LECTURE 3 ANALYSIS OF URINE AND BODY FLUIDS | MED 10

BIOCHEMICAL TESTS FOR URINE

QUALITATIVE TEST FOR PROTEINS  Benedicts solution:
I. SULFOSALICYLIC ACID TEST (EXTON’S TEST)  Copper sulfate
 Reference method  Sodium carbonate
 Sodium citrate buffer or citric acid
 Alternative: Trichloroacetic acid (TCA)
 NaOH
 Most abundant protein: Albumin (1st), Globulin (2nd)
 Procedure: Centrifuge  3 mL urine + 3 mL 3% SSA  Invert  Stand
Results:
for 10 minutes  Invert twice  Observe
Negative No color change (Blue)
Trace Pale green w/ slightly cloudy
Protein
1+ Cloudy green
Grade Turbidity range
2+ Yellow to orange
(mg/dL)
3+ Orange to red
Negative No increase in turbidity <6 4+

Trace Noticeable turbidity 6-30 II. FEHLINGS’S TEST

Results:
1+ Distinct turbidity with no granulation 30-100 - No change in color
+ Green opacity, no precipitate
+ Green solution with yellow precipitate
Turbidity with granulation with no
2+ 100-200 ++ Green to yellow solution with yellow precipitate
flocculation
+++ Muddy orange solution with yellow precipitate
Turbidity with granulation and ++++ Orange to brick red precipitate
3+ 200-400
flocculation
III. NYLANDER’S METHOD
4+ Clumps of protein >400
Procedure:
1. Place 5 ml of urine in a test tube
II. HELLER’S TEST 2. Add 0.5 ml of Nylander's reagent
 Positive Result: White ring at the point of contact 3. Heat for 3-5 minutes
4. Allow to stand for few minutes (let it cool)
III. ROBERT’S TEST
 Positive Result: White ring at the point of contact of the two fluids IV. IODINE-STARCH TEST
indicates albumin. The larger the ring, the greater the amount of  Blue to black reaction
albumin
Results:
QUANTITATIVE TEST FOR PROTEINS  Black indicates presence of sugar
I. KWILECKI’S MODIFICATION OF ESBACH’S METHOD  Brown indicates trace amount of sugar
 Procedure: Fill Esbach’s tube ‘til “U” mark w/urine  Add 10 drops of  No change of color: negative (colorless)
10% Ferric chloride  Mix  Fill ‘til “R” mark w/ Esbach’s reagent   If solution turns black after cooling, reaction due to substances other
Water bath at 72C for 5 minutes than sugar

II. KINGSBURY CLARK METHOD TEST FOR OTHER SUGARS

 Procedure: 1. SELIWANOFF’S TEST
a. Pipet 2.5 mL of centrifuged urine into a test tube graduated at  When heated with conc. HCl, fructose forms oxymethylfurfurol which
10 mL and add 3% sulfosalicylic acid to the 10 mL mark. gives a red color with resorcin.
b. Immediately after heating, read the height of the coagulum  Procedure:
formed and report in gm%. Height of coagulum is the number of a. 5 ml of urine in test tube
grams albumin in 1000 mL urine; obtain the amount of albumin b. Add 2 ml of Seliwanoff reagent
in gm%, divide by 10. c. Heat
c. Read according to the plus system below and report in gm%.
2. BORCHARDT’S TEST
Results:  Test used to detect fructose.
+ No definite flocculation, cloud is distinguishable but not  Procedure:
granular a. Take 5 ml of urine in a tube and add 5 mL of 25% HCI.
++ No definite flocculation, cloud is distinct and granular. b. Add a few crystals of resorcinol and boil for not more than half a
Represents 0.04-0.1 gm% albumin minute. A red color represents fructose.
+++ Marked flocculation with dense cloud. Represents 0.2-2.3
gm% albumin 3. RUBNER’S METHOD
++++ Heavy thick precipitate almost to boiling solid. Represents 0.5  Test for lactose (glucose+galactose)
gm% albumin or higher, 3 gm% albumin boils solid.
 Procedure:
a. 10 ml of urine, add 3 g lead acetate powder then shake
QUALITATIVE TESTS FOR SUGAR b. b) Filter off the precipitate and heat the filtrate for few minutes
I. BENEDICT’S TEST (yellowish-brown appears
 Detects the reducing sugars only c. c) Add ammonium hydroxide and continue heating
 Procedure: 5 mL of Benedict’s sol’n + 8-10 Drops of urine  Mix  d. d) Let it cool upon heating
Boil for 2-3 minutes  Cool

