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
SpecimenCollection– 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
Types of Analysis − Macroscopic Examination − Chemical Analysis (Urine Dipstick) − Microscopic Examination − Culture (not covered in this lecture) − Cytological Examination
Macroscopic ExaminationOdor:− Ammonia-like: (Urea-splitting bacteria)− Foul, offensive: Old specimen, pus or inflammation− Sweet: Glucose− Fruity: Ketones− Maple syrup-like: Maple Syrup Urine DiseaseColor:− 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 ExaminationTurbidity:− 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.
Uses and Limitations of Urine GlucoseDetection 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* byCuSO 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 GlucoseUrinalysis Glucose Result ++ + trace Negative 200 400 600 800 1000 Blood Glucose (mg/dL)
The UrineDipstick: Bilirrubin Negative Chemical Principle + (weak) Bilirubin + Diazo salt Acidic Azobilirubin ---------> ++ (moderate) Read at 30 seconds +++ (strong) RR: Negative
Uses and Limitations of Urine BilirrubinDetectionSignificance - 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 bilirubinOther Tests - Ictotest (more sensitive tablet version of same assay) - Serum test for total and direct bilirubin is more informative
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 UrineDipstick: 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 GravitySignificance - Diabetes insipidusLimitations - 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 UrineDipstick: 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)
The UrineDipstick: 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 UrineDipstick: 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
Uses and Limitations of Urine ProteinDetection 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” UrineProtein % of Total Daily MaximumAlbumin 40% 60 mgTamm-Horsfall 40% 60 mgImmunoglobulins 12% 24 mgSecretory IgA 3% 6 mgOther 5%
Uses and Limitations of UrobilinogenDetection 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 UrineDipstick: 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
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 AspectsPreservation - 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 FindingsPer High Power Field (HPF) (400x) – > 3 erythrocytes – > 5 leukocytes – > 2 renal tubular cells – > 10 bacteriaPer 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 CellsErythrocytes - “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 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.
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 yourUrobilinogen 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 youUrobilinogen 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: 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, voidUrobilinogen or 24 hrs.) Nitrite L.E.