MLS 419: AUBF LAB CHEMICAL EXAMINATION OF URINE

PRELIM WEEK 4 – LESSON 4  pH

Chemical Examination of Urine  Proteins
 Glucose
 Ketones
REAGENT STRIPS –aka DIPSTICK  Blood
2 known brands:  Bilirubin
 Multistix  Urobilinogen
 Chemstrip  Nitrite
 Leukocytes
What is it?  Specific gravity
 Chemical-impregnated absorbent pads attached
to a plastic strip
 Each pad contains reagents, chemicals and
enzymes that allow us to test for chemical
constituents of your urine
 Looks simple but modern-state-of-the-art
 A color-producing chemical reaction takes place
when the strip comes in contact with urine
 End result –change in color
 Reactions are interpreted by comparing the color
produced on the pad with the chart provided by
the manufacturer (Semiquantitative)
 Only able to provide approximate
concentrations and not extract
concentrations
 Very sensitive and specific

4 VS 10 PARAMETERS
Question: When to perform?
Urinalysis is a 3-step process:

 1st process = PHYSICAL EXAM

o Performed after receiving the sample
o After assessing the validity of the sample
o Color, clarity and specific gravity
o
 2nd process = CHEMICAL EXAM
 4 parameters (Most common since it is cheaper) o Where your reagent strip enters
include: o NOTE: only perform a C.E in a sample
o Glucose that has not been centrifugated yet
o pH
o Specific Gravity –part of chemical  3rd process = MICROSCOPIC EXAM
examination of urine but it still remains as a
physical parameter (more reliable) Final ans: after physical exam, before miscroscopic exam
o Protein
 10 parameters include: REAGENT STRIPS
o 4 parameters Procedure:
o +6 other parameters – 1. Dip the reagent COMPLETELY but BRIEFLY into a
 Ketones well-mixed specimen
 Blood 2. Remove the excess urine by running the edge of the
 Bilirubin strip on the container/test tube when withdrawing
 Urobilinogen it from the specimen and blotting it horizontally on
 Nitrite the absorbent medium (BACK PORTION ONLY –
 Leukocytes
 facilitate further drainage of excess fluid to ensure
na di siya masyadong wet)
3. Wait for the specified length of time (CRUCIAL) for
the reactions to take place and compare colored
results to the manufacturer’s chart under a good
source of light
 Leukocyte –read after 120s
 Specific Gravity –read after 45s
Mix specimine > Dip strip > remove excess > blot > time
> compare
 Transfer immediately your urine sample after
receiving it
 Containers –plastic and testube –glass (PERMITS
LIGHT TO ENTER =ALLOW BETTER
OBSERVATION)
ERRORS CAUSED BY IMPROPER TECHNIQUE
1. Formed elements such as red and white cells
sink to the bottom of the specimen and will be
undetected in an unmixed specimen
 No contact = no reaction = false negative
reaction
2. Allowing the strip to remain in the urine for an
extended period may cause leaching of reagents
from the pad.
  Metabolites of drugs –PHENAZOPYRIDINE
3. 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
color. To ensure against run-over, blotting the
edge of the strip on absorbent paper and
holding the strip horizontally while comparing it
with the chart.
 Should drain excess urine
 Too wet/too drench = there can be mixing of
2 pads and their color reactions
4. The timing for the reactions to take place varies
among manufacturers and ranges from immediate
reaction for pH to 120 seconds for the leukocyte
esterase (60-120 seconds)
5. A good light source is essential for accurate
interpretation of color reactions.
 Proper lightings daw po kasee mamser, chareng!
6. The strip must be held close to the color chart
without actually placing on the chart
7. Reagent strips and color charts from different
manufacturers are not interchangeable
 Be brand specific
 Winner jd sa layf ang mga loyal, hehe
8. Specimens that have been refrigerated must be
allowed to return to RT prior to reagent strip testing
as the enzymatic reactions are temperature-
dependent.
HANDLING AND STORING OF REAGENT STRIPS
 Reagent strips must be protected from moisture,
volatile chemicals, heat and light
 Packaged into opaque containers with desiccant
 Strips are removed just prior to testing and bottle is
resealed immediately
 Stored at room temperature below 30 degree Celsius
(but never refrigerated)
 Must not be used beyond expiration date
 Care must be taken not to touch the chemical pads
when removing the strips from the container
 Visual inspection should be done every use to detect
