Chemical Examination of Urine Ricki Otten MT(ASCP)SC uotten@unmc.edu CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Objectives: Review the objectives on page 1 and 2 of the lecture handout Objectives marked with ‘*’ will not be tested over during student lab rotation CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Historical Perspective: Urinalysis Physical examination of urine Odor Taste Color Clarity CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Historical Perspective Chemical examination of urine Limited reactions Required large volumes of urine Large volumes of reagent Performed in test tubes Time consuming and cumbersome Clinical usefulness was not realized Not routinely ordered CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Historical Perspective Microscopic examination of urine Not until invention of the microscope Then clinical usefulness realized CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Reagent Strip Testing Technology and necessity Chemical reactions ‘miniaturized’ Required less urine Test results within minutes Easy to perform Increased test utilization Brunzel, 2nd Ed, page 124 CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Reagent Strip Testing Ideal qualitative screening tool Sensitive: Low concentration of substances Negative result = normal Specific: Reacts with only one substance False negative and false positive Cost effective: Relatively inexpensive tool that provides information about the health status of the patient CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Reagent Strip Testing Chemically impregnated absorbent pads attached to an inert plastic strip Each pad is a specific chemical reaction that takes place upon contact with urine Chemical reaction causes the color of the pad to change Color compared to a color chart for interpretation CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Reagent Strip Testing Qualitative or semi-quantitative results Concentration units (mg/dl) Negative, small, moderate large Negative, 1+, 2+, 3+, 4+ Timing of chemical reactions is CRITICAL Shortest time requirement on one end of strip: 30 sec Longest time requirement on the other: 2 min CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Reagent Strip Testing Principle of chemical reactions False negative reactions False positive reactions Color interferences Alternative testing: used to confirm results that you may think are invalid due to Interfering substance Color interference (called color masking) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Confirmatory Testing (pg 6) Care and Storage (pg 4) Reading assignment: Textbook, chapter 7 Page 124-130 Confirmatory Testing (pg 6) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Confirmatory Testing Alternative testing establishes the correctness or accuracy of another procedure Often used when urine is highly pigmented Bilirubin reagent strip  ictotest CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Confirmatory Testing Ideallywant all 3 Characteristics: Differ in sensitivity Ictotest vs Bilirubin reagent strip Differ in specificity SSA vs Protein reagent strip Clinitest vs Glucose reagent strip Differ in methodology/reaction Ideallywant all 3 CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Differ in Specificity Clinitest reacts with all reducing substances Glucose reagent strip reacts with only one reducing substance: glucose CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Causes of invalid results 10 reagent strip tests Specific gravity pH Protein Glucose Ketones Blood Bilirubin Urobilinogen Nitrite Leukocyte Esterase Purpose of the test What is normal What is abnormal Reaction Causes of invalid results CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Specific Gravity: Purpose Evaluates the concentrating and diluting ability of the kidney Density is related to the amount of substances (solutes) in solution Increased density ~ increased solute in solution ~ hypertonic urine ~ concentrated urine Decreased density ~ decreased solute in solution ~ hypotonic urine ~ dilute urine CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Specific Gravity: Normal Majority of urines: 1.010 – 1.025 Physiologically impossible: 1.000 >1.040 Dependent upon hydration status CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Specific Gravity: Terms Isosthenuria Fixed at 1.010 Renal tubules lost absorption and secreting capability Hypersthenuria Increased specific gravity Concentrated urine Hyposthenuria Decreased specific gravity Dilute urine Sensitivity issues: Pregnancy testing Urinary tract infection CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Specific Gravity: Methods Methods of measurement Reagent strip test: indicates ionic solutes Refractometer: indicates amount of total solutes Two functions of the kidney Maintain water balance Maintain electrolyte homeostasis Performed by renal tubules through concentrating and diluting; reabsorbing and secreting water and electrolytes (ionic) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Specific Gravity: Reaction Based on a change in the pKa of a polyelectrolyte on the reagent pad Increased ions in solution causes the polyelectrolyte on the pad to produce free H+ Free H+ cause a change in pH on the reagent pad Change in pH: bromthymol blue indicator CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Specific Gravity: Reaction CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Specific Gravity Sensitivity: 1.000 Specificity: detects only ionic substances Radiographic dye Mannitol Glucose Does not interfere CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

pH: Purpose Kidneys regulate body’s acid-base balance by selective handling of H+ and HCO3- Urine pH reflects acid-base status of body Treatment protocol may require urine pH be maintained at a specific pH (Aids in identification of crystals (microscope)) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

pH: Normal Normal: ranges from 4.5 – 8.0 First morning void: acidic Physiologically impossible: <4.5 >8.0 Urine not handled properly Old urine Treatment induced CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

