WEEK 4 LEC
CHEMICAL EXAMINATION OF URINE
INTRODUCTION
This topic focuses on chemical examination of
urine through the reagent strips. Reagent strips currently
provide a simple, rapid means for performing medically
significant chemical analysis of urine, including pH,
protein, glucose, ketones, blood, bilirubin, urobilinogen,
nitrite, leukocyte and specific gravity.
REAGENT STRIPS
 Reagent strips are used to perform the routine
chemical tests on urine
 Strips consist of chemical-impregnated absorbent
pads on a plastic strip
 Test performed for: pH, protein, glucose, ketones,
blood, bilirubin, urobilinogen, nitrite, leukocyte
esterase, specific gravity (SG)
 Single and multitest strips available
 Color comparison charts are supplied by the
manufacturer
 Several degrees of color are shown to provide
semiquantitative readings of neg, trace, 1+, 2+, 3+,
and 4+
 Estimates of mg/dL are also provided for many of
the test areas
Reagent Strip Technique
1. Dip the reagent strip briefly into a well-mixed
uncentrifuged urine specimen at room temperature.
2. Remove excess urine by touching the edge of the strip
to the container as the strip is withdrawn.
3. Blot the edge of the strip on a disposable absorbent
pad.
4. Wait the specified amount of time for the reaction to
occur.
5. Compare the color reaction of the strip pads to the
manufacturer’s color chart in good lighting.
 Hold the strip horizontally when comparing colors
Improper Technique Errors
 Enzyme reactions on strip are based on room
temperature readings
 Reagents will leach off a strip remaining in the urine
too long - Dip briefly into specimen
 Formed elements such as red and white blood cells
sink to the bottom of the specimen and will be
undetected in an unmixed specimen.
 Allowing the strip to remain in the urine for an
extended period may cause leaching of reagents from
the pads.
 Excess urine remaining on the strip after its removal
from the specimen can produce a run-over between
chemicals on adjacent pads, producing distortion of
the colors. To ensure against run-over, blotting the
edge of the strip on absorbent paper and holding the
strip horizontally while comparing it with the color
chart is recommended.
 The timing for reactions to take place varies between
tests and manufacturers, and ranges from an
immediate reaction for pH to 120 seconds for
leukocyte esterase. For the best semiquantitative
results, the manufacturer’s stated time should be
followed; however, when precise timing cannot be
achieved, manufacturers recommend that reactions be
read between 60 and 120 seconds, with the leukocyte
esterase reaction read at 120 seconds.
 A good light source is essential for accurate
interpretation of color reactions.
 The strip must be held close to the color chart without
actually being placed on the chart. Automated reagent
strip instruments standardize the color interpretation
and timing of the reaction and are not subject to room
lighting deficiencies or inconsistency among
laboratory personnel
 Reagent strips and color charts from different
manufacturers are not interchangeable.
 Specimens that have been refrigerated must be
allowed to return to room temperature prior to reagent
strip testing, as the enzymatic reactions on the strips
are temperature dependent.
Handling and Storage of Strips
 Store with desiccant in an opaque, tightly closed
container—pads are very hydroscopic
 Store below 30C, do not freeze
 Do not expose to volatile fumes
 Do not use past the expiration date
 Do not use if pads are discolored
 Remove strips immediately prior to use
Quality Control of Reagent Strips
 Run positive and negative controls at least once per
24 hours or on each shift
 Run controls when a new bottle of strips is opened,
results are questionable, or there is concern over strip
integrity
 Record control results
 Manufactured positive and negative controls are
available
 Do not use distilled water as a negative control as
reactions are designed for urine ionic concentration
 All negative control readings should be negative
 Positive control readings should agree with published
control values by +/- one color block
 Be aware of manufacturer-stated limitations and
interfering substances ▪ Relate chemical readings to
each other and physical and microscopic readings
 WEEK 4 LEC
CHEMICAL EXAMINATION OF URINE
 They must also have positive and negative controls
performed whenever these tests are required:
o Protein: sulfosalycilic acid/acidify specimen
o Galactose: Clinitest
o Ketones: Acetest
o Bilirubin: Ictotest (primary confirmatory test)
SUMMARY OF REAGENT STRIP TESTING
CARE OF REAGENT STRIPS
1. Store with desiccant in an opaque, tightly closed
container
2. Store below 30°C; do not freeze.
3. Do not expose to volatile fumes.
4. Do not use past the expiration date.
5. Do not use if chemical pads become discolored.
6. Remove strips immediately prior to use.
TECHNIQUE
1. Mix specimen well.
2. Let refrigerated specimens warm to room
temperature before testing.