BARTOLOME, A.A. | BSMT 3 | BALIUAG UNIVERSITY | S.Y. 2023-2024

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  Results: TEST FOR CHLORIDE
 red color with red precipitate: presence of lactose I. FANTUS
 Red color with yellow precipitate: glucose  Procedure:
 Negative: milky appearance with flocculation a. Place 20 drops of urine.
b. Add 1 drop of potassium dichromate as an indicator.
4. BIAL ORCINOL METHOD c. Finally, add drop by drop 2.9 % silver nitrate solution until
 Procedure: permanent distinct red brown is produced.
a. Boil 5 ml Bial's Orcinol reagent in a test tube  Positive result:
b. Remove from the flame and add urine drop by drop until no  Brick red - the number of drops required to produce reddish
more than 1 ml urine has been used brown color expresses the NaCl content of the urine in gm/L.
 Results:  A urine with normal chloride content required 16-20 drops of
 Positive- turns green immediately silver nitrate to the end point.
 Negative – no reaction (yellow)
TEST FOR BLOOD
TEST FOR KETONES
I. BENZIDINE TEST
I. LEGAL’S TEST (ACETONE)
 Procedure:
 Principle: In reaction with the –NO group, sodium nitroprusside, a. Prepare about 3 mL saturated solution of benzidine in glacial
acetone and acetoacetic acid form isonitroacetone which remains acetic acid in a test tube.
trapped in the complex anion. b. Add an equal volume of 3% hydrogen peroxide, mix thoroughly.
 Positive: Purple/Violet-red (indicates acetone) c. Should the solution turn green or blue, it must be discarded.
 Procedure: d. Add about 1 mL of urine and shake
a. To few mL of urine, add enough NaOH or KOH solution to render  Positive: Green or blue color indicates presence of occult blood
it slightly alkaline.
b. Add a few drops of sodium nitroprusside solution. II. GUIAC TEST
c. Add a few drops of concentrated acetic acid.
 Procedure:
d. Observe the result and record as positive or negative.
a. To a small amount of powdered guiac in a test tube, add 2 mL of
95% alcohol and mix.
II. GUNNING’S TEST
b. Mix 5 mL of hydrogen peroxide with 5 mL of the guiac solution
 Procedure: prepared above.
a. Pour 5 mL of urine into a test tube c. Make the urine strongly acidic with acetic acid.
b. Add 5 drops of strong ammonia C. d. Apply the ring and contact test.
c. Add Lugol's solution enough to produce a black cloud which  Positive: Blue ring in the presence of blood
does not disappear immediately.
d. Let stand for a few minutes.
TEST FOR CALCIUM
e. Take a small quantity of the sediment and examine
I. SULKOWITCH TEST
microscopically. Iodoform crystals appear as yellowish or six-
pointed stars or six-sided plates. RESULT INTERPRETATIONS
Negative No precipitation (less than 5 mg / 100 mL)
f. Report as positive or negative.
+ Slight cloudiness (5-10 mg / 100 mL)
++ Definite cloudiness (10-15 mg / 100 mL)
III. GERHARDT’S TEST (AAA)
+++ Cloudiness throughout but not totally opaque (15-20 mg /
 Procedure: To a 5 ml of urine in a test tube, add drop by drop 10% 100 mL)
ferric chloride until no further precipitation occurs. ++++ Opaque precipitate (over 20 mg / 100 mL)
 Result: Procedure:
 Positive: Bordeaux red a. Pipette 3 mL of urine specimen. (Centrifuge urine if not clear).
 Negative: Rust color (no reaction) b. Add equal amount of sulkowitch reagent.
c. Mix and allow to stand for few minutes.
TEST FOR BILE PIGMENTS d. Observe and record result obtained
I. SMITH’S TEST
 Procedure: TEST FOR INDICAN
a. Take 5 mL of urine in a test tube. I. OBERMAYER’S TEST
b. Overlay the urine with the tincture of alcoholic iodine.  Procedure:
c. Observe the result and report as positive or negative. An a. To 5 mL urine in a test tube, add 5 mL Obermayer's reagent.
emerald green ring at the point of contact shows the presence b. Mix and add chloroform to settle. A blue coloration of the
of bile pigment. chloroform indicates the presence of indican.
 Positive: Emerald green ring  Positive: Blue coloration