pH: Interpretation Made in conjunction with Acid-base status Renal function Presence of infection in urinary tract Diet: high protein, low protein Medications Age of urine sample CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

pH: Abnormal Acid Alkaline Respiratory acidosis High protein diet Starvation UTI Alkaline Respiratory alkalosis Vegetarian diet Renal tubular acidosis UTI CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

pH: Reaction Double indicator system Methyl red Bromthymol blue Amount of free H+ influences acidity of urine and cause pH indicator to change color Needed to measure the wide pH range: acid to alkaline CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

pH: Invalid test results due to: Improper handling of urine sample Contamination of urine vessel prior to collection ‘Run-over’ phenomenon CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: Purpose Normal kidneys secrete LITTLE protein <15 mg/dl (or <150 mg/24 hours) The protein that is found in urine comes from Bloodstream Urinary tract Proteinuria is an indicator of early renal disease Proteinuria also caused by non-renal disease CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Renal Cause of Proteinuria: Glomerular damage: Most serious cause of proteinuria Most common cause of proteinuria Glomerulonephritis Nephrotic Syndrome Tubular dysfunction: Reabsorption capability decreased Toxin exposure, inherited disorder Fancon’s syndrome: heavy metal poisoning CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Classification of Proteinuria Functional Orthostatic (postural) Transient Pathologic Pre-renal (overflow) Renal: glomerular Renal: tubular Post-renal CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: Methods Reagent strip test SSA test Foam test Micro-albumin test CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: Reagent Strip The reagent pad is held at a constant pH of 3 by a buffer Proteins (anions) in solution cause an indicator dye to release H+ causing a color change ‘Protein error of indicators’ CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: Reagent Strip Sensitivity: ~ 10-25 mg/dl Specificity: reacts with albumin False positive: highly alkaline urine (pH > 8.0) False negative: Dilute urine Presence of other proteins (Tamm-Horsfall, globulins, myoglobin, free light chains, hemoglobin) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: SSA (Exton’s Test) Sulfosalicylic Acid (SSA) Precipitation Test Acid will precipitate proteins out of solution causing the solution to become cloudy Amount of cloudiness is related to the amount of protein present CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: SSA (Exton’s Test) Amount of cloudiness is evaluated, thus must use centrifuged urine Sensitivity: 5-10 mg/dl Specificity: detects all protein CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: SSA (Exton’s Test) False positive results: Radiographic dyes Turbid urine Uncentrifuged urine False negative results: Highly alkaline urine Dilute urine CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: Foam test Shake aliquot of urine and observe color of resulting foam White foam: protein present CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Protein: Micro-albumin test Measures very low concentration of albumin (better sensitivity than reagent strip test for albumin) Management of diabetic patient Methods vary: reagent strip test, immunochemical reaction CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Glucose: Purpose Healthy normal urine does not contain glucose Normally, glucose is filtered by the glomerulus and is reabsorbed back into the bloodstream through active transport mechanism Glucose in urine is pathologic CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Glucose: Purpose Glucosuria Glycosuria Caused by renal and non-renal disease Pre-renal glycosuria: plasma glucose level exceeds renal threshold (diabetes mellitus) Renal glycosuria: plasma glucose level below renal threshold, but tubules cannot reabsorb glucose back into bloodstream Terms used interchangeably CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Reducing Substances: Purpose Glucose Other sugars: galactosemia (inherited metabolic disorder) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Glucose, Reducing Substances Normal: negative Abnormal: Diabetes mellitus: glucose Impaired renal tubular reabsorption: glucose Inborn error of metabolism: galactosemia CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Methods Reagent strip: detects only glucose Copper Reduction: detects reducing substances CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Glucose: Reagent Strip Detects only glucose Double sequential enzyme reaction CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Glucose: Reagent Strip Sensitivity: ~ 30 mg/dl Specificity: Reacts only with glucose False positive: Strong oxidizing agents (bleach) Peroxides False negative: Ascorbic acid (reducing agent) Improperly stored urine: glycolysis CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Clinitest Reaction Copper Reduction Test: Reducing substances are able to reduce copper sulfate to cuprous oxide Pass-through phenomenon All children <2 years: metabolic disorder (galactosemia) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Clinitest Reaction Sensitivity: ~ 250 mg/dl Specificity: Reacts with all reducing substances Reducing sugars: glucose, galactose, fructose, lactose, maltose (NOT SUCROSE) False positive: any reducing substance (Ascorbic acid) False negative: radiographic dye CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Ketones: Purpose Ketones are intermediary products of fat metabolism CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Ketones Three ketone bodies Characteristic ‘fruity breath’ ~ acetone Acetoacetic acid 20% Beta-hydroxybutyric acid 78% Characteristic ‘fruity breath’ ~ acetone CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Ketones: Normal Normal: negative Abnormal: Inability to utilize carbohydrates Excessive loss of carbohydrates Inadequate intake of carbohydrates CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Ketones: Methods Reagent strip Acetest: tablet test CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Ketones: Method Glycine: also measures acetone Reagent strip: check package insert Acetest tablets: contain glycine CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Ketones Reagent strip Acetest Sensitivity: 5-10 mg/dl Specificity: acetoacetic acid and/or acetone False positive: highly pigmented urine False negative: improper specimen handling Acetest Specificity: acetoacetic acid and acetone False positive: highly pigmented urien CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Blood: Purpose Blood in urine indicates pathology Two forms found in urine Intact RBC Hemolyzed RBC CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Blood: Terms Hematuria Hemoglobinuria Myoglobinuria All will give a positive blood reaction CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Blood: Reagent strip Test can detect hemolyzed RBC Heme moiety imparts peroxidase activity and catalyzes the reaction CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Blood Sensitivity Specificity Intact RBC Hemolyzed RBC (hemoglobin) Myoglobin False positives: myoglobin, oxidizing agents False negatives: ascorbic acid CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Blood: Correlate reagent strip results Microscopic findings Color and clarity CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Bilirubin and Urobilinogen Bilirubin in urine is always pathologic: liver disease Urobilinogen in urine: normal to have a small amount: 0.2 – 1.0 mg/dl CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Three mechanisms Pre-hepatic: liver is healthy Hepatic: liver disease Post-hepatic: liver is healthy, obstruction indicated CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Bilirubin: Methods Reagent strip Ictotest: tablet test Foam test CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Bilirubin: Methods Reagent strip Ictotest: tablet test Same reaction Same specificity: conjugated bilirubin False positive: urine color False negative: low concentration, ascorbic acid, improper specimen handling CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Bilirubin: Methods Reagent strip Ictotest: tablet test Sensitivity differs Reagent strip: ~0.5 mg/dl Ictotest: 0.05 – 0.1 mg/dl CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Bilirubin: Methods Possible to have a negative reagent strip test and positive ictotest Difference in sensitivity levels Always perform Ictotest when Urine bilirubin test specifically ordered Urine appearance is amber: even if bilirubin reagent strip test is negative Positive reagent strip test CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Bilirubin: Foam Test Shake urine and observe resulting foam Yellow foam = bilirubin CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Urobilinogen: Methods Reagent strip test Two reactions dependent upon manufacturer Para-dimethylaminobenzaldehyde Diazonium salt Cannot determine absence of UBG Watson-Schwartz assay CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Urobilinogen: Methods Para-dimethylaminobenzaldehyde Sensitivity: 0.2 mg/dl Specificity: False positive: any ‘Ehrlich reactive compound’; color masking; urine at body temp False negative: improper specimen handling Diazonium salt Sensitivity: 0.4 mg/dl Specificity: reacts only with UBG False positive: color masking CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Urobilinogen: Watson Schwartz Classic method used to differentiate urobilinogen from porphobilinogen using a differential extraction method Para-dimethylaminobenzaldehyde CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Nitrite: Purpose Bacteria that contain a specific enzyme can reduce dietary nitrates to nitrites Rapid screening test for UTI CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Nitrite: Normal Normal: negative Abnormal: Cystitis: bladder Pyelonephritis: kidney CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Nitrite: Method Reagent strip test Nitrite + aromatic amine  diazonium salt Diazonium salt + aromatic compound  pink color Sensitivity: 0.06-0.1 mg/dl nitrite ~ 10,000 organisms CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Nitrite: Method Reagent strip test Specificity: False positive: improper specimen handling; color masking False negative: bacteria cannot reduce nitrates Bladder time not sufficient: need 4 hours Low nitrite levels Ascorbic acid Antibiotic inhibition of bacteria Further reduction of nitrites to nitrogen CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Leukocyte Esterase: Purpose Increased WBC in urine is pathologic Indicates inflammation, infection Neutrophils most common type of WBC found in urine Can detect intact WBC and lysed WBC CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Leukocyte Esterase: Normal Normal: negative Abnormal: Bacterial infection: cystitis, pyelonephritis, urethritis Non-bacterial infection: yeast, trichomonas CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Leukocyte Esterase: Method Reagent strip: Granules in cytoplasm of WBC contain an enzyme (esterase) Ester –esterase aromatic compound Aromatic compound + diazonium salt  Purple colored complex CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

Leukocyte Esterase Sensitivity: 5-15 WBC/hpf Specificity: False positive: vaginal contamination; color masking False negative: strong oxidizing agents (bleach); lymphocytes (no granules) CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine

CLS 426 Urine & Body Fluid Analysis; Student Lab Rotation: Chemical Exam of Urine