CHEMICAL ANALYSIS OF URINE

Chemical Analysis involves the study of the chemical components of a sample. may involve enzymatic & colorimetric methods of determination. Reagent Strips Provide a simple means of performing medical significant chemical analysis including: pH Protein Glucose Ketones Blood Bilirubin Urobilinogen Nitrite Leukocytes Specific gravity CHEMICAL ANALYSIS: pH Acid Urine Emphysema Diabetes mellitus Starvation Dehydration Diarrhea Presence of acid-producing bacteria (Escherichia coli) High-protein diet Cranberry juice Medications (methenamine mandelate [Mandelamine], fosfomycin tromethamine) Alkaline Urine Hyperventilation Vomiting Renal tubular acidosis Presence of urease-producing bacteria Vegetarian diet Old specimens

pH 5 - yellow and green pH 9 - final deep blue

CHEMICAL ANALYSIS: Protein Test: Protein Normal: Negative-trace Principle: Protein-error of indicator Significance: Renal Disease Source of Error: False-positive: highly buffered or alkaline urine, prolonged dipping. False-negative: Proteins other than albumin Comments: Buffered to maintain pH 3. Most sensitive to albumin. Blood, WBCs, bacteria can cause positive reaction: Orthostatitc proteinuria: a benign condition in which protein is negative in the first AM specimen and positive after standing. Summary of Clinical Significance of Urine Protein Prerenal Prerenal Intravascular hemolysis Muscle injury Acute phase reactants Multiple myeloma Renal Glomerular disorders Immune complex disorders Menstrual contamination Amyloidosis Toxic agents Diabetic nephropathy Strenuous exercise Dehydration Hypertension Pre-eclampsia Orthostatic or postural proteinuria Urine Protein Dipstick Readings Negative Trace 1 2 3 4 Indicator + Protein Yellow pH 3.0 Semiquantitative Values < 30 mg/dL 30 100 300 2000 Protein + H+ Indicator is H+ Blue-green Tubular Disorders Fanconi syndrome Toxic agents/heavy metals Severe viral infections Postrenal Lower urinary tract infections/Inflammation Injury/trauma Prostatic fluid/spermatozoa Vaginal secretions

Summary of Clinical Significance of Urine pH 1. Respiratory or metabolic acidosis/ketosis 2. Respiratory or metabolic alkalosis 3. Defects in renal tubular secretion and reabsorption of acids and basesrenal tubular acidosis 4. Renal calculi formation 5. Treatment of urinary tract infections 6. Precipitation/identification of crystals 7. Determination of unsatisfactory specimens Urine pH Test: pH Normal: First AM: 5-6 o Random: 4.5-8 Principle: Double Buffer System Significance: Useful in evaluation of acid-base balance, management of UTI and renal calculi Source of Error: Decreased: Acid run over from protein square. Increased: Specimen left at room temperature too long Comments: Acid with protein/ meat diet. Alkaline with vegetarian diet. Methyl red - pH range 4 to 6 Bromthymol blue - pH range 6 to 9 Methyl red H Bromthymol blue H (Red-Orange Yellow) (Green Blue) pH range 5 to 9 - orange

Reagents Multistix: Tetrabromphenol blue Chemstrip: 3', 3'', 5', 5'' tetrachlorophenol

3, 4, 5, 6-tetrabromosulfophthalein

Sensitivity Multistix: 1530 mg/dL albumin Chemstrip: 6 mg/dL albumin

Clinical 1. 2. 3. 4. 5. 6. 7. Results Results

Test: Ketones Normal: Negative Principle: Sodium nitro-prusside reaction Significance: Increased fat metabolism, e.g., diabetes mellitus, vomiting, starvation, low carbohydrate diet. Source of Error: Decreased in improperly stored specimens Comments: Most sensitive to acetoacetic acid Significance of Urine Ketones Diabetic acidosis Insulin dosage monitoring Starvation Malabsorption/pancreatic disorders Strenuous exercise 6.Vomiting Inborn errors of amino acid metabolism are reported qualitatively as: negative, trace, small (1), moderate (2) or large (3), are reported semiquantitatively: negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), large (80 to 160 mg/dL) Alkaline Purple Colored Complex