II. HARRISON’S SPOT TEST II. JAFFE’S METHOD

 Procedure:
a. Take 5 mL of urine and 5mL of barium chloride 10% solution in a
test tube. Mix and stand for a few minutes.
b. Filter. Spread the filter paper bearing the precipitate on another
piece of dry filter paper.
c. Place 3 drops of Fouchet's reagent (25% trichloroacetic acid and
0.9% ferric chloride) on the precipitate.
d. Observe the result and report as positive or negative.
 Positive: Blue to green; Negative: No changes
 Procedure:
a. To a small amount of urine in a test tube, add an equal volume
of HCl, C.P.
b. Mix and add 10-15 drops of chloroform.
c. Add drop by drop a strong fresh solution of calcium hypochlorite
shaking after the addition of each drop.
d. A 0.5% KMnO4 may be used in place of calcium hypochlorite if
preferred. Five drops of KMn04 are used with 10mL of urine
 Positive: Blue color

BARTOLOME, A.A. | BSMT 3 | BALIUAG UNIVERSITY | S.Y. 2023-2024

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 CHEMICAL EXAMINATION OF URINE ACIDIC URINE ALKALINE URINE
REAGENT STRIP Diabetes mellitus Renal Tubular Acidosis
Starvation Vegetarian Diet
 Provide a simple, rapid means for performing medically significant
High Protein Diet After meal
chemical analysis of urine
Cranberry juice Old specimen
 Consist of chemical-impregnated absorbent pads attached to a plastic
Emphysema Hyperventilation
strip
Dehydration Urease-producing bacteria
 Types:
Diarrhea
 4 parameter= glucose, protein, specific gravity, pH Acid-producing bacteria
 10 parameter Medication
 11 parameter
 13-14 parameter Principle: Double Indicator System
 Reagent: Methyl Red, Bromthymol Blue
TECHNIQUE
 Sources of Error: Runover, Old specimen
1. Dip the reagent strip briefly into a well-mixed uncentrifuged urine  Correlation: Nitrite, Leukocyte, Microscopic
specimen at room temperature.
2. Remove the excess urine by touching the edge of the strip to the SUMMARY OF CLINICAL SIGNIFICANCE OF URINE pH
container as the strip is withdrawn.
1. Respiratory or metabolic acidosis/ketosis
3. Blot the edge of the strip on a disposable reagent pad.
2. Respiratory or metabolic alkalosis
4. Wait the specified length of time for reactions to take place.
3. Defects in renal tubular secretion and reabsorption of acids and
5. Compare the colored reactions against the manufacturer’s chart using
bases—renal tubular acidosis
a good light source.
4. Renal calculi formation
5. Treatment of urinary tract infections
CARE
6. Precipitation/identification of crystals
1. Store with dessicant in an opaque, tightly closed container. 7. Determination of unsatisfactory specimens
2. Store below 30°C (room temperature); do not freeze.
3. Do not expose to volatile fumes
PROTEIN
4. Do not use past the expiration dates.
 Most indicative of Renal disease
6. Do not use if chemical pads become discolored.
 White foam in the urine
7. Remove strip immediately prior to use.
