazole (Flagyl) Darkens on standing, intestinal and vaginal infections CLARITY • Refers to TRANSPARENCY or TURBIDITY • Take note: color and clarity is done at the same time o Clear - readable text • Turbidity/cloudiness is related to amount of urine content • Urine Clarity o Clear - No visible particulates, transparent o Hazy - Few particulates, print easily seen through urine o Cloudy - Many particulates, print blurred through urine o Turbid - Print cannot be seen through urine o Milky - May precipitate or be clotted • Non-pathologic Cause of Turbidity o Squamous epithelial cells (usually in females) o Mucus (mucus threads) o Amorphous phosphates, carbonates, urates o Semen, spermatozoa o Fecal contamination o Radiographic contrast media o Talcum powder o Vaginal creams • Pathogenic Cause of Turbidity o RBCs - bleeding ▪ Fresh blood - bleeding in ureter, urethra ▪ Ghost cells - bleeding in nephron/kidney o WBCs - UTI o Bacteria - urethral contamination during collection o Yeast (fungi) - diabetes/poor hygiene ▪ C. albicans - usual specie of yeast o Nonsquamous epithelial cells (renal epithelial) o Abnormal crystals - liver cirrhosis/cancer o Lymph fluid o Lipids - excessive fats ▪ From cholesterol crystals, fat droplets SPECIFIC GRAVITY • Density of a solution compared with the density of a similar volume of distilled water (sg 1.000) at a similar temperature. • Plasma filtrate sp.gr : o Isosthenuric - 1.010 o Hypoisthenuric - below 1.010 o Hypersthenuric - above 1.010 • Normal random specimen : 1.002 to 1.035 • Most random specimen : 1.015 to 1.030 • Higher sp. gr. = more dense • Afternoon urine - expect 1.010 or less • Dipstick - 1.000 to 1.030 • Normal Values : depend on the patient’s degree of hydration • Methods o Urinometry ▪ Obsolete method ▪ Urinometer/Hydrometer - weighted float that is designed to sink to a level of 1.000 in distilled water; calibrated at 20°C; less accurate than other methods; requires large volume of urine ▪ Corrections • Temperature – for every 3°C that the urine temperature is above or below the calibration temperature, 0.001 is respectively added to or subtracted from the reading • Protein – subtract 0.003 for every g/dL; • Glucose – subtract 0.004 for every g/Dl o Refractometry ▪ Determines concentration of dissolved particles in a specimen by measuring refractive index ▪ Accurate measure for sp. gr. ▪ Refractometer/ TS meter - measures refractive index; compensated between 15°C and 38°C ▪ Corrections : protein and glucose only; temperature correction not done o Harmonic Oscillation Densitometry ▪ Mass gravity meter - used by Yellow IRIS automated workstations to measure sp. gr. ▪ Principle : Sound waves of specific frequency are generated at one end of the tube and as the sound waves oscillate through urine, their frequency is altered by the density of the specimen.
