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Urin Analysis

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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 – 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
Clean Catch
Specimen Collection Supra-pubic Needle Aspiration
Types of Analysis − Macroscopic Examination − Chemical Analysis (Urine Dipstick) − Microscopic Examination − Culture (not covered in this lecture) − Cytological Examination
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)
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.
Chemical Analysis
Chemical Analysis Urine Dipstick Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase
The Urine Dipstick: Glucose Chemical Principle Negative Glucose Oxidase Trace (100 mg/dL) Glucose + 2 H2O + O2 ---> + (250 mg/dL) Gluconic Acid + 2 H2O2 ++ (500 mg/dL) Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O +++ (1000 mg/dL) Read at 30 seconds ++++ (2000+ mg/dL) RR: Negative
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.
Detection of Reducing Sugars* by CuSO 4 Sugar Disease(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
Urine versus Blood Glucose Urinalysis Glucose Result ++ + trace Negative 200 400 600 800 1000 Blood Glucose (mg/dL)
The Urine Dipstick: Bilirrubin Negative Chemical Principle + (weak) Bilirubin + Diazo salt Acidic Azobilirubin ---------> ++ (moderate) Read at 30 seconds +++ (strong) RR: Negative
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
The Urine Dipstick: Ketones Negative Chemical Principle Trace (5 mg/dL) + (15 mg/dL) Acetoacetic Acid + Nitroprusside ------> Colored Complex ++ (40 mg/dL) +++ (80 mg/dL) Read at 40 seconds RR: Negative ++++ (160+ mg/dL)
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
The Urine Dipstick: Specific Gravity 1.000 Chemical Principle 1.005 X+ + Polymethyl vinyl ether / maleic anhydride 1.010 ---------------> X+-Polymethyl vinyl ether / maleic anhydride + H+ 1.015 H+ interacts with a Bromthymol Blue indicator to 1.020 form a colored complex. 1.025 Read up to 2 minutes RR: 1.003-1.035 1.030
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)
The Urine Dipstick: Blood Negative Chemical Principle Trace (non-hemolyzed) Lysing agent to lyse red blood cells Moderate (non-hemolyzed) Diisopropylbenzene dihydroperoxide + Tetramethylbenzidine Trace (hemolyzed) Heme ------------> Colored Complex + (weak) ++ (moderate) Read at 60 seconds RR: Negative Analytic Sensitivity: 10 RBCs +++ (strong)
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
The Urine Dipstick: pH 5.0 6.0 Chemical Principle H+ interacts with: 6.5 Methyl Red (at high concentration; low pH) and Bromthymol Blue (at low concentration; high 7.0 pH), to form a colored complexes (dual indicator system) 7.5 8.0 Read up to 2 minutes R.R.: 4.5-8.0 8.5
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
pH Run Over Effect Glucose Bilirubin Ketones Specific Gravity Buffers from the protein area of the strip (pH 3.0) spill over to the Blood pH area of the strip and make the pH pH of the sample appear more acidic than it really is. Protein Urobilinogen Nitrite Leukocyte Esterase
The Urine Dipstick: Protein Chemical Principle “Protein Error of Indicators Method” Negative Pr H Pr H Pr Trace H Pr Pr + (30 mg/dL) H H H Pr ++ (100 mg/dL) Tetrabromphenol Blue H+ H H + + (buffered to pH 3.0) H+ H H + + +++ (300 mg/dL) Pr Pr Pr Pr Pr ++++ (2000 mg/dL) Pr Read at 60 seconds RR: Negative
Causes of Proteinuria Functional Renal - Severe muscular exertion - Glomerulonephritis - Pregnancy - Nephrotic syndrome - Orthostatic proteinuria - Renal tumor or infection Pre-Renal Post-Renal - Fever - Cystitis - Renal hypoxia - Urethritis or prostatitis - Hypertension - Contamination with vaginal secretions
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
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
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%
The Urine Dipstick: Urobilinogen 0.2 mg/dL Chemical Principle 1 mg/dL Urobilinogen + Diethylaminobenzaldehyde (Ehrlich’s Reagent) 2 mg/dL -------> Colored Complex 4 mg/dL Read at 60 seconds 8 mg/dL RR: 0.02-1.0 mg/dL
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
The Urine Dipstick: Nitrite Chemical Principle Acidic Negative Nitrite + p-arsenilic acid -------> Diazo compound Diazo compound + Tetrahydrobenzoquinolinol Positive ----------> Colored Complex Read at 60 seconds RR: Negative
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
The Urine Dipstick: Leukocyte Esterase Chemical Principle Derivatized pyrrole amino acid ester Negative Esterases ------------> 3-hydroxy-5-phenyl pyrrole Trace + (weak) 3-hydroxy-5-phenyl pyrrole + diazo salt -------------> Colored Complex ++ (moderate) Read at 2 minutes +++ (strong) RR: Negative Analytic Sensitivity: 3-5 WBCs
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
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)
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
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
Microscopic Examination RBCs
Microscopic Examination RBCs
Microscopic Examination WBCs
Microscopic Examination Squamous Cells
Microscopic Examination Tubular Epithelial Cells
Microscopic Examination Transitional Cells
Microscopic Examination Transitional Cells
Microscopic Examination Oval Fat Body
Microscopic Examination LE Cell
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.