8. Do not touch the chemical pads.
ALBUMIN
9. Do not cut the reagents.
 Major serum protein found in the urine
QUALITY CONTROL  Normal values:
 Strips must be checked with both positive and negative controls a  <10 mg/dL or 100 mg/24 hrs (Strasinger)
minimum of once every 24 hours.  <150 mg/dL(Henry)
 Testing is also performed when:
 a new bottle of reagent strips is opened. OTHER PROTEINS
 Questionable results are obtained.  Serum and tubular microglobulins, Tamm Horsfall protein produced
 Distilled water is not recommended as a negative control by the tubules, and proteins from prostatic, seminal, and vaginal
secretions.
SUMMARY OF REAGENT STRIP
PRE-RENAL PROTEINURIA
PARAMETER TIME PRINCIPLE
 Caused by the conditions that affect the plasma prior its reaching the
Glucose 30 Double Sequential Enzyme Reaction
kidney.
Bilirubin 30 Diazo Reaction
 Intravascular hemolysis
Ketones 40 Sodium Nitroprusside
 Muscular injury
Specific Gravity 45 pka change of polyelectrolyte
 Severe infection and inflammation
pH 60 Double indicator system
 Multiple Myeloma = proliferation of immunoglobulin-producing
Protein 60 Protein (Sorensen’s) error of plasma cells (Bence Jones Proteins) - Immunoglobulin light
indicators chains
Blood 60 Pseudoperoxidase activity of  ID= Serum Immunoelectrophoresis
hemoglobin  Urine= precipitates at 40-60’C and dissolves at 100’C (clear)
Urobilinogen 60 Ehrlich reaction
Nitrite 60 Greiss’ reaction A. GLOMERULAR PROTEINURIA
Leukoyte 120 Leukocyte esterase I. DIABETIC NEPHROPATHY
 Decreased glomerular filtration
pH  May lead to renal failure
 Important in the identification of crystals and determination of  Indicator: Microalbuminuria
unsatisfactory specimen  Urine Albumin Excretion Rate (AER)= in ug/min or in mg/24 hrs
 Normal pH:  Normal AER= 0-20 ug/min
 Random Specimen: 4.5-8  Microalnuminuria: 20-200 ug/min (Perform micral test)
 First Morning: 5-6  Clinical Albuminuria= >200 ug/min
 After a meal: alkaline
 Unpreserved urine: 9 (increased pH) II. ORTHOSTATIC / CADET / POSTURAL PROTEINURIA
 Acidic: due to hypoventilation (pO2; pCO2=pH) leading to  Proteinuria when in vertical position due to increased pressure to
respiratory acidosis. renal veins.
 Specimen: first morning urine

BARTOLOME, A.A. | BSMT 3 | BALIUAG UNIVERSITY | S.Y. 2023-2024

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 CLINICAL SIGNIFICANCE OF URINE GLUCOSE
Hyperglycemia-associated
Inc Blood glucose
Inc Urine Glucose
Causes:
 DM
 Cushing’s Syndrome
 Pheochromocytoma
 Acromegaly
 Hyperthyroidism
GLUCOSE
Renal-associated  Most frequently tested in urine
Normal Blood glucose  Renal Threshold - plasma concentration of a substance at which
Inc Urine Glucose tubular reabsorption stops
 160-180 mg/dL - renal threshold for glucose
Impaired tubular reabsorption
of glucose Principle: Double sequential enzyme reaction
Causes:
Fanconi Syndrome:
Defective tubular reabsorption
of glucose and amino acids