CHEMICAL EXAMINATION OF URINE URINE pH • pH Reagent Strip o Reagents : Methyl red, bromthymol blue o Sensitivity : Multistix : 5.0 to 8.5 in 0.5 increments Chemstrip : 5.0 to 9.0 in 1.0 increments o Interference : ▪ No known interfering substances ▪ Run-over from adjacent pads ▪ Old specimens o Reactions : Methyl red + H+ → bromthymol blue - H+ (Red-orange → yellow) (green → blue) • Causes of Acid Urine o Emphysema o Diabetes mellitus o Starvation o Dehydration, Diarrhea o Presence of acid-producing bacteria ( Escherichia coli ) o High-protein diet o Cranberry juice o Medications (methenamine mandelate [Mandelamine], fosfomycin tromethamine [Monurol]) • Causes of Alkaline Urine o Hyperventilation o Vomiting o Renal tubular acidosis o Presence of urease producing bacteria o Vegetarian diet o Old specimens • Clinical Significance o Respiratory or metabolic acidosis/ketosis o Respiratory or metabolic alkalosis o Defects in renal tubular secretion and reabsorption of acids and bases —renal tubular acidosis o Renal calculi formation and prevention o Treatment of urinary tract infections o Precipitation/identification of crystals o Determination of unsatisfactory specimens PROTEIN • Normal : <10 mg/dl or 100mg/24hrs(150mg/24hrs) • Albumin – major serum protein found in urine • Proteins found in urine : albumin, serum and tubular microglobulins, Tamm-Horsfall Protein, and Protein from prostatic, seminal and vaginal secretions • Principle : Protein error of indicators • Reagents : o Multistix : Tetrabromphenol blue o Chemstrip : 3’3”5’5” tetrachlorophenol 3,4,5,6- tetrabromosulfophthalein • Sensitivity : Multistix : 15-30 mg/dl albumin Chemstrip : 6 mg/dl albumin • Sources of error o False positive ▪ Highly buffered alkaline urine ▪ Pigmented specimens, phenazopyridine ▪ Quaternary ammonium compounds ▪ Antiseptics, chlorhexidine ▪ High specific gravity ▪ Loss of buffer from prolonged exposure of the reagent strip to the specimen. o False negative ▪ Proteins other than albumin ▪ Microalbuminuria • 3 major categories of proteinuria o Pre-Renal Proteinuria – caused by increased levels of low molecular-weight plasma proteins such as hemoglobin, myoglobin, and the acute phase reactants. ▪ Bence-Jones Protein -abnormal protein; monoclonal immunoglobulin light chains. • precipitates at 40- 60⁰C and disappears at 90- 100⁰C and precipitates again upon cooling. ▪ Multiple Myeloma – proliferative disorder of the immunoglobulin – producing plasma cells producing high levels of BJP in serum. o Renal Proteinuria – proteinuria associated with true renal disease may be the result of either glomerular or tubular damage. ▪ Glomerular Proteinuria – Increased pressure from the blood entering the glomerulus may override the selective filtration of the glomerulus, causing increased albumin to enter the filtrate. ▪ Tubular Proteinuria - Increased albumin is also present in disorders affecting tubular reabsorption because the normally filtered albumin can no longer be reabsorbed. ▪ Orthostatic (postural) Proteinuria – a persistent benign proteinuria occurs frequently in young adults; occurs following periods spent in a vertical posture and disappears when a horizontal position is assumed. ▪ Microalbuminuria – associated with an increased risk of cardiovascular disease; can be determined by Micral Test o Postrenal Proteinuria – Protein can be added to a urine specimen as it passes through the structures of the lower urinary tracts. • Clinical Significance of Urine Protein o Prerenal – Intravascular hemolysis, Muscle injury, Acute phase reactants, Multiple myeloma o Renal – Glomerular disorder, Immune complex disorders, Amyloidosis, Toxic agents, Diabetic Nephropathy, Strenuous Exercise, Dehydration, Hypertension, Preeclampsia, orthosthatic or postural proteinuria o Tubular Disorder – Fanconi syndrome, Toxic Agents/heavy metals, Severe viral infections o Postrenal – Lower UTI/inflammation, Injury/trauma, Menstrual Contamination, Prostatic Fluid/ Spermatozoa, Vaginal Secretions • Testing for Microalbuminuria o Micral Test – Contain a gold labeled antihuman albumin antibody-enzyme conjugate. o Immunodip Reagent Strip - uses immunochromographic technique o Sulfosalicylic Acid Precipitation Test – most proteins are precipitated by dilute SSA. • Reporting Sulfosalicylic Turbidity GRADE TURBIDITY PROTEIN RANGE (mg/dl) Negative No increase in turbidity <6 Trace Noticeable turbidity 6-30 1+ Distinct turbidity with no granulation 30-100