Microscopic Examination Bacteria
Microscopic Examination Yeasts
Microscopic Examination Yeasts
Microscopic Examination Cytomegalovirus
Microscopic Examination Casts Erythrocyte Casts: Glomerular diseases Leukocyte Casts: Pyuria, glomerular disease Degenerating Casts: - Granular casts Nonspecific (Tamm-Horsfall protein) - Hyaline casts Nonspecific (Tamm-Horsfall protein) - Waxy casts Nonspecific - Fatty casts Nephrotic syndrome (oval fat body casts)
Microscopic Examination Casts
Microscopic Examination RBCs Cast - Histology
Microscopic Examination RBCs Cast
Microscopic Examination RBCs Cast - Histology
Microscopic Examination WBCs Cast
Microscopic Examination Tubular Epith. Cast
Microscopic Examination Tubular Epith. Cast
Microscopic Examination Granular Cast
Microscopic Examination Hyaline Cast
Microscopic Examination Waxy Cast
Microscopic Examination Fatty Cast
Significance of Cellular Casts Erythrocyte Casts Leukocyte Casts Bacterial Casts Single Erythrocytes Single Leukocytes Single Bacteria Verrier-Jones & Asscher, 1991.
Microscopic Examination Crystals - Urate Ammonium biurate Uric acid - Triple Phosphate - Calcium Oxalate - Amino Acids Cystine Leucine Tyrosine - Sulfonamide
Microscopic Examination Calcium Oxalate Crystals
Microscopic Examination Calcium Oxalate Crystals Dumbbell Shape
Microscopic Examination Triple Phosphate Crystals
Microscopic Examination Urate Crystals
Microscopic Examination Leucine Crystals
Microscopic Examination Cystine Crystals
Microscopic Examination Ammonium Biurate Crystals
Microscopic Examination Cholesterol Crystals
Cytological Examination Staining: – Papanicolau – Wright’s – Immunoperoxidase – Immunofluorescence
Cytology: Normal
Cytology: Normal
Cytology: Reactive
Cytology: Reactive
Cytology: Polyoma (Decoy Cell)
Cytology: Polyoma (Decoy Cell) Immunoperoxidase to SV40 ag
Cytology: TCC Low Grade
Cytology: TCC Low Grade
Cytology: TCC High Grade
Cytology: TCC High Grade
Cytology: Squamous Cell Ca.
Cytology: Renal Cell Ca.
