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
Urine Specimens Collected for a number of tests: – Clean voided specimens For routine urinalysis – Clean-catch or midstream urine specimens For urine culture – Timed urine specimens For a variety of tests that depend of the client’s specific health problem
Obtaining Samples Clients need varying degrees of instruction and assistance to provide clean voided specimens. About 120 ml (4 oz) of urine is generally required. Clients who are seriously ill, physically incapacitated, or disoriented may need to use a bedpan or urinal in bed. Others may require supervision and/or assistance in the bathroom.
Directions for Collection The nurse should explain that all specimens must be free of fecal contamination, so voiding needs to occur at a different time from defection. Instruct female clients to discard toilet tissue in the toilet or in a waste bag rather than in the bedpan or hat, since tissue in the specimen makes laboratory analysis more difficult.
Obtaining Specimen When the specimen is obtained, put the lid tightly on the container to prevent spillage and contamination. Label the specimen.
Obtaining TimedSpecimens All timed urine specimens should be refrigerated to prevent bacterial growth and decomposition of the urine components. Each voiding of urine is collected in a clean container and then emptied immediately into the large refrigerated bottle.
Obtaining TimedSamples Alert signs are placed in the client’s room to remind staff of the test in progress. Specimen identification labels need to indicate the date and time of each voiding in addition to the usual identification information.
Obtaining TimedSamples Clients need to be told why the test is being done and how they can assist. Instructions should include the following: – All urine must be saved and placed in the specimen containers once the test starts. – The urine must be free of fecal contamination and toilet tissue. – Each specimen must be given to the nursing staff immediately so that it can be placed in the appropriate specimen bottle.
Obtaining TimedSamples The collection period is started by having the client void in the toilet, bedpan, or urinal. – This urine is usually discarded, but check facility procedure. All subsequent urine specimens are collected.
SpecimenCollection Supra-pubic Needle Aspiration
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.
Appearance Including color and clarity Color : normally , pale to dark yellow (urochrome) Abnormal color : some drugs cause color changes 1. red urine : causes: hematuria hemoglobinuria myoglobinuria 2. yellow-brown or green-brown urine : bilirubin cause : obstructive jaundice
Red Urine Causes of Asymptomatic Gross Hematuria by Incidence Acute Cystitis (23%) Bladder Cancer (17%) Benign Prostatic Hyperplasia (12%) Nephrolithiasis (10%) Benign essential hematuria (10%) Prostatitis (9%) Renal cancer (6%) Pyelonephritis (4%) Prostate Cancer (3%) Urethral stricture (2%)
Appearance Clarity : normally, clear Abnormal color : cloudy urine Causes: 1. crystals or nonpathologic salts phosphate, carbonate in alkaline urine (dissolve---add acetic acid) uric acid in acid urine (dissolve---warming to 60℃) 2. various cellular elements: leukocytes, RBCs, epithelial cells
Urine volume The average adult : 1000ml to 2000ml/24h Increase polyuria ---more than 2000ml of urine in 24 hours 1. physiological states: water intake, some drugs, intravenous solutions 2. pathologic states: diabetes mellitus, diabetes insipidus
Urine volume Decrease Oliguria ---less than 400ml of urine in 24 hours Anuria ---less than 100ml of urine in 24 hours 1. prerenal: hemorrhage, dehydration, congestive heart failure 2. postrenal: obstruction of the urinary tract (may be stones, carcinoma) 3. renal parenchymal disease: acute tubular necrosis, chronic renal failure
Chemical Analysis Urine Dipstick Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase
Typical Test Strip Test Sensitivity_ Glucose – 4 to 7 mmol/L Bilirubin – 7 to 14 µmol/L Ketone – 0.5 to 1.0 mmol/L (Acetoacetic acid) Blood – 150 to 620 µg/L (Hemoglobin) Protein – 0.15 to 0.3 g/L (Albumin) Nitrite – 13 to 22 µmol/L Leukocytes – 5 to 15 cells/ µL pH – pH 5.0 to 8.5Specific Gravity – 1.000 to1.030 Urobilinogen – 0.2 to 8 µmol/L BioMedica Diagnostics Inc. / D. Jette / March 25 2003
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%
The UrineDipstick: 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 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 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.
Cytology: Polyoma (DecoyCell)Immunoperoxidase to SV40 ag
Cytology: TCC Low Grade
Cytology: TCC Low Grade
Cytology: TCC HighGrade
Cytology: TCC HighGrade
Cytology: Squamous CellCa.
Cytology: Renal Cell Ca.
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 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.