CHROMOGENS
B. TUBULAR PROTEINURIA
1. Fanconi’s syndrome (problem with PCT, cannot absorb)  O-toluidine (pink to purple)
2. Toxic Agents/ heavy metals  Potassium iodide (blue to green to brown)
3. Severe Viral Infections  Aminopropylcarbazole (yellow to orange-brown)
POST RENAL PROTEINURIA  Tetramethylbenzidine (yellow to green)
1. Lower UTI/ inflammation
2. Injury or trauma Reported in terms of negative, trace, 1, 2, 3, and 4; however, the color
3. Menstrual contamination charts also provide quantitative measurements ranging from 100 mg/dL to
5. Prostatic fluid/ spermatozoa 2 g/dL, or 0.1% to 2%.
6. Vaginal secretions

CLINITEST/BENEDICT’S TEST
 Test: Nonspecific test for reducing sugars
 Galactose, lactose, fructose, maltose, pentoses, ascorbic acid, certain
drug metabolites, and antibiotics such as the cephalosporins.
 Principle: Copper reduction
Principle: Protein Error of Indicator

Indicators: CLINITEST TABLET PROCEDURE:

Tetrabomphenol blue; Tetrachlorophenol tetrabromosulfonphthalein
 5 drops + 10 drops of H2O + Clinitest tablet
Note: Indicator is sensitive to albumin only.
 The tablet contains:
 CuSO4
Readings are reported in terms of negative, trace, 1, 2, 3, and 4; or the
 NaCO3
semiquantitative values of 30, 100, 300, or 2000 mg/dL corresponding to
 Sodium Citrate
each color change.
 NaOH

PASS THROUGH PHENOMENON

 Occurs when > 2 g/dL sugar is present
 Blue > Green > Yellow > Brick red > Blue
 To prevent pass through, use 2 drops of urine

BARTOLOME, A.A. | BSMT 3 | BALIUAG UNIVERSITY | S.Y. 2023-2024

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 KETONES
 Result from an increased Fat metabolism due to inability to
metabolize carbohydrates.

 B-hydroxybutyric Acid
 Acetoacetic acid/Diacetic acid
 Acetone HEMOGLOBIN VS. MYOGLOBIN
 Principle: sodium nitroprusside TEST HEMOGLOBIN MYOGLOBIN
PLASMA Red/pink plasma Pale yellow
EXAMINATION
Note: Acetone is detected only when Glycine is present TEST
Blondheim  haptoglobin  CK and
Results are reported qualitatively as negative, trace, small (1), moderate (2), (ammonium (precipitated) aldolase (not
or large (3), or semiquantitatively as negative, trace (5 mg/dL), small (15 sulfate) precipitated)
mg/dL), moderate (40 mg/dL), or large (80 to 160 mg/dL) Procedure:
Urine + 2.8 g
NH4 Sulfate
(Filter/
Centrifuge)
Test
Supernatant for
blood reagent
strip

BILIRUBIN
 Early indication of liver disease
 Amber urine with Yellow foam
TABLET CONTAINS:
 Sodium nitroprusside CLINICAL SIGNIFICANCE
 Glycine
 Hepatitis
 Disodium phosphate,
 Cirrhosis
 Lactose
 Biliary Obstruction (gallstone, carcinoma)
 Other liver disorders

REAGENT STRIP FOR BILIRUBIN

 Principle: Diazo reaction

 (+) result
 Tan or Pink to Violet

REAGENT STRIP FOR BLOOD OTHER TEST

 Principle: Pseudoperoxidase activity of hemoglobin I. ICTOTEST (TABLET)
 Contains p-nitrobenzene-diazonium p-toluene sulfonate, SSA, sodium
carbonate, boric acid
 (+) blue to purple
CHROMOGEN:
 Tetramethylbenzidine
 (-) yellow
 (+) Green () to Blue ()
 Notes:
Uniform green/blue = Hemoglobin/ Myoglobin
Speckled/Spotted= Hematuria

