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Course: IDPT 5005 School of Medicine, UCDHSC

Urinalysis. Course: IDPT 5005 School of Medicine, UCDHSC. Francisco G. La Rosa, MD Francisco.LaRosa@uchsc.edu Assistant Professor, Department of Pathology University of Colorado at Denver Health Science Center, Denver, Colorado. Specimen Collection.

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Course: IDPT 5005 School of Medicine, UCDHSC

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  1. Urinalysis Course: IDPT 5005 School of Medicine, UCDHSC Francisco G. La Rosa, MD Francisco.LaRosa@uchsc.edu Assistant Professor, Department of Pathology University of Colorado at Denver Health Science Center, Denver, Colorado

  2. Specimen Collection • First morning voiding (most concentrated) • Record collection time • Type of specimen (e.g. “clean catch”) • Analyzed within 2 hours of collection • Free of debris or vaginal secretions

  3. Clean Catch

  4. Specimen Collection Supra-pubic Needle Aspiration

  5. Types of Analysis • Macroscopic Examination • Chemical Analysis (Urine Dipstick) • Microscopic Examination • Culture (not covered in this lecture) • Cytological Examination

  6. Macroscopic Examination Odor: • Ammonia-like: (Urea-splitting bacteria) • Foul, offensive: Old specimen, pus or inflammation • Sweet: Glucose • Fruity: Ketones • Maple syrup-like: Maple Syrup Urine Disease Color: • Colorless Diluted urine • Deep Yellow Concentrated Urine, Riboflavin • Yellow-Green Bilirubin / Biliverdin • Red Blood / Hemoglobin • Brownish-red Acidified Blood (Actute GN) • Brownish-black Homogentisic acid (Melanin)

  7. Macroscopic Examination Turbidity: • Typically cells or crystals. • Cellular elements and bacteria will clear by centrifugation. • Crystals dissolved by a variety of methods (acid or base). • Microscopic examination will determine which is present.

  8. Chemical Analysis

  9. Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase Chemical Analysis Urine Dipstick

  10. Negative Trace (100 mg/dL) + (250 mg/dL) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) The Urine Dipstick: Glucose Chemical Principle Glucose Oxidase Glucose + 2 H2O+ O2---> Gluconic Acid + 2 H2O2 Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O Read at 30 seconds RR: Negative

  11. Uses and Limitations of Urine Glucose Detection Significance • Diabetes mellitus. • Renal glycosuria. Limitations • Interference: reducing agents, ketones. • Only measures glucose and not other sugars. • Renal threshold must be passed in order for glucose to spill into the urine. Other Tests • CuSO4 test for reducing sugars.

  12. Detection of Reducing Sugars* by CuSO4 SugarDisease(s) - Galactose Galactosemias - Fructose Fructosuria, Fructose Intolerance, etc. - Lactose Lactase Deficiency - Pentoses Essential Pentosuria - Maltose Non-pathogenic * NOT Sucrose because it is not a reducing sugar

  13. Urine versus Blood Glucose ++ + Urinalysis Glucose Result trace Negative 200 400 600 800 1000 Blood Glucose (mg/dL)

  14. Negative + (weak) ++ (moderate) +++ (strong) The Urine Dipstick: Bilirrubin Chemical Principle Acidic Bilirubin + Diazo salt ---------> Azobilirubin Read at 30 seconds RR: Negative

  15. Uses and Limitations of Urine Bilirrubin Detection Significance - Increased direct bilirubin (correlates with urobilinogen and serum bilirubin) Limitations - Interference: prolonged exposure of sample to light - Only measures direct bilirubin--will not pick up indirect bilirubin Other Tests - Ictotest (more sensitive tablet version of same assay) - Serum test for total and direct bilirubin is more informative

  16. Negative Trace (5 mg/dL) + (15 mg/dL) ++ (40 mg/dL) +++ (80 mg/dL) ++++ (160+ mg/dL) The Urine Dipstick: Ketones Chemical Principle Acetoacetic Acid + Nitroprusside ------> Colored Complex Read at 40 seconds RR: Negative

  17. Uses and Limitations of Urine Ketone Detection Significance - Diabetic ketoacidosis - Prolonged fasting Limitations - Interference: expired reagents (degradation with exposure to moisture in air) - Only measures acetoacetate not other ketone bodies (such as in rebound ketosis). Other Tests - Ketostix (more sensitive tablet version of same assay) - Serum glucose measurement to confirm DKA

