CHEMICAL EXAMINATION OF URINE January 24, 2018

LEARNING OBJECTIVES: Cranberry juice

 State the proper care and storage of commercial reagent Medications (methenamine
strip and cite atleast 3 potential causes of their mandelate [Mandelamine],
deterioration. fosfomycin tromethamine)
 Summarize the clinical significance of the following:
blood, L.E, nitrite, protein, glucose, ketones, bilirubin, Summary of Clinical Significance of Urine pH
urobilinogen and ascorbic acid. 1. Respiratory or metabolic acidosis/ketosis
 Compare and contrast the mechanisms for the clinical 2. Respiratory or metabolic alkalosis
significance of following types of proteinuria: Overflow 3. Defects in renal tubular secretion and reabsorption of
proteinuria, glomerular, postural, tubular, post renal acids and bases—renal tubular acidosis
proteinuria. 4. Renal calculi formation
5. Treatment of urinary tract infections
Chemical Analysis 6. Precipitation/identification of crystals
 involves the study of the chemical components of a 7. Determination of unsatisfactory specimens
sample.
 may involve enzymatic & colorimetric methods of Urine pH
determination.  Test: pH (potential Hydrogen)
 Normal: First AM: 5-6
Reagent Strips PRINCIPLE (reflectance Photometry) o Random: 4.5-8
 inert plastic strip onto which reagent-impregnated test  Principle: Double Buffer System/ indicator
pads are bonded.  Significance: Useful in evaluation of acid-base balance,
 Each reaction results in a color change that can be management of UTI and renal calculi
assessed visually or mechanically  Source of Error: Decreased: Acid run over from protein
 Provide a simple means of performing medical significant square.
chemical analysis including: Increased: Specimen left at room temperature
- pH too long
- Protein  Comments: Acid with protein/ meat diet. Alkaline
- Glucose with vegetarian diet.
- Ketones  During and after meal, urine produced less acidic “alkaline
- Blood tide”
- Bilirubin Three most common factor for Urine >8.0 pH:
- Urobilinogen 1. Improperly preserved urine (proliferation of urease-
- Nitrite producing bacteria)
- Leukocytes 2. Adulterated Urine
- Specific gravity 3. Taking of Highly alkaline substance (medications)

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

CHEMICAL ANALYSIS: pH

Acid Urine Alkaline Urine

Emphysema Hyperventilation
Diabetes mellitus Vomiting
Starvation Renal tubular acidosis
Dehydration Diarrhea Presence of urease-producing
Presence of acid-producing bacteria
bacteria (Escherichia coli) Vegetarian diet
High-protein diet Old specimens

DAYLE DANIEL G. SORVETO, RMT 1

CHEMICAL EXAMINATION OF URINE
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.
 Most indicative test for renal disease
 Normal urine contains up to 150mg (1 to 14mg/dL) each day
 Only small amount of albumin is present in normal urine.
 3 types of protein that originate from the urinary tract itself:
o 1. Uromodulin (tamm-horsfall protein) mucoprotein
synthesized in distal tubular cells and involved cast
formation.
o 2. Urokinase fibrinolytic enzyme secreted by tubular
cells.
o 3. IgA, synthesized by renal tubular epithelial cells.
Protenuria:
1. Increase in the quantity of plasma protein that are filtered
2. Filtering of the normal quantity of proteins but with
reduction in the reabsorptive ability of renal tubules.
Overflow proteinuria (pre-renal)
January 24, 2018
Increased quantities of plasma protein in the blood readily
passing through the glomerular filtration barriers into the
urine.
 Increased excretion of low-molecular-weight plasma
proteins.
 Monoclonal Immunoglobulin light chains (Bence Jones
Protein) coagulate at 40- 60 °C and redissolved at 100 °C
 Increase secretion of BJP in patients with multiple
myeloma.
Renal Proteinuria:
 Associated with true renal disease may be the result of
either glomerular or tubular damage
 Glomerular proteinuria; glomerular membrane is damage,
selective filtration is impaired and increased amounts of
serum protein, RBC, WBC pass through the membrane and
excreted to the urine.
o The proteinuria is usually heavy exceeding
2.5/day of total protein and can be as much as
20g/day.
Microalbuminuria: specific test for albumin only for the detection of
development of diabetic nephropathy (DM I or DM II) micral testing
Postural (orthostatic) proteinuria:
 Considered to be as functional proteinuria.
 Excretion of protein only when the individual is in upright
(orthostatic) position)
 The first morning urine specimen is normal in protein, and
during the day the urine protein is elevated in second
collection.
 When the px is in upright position, increased renal
venous pressure causes renal congestion and glomerular
changes.
Tubular Proteinuria:
 When tubular reabsorptive function is altered or impaired.
 Total urine protein concentration is less than 2.5g/day, w/
LMW-proteins is predominating
 Quantitative urine total protein method should be
employed or SSA precipitation test.
 Fanconi syndrome: This syndrome altered tubular
transport mechanism retains normal glomerular function.
Postrenal Proteinuria:
 Inflammation in Urinary tract, renal pelvis, Ureters,
bladder (Cystitis), prostate, urethra
 Renal hemorrhage.
 Vaginal secretions or seminal fluid can cause positive
protein or proteinuria.
DAYLE DANIEL G. SORVETO, RMT 2
CHEMICAL EXAMINATION OF URINE January 24, 2018