Cytology: Prostatic Carcinoma
Urinalysis Disease Diagnosis
Case Diluted urine, request a voided urine in the morning If persisting low SG, possible diabetes insipida 1 A microscopic may give negative results Glucose Negative Bilirubin Negative A 35-year old man undergoing routine pre employment drug screening. Ketones Negative Physical characteristics: Clear. S.G. 1.001 Microscopic: Not performed. Drugs Identified: None. Blood Negative pH 5.5 Questions: Protein Negative - What is your differential diagnosis? - What would you do next to confirm your Urobilinogen 0.2 mg/dL suspicion? - Would you order a microscopic analysis Nitrite Negative on this sample? L.E. Negative
Case Possible gallbladder or hepatic disease. No hemolytic anemia. Perform bilirubins in serum 2 Microscopic unlikely to provide additional info Glucose Negative Bilirubin +++ A 42-year old woman presents with “dark urine” Ketones Negative Physical characteristics: Red-brown. S.G. 1.020 Microscopic: Not performed. Blood Negative Questions: pH 5.5 - What is your differential diagnosis? Protein Negative - Could this be a case of hemolytic anemia? - How would you rule it out? Urobilinogen 0.2 mg/dL - What tests would you order next? Why? Nitrite - Would you order a microscopic analysis? Negative L.E. Negative
Case Possible UTI, request culture and antibiotic sensitivity Negative Nitrite test: Gram positive bacteria 3 Lower SG may show less number of cells and bacteria Un-common diagnosis in this type of patient Glucose Negative A 42-year old man presents painful urination Bilirubin Negative Ketones Physical characteristics: dark red, turbid Negative Microscopic: leukocytes = 30 per HPF S.G. 1.030 RBCs = >100 per HPF Bacteria = >100 per HPF Blood +++ Questions: pH 6.5 - What is your suspected diagnosis? - What would you do next? Protein Trace - What do you make of the nitrite test? Urobilinogen 1.0 mg/dL - How would the microscopic exam differ if the S.G. were 1.003? Nitrite Negative - Is this a common diagnosis for this type of patient? L.E. +++
Case Diabetes May be decompensated and with ketoacidosis 4 Ketones should become negative after treatment Glucose ++ Bilirubin Negative Ketones Trace A 27-year old woman presents with severe abdominal pain. S.G. 1.015 Physical characteristics: clear-yellow. Blood Negative Microscopic: Not performed. pH 6.0 Questions: Protein Negative - What is the most likely diagnosis? - What do you make of the ketone result? Urobilinogen 1.0 mg/dL - What do you expect to happen to the ketone measurement when treatment begins? Nitrite Negative L.E. Negative
Case Glomerulonephritis RBC casts reveals renal cortex involvement 5 RBC cast are not always present in GN Glucose Negative 8-year old boy presents with discolored urine Bilirubin Negative Ketones Physical characteristics: Red, turbid. Negative Microscopic: erythrocytes = >100 per HPF S.G. 1.015 (almost all dysmorphic) Red cell casts present. Blood +++ Questions: pH 6.5 - What is the most likely diagnosis in this Protein case? + - Does the presence of red cell casts help you Urobilinogen 1.0 mg/dL in any way? - If the erythrocytes were not dysmorphic Nitrite Negative would that change your diagnosis? L.E. Negative
Case “Functional” proteinuria? Microscopic may reveal a few leukocytes 6 Request protein concentration in 24 h urine Glucose Negative 22-year old man presenting for a routine Bilirubin Negative physical required for admission to medical Ketones Negative school S.G. 1.010 Physical characteristics: Yellow Microscopic: Not performed Blood Negative Questions: pH 5.0 - What is your differential diagnosis? Protein + - Would you order a microscopic analysis on this sample? Urobilinogen 0.2 mg/dL - What would you do next to confirm the diagnosis? Nitrite Negative L.E. Negative
Common Findings in: Acute Tubular Necrosis Glucose Bilirubin Ketones S.G. Decreased Microscopic: Blood +/- • Renal tubular epithelial cells pH • Pathological casts Protein +/- Urobilinogen Nitrite L.E.
Common Findings in: Acute Glomerulonephritis Glucose Bilirubin Ketones Microscopic: S.G. Blood Increased • Erythrocytes (dysmorphic) pH • Erythrocyte casts • Mixed cellular casts Protein Increased Urobilinogen Nitrite L.E.
Common Findings in: Chronic Glomerulonephritis Glucose Bilirubin Ketones Decreased Microscopic: S.G. Blood Increased • Pathological casts pH (broad waxy casts, RBCs) Protein Increased Urobilinogen Nitrite L.E.
Common Findings in: Acute Pyelonephritis Glucose Bilirubin Ketones Microscopic: S.G. • Bacteria Blood • Leukocytes pH • Leukocyte, granular, and Protein Trace waxy casts • Renal tubular epithelial Urobilinogen cell casts Nitrite Positive L.E. Positive
Common Findings in: Nephrotic Syndrome Glucose Bilirubin Ketones S.G. Microscopic: Blood • Oval fat bodies pH • Fatty casts Protein ++++ • Waxy casts Urobilinogen Nitrite L.E.
