URINALYSI SBy Roderick D. Balce, RMT ANATOMY OF THE KIDNEY RENAL FUNCTIONS 1. Renal Blood Flow Afferent arterioles Efferent arterioles Peritubular capillaries Proximal convoluted tubule Distal convoluted tubule Vasa recta Ascending loop of Henle Descending loop of Henle 2. Glomerular Filtration Glomerulus GFR = 120 ml/min Factors Affecting Glomerular Filtration a. Cellular structure Capillary Wall Membrane Basement Membrane Visceral Epithelium b. Hydrostatic pressure Blood Colloidal Oncotic Pressure Capsular Pressure c. Renin-Angiotensin-Aldosterone Mechanism 3. Tubular Reabsorption Mechanisms involved: a. Active Transport Actively transported: Glucose, Amino acids Salts Chloride Sodium Renal threshold –important in distinguishing between: Excess solute filtration Renal tubular damage b. Passive Transport Passively Transported: Water Urea Sodium c. Counter-Current Mechanism Osmotic Gradient of Medulla Vasopressin 4. Tubular Secretion Major Functions: a. Elimination of waste products not filtered by the glomerulus b. Regulation of acid-base balance through secretion of H ions Renal Function Tests 1. Glomerular Filtration Test Clearance test a. Urea b. Radioisotope c. B2 microglobulin d. Creatinine C = UV/P Normal values: Creatinine Clearance: F = 75-112 ml/m M = 85-125 ml/m Plasma Creatinine = 0.5-1.5 mg/dl e. Inulin 2. Tubular Reabsorption Tests a. Fishberg Test b. Mosenthal Test c. Osmometry 3. Tubular Secretion and Renal Blood Flow Tests a. PAH b. PSP c. Indigo Carmine Test Renal Diseases A. Glomerular Disorders: 1. Glomerulonephritis – blood, protein, and casts in urine a. Acute Poststreptococcal – caused by deposition of immune complexes and antibodies in the glomerular membrane following group A streptococcal infection. b. Rapidly Progressive ( Crescentic) – more serious, can lead to renal failure; arises as a result of another form of glomerulonephritis or an immune systemic disorder. c. Good Pasture’s Syndrome – attachment of cytotoxic autoantibody (anti-glomerular basement membrane antibody) to the glomerular and alveolar basement membranes during viral respiratory infections. Lab Findings:proteinuria, hematuria, RBC casts 2. Vasculitis - immune-mediated disoders affecting the systemic vascular system resulting to glomerular damage. a. Wegener’s granulomatosis – granulomaproducing inflammation of the small blood vessels in the lungs and kidney.The antibody causing the damage is the antineutrophilc cytoplasmic antibody (ANCA) Lab findings: hematuria, proteinuria, rbc casts, and increased BUN and crea b. Henoch-Schonlein Purpura – characterized by a decrease in platelets that causes disruption of vascular integrity Lab findings: heavy proteinuria and hematuria with rbc casts Wegener’s Granulomatosis 3. Immunoglobulin A nephropathy - deposition of immune complexes on the glomerular membrane resulting from increased levels of serum IgA Lab findings: macroscopic/microscopic hematuria and increased IgA 4. Membranous Glomerulonephritis - thickening of the glomerular membrane due to deposition of IgG immune complexes. Lab findings: microscopic hematuria and increased IgG and protein excretion 5. Membranoproliferative Glomerulonephritis – immune mediated disorder characterized by cellular proliferation in capillary walls or glomerular basement membrane. Lab findings: hematuria, proteinuria, and decreased serum complement levels 6. Chronic Glomerulonephritis – glomerular damage as a result of other renal disorders leads to marked decrease in renal functions and eventually to renal failure Lab findings: hematuria, proteinuria, glucosuria, varieties of casts including broad cast, markedly decreased GFR with increased BUN and crea levels, and electrolyte imbalance 7. Nephrotic Syndrome – disruption in the electrical charges in the basal lamina and podocytes, producing a less tightly connected