3. Dip the strip completely, but briefly, into specimen.
4. Remove excess urine by withdrawing the strip
against the rim of the container and by blotting the edge
of the strip.
5. Compare reaction colors with the manufacturer’s
chart under a good light source at the specified time.
6. Perform backup tests when indicated.
7. Be alert for the presence of interfering substances.
8. Understand the principles and significance of the
test; read package inserts.
9. Relate chemical findings to each other and to the
physical and microscopic urinalysis results.
QUALITY CONTROL
1. Test open bottles of reagent strips with known
positive and negative controls every 24 hours.
2. Resolve control results that are out of range by
further testing.
3. Test reagents used in backup tests with positive and
negative controls.
4. Perform positive and negative controls on new
reagents and newly opened bottles of reagent strips
5. Record all control results and reagent lot numbers.
URINE PH
 Determined by the concentration of the free H⁺
o As H⁺ increases, pH decreases
o As H⁺ decreases, pH increases
 Kidneys are major regulator of acid-base balance
 Normal range 4.5–8.0
 Normal fresh urine cannot reach pH 9
 Diet and medication regulation
o Meat = acid pH
o Vegetables = alkaline pH
 Exception = cranberry juice
o Medications for urinary tract infection
 Maintain an acid pH
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 bases—renal tubular
acidosis
4. Renal calculi formation
5. Treatment of urinary tract infections
6. Precipitation/identification of crystals
7. Determination of unsatisfactory specimens
PROTEIN
 Most indicative of renal disease
 Proteinuria seen in early renal disease
 Proteins normally found in urine:
o Albumin: major serum protein found in urine
o Tamm-Horsfall protein: mucoprotein produced by
the renal tubules
o Serum and tubular microglobulins
o Vaginal, prostatic, and semen proteins
 Normal = < 10 mg/dL or 100 mg/24 hr
 Low molecular weight serum proteins are filtered;
many are reabsorbed
Clinical Significance
 Clinical proteinuria = > 30 mg/dL, 300 mg/L
 Variety of causes:
o Prerenal or Overflow
o Renal
o Postrenal
Prerenal Proteinuria
 Conditions affecting the plasma, not the kidney
 Not indicative of actual renal disease & not
detected by reagent strip
 Transient, increase levels of low molecular weight
plasma proteins, acute phase reactants, exceed
reabsorptive capacity
 Conditions associated:
o Septicemia/ severe infection or inflammation
o Hemoglobinura: after a hemolytic episode
o Myoglobinuria: follows muscle injury
o Immunoglobulins paraproteins: abnormally
produced in multiple myeloma and
macroglobulinemia.