Common Findings in: Eosinophilic Cystitis Glucose Bilirubin Ketones S.G. Microscopic: Blood + • Numerous eosinophils pH (Hansel’s stain) Protein • NO significant casts. Urobilinogen Nitrite L.E.
Common Findings in: Urothelial Carcinoma Glucose Bilirubin Ketones S.G. Microscopic: Blood + • Malignant cells on pH urine cytology (urine Protein sample should be submitted separately to cytology, void Urobilinogen or 24 hrs.) Nitrite L.E.
Acknowledgment: Dr. Brad Brimhall
Questions ?

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Urin Analysis

  • 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
  • 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.
  • 9. Chemical Analysis Urine Dipstick Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase
  • 10. The Urine Dipstick: Glucose Chemical Principle Negative Glucose Oxidase Trace (100 mg/dL) Glucose + 2 H2O + O2 ---> + (250 mg/dL) Gluconic Acid + 2 H2O2 ++ (500 mg/dL) Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O +++ (1000 mg/dL) Read at 30 seconds ++++ (2000+ mg/dL) 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 CuSO 4 Sugar Disease(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. The Urine Dipstick: Bilirrubin Negative Chemical Principle + (weak) Bilirubin + Diazo salt Acidic Azobilirubin ---------> ++ (moderate) Read at 30 seconds +++ (strong) 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. The Urine Dipstick: Ketones Negative Chemical Principle Trace (5 mg/dL) + (15 mg/dL) Acetoacetic Acid + Nitroprusside ------> Colored Complex ++ (40 mg/dL) +++ (80 mg/dL) Read at 40 seconds RR: Negative ++++ (160+ mg/dL)
  • 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. The Urine Dipstick: Specific Gravity 1.000 Chemical Principle 1.005 X+ + Polymethyl vinyl ether / maleic anhydride 1.010 ---------------> X+-Polymethyl vinyl ether / maleic anhydride + H+ 1.015 H+ interacts with a Bromthymol Blue indicator to 1.020 form a colored complex. 1.025 Read up to 2 minutes RR: 1.003-1.035 1.030
  • 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 Trace (hemolyzed) Heme ------------> Colored Complex + (weak) ++ (moderate) Read at 60 seconds RR: Negative Analytic Sensitivity: 10 RBCs +++ (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. The Urine Dipstick: pH 5.0 6.0 Chemical Principle H+ interacts with: 6.5 Methyl Red (at high concentration; low pH) and Bromthymol Blue (at low concentration; high 7.0 pH), to form a colored complexes (dual indicator system) 7.5 8.0 Read up to 2 minutes R.R.: 4.5-8.0 8.5
  • 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. pH Run Over Effect Glucose Bilirubin Ketones Specific Gravity Buffers from the protein area of the strip (pH 3.0) spill over to the Blood pH area of the strip and make the pH pH of the sample appear more acidic than it really is. Protein Urobilinogen Nitrite Leukocyte Esterase
  • 25. The Urine Dipstick: Protein Chemical Principle “Protein Error of Indicators Method” Negative Pr H Pr H Pr Trace H Pr Pr + (30 mg/dL) H H H Pr ++ (100 mg/dL) Tetrabromphenol Blue H+ H H + + (buffered to pH 3.0) H+ H H + + +++ (300 mg/dL) Pr Pr Pr Pr Pr ++++ (2000 mg/dL) Pr Read at 60 seconds RR: Negative
  • 26. Causes of Proteinuria Functional Renal - Severe muscular exertion - Glomerulonephritis - Pregnancy - Nephrotic syndrome - Orthostatic proteinuria - Renal tumor or infection Pre-Renal Post-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%
  • 30. The Urine Dipstick: Urobilinogen 0.2 mg/dL Chemical Principle 1 mg/dL Urobilinogen + Diethylaminobenzaldehyde (Ehrlich’s Reagent) 2 mg/dL -------> Colored Complex 4 mg/dL Read at 60 seconds 8 mg/dL 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. The Urine Dipstick: Nitrite Chemical Principle Acidic Negative Nitrite + p-arsenilic acid -------> Diazo compound Diazo compound + Tetrahydrobenzoquinolinol Positive ----------> 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. The Urine Dipstick: Leukocyte Esterase Chemical Principle Derivatized pyrrole amino acid ester Negative Esterases ------------> 3-hydroxy-5-phenyl pyrrole Trace + (weak) 3-hydroxy-5-phenyl pyrrole + diazo salt -------------> Colored Complex ++ (moderate) Read at 2 minutes +++ (strong) 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
  • 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
  • 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.
