TEST DETERMINATION FOR FALSE POSITIVE RESULTS

1. Strong oxidizing agents (sodium hypochlorite) :

FALSE-POSITIVE RESULTS
1. chlorpromazine and iodine metabolites: can react
BLOOD, BILIRUBIN, oxidizes the chromogen instead of the heme moiety
2. Bacterial peroxidases (by E. coli): catalyzes the
directly with diazonium salt
2. phenazopyridine-colors the urine red: result to a
UROBILINOGEN reaction of the reagent and chromogen despite the
absence of the pseudoperoxidase activity of heme
highly pigmented urine making the interpretation of the
result difficult
HEMATURIA vs. HEMOGLOBINURIA 3. Blood contamination (hemorrhoidal or menstrual blood) 3. Presence of indican (in intestinal disorders): makes
● Done by observing: clarity, color, pH, presence of a cell the urine highly pigmented
button after centrifugation FALSE-NEGATIVE RESULTS
● Hematuria: 1. Ascorbic acid and other reducing substances: react FALSE-NEGATIVE RESULTS
○ cloudy/smoky appearance directly with peroxide (H202) impregnated on blood 1. Improper specimen storage: must be protected from
○ Alkaline pH: Lysis of intact red cells reagent pad and removes it from the intended reaction → light as it causes rapid oxidation of bilirubin to biliverdin
○ Cell button after centri preventing oxidation of the chromogen or hydrolysis to free bilirubin
● Hemoglobinuria: ● Multistix is affected by ascorbic acid 2. Ascorbic acid: combines with the diazonium salt,
○ Clear concentration of ≥9 mg/dL and vChem at ≥ 5 preventing its reaction with bilirubin
○ Alkaline pH: hemoglobin oxidation mg/dl 3. High concentrations of nitrite
○ Uniform clear red urine after centri ● Multistix and vChem have modified their reagent
● Chemical methods: strips to reduce the interference to very high REAGENT STRIP METHOD FOR UROBILINOGEN
○ detect heme levels (25 mg/dl) of ascorbic acid while
● Multistix: Ehrlich’s aldehyde reaction
○ also detect other compounds containing it such Chemstrip is unaffected
○ light pink to dark pink
as myoglobin and cytochrome 2. High urine specific gravity, increased urine nitrite
○ Sensitivity: 0.2 mg/dL
(>10 mg/dL) and presence of formalin
○ False +: porphobilinogen, methydopa and
REAGENT STRIP METHOD FOR BLOOD ● high specific gravity causes intact red cells in
indican
urine to become crenated and resist lysis which
● impregnated with tetramethylbenzidine (chromogen) and is necessary to detect heme.
peroxide
● pseudoperoxidase activity of the heme moiety → REAGENT STRIP METHOD FOR BILIRUBIN
reduction of peroxide + oxidation of the chromogen → ● Chemstrip and vChem: azocoupling reaction
reaction pad from yellow to green ● based on the coupling reaction between bilirubin and the ○ white to pink
diazonium salt impregnated in the reagent pad in acid ○ Sensitivity:
medium ■ Chemstrip: 0.4 mg/dL, more specific
● produces a color change from light tan to beige or pink ■ vChem: 1 mg/dl
● Homogenous color change: hemoglobin
● Mottled/ speckled: intact RBC, lysed on the reax pad
● Sensitivity:
○ Multistix: diisopropylbenzene dihydroperoxide ● Multistix: 2,4-dichloroanilinediazonium salt ● urobilinogen result of 1 mg/dL or less is considered
tetramethylbenzidine ○ Sensitivity: 0.4-0.8 mg/dL normal
■ 6-20 RBCs/uL ● Chemstrip: 2,6-dichlorobenzine diazonium salt ○ neither of this method can detect its absence
■ 0.02-0.06 mg/dL hemoglobin ○ Sensitivity: 0.5 mg/dL ● FALSE POSITIVE: due to drugs (phenazopyridine) or
○ Chemstrip: dimethyldihydroperoxyhexane ● vChem:2,4-dichlorobenzene diazonium tetrafluoroborate atypical urine color (ingestion of red beets) masking the
tetramethylbenzidine reaction pad color change
■ 5-10 RBCs/uL ● FALSE NEGATIVE: improper preservation resulting to
■ 0.02-0.03 mg/dL hemoglobin the oxidation of urobilinogen to urobilin
○ presence of formalin and nitrites would also
inhibit the reaction.
Bravo J. 2023 AUBF Midterms
 QUALITATIVE TEST FOR BLOOD