Summary of Clinical Significance of Urine Protein Sulfosallcylic Acid Precipitation:

Two methods:
Prerenal Tubular Disorders 1. 3%SSA to 3mL urine
Prerenal Fanconi syndrome 2. 7.0% SSA to 11 mL urine
Intravascular hemolysis Toxic agents/heavy metals a. Mix inversion and observed for
Muscle injury Severe viral infections cloudiness.(10mins)
Acute phase reactants b. Grade of turbidity.
Multiple myeloma c. Sensitive to 5-10mg/dL of protein in any type of
Renal Postrenal protein.
Glomerular disorders Lower urinary tract
Immune complex disorders infections/Inflammation
Menstrual contamination Injury/trauma
Amyloidosis Prostatic fluid/spermatozoa
Toxic agents Vaginal secretions
Diabetic nephropathy
Strenuous exercise
Dehydration
Hypertension
Pre-eclampsia
Orthostatic or postural
proteinuria

Urine Protein Dipstick

Readings Semiquantitative Values

Negative
Trace < 30 mg/dL
1 30
2 100
3 300
4 2000

CHEMICAL ANALYSIS: Urine Glucose

Reagents  Test: Glucose
Multistix:  Normal: Negative
 Tetrabromphenol blue  Principle: Glucose oxidase/peroxidase
Chemstrip:  Significance: Diabetes mellitus
 3', 3'', 5', 5'' tetrachlorophenol  Source of Error: False-Postive: Contamination with
 3, 4, 5, 6-tetrabromosulfophthalein peroxide or oxidizing detergents (bleach).
False-negative: High levels of ascorbic acid,
Sensitivity glycolysis.
 Multistix: 15–30 mg/dL albumin  Comments: Specific for glucose. More sensitive and
 Chemstrip: 6 mg/dL albumin specific than copper reduction test. For
diabetic monitoring, specimen collected 2
hours after eating is preferred. Normal renal
threshold = 160-180 mg/dL.
 Cause of glucosuria:
1. Pre-renal condition (hyperglycemia)
2. Renal condition (defective tubular absorption)

DAYLE DANIEL G. SORVETO, RMT 3

CHEMICAL EXAMINATION OF URINE January 24, 2018

Hyperglycemia-Associated Renal-Associated
Diabetes mellitus Fanconi syndrome
Pancreatitis Advanced renal disease
Pancreatic cancer Osteomalacia
Acromegaly Pregnancy
Cushing syndrome
Hyperthyroidism
Pheochromocytoma
Central nervous system
damage
Stress
Gestational diabetes

Reagent Strip (Glucose Oxidase) Reactions

Other glucose in urine:

 Galactose
o Most significant and can cause galactosemia
o Inherited disorder, in which unable to
metabolize galactose to glucose.
o Absent or deficit of Galactose 1-phosphate
uridyl transferase (GALT).
 Fructose
o Excess fruit or honey ingestion
 Lactose
o Increased concentration of toxic intermediate
products Galactonate and Galactitol can cause
brain damage.
 Maltose
 Pentose
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: 75–125 mg/dL
 Chemstrip: 40 mg/dL
Copper reduction tests (Benedicts test).
 Ability of reducing substances to convert cupric sulfate to
cuprous oxide
 Blue to green to orange.