CHEMICAL ANALYSIS: Urine Glucose Test: Glucose Normal: Negative Principle: Glucose oxidase/peroxidase Significance: Diabetes mellitus Source of Error: False-Postive: Contamination with peroxide or oxidizing detergents (bleach). False-negative: High levels of ascorbic acid, glycolysis. Comments: Specific for glucose. More sensitive and specific than copper reduction test. For diabetic monitoring, specimen collected 2 hours after eating is preferred. Normal renal threshold = 160-180 mg/dL. Summary of Clinical Significance of Urine Glucose Hyperglycemia-Associated Diabetes mellitus Pancreatitis Pancreatic cancer Acromegaly Cushing syndrome Hyperthyroidism Pheochromocytoma Central nervous system damage Stress Gestational diabetes Renal-Associated Fanconi syndrome Advanced renal disease Osteomalacia Pregnancy

Acetoacetate + Sodium nitroprusside + (glycine and acetone)

Reagent Strip (Glucose Oxidase) Reactions Glucose + O2 (air) glucose oxidase gluconic acid + H2O2 peroxidase

Summary of Ketone Reagent Strip Reagents Sodium nitroprusside Glycine (Chemstrip) Sensitivity Multistix: 510 mg/dL acetoacetic acid Chemstrip: 9 mg/dL acetoacetic acid; 70 mg/dL acetone CHEMICAL ANALYSIS: Urine Blood Test: Blood Normal: Negative Principle: Peroxidase-like activity of hemoglobin Significance: Renal calculi, glomerular disease, tumors, trauma, pyelonephritis, hemolytic anemia, hemolytic transfusion reaction, burns, infections, strenuous exercise Source of Error: Decreased: High levels of ascorbic acid, nitrites, protein, specific gravity. Failure to mix specimen. False positive: Menstruation, oxidizing detergents, bacterial peroxidase. Comments: Detects RBCs, hemoglobin, and myoglobin (muscle destruction) Summary of Clinical Significance of a Positive Reaction for Blood Hematuria 1. Renal calculi 2. Hemoglobinuria 1.Transfusion reactions Myoglobinuria 1. Muscular trauma/

H2O2 + chromogen chromogen + H2O

Oxidized colored

Summary of Glucose Reagent Strip Reagents Multistix: Glucose oxidase, Peroxidase, Potassium iodide (green to brown) Chemstrip: Glucose oxidase, Peroxidase, Tetramethylbenzidine (yellow to green) Sensitivity Multistix: 75125 mg/dL Chemstrip: 40 mg/dL CHEMICAL ANALYSIS: Ketones

Glomerulonephritis 3. Pyelonephritis 4.Tumors 5.Trauma 6. Exposure to toxic chemicals 7. Anticoagulants 8. Strenuous exercise

2. Hemolytic anemias 3. Severe burns 4. Infections/malaria 5. Strenuous exercise/ red blood cell trauma 6. Brown recluse spider bites

crush syndromes 2. Prolonged coma 3. Convulsions 4. Muscle-wasting diseases 5. Alcoholism /overdose 6. Drug abuse 7. Extensive exertion 8. Cholesterollowering statin medications

Principle: Ehrlichs reaction (-dimethyl-aminobenzaldehyde) Significance: Liver disease, hemolysis Source of Error: False-positive: Porphobilinogen (with some brands of reagent strips) Comments: Reagent strips do not detect absence of urobilinogen, only increase.