barrier. Lab Findings: massive proteinuria, low albumin, high serum levels of lipids, and pronounced edema 8. Minimal Change Disease – podocytes appear to be less tightly fitting allowing increased filtration of protein; seen in children following allergic reaction and immunization. Lab Findings: edema, heavy proteinuria, transient hematuria 9. Focal Segmental Glomerulosclerosis - only a certain number and areas of glomeruli are affected. The disease is caused by disruption of the podocytes associated with analgesics and heroin abuse and AIDS. Immunoglobulins M and C3 are seen in undamaged glomeruli. •Lab Findings: heavy proteinuria, microscopic hematuria II. Tubulointerstitial Disorders 1. Acute Tubular Necrosis – damage to the renal tubular cells by toxic agents or ischemia 2. Fanconi’s Syndrome – generalized failure of tubular reabsorption in the PCT; may be hereditary or acquired 3. Cystitis – ascending bacterial infection of the bladder 4. Acute Pyelonephritis – infection of the upper urinary tract involving the interstitium and tubules due to interference of urine flow to the bladder, reflux of urine from the bladder, or untreated cystitis 5. Chronic Pyelonephritis – recurrent infection of the tubules and interstitium caused by structural abnormalities affecting urine flow 6. Acute Interstitial Nephritis – inflammation of the renal interstitium associated with allergic reaction to medications C. Vascular Disorders 1. Renal Failure – may be gradual progression from the original disorder to chronic renal failure or end-stage renal disease. 2. Renal Lithiasis – deposition of renal calculi or kidney stones in the calyces and pelvis of the kidney, ureters and urinary bladder. Chemical Composition of Renal Calculi: a. Calcium oxalate or phosphate b. Magnesium ammonium phosphate c. Uric acid d. Cystine Lab Findings: microscopic hematuria Composition of Urine 1. water 2. analytes organic inorganic 3. hormones, vitamins, medications, formed elements, etc. Types of Urine Specimens Timed Specimen: 24-hour 12-hour 2-hr Postprandial Afternoon Specimen Quantitative chemical tests, hormone studies Addis count Diabetic monitoring Urobilinogen determination Glucose Tolerance Test Accompaniment to blood samples in GTT Random First Morning Routine screening Routine screening Pregnancy Tests Orthostatic Proteinuria Fasting/Second Morning Diabetic screening / monitoring Catheterized Bacterial culture Midstream clean-catch Routine screening Bacterial culture Suprapubic aspiration Bladder urine for bacterial culture Cytology Three-glass collection Prostatic infection Drug Specimen Drug testing Voiding cystourethrogram Examining bladder function by injecting dye that is visible on X-rays through a catheter to fill the bladder. X-rays are taken while the bladder is full and while the patient is urinating to determine if fluid is forced out of the bladder through the urethra (normal) or up through the ureters into the kidney (vesicoureteral reflux). DRUG SPECIMEN COLLECTION Chain of Custody (COC) Form the process that provides the documentation of proper sample identification from the time of collection to the receipt of laboratory results a standardized form that documents that the specimen collected by the patient is the same one that is analyzed and reported Urine Specimen Collection - the most vulnerable part of a drug testing program - may be witnessed or unwitnessed; a same-gender collector is required in witnessed collection Required Volume - 30-45 ml taken within 4 minutes Temperature - 32.5ºC to 37.7ºC Procedure: The collector washes hands, wears gloves, adds bluing agent or dye to the toilet water reservoir, and tapes the toilet lid and faucet handles. The donor provides identification from employer. The collector completes step 1 COC Form and has the donor sign the form. The donor leaves all