  • 52. Microscopic Examination Cytomegalovirus
  • 53. Microscopic Examination Casts Erythrocyte Casts: Glomerular diseases Leukocyte Casts: Pyuria, glomerular disease Degenerating Casts: - Granular casts Nonspecific (Tamm-Horsfall protein) - Hyaline casts Nonspecific (Tamm-Horsfall protein) - Waxy casts Nonspecific - Fatty casts Nephrotic syndrome (oval fat body casts)
  • 55. Microscopic Examination RBCs Cast - Histology
  • 57. Microscopic Examination RBCs Cast - Histology
  • 59. Microscopic Examination Tubular Epith. Cast
  • 60. Microscopic Examination Tubular Epith. Cast
  • 61. Microscopic Examination Granular Cast
  • 62. Microscopic Examination Hyaline Cast
  • 65. Significance of Cellular Casts Erythrocyte Casts Leukocyte Casts Bacterial Casts Single Erythrocytes Single Leukocytes Single Bacteria Verrier-Jones & Asscher, 1991.
  • 66. Microscopic Examination Crystals - Urate Ammonium biurate Uric acid - Triple Phosphate - Calcium Oxalate - Amino Acids Cystine Leucine Tyrosine - Sulfonamide
  • 67. Microscopic Examination Calcium Oxalate Crystals
  • 68. Microscopic Examination Calcium Oxalate Crystals Dumbbell Shape
  • 69. Microscopic Examination Triple Phosphate Crystals
  • 70. Microscopic Examination Urate Crystals
  • 71. Microscopic Examination Leucine Crystals
  • 72. Microscopic Examination Cystine Crystals
  • 73. Microscopic Examination Ammonium Biurate Crystals
  • 74. Microscopic Examination Cholesterol Crystals
  • 75. Cytological Examination Staining: – Papanicolau – Wright’s – Immunoperoxidase – Immunofluorescence
  • 86.
  • 87.
  • 91. Urinalysis Disease Diagnosis
  • 92. Case Diluted urine, request a voided urine in the morning If persisting low SG, possible diabetes insipida 1 A microscopic may give negative results Glucose Negative Bilirubin Negative A 35-year old man undergoing routine pre employment drug screening. Ketones Negative Physical characteristics: Clear. S.G. 1.001 Microscopic: Not performed. Drugs Identified: None. Blood Negative pH 5.5 Questions: Protein Negative - What is your differential diagnosis? - What would you do next to confirm your Urobilinogen 0.2 mg/dL suspicion? - Would you order a microscopic analysis Nitrite Negative on this sample? L.E. Negative
  • 93. Case Possible gallbladder or hepatic disease. No hemolytic anemia. Perform bilirubins in serum 2 Microscopic unlikely to provide additional info Glucose Negative Bilirubin +++ A 42-year old woman presents with “dark urine” Ketones Negative Physical characteristics: Red-brown. S.G. 1.020 Microscopic: Not performed. Blood Negative Questions: pH 5.5 - What is your differential diagnosis? Protein Negative - Could this be a case of hemolytic anemia? - How would you rule it out? Urobilinogen 0.2 mg/dL - What tests would you order next? Why? Nitrite - Would you order a microscopic analysis? Negative L.E. Negative
  • 94. Case Possible UTI, request culture and antibiotic sensitivity Negative Nitrite test: Gram positive bacteria 3 Lower SG may show less number of cells and bacteria Un-common diagnosis in this type of patient Glucose Negative A 42-year old man presents painful urination Bilirubin Negative Ketones Physical characteristics: dark red, turbid Negative Microscopic: leukocytes = 30 per HPF S.G. 1.030 RBCs = >100 per HPF Bacteria = >100 per HPF Blood +++ Questions: pH 6.5 - What is your suspected diagnosis? - What would you do next? Protein Trace - What do you make of the nitrite test? Urobilinogen 1.0 mg/dL - How would the microscopic exam differ if the S.G. were 1.003? Nitrite Negative - Is this a common diagnosis for this type of patient? L.E. +++