Summary of Clinical Significance of Urine Urobilinogen 1. Early detection of liver disease 2. Liver disorders, hepatitis, cirrhosis, carcinoma 3. Hemolytic disorders Reagent Strip Summary for Urobilinogen Reagents Multistix: p-dimethylaminobenzaldehyde Chemstrip: 4-methoxybenzenediazoniumtetrafluoroborate Sensitivity Multistix: 0.2 mg/dL urobilinogen Chemstrip: 0.4 mg/dL urobilinogen Watson-Schwartz Differentiation Test 1. Label 2 tubes #1 and #2 Tube 1 2 mL urine 2 mL chloroform 4 mL sodium acetate Tube 2 2 mL urine 2 mL butanol 4 mL sodium acetate 2. Vigorously shake both tubes. 3. Place in a rack for layers to settle. 4. Observe both tubes for red color in the layers. Interpretation: Tube 1 Upper layer=urine; if colorless= porphobilinogen or Ehrlich-reactive compounds. Bottom layer=chloroform; if red=urobilinogen. If both layers are red re-extract the urine layer from tube 1. Place 2 mL of urine layer from tube 1 and 2 mL chloroform and 4 mL sodium acetate into a new tube. Repeat procedure. Interpretation: Upper layer urine colorless Bottom layer chloroformred =excess urobilinogen Both layers red =porphobilinogen and urobilinogen Tube 2 Upper layer =butanol If red =urobilinogen or Ehrlich-reactive compounds Bottom layer =urine If colorless =porphobilinogen CHEMICAL ANALYSIS: Urine Nitrite Test: Nitrites Normal: Negative Principle: Greiss reaction Significance: Urinary Tract Infection Source of Error: False-negative: Non-nitritereducing bacteria, insufficient dietary nitrate, high levels of ascorbic acid, some antibiotics, reduction of nitrites to nitrogen, insufficient bladder incubation. False-positive: Bacterial contamination, medications that color urine red Comments: Test first AM specimen.

Hemoglobin Peroxidase H2O2 + Chromogen Oxidized Chromogen + H2O *Pseudoperoxidase activity of hemoglobin Reagents Multistix: Diisopropylbenzene dehydroperoxide tetramethylbenzidine Chemstrip: dimethyldihydroperoxyhexane tetramethylbenzidine Sensitivity Multistix: 520 RBCs/mL, 0.0150.062 mg/dL hemoglobin Chemstrip: 5 RBCs/mL, hemoglobin corresponding to 10 RBCs/mL CHEMICAL ANALYSIS: Bilirubin Test: Bilirubin Normal: Negative Principle: Diazo reaction Significance: Liver disease, biliary obstruction Source of Error: False-negative: Exposure to light, oxidation to biliverdin, hydrolysis of bilirubin diglucuronide, high levels of ascorbic acid or nitrites, drugs causing atypical colors. False-positive: Urine pigments Comments: only conjugated bilirubin is excreted in urine Summary of Clinical Significance of Urine Bilirubin 1. Hepatitis 2. Cirrhosis 3. Other liver disorders 4. Biliary obstruction (gallstones, carcinoma) Reagent Strip (Diazo) Reactions Reagents Multistix: 2,4-dichloroaniline diazonium salt Chemstrip: 2,6-dichlorobenzene-diazonium salt Sensitivity Multistix: 0.40.8 mg/dL bilirubin Chemstrip: 0.5 mg/dL bilirubin

Urine Bilirubin and Urobilinogen in Jaundice Urine Bilirubin Bile Duct Obstruction Liver Damage Hemolytic Disease +++ + or Negative Urine Urobilinogen Normal ++ +++

CHEMICAL ANALYSIS: Urine Urobilinogen Test: Urobilinogen Normal: 1 mg/dL or 1 Ehrlich unit

Summary of Clinical Significance of Urine Nitrite 1. Cystitis 2. Pyelonephritis 3. Evaluation of antibiotic therapy 4. Monitoring of patients at high risk for urinary tract infection 5. Screening of urine culture specimens