TESTS PRINCIPLE REAGENT RESULTS REMARKS

SALT PRECIPITATION Purpose: Differentiate hemoglobinuria from Procedure:

METHOD (BLONDHEIM myoglobinuria 1. Add 2.8g of ammonium sulfate
+ supernatant remains red in myoglobin
TEST) to 5 mL of centrifuged urine
color and the blood reagent
Principle: Larger hemoglobin molecules are 2. Mix and stand for 5 minutes
strip is positive
precipitated by ammonium sulfate and myoglobin 3. The urine is now 80%
remains in the supernatant. saturated with ammonium
- red precipitate is observed hemoglobin
sulfate – this is optimal for
and the supernatant has a
hemoglobin precipitation
negative blood reagent strip
4. Filter or centrifuge urine
5. Test supernatant with reagent
strip

BENZIDINE TEST In the presence of the pseudoperoxidase activity of Benzidine powder, Glacial acetic acid, Green to blue color
hemoglobin or myoglobin, hydrogen peroxide is 3% Hydrogen Peroxide
decomposed to water and oxygen. The liberated
oxygen oxidizes benzidine to a green or blue color

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GUAIAC TEST In the presence of the pseudoperoxidase activity of Guaiac gum solution, Glacial acetic Green to blue color at the zone of contact
hemoglobin or myoglobin, hydrogen peroxide is acid, 3% Hydrogen Peroxide
decomposed to water and oxygen. The liberated
oxygen oxidizes the phenols present in guaiac to
quinones.

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OCCULT TABLET TEST Sodium bicarbonate reacts with tartaric acid to o-toluidine, Strontium peroxide, Blue color within 2 minutes
liberate carbon dioxide which causes Calcium acetate, Tartaric acid, Sodium
effervescence to facilitate the reaction. Tartaric acid bicarbonate, red dye
and calcium acetate reacts with strontium peroxide
to form hydrogen peroxide. In the presence of the
pseudoperoxidase activity of hemoglobin or
myoglobin, hydrogen peroxide is decomposed to
water and oxygen. The liberated oxygen oxidizes
o-toluidine to a blue color. A red dye is added to
mask discoloration of the tablet by blood.

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ORTHOTOLUIDINE Prepare a urine sediment and add orthotoluidine orthotoluidine in 1 % pure methyl
TEST and acid-peroxide mixture. A greenish blue to deep alcohol + mixture of glacial acetic acid
blue color that last for 1 minute or longer is a positive and H202
result.

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 QUALITATIVE TEST FOR BILIRUBIN

TESTS PRINCIPLE REAGENT/ PROCEDRE RESULTS

DIAZO TABLET TEST ● based on the same azocoupling reaction with a 1. 10 drops of urine are added to a
(ICTOTEST METHOD) diazonium salt on which the reagent strips are based special absorbent pad
+ purple or blue coloration is
● confirmatory test for bilirubin 2. An ictotest tablet is placed on top of
observed on the absorbent
● can detect bilirubin at a concentration as low as the pad then 2 drops of water is
pad
0.05-0.1 mg/dL added
● shares the same interferences as the reagent strip 3. After 30 seconds, the tablet is
- any other color such as red
removed and if bilirubin is present, a
or pink
purple or blue coloration is observed
on the absorbent pad.
4. Observation of any other color such
as red or pink is still reported as
negative.

(A) Negative
(B) Positive
(C)Negative result with
atypical color

GMELIN’S TEST In the presence of an acid, bilirubin undergoes varying degrees Nitric Acid Play of colors such as green
of oxidation. (biliverdin), blue (bilicyanine) and
yellow (choletelin)

HARRISON’S SPOT Barium chloride combines with sulfate radicals in urine to form 10% Barium Chloride, Fouchet’s Reagent Blue to green color
TEST barium sulfate precipitates to which bilirubin will adheres. Ferric (25% Trichloracetic acid, 10% Ferric
chloride, in the presence of trichloroacetic acid, oxidizes Chloride)
bilirubin to biliverdin resulting to a color change.