  • 95. Case Diabetes May be decompensated and with ketoacidosis 4 Ketones should become negative after treatment Glucose ++ Bilirubin Negative Ketones Trace A 27-year old woman presents with severe abdominal pain. S.G. 1.015 Physical characteristics: clear-yellow. Blood Negative Microscopic: Not performed. pH 6.0 Questions: Protein Negative - What is the most likely diagnosis? - What do you make of the ketone result? Urobilinogen 1.0 mg/dL - What do you expect to happen to the ketone measurement when treatment begins? Nitrite Negative L.E. Negative
  • 96. Case Glomerulonephritis RBC casts reveals renal cortex involvement 5 RBC cast are not always present in GN Glucose Negative 8-year old boy presents with discolored urine Bilirubin Negative Ketones Physical characteristics: Red, turbid. Negative Microscopic: erythrocytes = >100 per HPF S.G. 1.015 (almost all dysmorphic) Red cell casts present. Blood +++ Questions: pH 6.5 - What is the most likely diagnosis in this Protein case? + - Does the presence of red cell casts help you Urobilinogen 1.0 mg/dL in any way? - If the erythrocytes were not dysmorphic Nitrite Negative would that change your diagnosis? L.E. Negative
  • 97. Case “Functional” proteinuria? Microscopic may reveal a few leukocytes 6 Request protein concentration in 24 h urine Glucose Negative 22-year old man presenting for a routine Bilirubin Negative physical required for admission to medical Ketones Negative school S.G. 1.010 Physical characteristics: Yellow Microscopic: Not performed Blood Negative Questions: pH 5.0 - What is your differential diagnosis? Protein + - Would you order a microscopic analysis on this sample? Urobilinogen 0.2 mg/dL - What would you do next to confirm the diagnosis? Nitrite Negative L.E. Negative
  • 98. Common Findings in: Acute Tubular Necrosis Glucose Bilirubin Ketones S.G. Decreased Microscopic: Blood +/- • Renal tubular epithelial cells pH • Pathological casts Protein +/- Urobilinogen Nitrite L.E.
  • 99. Common Findings in: Acute Glomerulonephritis Glucose Bilirubin Ketones Microscopic: S.G. Blood Increased • Erythrocytes (dysmorphic) pH • Erythrocyte casts • Mixed cellular casts Protein Increased Urobilinogen Nitrite L.E.
  • 100. Common Findings in: Chronic Glomerulonephritis Glucose Bilirubin Ketones Decreased Microscopic: S.G. Blood Increased • Pathological casts pH (broad waxy casts, RBCs) Protein Increased Urobilinogen Nitrite L.E.
  • 101. Common Findings in: Acute Pyelonephritis Glucose Bilirubin Ketones Microscopic: S.G. • Bacteria Blood • Leukocytes pH • Leukocyte, granular, and Protein Trace waxy casts • Renal tubular epithelial Urobilinogen cell casts Nitrite Positive L.E. Positive
  • 102. Common Findings in: Nephrotic Syndrome Glucose Bilirubin Ketones S.G. Microscopic: Blood • Oval fat bodies pH • Fatty casts Protein ++++ • Waxy casts Urobilinogen Nitrite L.E.
  • 103. Common Findings in: Eosinophilic Cystitis Glucose Bilirubin Ketones S.G. Microscopic: Blood + • Numerous eosinophils pH (Hansel’s stain) Protein • NO significant casts. Urobilinogen Nitrite L.E.
  • 104. Common Findings in: Urothelial Carcinoma Glucose Bilirubin Ketones S.G. Microscopic: Blood + • Malignant cells on pH urine cytology (urine Protein sample should be submitted separately to cytology, void Urobilinogen or 24 hrs.) Nitrite L.E.
  • 105. Acknowledgment: Dr. Brad Brimhall