his belongings outside, washes hands, and receives a specimen cup. The collector remains in the restroom, outside the stall (unwitnessed) listening for unauthorized water use. The donor hands specimen cup to the collector and the latter checks the urine for abnormal color and for the required amount. The collector checks the temperature using a temperature strip, records the reading on the COC Form (step 2). If the temperature is out of range, recollection is needed. With the donor watching, the collector seals the capped bottle with identification strips (COC step 3) covering both sides of the cup. The seals contain the date and time. The donor initials the seals and completes COC step 4 after which, the collector accomplishes COC step 5. Each time the specimen is handled, transferred or stored, every individual must be identified, the date and purpose of the change must be recorded, and specific instructions on labeling, packaging or transport must be followed. Methods of Preservation 1. Physical (Refrigeration) 2. Chemical Phenol – causes an odor change Toluene –not effective for bacteria and molds Thymol crystals – preserves glucose and sediments well Formalin – excellent sediment preservative Boric acid – preserves protein well Sodium fluoride – good for drug analysis Sacomanno’s fixative – preserves cellular elements Changes in Unpreserved Urine Color - modified Decreased: Clarity Glucose Ketones Bilirubin Urobilinogen Red or darkened Increased: Odor pH Nitrite Bacteria blood cells White blood cells Casts Clinical Utilities of Routine Urinalysis Indicators of the State of the Kidney or Urinary Tract Appearance Specific Gravity Chemical tests Leukocyte Esterase Urinary Sediment Indicators of Metabolic and Other Conditions or Disease pH Appearance Glucose Bilirubin Urobilinogen and Ketones Indications of Other Systemic (Nonrenal) Conditions or Disease Hemoglobin Myoglobin Light-chain proteins Porphobilinogen PHYSICAL EXAMINATION I. Volume Average daily output: 1,200-1,500 ml Variations: a. Polyuria Indications: Diabetes mellitus Diabetes insipidus b. Diuresis c. Oliguria d. Anuria e. Nocturia II. Specific Gravity Performed using: a. Urinometer Corrections done b. Refractometer Instrument Calibration c. Reagent strip d. Harmonic Oscillation Densitometry Variations in S.G.: a. Hypersthenuria b. Hyposthenuria c. Isosthenuria III. pH IV. Color • Pigments Variations in Color: Colorless Pale yellow Dark yellow Orange Red/ Yellow green/ Reddish brown Amber/Orange Yellow brown Green Blue green Milky white Pink/Red Brown/Black V. Odor Normal: Aromatic Variations in Urine Odor: Amoniacal/Putrid/ Foul Mercaptan Fecaloid Fruity, sweet Maple syrup Sulfur odor Mousy Rancid Sweaty feet Rotting fish Cabbage Bleach VI. Transparency Urine Clarity Clear Hazy Cloudy Turbid Milky No visible particulates, transparent Few particulates, print easily seen through urine Many particulates, print blurred through urine Print cannot be seen through urine May precipitate or be clotted Nubecula Nonpathologic Causes of Urine Turbidity Epithelial cells Normal crystals Bacteria (old urine) Semen, prostatic fluid Fecal contamination Radiographic contrast media, mucus, talcum powder Vaginal creams Pathologic Causes of Urine Turbidity Red blood cells White blood cells Bacteria Yeasts Nonsquamous epithelial cells Abnormal crystals Casts Lymph fluid/Chyle Lipids Fecal matter CHEMICAL ANALYSIS I. pH Normal values: average-6; random-4.5-8.0; fasting-5.5-6.5 Clinical Significance a. respiratory or metabolic acidosis/ketosis b. respiratory or metabolic alkalosis c. renal tubular acidosis d. renal calculi formation e. treatment of UTI f. precipitation/identification of crystals g. determination of unsatisfactory specimen Causes of Acid and Alkaline Urine Acid Urine Emphysema Diabetes mellitus Starvation Dehydration Diarrhea