 Bence Jones Protein (BJP)
 Multiple myeloma
 Monoclonal Immunoglobulin Light Chains
 Screening test: BJP coagulates between 40-60°C
and dissolves at 100 °C
 Not all patients with multiple myeloma will
excrete detectable levels of BJP
 WEEK 4 LEC
CHEMICAL EXAMINATION OF URINE
Renal Protenuria
 Glomerular proteinuria Tests for Albumin
o Occurs in primary glomerular disease or
disorders that cause glomerular damage Heat and acetic acid test
o Most common type of proteinuria encountered
and most serious clinically  Principle: Urine is coagulated by heat and
o Immune complex disorders, amyloidosis, precipitated by acetic acid (5-10%) and the degree
toxic agent, diabetic nephropathy, strenuous of turbidity produced is proportionanl to the amount
exercise, dehydration, hypertension, of protein present
orthostatic proteinuria  Positive Results:
 Tubular proteinuria o 1+: diffuse cloud
o Occurs when normal tubular reabsorptive o 2+: granular cloud
function is altered or impaired o 3+: distinct floccule
o Fanconi’s syndrome, toxic agents/ heavy o 4+: large floccule, dense, something solid
metals, pyelonephritis, acute tubular necrosis,
polycystic kidney disease, phenacetin
damage. Sulfosalicylic Acid (SSA)Test

 Microalbuminuria  Cold precipitation test that reacts equally with all

 Proteinuria not detected by reagent strip forms of protein
 Signifies onset of renal complications of Diabetes  Precipitates albumin, globulin, and other proteins
mellitus  SSA precipitates proteins without heat
 Associated with increased risk of cardiovascular  Use centrifuged specimen
disease  Reagent: 3% SSA
 Methods:  Principle: most proteins are precipitated in dilute SSA
 Quantitative procedures for albumin using 24  False positive: radiographic dye, tolbutamide,
hr specimen cephalosporins, penicillins, and sulfonamides
o 30-300 mg/24hrs  False negative: Highly alkaline urine and very dilute
o Albumin excretion rate (AER) of 20 to 200 samples
ug/min
 Micral test REPORTING SSA TURBIDITY
o Principle: Enzyme immunoassay GRADE TURBIDITY PROTEIN
o Sensitivity:0-10 mg/dl RANGE (mg/dL)
Negative No increase in Less than 6
 Immunodip
turbidity
o Principle: Immunochromagraphics
Trace Noticeable turbidity 6–30
o Sensitivity:1.2-8.0 mg/dl
1+ Distinct turbidity, no 30–100
 Clinitest microalbumin strips/Multistix-Pro granulation
o Principle: Sensitive albumin test related to 2+ Turbidity, 100–200
creatinine concentration to correct for granulation, no
patient dehydration flocculation
o Sensitivity: Albumin:10-150 mg/L 3+ Turbidity, 200–400
Creatinine: 10-300 mg/dl, 0.9-26.5 mmol/L granulation,
o Significant: albumin: creatine= >3.4 flocculation
mg/mmol 4+ Clumps of protein Greater than 400

Postrenal Proteinuria
 Protein added in the lower urinary and GLUCOSE
genitourinary tract
 Microbial infections causing inflammations and
release of interstitial fluid protein  Most frequent chemical analysis performed on urine
 Urine contaminated with proteins during  Assessment of hyperglycemia-associated glucosuria
excretion: and renal associated glucosuria
o Menstrual contamination  Clinical significance-major screening test for diabetes
o Semen / Prostatic fluid mellitus
o Vaginal secretions o Mellituria: presence of any sugar (reducing or non-
o Traumatic injury reducing) in urine
o Glycosuria: presence of any reducing sugar in
urine
 WEEK 4 LEC
CHEMICAL EXAMINATION OF URINE
CLINICAL SIGNIFICANCE OF URINE  10 drops water
GLUCOSE  2 drops urine
Hyperglycemia- Renal-Associated  Values up to 5 g/L vs. 2 g/L
Associated  Sensitivity: 200 mg/dL
Increased blood glucose Fanconi syndrome  Reaction Interference:
Increased urine glucose Advanced renal disease o Galactose, lactose, fructose, maltose,
Diabetes mellitus Osteomalacia pentoses, ascorbic acid, cephalosporins
Pancreatitis Pregnancy
Pancreatic cancer SUMMARY OF GLUCOSE OXIDASE AND
Acromegaly CLINITEST REACTIONS
Cushing syndrome Glucose Clinitest Interpretation
Hyperthyroidism Oxidase
Pheochromocytoma 1+ positive Negative Small amount of
Central nervous system glucose present
damage 4+ positive Negative Possible
Stress oxidizing agent
Gestational diabetes interference on
reagent strip
Negative Positive Nonglucose
Tests for Glucose reducing
substance
present
Benedict’s Test Possible
 General test for glucose and other reducing sugars interfering
substance for
 Reagents: Benedict’s Solution
reagent strip
 Principle: Relies on the ability of the glucose and
other reducing substances to reduce copper
sulfate to cuprous oxide in the presence of alkali
and heat. KETONES
NEGATIVE Clear blue color, blue
precipitate may form  Presence of ketone bodies in urine results from
TRACE Bluish-green color increased fat metabolism due to abnormal
1+ Green color, green or carbohydrate utilization
yellow ppt  Three intermediate products of fat metabolism
2+ Yellow to green color, o Acetone
yellow ppt o Acetoacetic acid
3+ Yellow-orange color, o Beta-hydroxybutyric acid
yellow-orange ppt
4+ Reddish-yellow color,
brick red or red ppt. Clinical Significance
Copper Reduction Method-Clinitest tablet  Primary causes:
o Diabetes mellitus
 Non-specific for glucose
o Vomiting (loss of carbohydrates)
 Sensitivity: 200mg/dl
o Starvation, malabsorption, dieting (↓intake)
 Reagents:
 Ketonuria shows inadequate insulin
o Copper sulfate
o Monitor diabetes
o Sodium Carbonate and Citric acid
o Can result in diabetic acidosis
o Sodium Hydroxide
o Hospitalized patients are often positive
o Sodium Hydroxide with water and citric acid
o Illness = ↓intake, poor absorption
 5 ggts urine + 10 ggts of H2O + Clinitest tablet
then wait for 15 seconds
Acetest
 Reported as: Negative, ¼ %(Trace), ½ % (1+), ¾
 Tablet test for ketone bodies
% (2+), 1%(3+), 2%(4+)
 Not a confirmatory test
 Pass through phenomenon
 Can perform serial dilutions and use serum
o High levels of reducing substance
 Tablet = sodium nitroprusside, glycine, disodium
o Color from blue through red back to green-
phosphate, lactose
brown. Rapid reaction
o Occur if >2 g/dL is present in urine  Acetoacetic acid (acetone) + sodium nitroprusside
o Repeat with two-drop procedure + glycine → purple
 WEEK 4 LEC
CHEMICAL EXAMINATION OF URINE
 Blondheim’s Test: test for the identification of
myoglobinuria from hemoglobinuria
o Hemoglobinuria: Hemoglobin + NH4SO4 =
BLOOD clear supernatant
o Myoglobinuria: Myoglobin + NH4SO4 = red
 Reagent strip is more accurate than microscopic for supernatant
detecting blood
 Normally no blood should be detected in the form of
hematuria, hemoglobinuria, and myoglobinuria. BILIRUBIN
 Presence of >5rbcs/ul is clinically significant
 Yellow pigmented degradation of hemoglobin
 Urine bilirubin early indicator of liver disease
Clinical Significance  Assessment of hepatic conditions, and biliary
obstruction
SUMMARY OF CLINICAL SIGNIFICACE OF  Clinical Significance: Screening of Abnormal
BLOOD IN URINE Hepatobiliary Function
HEMATURIA HEMOGLOI MYOGLOBI  Pre-hepatic jaundice (hemolytic anemia)
MURIA NURIA
 Hepatic jaundice(hepatitis, cirrhosis)
Intact red cells No red cells No red cells
Cloudy red urine Clear red urine Clear red urine  Post hepatic jaundice (biliary obstruction, gallstones,
Bleeding is renal or Lysis of RBC Characterized carcinoma)