  18. 1.000 1.005 1.010 1.015 1.020 1.025 1.030 The Urine Dipstick: Specific Gravity Chemical Principle X+ + Polymethyl vinyl ether / maleic anhydride ---------------> X+-Polymethyl vinyl ether / maleic anhydride + H+ H+ interacts with a Bromthymol Blue indicator to form a colored complex. Read up to 2 minutes RR: 1.003-1.035

  19. Uses and Limitations of Urine Specific Gravity Significance - Diabetes insipidus Limitations - Interference: alkaline urine - Does not measure non-ionized solutes (e.g. glucose) Other Tests - Refractometry - Hydrometer - Osmolality measurement (typically used with water deprivation test)

  20. The Urine Dipstick: Blood Negative Chemical Principle Trace (non-hemolyzed) Lysing agent to lyse red blood cells Moderate (non-hemolyzed) Diisopropylbenzene dihydroperoxide + Tetramethylbenzidine ------------> Colored Complex Trace (hemolyzed) Heme + (weak) Read at 60 seconds RR: Negative Analytic Sensitivity: 10 RBCs ++ (moderate) +++ (strong)

  21. Uses and Limitations of Urine Blood Detection Significance - Hematuria (nephritis, trauma, etc) - Hemoglobinuria (hemolysis, etc) - Myoglobinuria (rhabdomyolysis, etc) Limitations - Interference: reducing agents, microbial peroxidases - Cannot distinguish between the above disease processes Other Tests - Urine microscopic examination - Urine cytology

  22. 5.0 6.0 6.5 7.0 7.5 8.0 8.5 The Urine Dipstick: pH Chemical Principle H+ interacts with: Methyl Red (at high concentration; low pH) and Bromthymol Blue (at low concentration; high pH), to form a colored complexes(dual indicator system) Read up to 2 minutes R.R.: 4.5-8.0

  23. Uses and Limitations of Urine pH Detection Significance - Acidic (less than 4.5): metabolic acidosis, high-protein diet - Alkaline (greater than 8.0): renal tubular acidosis (>5.5) Limitations - Interference: bacterial overgrowth (alkaline or acidic), “run over effect” effect of protein pad on pH indicator pad Other Tests - Titrable acidity - Blood gases to determine acid-base status

  24. Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase pH Run Over Effect Buffers from the protein area of the strip (pH 3.0) spill over to the pH area of the strip and make the pH of the sample appear more acidic than it really is.

  25. Negative Trace + (30 mg/dL) Pr H Pr ++ (100 mg/dL) H Pr H +++ (300 mg/dL) Pr Pr H H Pr H ++++ (2000 mg/dL) H+ H+ H+ H+ H+ H+ The Urine Dipstick: Protein Chemical Principle “Protein Error of Indicators Method” Tetrabromphenol Blue (buffered to pH 3.0) Pr Pr Pr Pr Pr Pr Read at 60 seconds RR: Negative

  26. Causes of Proteinuria FunctionalRenal - Severe muscular exertion - Glomerulonephritis - Pregnancy - Nephrotic syndrome - Orthostatic proteinuria - Renal tumor or infection Pre-RenalPost-Renal - Fever - Cystitis - Renal hypoxia - Urethritis or prostatitis - Hypertension - Contamination with vaginal secretions

  27. Nephrotic Syndrome (> 3.5 g/dL in 24 h) Primary - Lipoid nephrosis (severe) - Membranous glomerulonephritis - Membranoproliferative glomerulonephritis Secondary - Diabetes mellitus (Kimmelsteil-Wilson lesions) - Systemic lupus erythematosus - Amyloidosis and other infiltrative diseases - Renal vein thrombosis

  28. Uses and Limitations of Urine Protein Detection Significance - Proteinuria and the nephrotic syndrome. Limitations - Interference: highly alkaline urine. - Much more sensitive to albumin than other proteins (e.g., immunoglobulin light chains). Other Tests - Sulfosalicylic acid (SSA) turbidity test. - Urine protein electrophoresis (UPEP) - Bence Jones protein