Summary of Clinical Significance of Urine Glucose

DAYLE DANIEL G. SORVETO, RMT 4

CHEMICAL EXAMINATION OF URINE
Clinitest:
 Reagents: anhydrous copper sulfate, Sodium hydroxide,
citric acid and sodium bicarbonate.
 Read for 15 secs
 False low result due to “pass through phenomenon”
o High concentration and change back to low
concentration.
o Because of reoxidation of cuprous oxide to
cupric oxide
CHEMICAL ANALYSIS: Ketones
 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
Three intermediate products of fatty acid:
1. B-hydroxybutyrate 70%
2. Acetone 2%
3. Acetoacetate 20%
Renal threshold of ketones in blood = 70 mg/dL.
3 factors of increase ketone levels:
1. Inability to use carbohydrates
2. Inadequate carbohydrate intake
3. Loss of carbohydrates
“Only acetoacetate and acetone can be detected in urine reagent
strips.”
Clinical Significance of Urine Ketones
1. Diabetic acidosis
2. Insulin dosage monitoring
3. Starvation
4. Malabsorption/pancreatic disorders
5. Strenuous exercise
6. 6.Vomiting
7. Inborn errors of amino acid metabolism
Results are reported qualitatively as:
 negative, trace, small (1), moderate (2) or large (3),
Results are reported semiquantitatively:
 negative, trace (5 mg/dL), small (15 mg/dL),
 moderate (40 mg/dL), large (80 to 160 mg/dL)
January 24, 2018
Summary of Ketone Reagent Strip
Reagents
 Sodium nitroprusside
 Glycine (Chemstrip)
Sensitivity
 Multistix: 5–10 mg/dL acetoacetic acid
 Chemstrip: 9 mg/dL acetoacetic acid; 70 mg/dL acetone
ACETEST:
 Tablet test for detection of ketones in urine.
 More sensitive than reagent strip test.
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)
Hematuria: blood in the urine
 Cloudy or smoky urine
Hemoglobinuria: hemoglobin in urine
 Presence due to intravascular hemolysis
 Within the renal cells, ferritin is denatured to form
hemosiderin, it appears in urine 2 to 3 days after
Hemolytic episode and appear as yellow brown granules
1. Within sloughed Renal tubular cells.
2. Free-floating granules
3. Within casts
Hemoglobin vs myoglobin
 Ammonium sulfate precipitation method
o 2.8g ammonium sulfate add to 5ml urine and
stand for 5 mins
o Positive= hemoglobin precipitated
o Negative= myoglobin remains in supernatant.
Summary of Clinical Significance of a Positive Reaction for Blood
DAYLE DANIEL G. SORVETO, RMT 5
CHEMICAL EXAMINATION OF URINE January 24, 2018

Hematuria Hemoglobinuria Myoglobinuria Reagent Strip (Diazo) Reactions

1. Renal calculi 1.Transfusion 1. Muscular Reagents
2. reactions trauma/  Multistix: 2,4-dichloroaniline diazonium salt
Glomerulonephritis 2. Hemolytic crush syndromes  Chemstrip: 2,6-dichlorobenzene-diazonium salt
3. Pyelonephritis anemias 2. Prolonged coma Sensitivity
4.Tumors 3. Severe burns 3. Convulsions  Multistix: 0.4–0.8 mg/dL bilirubin
5.Trauma 4. 4. Muscle-wasting  Chemstrip: 0.5 mg/dL bilirubin
6. Exposure to toxic Infections/malaria diseases
chemicals 5. Strenuous 5. Alcoholism
7. Anticoagulants exercise/ /overdose
8. Strenuous exercise red blood cell 6. Drug abuse
Urine Bilirubin and Urobilinogen in Jaundice
9. Cystitis trauma 7. Extensive
10. Medications 6. Brown recluse exertion
Urine Bilirubin Urine Urobilinogen
(cyclophosphamide) spider bites 8. Cholesterol-
7. PNH lowering statin Bile Duct +++ Normal
8. syphilis, medications Obstruction
mycoplasma, 9. toxins; snake Liver Damage + or - ++
C.perfringens venom, spider Hemolytic Disease Negative +++
9. chemicals; bites
copper, nitrites, CHEMICAL ANALYSIS: Urine Urobilinogen
nitrates  Test: Urobilinogen
 Normal: 1 mg/dL or 1 Ehrlich unit
 Principle: Ehrlich’s reaction (-dimethyl-aminoben-
zaldehyde)
 Significance: Liver disease, hemolysis
 Source of Error: False-positive: Porphobilinogen (with
some brands of reagent strips)
*Pseudoperoxidase activity of hemoglobin  Comments: Reagent strips do not detect absence of
urobilinogen, only increase.
Reagents
 Multistix: Diisopropylbenzene dehydroperoxide Summary of Clinical Significance of Urine Urobilinogen
tetramethylbenzidine 1. Early detection of liver disease
 Chemstrip: dimethyldihydroperoxyhexane 2. Liver disorders, hepatitis, cirrhosis, carcinoma
tetramethylbenzidine 3. Hemolytic disorders