GRADE TURBIDITY PROTEIN RANGE (mg/dl) 2+ Turbidity with granulation with no flocculation 100-200 3+ Turbidity with granulation and flocculation 200-400 4+ Clumps of protein >400 GLUCOSE • Renal Threshold : 160 – 180 mg/dl • Other sugar in urine : fructose, galactose, lactose, pentose • Principle : Double sequential enzyme reaction • Reagents : o Multistix : Glucose oxidase, peroxidase, Potassium iodide o Chemstrip : Glucose oxidase, Peroxidase, Tetramethylbenzidine • Sensitivity : Multistix : 75 – 125 mg/dl Chemstrip : 40 mg/dl • Interference o False positive ▪ Contamination by oxidizing agents and detergents o False negative ▪ High levels of ascorbic acid ▪ High levels of ketones ▪ High specific gravity ▪ Low temperatures ▪ Improperly preserved specimens • Test for Glucose o Benedict’s test – general test for glucose and other reducing sugars. ▪ relies on the ability of the glucose and other reducing substances to reduce copper sulfate to cuprous oxide in the presence of alkali and heat. o Copper Reduction Method – Clinitest Tablet o Glucose Oxidase/Reagent Strip – Specific test for glucose than benedict’s/clinitest • Clinical Significance of Urine Glucose , o Hyperglycemia Associated ▪ Diabetes Mellitus ▪ Pancreatic cancer ▪ Cushing Syndrome ▪ Pheochromocytoma ▪ Central Nervous System damage ▪ Gestational diabetes ▪ Pancreatitis ▪ Acromegaly ▪ Hyperthyroidism ▪ Stress o Renal Associated ▪ Fanconi’s Syndrome ▪ Advanced renal disease ▪ Pregnancy ▪ Osteomalacia KETONES • Result from increased fat metabolism o 78% betahyroxybutyric acid o 20% acetoacetic acid o 2% Acetone • Principle : Sodium Nitroprusside Reaction • Reagents : Sodium Nitroprusside Glycine (chemstrip) • Sensitivity : o Multistix : 5-10 mg/dl acetoacetic acid o Chemstrip : 9mg/dl acetoacetic acid; 70mg/dl acetone • Interference o False Positive ▪ Phthalein dyes ▪ Highly pigmented red urine ▪ Levodopa ▪ Medications containing free sulfhydryl groups o False Negative ▪ Improperly preserved specimens • Clinical Significance of Urine Ketones o Diabetic acidosis o Insulin dosage monitoring o Starvation o Malabsorption/ Pancreatic disorder o Strenuous Exercise o Vomiting o Inborn errors of amino acid metabolism BLOOD • Principle : Pseudoperoxidase activity of hemoglobin tetramethybenzidine (chromogen) • Reagents : o Multistix : Diisopropylbenzene dehydroperoxide tetramethybenzidine o Chemstrip : dimethyldihydroperoxyhexane tetramethybenzidine • Sensitivity : o Multistix : 5-20 RBCs/ml, 0.015-0.062mg/dl Hgb o Chemstrip : 5 RBCs/ml, Hgb corresponding to 10 RBCs/ml • Interference o False Positive ▪ Strong oxidizing agent ▪ Bacterial peroxidases ▪ Menstrual contamination o False Negative ▪ High specific gravity/ crenated cells ▪ Formalin ▪ Captopril ▪ High concentrations of nitrite ▪ Ascorbic Acid >25 mg/dl ▪ Unmixed specimens • Hematuria – macroscopic cloudy red urine o Signifies bleeding which is of renal or genitourinary origin which may be due to trauma or damage to the organ systems • Hemoglobinuria – macroscopic clear red urine o Result from lysis of rbc produced in urinary tract particularly in dilute alkaline o Removed from the circulation through the binding of haptoglobin with hemoglobin o May result from intravascular hemolysis; no RBCs • Myoglobinuria – presence in the urine of a heme- containing protein found in muscle tissue. o Reacts positively with the reagent strip test for blood o Not bound to haptoglobin in plasma and quickly excreted in the kidneys. o Increase creatinine kinase and lactic dehydrogenase enzyme with clear plasma.