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ROSENBACH’S A drop of nitric acid containing yellow nitrous acid is applied to (+): band of colored ring around the nitric
MODIFICATION OF a filter paper soaked in urine acid
GMELIN’S TEST

HUPPERT’S TEST ● bile pigments are precipitated as a compound with

calcium hydroxide
● the compound is dissolved and the bile pigments are
oxidized to a green colored compound in an
acid-alcohol solution (conc. HCl and ethyl alcohol)

FERRIC CHLORIDE Bilirubin in the urine is concentrated by absorption on barium

TEST sulfate and phosphate and then is oxidized by ferric chloride in
acid solution to biliverdin (green) and cholecyanin

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FOUCHET TEST ● Barium chloride solution is added to the urine

● A precipitate of barium sulfate is produced on to which
the bilirubin is absorbed
● The barium sulfate absorbed bilirubin is filtered off and
a drop of Fouchet’s ferric chloride solution added to the
precipitate.
● The ferric chloride oxidizes the bilirubin to biliverdin,
producing a greenish-blue spot

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 QUALITATIVE TEST FOR UROBILINOGEN

TESTS PRINCIPLE REAGENT/ PROCEDURE RESULTS

WALLACE-DIAMOND Urobilinogens (urobilinogen, stercobilinogen, mesobilinogen) Ehrlich’s aldehyde reagent (+): Rose red color
METHOD condense in the presence of Ehrlich’s reagent in an acid
solution, causing a color reaction.

SCHLESINGER’S Urobilinogen is first oxidized to urobilin by iodine. Then, a Lugol’s solution (Potassium triiodide) and (+): Greenish fluorescence
METHOD zincurobilin complex is formed by the addition of zinc acetate. Schlesinger’s reagent (Zinc acetate in
absolute alcohol)

TESTS FOR BILE SALTS

TESTS PRINCIPLE REAGENT/ PROCEDURE RESULTS

HAY’S TEST Bile salts lower the surface tension of a liquid causing sulfur Sulfur powder ● Sulfur powder sinks at once:
powder to sink when placed on the solution 0.01% or more
● Sulfur powder sinks after
gentle agitation: 0.0025% or
more
● Sulfur powder remains
floating even after agitation:
Absence of bile acids

PETTENKOFER’S TEST In the presence of a dehydrating agent, sucrose form Sucrose and Sulfuric Acid (+) Red ring at the point of contact
hydroxymethylfurfural that forms a red compound when
complexed with bile salts.
 QUALITATIVE TEST FOR PORPHOBILINOGEN

TESTS PRINCIPLE REAGENT/ PROCEDURE RESULTS

HOESCH TEST ● screening method that can detect as low as 2

mg/dL of porphobilinogen in urine
● based on Ehrlich’s reaction but the ratio of urine
volume to reagent is inversed
● creates a highly acidic mixture preventing the
interference of urobilinogen except when
present in concentrations greater than 20 mg/dL
● deep pink or red color develops and disperses
uniformly in the urine when shaken.
○ intensity of the color directly relates to
the amount porphobilinogen present in
urine.
● Urine with atypical colors may make it difficult to
interpret
○ false-positive results: same as those
that affect the Ehrlich’s reaction
however, the presence of drugs (ex.
phenazopyridne) and indoles
● Hoesch test is preferred over Watson-Schwartz
Test

WATSON-SCHWARTZ ● modification of Erhlich’s reaction Procedure:
TEST ● allows differentiation of urine porphobilinogen 1. mix equal parts of urine and Ehrlich’s
from other Ehrlich reactive substances such as reagent (2 mL each)
urobilinogen 2. equal amount of saturated sodium
● based on the solubility characteristics of acetate (4 mL) is added to the tube
porphobilinogen with respect to pH and 3. red or magenta color, is observed in the
solvent type tube if Ehrlich reactive substances are
● Ehrlich’s reaction is performed on solvents with present
different polarity; a. If negative, no further testing is
○ sodium acetate done–absence of
○ Chloroform porphobilinogen and other
○ Butanol Ehrlich reactive substances
4. if the result is positive chloroform
extraction is done
a. 2-5 mL chloroform is added in
the tube and shaken vigorously
5. solvents are then allowed to separate
upon standing or can be hastened by
centrifugation
● If the red color is found in both
layers, chloroform extraction must
be repeated on the aqueous layer
● red color is only found in the
chloroform layer (bottom),
urobilinogen is present and
extracted
● red color is only found in the
aqueous layer (top),
porphobilinogen and other Ehrlich
reactive substances are present
indicating the need to perform
butanol extraction
● The aqueous layer from the
chloroform extraction set up is
transferred to another tube and an
equal volume of butanol is added
○ If the red color is only
 found in the aqueous
layer (bottom),
porphobilinogen is
present.
○ If the red color is only
found in the butanol
layer (top), other Ehrlich
reactive substances are
present