genitourinary orgin produced in by “cola
urinary tract drink” or
particularly in “black coffee Ictotest: Tablet test for bilirubin
dilute, alkaline urine”  More sensitive than reagent strip
urine  Less subject to interference
May result
 Use specified mat for test containing p-
from
intravascular nitrobenzene-diazonium-ptoluenesulfonate,
hemolysis SSA, sodium carbonate, and boric acid
1. Renal calculi 1. Transfusion 1. Muscular  Positive: Presence of a blue-to-purple color on the
reactions trauma/crush mat for 30seconds
syndromes  Negative: colors other than blue or purple
2. 2. Hemolytic 2. Prolonged
Glomerulonephritis anemias coma
3. Pyelonephritis 3. Severe 3. Convulsions UROBILINOGEN
burns
4. Tumors 4. 4. Muscle-  Colorless pigment formed from the breakdown of
Infections/mal wasting bilirubin in the intestines
aria diseases  There is always a small amount of urobilinogen in the
5. Trauma 5. Strenuous 5. urine < 1 mg/dL
exercise/red Alcoholism/ov
 Urobilinogen excretion reaches peak levels between
blood cell erdose
2pm and 4pm
trauma
6. Exposure to 6. Brown 6. Drug abuse  Never reported as negative in urinalysis report.
toxic chemicals recluse spider
bites Clinical Significance
7. Anticoagulants 7. Extensive
exertion
8. Strenuous 8. Cholesterol-  Early detection of liver disease
exercise lowering statin  Liver disorders, hepatitis, cirrhosis, carcinoma
medications  Hemolytic disorders
9. Schistosoma  No urobilinogen is seen in the urine with bile duct
hematobium obstruction; strip will give a normal result
infection
TYPE OF URINE URINE
JAUNDICE BILIRUBIN UROBILINOGEN
Hemoglobinuria vs. Myoglobinuria Pre-hepatic Negative +++
 Plasma examination: jaundice
o Hemoglobin: Red/Pink (hemolytic
o Myoglobin: Pale Yellow disease)
 WEEK 4 LEC
CHEMICAL EXAMINATION OF URINE
Hepatic + or - ++
jaundice (liver Clinical Significance
damage)
Post-hepatic +++ Normal  Cystitis (bladder)
jaundice (bile
 Pyelonephritis (tubules)
duct
obstruction)  Evaluation of antibiotic therapy
 Monitoring of patients at high risk for urinary tract
infection
 Screening of urine culture specimens (in combination
Tests for Urobilinogen with LE test)

Ehrlich’s Tube test

 Differentiation among urobilinogen,
LEUKOCYTE ESTERASE (LE)
porphobilinogen, and Ehrlich reactives  Purpose is to detect leukocytes so as not to rely on
 Addition of Ehrlich reagent to urine produces a microscopic
cherry red color enhanced by sodium acetate  Not considered a quantitative test – do microscopic if
 Reagent: p-dimethylaminobenzaldehyde, sodium positive
acetate  Advantage: detects presence of lysed leukocytes
 Detects the presence of esterase in the:
Hoesch Screening Test o Granulocytic WBC
 Rapid screening test for porphobilinogen o Monocytes
(>2mg/dl) o Trichomonas
 Maintains an acid solution by adding a small urine o Histiocytes
volume to relatively large reagent volume
 Reagent: Hoesch Reagent (Ehrlich reagent Clinical Significance
dissolved in 6M HCl)  Bacterial and nonbacterial urinary tract infection
 Result: RED upon addition of reagent  Inflammation of the urinary tract
 False-positive: methyldopa, indican, pigmented  Screening of urine culture specimens in conjunction
urine with nitrite but a better predictor than nitrite
 Also seen with Trichomonas, Chlamydia, yeast,
Schwartz-Watson Differentiation Test
interstitial nephritis
 Classic test to differentiate urobilinogen from
porphobilinogen
 Urobilinogen extracted into both butanol and SPECIFIC GRAVITY
chloroform
 Porphobilinogen insoluble in both butanol and  Based on pKa (dissociation constant) of a
chloroform polyelectrolyte in alkaline medium
 Ehrlich Reactives extracted into butanol and  Polyelectrolyte ionizes releasing H+ in relation to
insoluble in chloroform concentration of urine
 ↑concentration = more H+ released
EXTRAC UROBI PORPH OTHER
TION LINOG OBILIN ERLICH  Indicator bromthymol blue measures pH change
WITH EN OGEN REACTIVE  Blue (alkaline) through green to yellow (acid)
COMPOUND
BUTANOL
Top – Red Colorless Red
ASCORBIC ACID/ VITAMIN C
butanol
Bottom – Colorless Red Colorless  Source of Interference due to strong reducing
urine property leading to false negative results
CHLOROFORM  Affects blood, bilirubin, leukocyte esterase, nitrite
Top – urine Colorless Red Red and glucose
Bottom – Red Colorless Colorless  Principle used in some reagent strips:
chloroform o C-stix: impregnated with phosphomolydates
buffered in acid medium. Detects 5mg/dl of
ascorbic acid in urine after 10 seconds
NITRITE o Stix and Multi-stix: impregnated with methylene
green Detects 25mg/dl of ascorbic acid in urine at
60 seconds.
Rapid, indirect method for the detection of bacteria
capable of reducing nitrate to nitrite.