Presence of Alkaline Urine Hyperventilation Vomiting Renal tubular acidosis Presence of ureaseproducing bacteria Vegetarian diet Old specimens acid-producing bacteria High protein diet Cranberry juice Medications II. Protein Most indicative of renal disease Normal urine contains <10 mg/dl or 100 mg/24 hours Types of Proteinuria (according to the amount of protein excreted per day) Degree of Proteinuria Mild Moderate Heavy Grams Excreted per 24 hours <1 g/day 1- 4 g/day >4 g/day Types of Proteinuria (according to cause) 1. Pre-renal Intravascular Muscle hemolysis injury Severe infection and inflammation Multiple myeloma 2. Renal a. Glomerular Conditions that cause damage to the glomerulus Orthostatic or Postural Proteinuria b. Tubular • Fanconi’s Syndrome • Toxic agents/Heavy metals • Severe viral infections 3. Post-renal a. lower UTI/inflammation b. injury/trauma c. menstrual contamination d. prostatic fluid/spermatozoa e. vaginal secretions Tests for Albumin Heat and Acetic Acid Test Positive Results: 1+ - diffused cloud 2+ - granular cloud 3+ - distinct flocculi 4+ - large flocculi Reagent Strips SSA/Cold Protein Precipitation Correlation of Reagent Strip and SSA Results (+) Rgt. Strip, (-) SSA = albumin present (+) Rgt. Strip, (+)SSA = proteinuria (-) Rgt. Strip, (+) SSA = BJP, globulins, etc. III. Glucose Normal concentration: 15 mg/dl Melituria Glycusoria Clinical significance a. DM b. endocrine disorders c. pancreatitis, carcinoma, cystic fibrosis, hemochromatosis d. CNS disorders e. disturbance in metabolism f. liver disease g. renal glycusoria h. drugs Glycusoria without hyperglycemia a. renal tubular dysfunction b. tubular necrosis c. Fanconi’s syndrome Tests for Glucose 1. Benedict’s Test – general test for glucose and other reducing sugars 2. Clinitest – subject to interference from other reducing sugars • Pass-though phenomenon 3. Glucose Oxidase – specific for glucose Correlation of Glucose Oxidase and Clinitest GOD 1+ 4+ strip neg 1+ Clinitest neg neg Interpretation small amount of glucose present possible oxidizing agent interference on rgt non-glucose reducing substance present IV. Ketones Represents 3 intermediate products of fat metabolism: a. acetone b. acetoacetic acid c. β-hydroxybutyric acid Accumulation in blood leads to: a. electrolyte imbalance b. dehydration c. acidosis d. diabetic coma Clinical significance: a. DM b. insulin dose monitoring c. diabetic acidosis d. starvation/fasting e. weight reduction/dieting/strenuous exercise f. vomiting g. malabsorption/pancreatic disorders h. inborn error of amino acid metabolism V. Blood Hematuria Clinical Significance: a. renal calculi b. glomerulonephritis c. tumors d. trauma e. pyelonephritis f. toxic chemicals g. strenuous sxercise h. menstrual contamination Hemoglobinuria Clinical significance: a. hemolytic anemia b. transfusion reaction c. PNH d. severe burns and infections e. malaria f. strenuous exercise Myoglobinuria Clinical significance: a. muscular trauma/crush syndrome b. prolonged coma c. convulsions d. muscle-wasting diseases e. alcoholism/overdose f. drug abuse g. extensive exertion Tests to Differentiate Hemoglobin and Myoglobin Ammonium Sulfate Method Hb is precipitated by ammonium sulfate Absorption Spectrophotometry Immunodiffusion Technique Protein Electrophoresis Differentiation of Hematuria, Hemoglobinuria, and Myoglobinuria Finding Rgt. Strip Microscopic (RBCs) Red Cells Positive Present Hemoglobin Positive Myoglobin Positive Absent or few Absent or few Clear red Pink to red Slight elevation Elevated Elevated Normal Clear red Normal Marked elevation Elevated Normal Elevated Urine Appearance Cloudy red Plasma Appearance Total serum CK Total serum LD LD1 and LD2 LD4 and LD5 Normal Normal Normal Normal Normal VI. Bilirubin Clinical Significance Hepatitis, cirrhosis, other liver disorders Biliary