  29. Proteins in “Normal” Urine Protein % of Total Daily Maximum Albumin 40% 60 mg Tamm-Horsfall 40% 60 mg Immunoglobulins 12% 24 mg Secretory IgA 3% 6 mg Other 5% 10 mg TOTAL 100% 150 mg

  30. 0.2 mg/dL 1 mg/dL 2 mg/dL 4 mg/dL 8 mg/dL The Urine Dipstick: Urobilinogen Chemical Principle Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex (Ehrlich’s Reagent) Read at 60 seconds RR: 0.02-1.0 mg/dL

  31. Uses and Limitations of Urobilinogen Detection Significance - High: increased hepatic processing of bilirubin - Low: bile obstruction Limitations - Interference: prolonged exposure of specimen to oxygen (urobilinogen ---> urobilin) - Cannot detect low levels of urobilinogen Other Tests - Serum total and direct bilirubin

  32. Negative Positive The Urine Dipstick: Nitrite Chemical Principle Acidic Nitrite + p-arsenilic acid -------> Diazo compound Diazo compound + Tetrahydrobenzoquinolinol ----------> Colored Complex Read at 60 seconds RR: Negative

  33. Uses and Limitations of Nitrite Detection Significance - Gram negative bacteriuria Limitations - Interference: bacterial overgrowth - Only able to detect bacteria that reduce nitrate to nitrite Other Tests - Correlate with leukocyte esterase and - Urine microscopic examination (bacteria) - Urine culture

  34. Negative Trace + (weak) ++ (moderate) +++ (strong) The Urine Dipstick: Leukocyte Esterase Chemical Principle Derivatized pyrrole amino acid ester ------------> 3-hydroxy-5-phenyl pyrrole Esterases 3-hydroxy-5-phenyl pyrrole + diazo salt -------------> Colored Complex Read at 2 minutes RR: Negative Analytic Sensitivity: 3-5 WBCs

  35. Uses and Limitations of Leukocyte Esterase Detection Significance - Pyuria - Acute inflammation - Renal calculus Limitations - Interference: oxidizing agents, menstrual contamination Other Tests - Urine microscopic examination (WBCs and bacteria) - Urine culture

  36. Microscopic Examination General Aspects Preservation - Cells and casts begin to disintegrate in 1 - 3 hrs. at room temp. - Refrigeration for up to 48 hours (little loss of cells). Specimen concentration - Ten to twenty-fold concentration by centrifugation. Types of microscopy - Phase contrast microscopy - Polarized microscopy - Bright field microscopy with special staining (e.g., Sternheimer-Malbin stain)

  37. Microscopic Examination Abnormal Findings Per High Power Field (HPF) (400x) • > 3 erythrocytes • > 5 leukocytes • > 2 renal tubular cells • > 10 bacteria Per Low Power Field (LPF) (200x) • > 3 hyaline casts or > 1 granular cast • > 10 squamous cells (indicative of contaminated specimen) • Any other cast (RBCs, WBCs) Presence of: • Fungal hyphae or yeast, parasite, viral inclusions • Pathological crystals (cystine, leucine, tyrosine) • Large number of uric acid or calcium oxalate crystals

  38. Microscopic Examination Cells Erythrocytes - “Dysmorphic” vs. “normal” (> 10 per HPF) Leukocytes - Neutrophils (glitter cells) More than 1 per 3 HPF - Eosinophils Hansel test (special stain) Epithelial Cells - Squamous cells Indicate level of contamination - Renal tubular epithelial cells Few are normal - Transitional epithelial cells Few are normal - Oval fat bodies Abnormal, indicate Nephrosis

  39. Microscopic Examination RBCs

  40. Microscopic Examination RBCs

  41. Microscopic Examination WBCs

  42. Microscopic Examination Squamous Cells

  43. Microscopic Examination Tubular Epithelial Cells

  44. Microscopic Examination Transitional Cells

  45. Microscopic Examination Transitional Cells

  46. Microscopic Examination Oval Fat Body

  47. Microscopic Examination LE Cell

  48. Microscopic Examination Bacteria & Yeasts Bacteria - Bacteriuria More than 10 per HPF Yeasts - Candidiasis Most likely a contaminant but should correlate with clinical picture. Viruses - CMV inclusions Probable viral cystitis.

  49. Microscopic Examination Bacteria

  50. Microscopic Examination Yeasts

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