Sensitivity Multistix: 515 WBC/hpf Chemstrip: 1025 WBC/hpf

The Reagent Strip Color Comparison Chart

CHEMICAL ANALYSIS: Urine Specific Gravity Test: Specific gravity Normal: Random specimen: 1.003-1.030 Principle: PKa change of polyelectrolyte Significance: Indication of kidneys concentrating ability and state of hydration Source of Error: Increased: Protein. Decreased: Alkaline urine. Comments: Measures ionizable substance only, not specific gravity by refractometer. *Sources of error may vary with brand of reagent strip. Refer to manufacturers package insert. Clinical 1. 2. 3. 4. Significance of Urine Specific Gravity Monitoring patient hydration and dehydration Loss of renal tubular concentrating ability Diabetes insipidus Determination of unsatisfactory specimens due to low concentration

Reagents Multistix: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue Chemstrip: Ethyleneglycoldiaminoethylethertetraacetic acid, bromthymol blue Sensitivity= 1.0001.030 Specific gravity blue (1.000 [alkaline]).shades of green .yellow (1.030 [acid]) CHEMICAL ANALYSIS: Urine Leukocyte Esterase Test: Leukocyte esterase Normal: negative Principle: Granulocytic esterase reaction Significance: Urinary tract infection Source of Error: False-positive: Oxidizing agents. Decreased reaction: High glucose, protein, specific gravity, or ascorbic acid. Comments: Will detect intact and lysed polys. Lymphos do not react. Summary of Clinical Significance of Urine Leukocytes 1. Bacterial and nonbacterial urinary tract infection 2. Inflammation of the urinary tract 3. Screening of urine culture specimens Reagents Multistix: Derivatized pyrrole amino acid ester Diazonium salt Chemstrip: Indoxylcarbonic acid ester Diazonium salt Confirmatory/ Supplement Urine Chemistry Tests Sulfosalicylic Acid Test

Test: Sulfosalicylic acid Substance(s) Detected: Protein Principle: Acid precipitation Sources of Error: False-positive: Radiographic dyes, tolbutamide, some antibiotics, turbid urine. False-negative: Highly buffered alkaline urine. Comments: Detects all proteins, including Bence Jones proteins.

Copper Reduction Test (Benedicts Test)

Test: Watson-Schwartz Test Substance(s) Detected: Urobilinogen, porphobilinogen Principle: Ehrlichs aldehyde reaction Sources of Error: Decreased: Exposure to light, more than 1 hour at room temperature. Falsepositive: Warm aldehyde reaction. (Urine should be at room temperature.) Comments: Collect specimen from 2-4 PM. Store in dark. Urobilinogen is soluble in chloroform and butanol. Porphobilinogen is not soluble in either. Test: Hoesch Test Substance(s) Detected: Porphobilinogen Principle: Ehrlichs aldehyde reaction Sources of Error: Similar to Watson-Schwartz Comments: Urobilinogen doesnt react unless very high.

Test: Clinitest Substance(s) Detected: Reducing substances Principle: Copper reduction Sources of Error: False-positive: High levels of ascorbic acid. False-negative: Glycolysis, pass through. (Color goes through orange and returns to blue or blue-green. Repeat using two-drop method and two-drop color chart.) Comments: Non-specific. Reacts with glucose, galactose, fructose, maltose, lactose. (Sucrose is not re-ducing sugar.) Test all infants to diagnose galactosemia. Not as sensitive for glucose as reagent strip. Self-heating method. Perform in rack to avoid burning. Acetest

Effect of High Levels of Ascorbic Acid on Urinalysis Tests *May vary with brand of reagent strip. Refer to manufacturers package insert. False-positive Clinitest False-Negative or Decrease Glucose Blood Bilirubin Nitrite Leukocyte esterase

Test: Acetest Substance(s) Detected: Ketones Principle: Sodium nitroprusside reaction Sources of Error: False-negative: Improperly stored specimen Comments: Most sensitive to acetoacetic acid Ictotest

Test: Ictotest Substance(s) Detected: Bilirubin Principle: Diazo reaction Sources of Error: Decreased: Exposure to light, improperly stored specimen, high levels of ascorbic acid, nitrites. False-positive: Urine pigments. Comments: More sensitive than reagent strip. Less affected by interfering substances.