obstruction Tests Foam-Shake Test Oxidation Test – acidic oxidation of bilirubin into rainbow array of colors Diazotization Test Ictotest Reagent Strip VII. Urobilinogen found in urine in small amounts Clinical Significance Early detection of liver disease Hemolytic disorders Hepatitis, cirrhosis, carcinoma Tests Ehrlich’s Tube Test Schwartz-Watson Differentiation Test Reagent Strip Watson-Schwartz Test Interpretation Urobilinogen Ehrlich-reactive Substances Porphobilinogen Chloroform Extraction Urine (Top layer) Chloroform (Bottom layer) Colorless Red Red Colorless Butanol Extraction Butanol (Top layer) Urine (Bottom layer) Red Colorless Red Colorless Colorless Red Red Colorless Laboratory Findings in Various Types of Jaundice Blood Bilirubin Normal Hemolytic Hepatic 0–1.3 mg/dL Increased Increased Urine Bilirubin Negative Negative + or +++ Urine Urobilinogen ≤1 mg/dL +++ ++ Normal Obstructive Normal Urobilin – brown pigment which result from oxidation of urobilinogen upon exposure to light; tested when urobilinogen is negative Porphyrins – cyclic compounds derived from ALA Clinical Significance: Porphyrias Tests for Porphyrinuria Ehrlich Test – requires the addition of acetylacetone prior to performing the test Fluorescence – involves extraction into a mixture of glacial HAc and ethyl acetate and examination of solvent layer faint blue fluorescence = (-) violet, pink, or red = (+) VIII. Nitrite Clinical Significance Cystitis Pyelonephritis Evaluation of antibiotic therapy Monitoring of patients at high risk for UTI Screening of urine culture specimens IX. Leukocyte Esterase Clinical Significance Bacterial and nonbacterial UTI Inflammation of the urinary tract Screening of urine culture specimens Urine Reference Values Property Color Transparency pH Specific Gravity Protein (albumin) Blood (hemoglobin) Nitrite LE Glucose Ketones Bilirubin Urobilinogen Reference Value Yellow Clear 5-7 1.001-1.035 Negative or Trace Negative Negative Negative Negative Negative Negative ≤1 mg/dL Confirmatory Urinalysis Tests Substance Protein Blood Hemoglobin, Myoglobin Test SSA Test Microscopic Examination Centrifugation; then test supernatant with rgt. strip for blood Rous Test Clinitest Ictotest Watson-Schwartz Test Hoesch Test EM Quant Hemosiderin Glucose Bilirubin Urobilinogen Porphobilinogen Ascorbic Acid SPECIAL URINALYSIS SCREENING TESTS Amino Acid Disorders 1. Phenylalanine-Tyrosine Disorders a. Phenylketonuria Tests: Ferric Chloride Test Phenistix DPNH Robert Guthrie Bacterial Inhibition Test b. Tyrosyluria Tests: Millon’s Test Nitrosonaphthol Test Ferric Chloride Test Phenistix DPNH c. Melanuria Tests: Sodium nitroprusside Test - interference due to a red color from acetone and creatinine can be adding glacial HAc avoided by Ferric Chloride Test Acetest Blackberg and Wanger Test Ehrlich’s Test d. Alkaptonuria Tests: Ferric chloride Test Alkali Test Addition of silver nitrate or ammonium hydroxide Benedict’s Test or Clinitest 2. Branched Chain Amino Acid Disorders a. Maple Syrup Urine Disease Tests: DNPH Amino Acid Chromatography Ferric Chloride Test Nitrosonaphthol Acetest b. Organic Acidemias Isovaleric Acidemia DNPH Acetest Chromatography Propionic Acidemia DNPH Acetest Methylmalonic Acidemia DNPH Acetest p-nitroaniline 3. Tryptophan Disorders a. Indicanuria Clinical Significance: Obstruction Presence of bacteria Malabsorption syndrome b. Hartnup’s Disease production of blue color when indican is oxidized upon exposure to air c. 5-HIAA Tests: Ferric Chloride Test Sjoerdsma Test. Clinical Significance: Elevated in malignancy involving Argentaffin cells 4. Cystine Disorders a. Cystinuria Tests: + Cystine crystals Cyanide nitroprusside b. Cystinosis Tests: Cyanide nitroprusside Clinitest c. Homocystinuria Tests: Cyanide nitroprusside Silver nitroprusside Mucopolysaccharide Disorders Hurler’s Syndrome