• Clinical Significance of a Positive Reaction for blood o Hematuria – Renal calculi, Glomerulonephritis, Pyelonephritis, Tumors, Trauma, Exposure to toxic chemicals, Anticoagulants, Strenuous Exercise o Hemoglobinuria – Transfusion reaction, Hemolytic anemias, Severe burns, Infections/malaria, Strenuous exercise/RBC trauma, Brown recluse spider bites o Myoglobinuria – Muscular trauma/ crush syndrome, prolonged coma, convulsion, muscle-wasting disease, alcoholism/overdose, drug abuse, Extensive exertion, cholesterol-lowering statin BILIRUBIN • Highly pigmented yellow compound which is a degradation product of hemoglobin - Used as a screen for abnormal hepatobiliary function • Principle : Diazo reaction • Reagent : o Multistix : 2,4- dichloroaniline diazonium salt o Chemstrip : 2,6- dichlorobenzene-diazonium salt • Sensitivity : Multistix : 0.4-0.8 mg/dl bilirubin Chemstrip : 0.5 mg/dl bilirubin • Interference o False positive ▪ Highly pigmented urines, phenazopyridine ▪ Indican (intestinal disorders) ▪ Metabolites of Lodine o False negative ▪ Specimen exposure to light ▪ Ascorbic acid >25 mg/dl ▪ High concentrations of nitrite • Production of Bilirubin • Test o Foam-Shake Test o Oxidation Test ▪ Gmelin’s test ▪ Fouchet test ▪ Harrison spot test o Diazotozation test ▪ Ictotest Tablet • Clinical Significance o Hepatitis o Cirrhosis o Biliary Obstruction o Other liver disorder UROBILINOGEN • bile pigment that result from hemoglobin degradation • small amount of urobilinogen (<1mg/dl or 1 Ehrlich unit) is normally found in the urine. • Reagent : o Multistix : pdimethylaminobenzaldehyde o Chemstrip : 4- methoxybenzenediazonium- tetrafluoroborate • Sensitivity : o Multistix : 0.2 mg/dl o Chemstrip : 0.4 mg/dl • Interference o False Positive ▪ Multistix • Porphobilinogen • P-aminosalicylic acid • Sulfonamides • Methyldopa • Procaine • Chlorpromazine • Highly pigmented urine ▪ Chemstrip • Highly pigmented urine o False Negative ▪ Multistix • Old specimen • Preservation in formalin ▪ Chemstrip • Old specimen • Preservation in formalin • High concentration of nitrate • Clinical Significance o Early detection of liver disease o Liver disorders, hepatitis, cirrhosis, carcinoma o Hemolytic disorders • Watson Schwartz Differentiation Test - For diffentiating urobilinogen and porphobilinogen (contaminant) • Hoesch Test – rapid screening test for urine porphobilinogen (≥2mg/dl) o Hoesch rgt (Ehrlich rgt dissolved in 6M HCl) • Urine Bilirubin and Urobilinogen in Jaundice Urine Bilirubin Urine Urobilinogen Hemolytic disease Negative +++ Liver damage + or - ++ Bile duct obstruction +++ Normal • Watson-Schwartz Test Interpretation Urobilinogen Porpho- bilinogen Other Ehrlich – Reactive Substances Chloroform Extraction Urine (Top Layer) Colorless Red Red Chloroform (bottom layer) Red Colorless Colorless Butanol Extraction Butanol (Top Layer) Red Colorless Red Urine (Bottom Layer) Colorless Red Colorless