Sensitivity Reagent Strip Summary for Urobilinogen

 Multistix: 5–20 RBCs/mL, 0.015–0.062 mg/dL hemoglobin Reagents
 Chemstrip: 5 RBCs/mL, hemoglobin corresponding to 10  Multistix: p-dimethylaminobenzaldehyde
RBCs/mL  Chemstrip: 4-methoxybenzene-
diazoniumtetrafluoroborate
CHEMICAL ANALYSIS: Bilirubin
 Test: Bilirubin Sensitivity
 Normal: Negative  Multistix: 0.2 mg/dL urobilinogen
 Principle: Diazo reaction  Chemstrip: 0.4 mg/dL urobilinogen
 Significance: Liver disease, biliary obstruction
 Source of Error: False-negative: Exposure to light, Watson-Schwartz Differentiation Test
oxidation to biliverdin, hydrolysis of bilirubin 1. Label 2 tubes #1 and #2
diglucuronide, high levels of ascorbic acid or Tube 1
nitrites, drugs causing atypical colors.  2 mL urine
False-positive: Urine pigments  2 mL chloroform
 Comments: only conjugated bilirubin is excreted in  4 mL sodium acetate
urine Tube 2
 2 mL urine
Summary of Clinical Significance of Urine Bilirubin  2 mL butanol
1. Hepatitis  4 mL sodium acetate
2. Cirrhosis 2. Vigorously shake both tubes.
3. Other liver disorders 3. Place in a rack for layers to settle.
4. Biliary obstruction (gallstones, carcinoma) 4. Observe both tubes for red color in the layers.

DAYLE DANIEL G. SORVETO, RMT 6

CHEMICAL EXAMINATION OF URINE
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 – chloroform—red =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-nitrite-
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.
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
CHEMICAL ANALYSIS: Urine Specific Gravity
 Test: Specific gravity
 Normal: Random specimen: 1.003-1.030
 Principle: PKa change of polyelectrolyte
 Significance: Indication of kidney’s concentrating ability
and state of hydration
 Source of Error: Increased: Protein.
January 24, 2018
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
manufacturer’s package insert.
Clinical Significance of Urine Specific Gravity
1. Monitoring patient hydration and dehydration
2. Loss of renal tubular concentrating ability
3. Diabetes insipidus
4. Determination of unsatisfactory specimens due to low
concentration
5. 1.000 = same as pure water, suspect adulteration of urine
specimen.
6. 1.001-1.009 = dilute urine; associated with increase water
intake or water diuresis.
7. 1.010-1.025 = indicates average solute and water intake
and excretion.
8. 1.025-1.034 (1.040)= concentrated urine; associated with
dehydration, fluid restriction, profuse sweating, osmotic
diuresis
9. >1.040= Indicates presence of iatrogenic substances
(radiographic contrast media, mannitol)
Additional notes:
reducing if urine SG is 1.000 test the sample urea and creatinine
test to make sure the specimen is urine.
>1.040 used osmometry
Reagents
Multistix:
 Poly (methyl vinyl ether/maleic anhydride) bromthymol
blue
Chemstrip:
 Ethyleneglycoldiaminoethylethertetraacetic acid,
bromthymol blue
Sensitivity= 1.000–1.030
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
DAYLE DANIEL G. SORVETO, RMT 7
CHEMICAL EXAMINATION OF URINE January 24, 2018

 Principle: Acid precipitation

Reagents  Sources of Error: False-positive: Radiographic dyes,
 Multistix: Derivatized pyrrole amino acid ester Diazonium tolbutamide, some antibiotics, turbid urine.
salt False-negative: Highly buffered alkaline urine.
 Chemstrip: Indoxylcarbonic acid ester Diazonium salt  Comments: Detects all proteins, including Bence Jones
proteins.
Sensitivity
 Multistix: 5–15 WBC/hpf Copper Reduction Test (Benedict’s Test)
 Chemstrip: 10–25 WBC/hpf

The Reagent Strip Color Comparison Chart

 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.

Confirmatory/ Supplement Urine Chemistry Tests

Sulfosalicylic Acid Test Acetest

 Test: Sulfosalicylic acid
 Substance(s) Detected: Protein
 Test: Acetest
 Substance(s) Detected: Ketones
 Principle: Sodium nitroprusside reaction
 Sources of Error: False-negative: Improperly stored
specimen

DAYLE DANIEL G. SORVETO, RMT 8

CHEMICAL EXAMINATION OF URINE January 24, 2018

 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.

 Test: Watson-Schwartz Test

 Substance(s) Detected: Urobilinogen, porphobilinogen
 Principle: Ehrlich’s aldehyde reaction
 Sources of Error: Decreased: Exposure to light, more than
1 hour at room temperature. False-positive: 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: Ehrlich’s aldehyde reaction
 Sources of Error: Similar to Watson-Schwartz
 Comments: Urobilinogen doesn’t react unless very high.

Effect of High Levels of Ascorbic Acid on Urinalysis Tests

*May vary with brand of reagent strip. Refer to manufacturer’s

package insert.

False-positive False-Negative or Decrease

Clinitest Glucose
Blood
Bilirubin
Nitrite
Leukocyte esterase

DAYLE DANIEL G. SORVETO, RMT 9