Hunter’s Syndrome San Filippo’s Syndrome Tests: Acid Albumin CTAB Turbidity Test Metachromatic Staining Spot Test Purine Disorders Lesch-Nyhan Disease lack of the enzyme HGPT Manifestations: severe motor defects, mental retardation, tendency toward self-destruction, gout and renal calculi, orange sand in diapers MICROSCOPIC EXAMINATION Types of Microscope Bright Field – objects appear dark against a light background Phase Contrast – works by retardation of light rays diffused by the object in focus Polarizing – used to confirm the identification of fat droplets, oval fat bodies, and fatty casts Interference Contrast – object appears bright against a dark background but without the diffraction halo associated with Phase Contrast Microscope Microscopic Sediment Stains Sternheimer-Malbin 0.5% Toluidine Blue Sudan III Oil Red O Prussian Blue Hansel Stain Reference Values for Urine Sediment Constituent Red Blood Cells White Blood Cells Casts Squamous Epithelial Cells Transitional Epithelial Cells Renal Tubular Epithelial Cells Bacteria Yeast Abnormal crystals Reference Value 0-2/hpf 0-5/hpf (female>male) 0-2 hyaline/hpf Few/hpf Few/hpf Few/hpf Negative Negative Negative Standardization of Procedure 1. Urine Volume – 12 mL 2. Time of Centrifugation – 5 minutes 3. Speed of Centrifugation Relative Centrifugal Force of 400 g 4. Volume of Sediment Examined – 20 μL 5. Reporting Format Reporting System for Urine Sediment Average Number per Low-Power Field Casts Abnormal crystals Squamous ECs Mucus Red Blood Cells Normal Crystals Epithelial Cells Miscellaneous Sperm 0-2 2-5 2-5 Few Few Few Neg Neg 0-2 0-2 2-5 2-5 5-10 10-25 25-50 >50 5-10 10-25 Moderate Present 25-50 >50 Many Few Average Number per High-Power Field 5-10 10-25 25-50 50-100 >100 5-10 10-25 25-50 50-100 >100 Moderate Moderate Moderate Present Many Many Many White Blood Cells 0-2 Organized Sediments Red Blood Cells •Normal appearance •N.V. •Variations in shape and appearance •Clinical Significance Non-glomerular hematuria: RBCs are uniform in size and shape but show two populations of cells because a small number have lost their hemoglobin pigment. Glomerular hematuria: RBCs are small and vary in size, shape, and hemoglobin content. White Blood Cells Bacteria Amoeboid •Normal appearance •N.V. •Clinical Significance •Glitter cells •Eosinophils •Mononuclear cells Squamous Epithelial Cells • Clue Cells Transitional Epithelial Cells smaller than squamous cells, spherical, caudate, or polyhedral with central nucleus Clinical Significance Transitional that appear singly, in pairs, or in clumps Transitional cells with abnormal morphology Renal Tubular Epithelial Cells •Clinical Significance •Oval Fat Bodies •Bubble Cells Casts Mechanisms of Cast Formation Hyaline Cast •Strenuous exercise, dehydration, heat exposure, emotional stress •Acute glomerulonephritis, pyelonephritis, chronic renal disease, CHF Red Blood Cell Cast WBC Cast •Epithelial Cell Cast Coarsely Granular Cast Finely Granular Cast Waxy Cast •Fatty Cast •Broad Cast Bacteria Yeast Cells There are no PMNs seen suggestive of contamination and not of UTI. Mucus Thread Unorganized Sediments Crystals Normal Crystals in Acidic Urine 1. Amorphous Urates 2. Uric Acid 3. Sodium Urates 4. Calcium Sulfates 5. Calcium Oxalate 6. Hippuric Acid Amorphous Urates Uric Acid Dihydrate Calcium Oxalate Normal Crystals in Alkaline Urine 1. Amorphous Phosphate 2. Calcium Carbonate 3. Ammonium biurate 4. Calcium Phosphate 5. Triple Phosphate Amorphous Phosphate Calcium Carbonate Ammonium biurate Calcium phosphate Triple phosphate Abnormal Crystals of Metabolic Origin Cystine Abnormal Crystals of Iatrogenic Origin Sulfonamides Radiographic contrast Cholesterol Leucine Tyrosine Bilirubin Hemosiderin media Ampicillin Acyclovir Indinavir sulfate Cystine Cholesterol Leucine Tyrosine Sulfa Crystals Starch