NITRITE • Provides rapid screening test for the presence of UTI or assymptomatic bacteruria • Principle : Greiss Reaction • Reagents : o Multistix : p-arsanilic acid tetrahydrobenzo(h)- quinolin-3-ol o Chemstrip : Sulfanilamide, hydroxytetrahydro benzoquinoline • Sensitivity : o Multistix : 0.06-0.1mg/dl nitrite ion o Chemstrip : 0.05 mg/dl nitrite ion • Interference o False positive ▪ Improperly preserved specimen ▪ Highly pigmented urine o False negative ▪ Non reductase containing bacteria ▪ Insufficient contact time between bacteria and urinary nitrate ▪ Lack of urinary nitrate ▪ Large quantities of bacteria converting nitrite to nitrogen ▪ Presence of antibiotics ▪ High concentrations of ascorbic acid ▪ High specific gravity • Factors influencing the reliability of the nitrite test o Bacteria that lack the enzyme reductase can’t reduce nitrate to nitrite. o Bacteria capable of reducing nitrate must remain in contact with the urinary nitrate long enough to produce nitrite o Further reduction of nitrite to nitrogen when large numbers of bacteria are present and may cause false negative o Reliability depends on the presence of adequate amounts of nitrate in the urine • Clinical Significance o Cystitis o Pyelonephritis o Evaluation of antibiotic therapy o Monitoring of patients at high risk for UTI ▪ P. aeruginosa - cause nitrate to nitrite, uses different antibiotic o Screening of urine culture specimens LEUKOCYTE ESTERASE • Presence in urine implies that an inflammatory process is occurring in the kidney or urinary tract • Principle : Leukocyte Esterase • Reagents : o Multistix : Derivatized pyrrole amino acid ester, Diazonium salt o Chemstrip : Indoxylcarbonic acid ester, Diazonium salt • Sensitivity : o Multistix : 5-15 WBC/hpf o Chemstrip : 10-25 WBC/hpf • Interference o False positive ▪ Strong oxidizing agent ▪ Formalin ▪ Highly pigmented urine nitrofurantoin o False negative ▪ High concentrations of protein, glucose, oxalic acid, ascorbic acid, gentamicin, cephalosporins, tetracyclines, inaccurate timing • Clinical Significance o Bacterial and nonbacterial urinary tract infection o Inflammation of the urinary tract o Screening of urine culture specimens SUMMARY OF REAGENT STRIP TESTING Reagent Strip Test Principle Reagents Read Time Color reaction Specific gravity pKa change of polyelectrolytes M: Poly(methylvinyl ether) maleic anhydride BTB C: Ethyleneglycol-bis(aminoethylether) BTB 45s Blue-Green- Yellow pH Double indicator system Methyl red, Bromthymol Blue 60s Red-Yellow- Blue Protein Protein error of indicator M: Tetrabromphenol blue C: Tetrachlorophenol tetrabromosulfonphthalein 60s Blue-green Glucose Glucose oxidase reaction M: Glucose oxidase, peroxidase, KI C: Glucose oxidase, peroxidase, TMB 30s Green- Brown or Yellow- Green Ketones Sodium nitroprusside reaction M: Sodium nitroprusside C: Sodium nitroprusside and glycine 40s Purple Blood Pseudo-peroxidase activity of heme M: Diisoproplbenzene dihydroperoxide TMB C: 2,5-dimethyl-2,5-dihydroperoxyhexane, TMB 60s Blue-green Bilirubin Diazo reaction M: 2,4-dichloroaniline diazonium salt C:2,6-dichlorobenzene diazoniumtetrafluoroborate 30s Tan, pink, or violet Urobilinogen Ehrlich reaction M: paraethylaminobenzaldehyde C: 4-methoxybenzene diazonium tetrafluoroborate 60s cherry red Nitrite Greiss reaction M: p-arsanilic acid tetrahydrobenzoquinolinol C: sulfanilamide, 3-hydroxy-1,2,3,4-tetrahydro-7,8-benzoquinoline 60s Pink Leukocyte esterase Granulocytic esterase reaction M: derivatized pyerole amino acid ester diazonium salt C: indoxycarbonic acid ester, diazonium salt 2m Pink