BARTOLOME, A.A. | BSMT 3 | BALIUAG UNIVERSITY | S.Y. 2023-2024

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 UROBILINOGEN
 Bile pigment that result from Hgb degradation
 NV= < 1 mg/dL or Ehrlich units
 Specimen: Afternoon urine or 4-hour

CLINICAL SIGNIFICANCE
1. Early Detection of Liver Disease HOESCH TEST
2. Liver Disorders, hepatitis, cirrhosis, carcinoma  Principle: Inverse erlich reaction
3. Hemolytic disorders  Rapid screening test for porphobilinogen
 (≥ 2 mg/dL)
REAGENT STRIP  Procedure:
 Principle: Ehrlich reaction 1. 2 drops of urine + 2 ml hoesch reagent
2. Hoesch reagent: Ehrlich’s reagent in 6M or 6N HCl
3. (+) result is red

NITRITE
 Rapid Screening test for UTI
 Specimen: 1st morning urine 4-hour urine

 Reagent: Strip or Ehrlich’s reagent

Note:
 Also (+) Ehrlich reactive compounds:
REAGENT STRIP FOR NITRITE
 Porphobilinogen, indican, p-aminosalicylic acid, sulfonamides,
methyldopa, procaine, chlorpromazine  Principle: Grelss reaction
 Multistix results are reported as Ehrlich units (EU), which are equal to
mg/dL, ranging from normal readings of 0.2 and 1 through abnormal
readings of 2, 4, and 8.
 Chemstrip results are reported in mg/dL  (+) result (uniform pink)
 Reagent strip tests cannot determine the absence of urobilinogen, Note:
which is significant in biliary obstruction  Pink spot/ edges are considered negative
 (+) nitrite corresponds to 100,000 organisms/ml

LEUKOCYTE
WATSON-SCHWARTS TEST
 Differentiates urobilinogen, porphobilinogen and Ehrlich-reactive
compounds
 Uses two extraction with organic solvents: chloroform and butanol

REAGENT STRIP FOR LEUKOCYTE

 Principle : leukocyte esterase
 (+) result (purple)

BARTOLOME, A.A. | BSMT 3 | BALIUAG UNIVERSITY | S.Y. 2023-2024

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Note:  Brands:
 With esterase (neutrophils, eosinophils, basophils, monocytes, A. C-stix- 10 seconds, Stix – 60 seconds
histiocytes and Trichomonas) B. GC-MS – More accurate quantitative method
 Without esterase (Lymphocytes)  Gas chromatograph mass spectrometry
 Strip can even detect lysed WBC

SPECIFIC GRAVITY
 Density of the solution compared with the density of similar volume
of distilled water at a similar temperature
 Normal SG: 1.003 – 1.035 (random)
 SG < 1.003 : not a urine
 SG > 1.040: radiographic dye
 Isosthenuria: 1.008 – 1.024
 Hyposthenuria: <1.008
 Hypersthenuria: 1.025-1.040

REAGENT STRIP FOR SPECIFIC GRAVITY

 PRINCIPLE: pka change in polyelectrolyte
 Blue to Green to Yellow
 Reagent is sensitive to number of ions in the urine specimen, indicator
changes in relation to ionic concentration
 Indicator: Bromothymol blue

Note:
 Add 0.005 to SG reading when pH is ≥ 6.5 due to interference with
bromothymol blue indicator
 Not affected by glucose, protein and radiographic dye

ASCORBIC ACID
 Water soluble vitamin
 Strong reducing agent
 Interferes with reagent strip that use H2O2 or diazonium salt
 Causes False negative reactions on:
 Blood
 Bacteria
 Leukocyte
 Nitrite
 Glucose
 11th reagent pad
 Ascorbic acid (>/= 5 mg/dL) + Phosphomolybdate  + molybdenum
blue

BARTOLOME, A.A. | BSMT 3 | BALIUAG UNIVERSITY | S.Y. 2023-2024

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