deterioration. Do not use if pads are discoloured.
QUALITY CONTROL OF REAGENT STRIPS –WHEN?
 Reagent strips must be tested with positive and
negative controls
1.) minimum of once every 24 hours
2.) every after new bottle is opened
3.) questionable results are obtained
4.) when there is concern about the integrity of the
strip.
 Record all control results –for referencing
 Distilled water should not be used as negative
control. The manufacturers will provide the controls.
 Because the reagent strips are designed to
work at ionic concentration and since yung
dh20 nag undergone nan g distillation so
meaning deionize na siya
 Does not mimic the actual testing conditions
ERROR SOURCES IN READING THE REAGENT STRIP
1. Interfering substances in the urine
 Its pigment will interfere in the color
reaction
2. Technical carelessness
 Wag daw kasi tanga
 Sorry Sir K
3. Colorblindness
:<
VALUE AND APPLICATION
“Results should coincide with the physical examination
results and must be suggestive of the probable sediments
that can be seen during microscopic exam.”
*most of the time but not all the time!
 Example
o PE –red, cloudy
o CE –positive for blood na test
o ME –rbcs
REPORTING OF RESULTS
 Depending on the test performed, the results are
reported in the following manner:
 in concentration (mg/dL)
 as small, moderate or large
 using the plus system (1+,2+,3+,4+)
 as positive, negative or normal
 specific gravity and pH
 results are estimated in their
respective unit
pH
 The average adult on a normal diet excretes about
50–100 mEq of hydrogen ions in 24 hours to produce
urine of about pH 6. In healthy individuals, urine pH
may vary from 4.5–8.
 If 8.5-9 pH –indicates that the sample is old
and not suitable for testing = reject!
(deserve)
 Reagent Strip Reaction
 Double Indicator System
 Should detect pH changes
 Indicators should indicate pH
change in that together with the
change in pH, indicators should also
change in color
 Measurement of urine pH and acidity must always be
made on freshly voided specimens.
 The container should be filled to minimize the
amount of dead space, and the urine covered tightly.
 The container should be kept cold, preferably on
ice, but not frozen.
Protein
 The presence of increased amounts of protein in the
urine can be an important indicator of renal disease.
 High protein concentration = damaged
kidneys
 Reagent Strip Reaction
 Protein Error of Indicator
 Protein error of indicators
 Certain indicators can change color in the
presence of proteins even though the pH of
the medium remains constant
 NO pH change!!
 Proteins (amino group) accepts hydrogen
ions from the indicator. Albumin has a lot of
amino groups in its structure so it accepts
more hydrogen ions.
 Major protein detected in reagent
strip
 Multistix indicator = Tetrabromophenol blue
 Chemstrip indicator =3’,3”,5’,5”-tetrachlorophenol
3,4,5,6-tetrabromosulfonphthalein
*at acid pH = yellow
*at alkaline pH =bluish green
 PLUS BUFFER –maintains pH at constant level
 pH 3 =acid always!
 Default color =yellow
How will the indicator change in color?
Case 1
-urine has a lot of proteins (albumin) > many amino
groups > indicator in itself will lose > donates H ions
> accepted by albumins
*the more that you have albumin the more that
the indicators will lose Hydrogen > pH will be
alkaline > bluish green strip
Case 2
-urine has no proteins (no albumin) > no acceptance
of H ions > indicators will not lose hydrogen > pH
will remain acidic > yellow strip
 Very sensitive to albumin
 Other urine proteins such as gamma globulin,
glycoprotein, ribonuclease, lysozyme, hemoglobin,
Tamm–Horsfall mucoprotein, and Bence-Jones
protein are much less readily detected than albumin.
 Therefore, a negative urinary dipstick result does not
necessarily rule out the presence of these proteins.
Glucose
 When the blood glucose exceeds the renal
threshold, the tubules cannot reabsorb all of the
filtered glucose, and so glycosuria occurs
 Aids in the diagnosis of diabetes mellitus
 Therefore, a negative urinary dipstick result does not
necessarily rule out the presence of these proteins.
 Reagent Strip Reaction:
 Double Sequential Enzymatic Reaction
 Chromogens used are the following (it indicates
reaction by changing color)
 Multistix: Potassium iodide (green to
brown)
  Chemstrip : Tetramethylbenzidine  No method can detect BHA
(yellow to green)  Acetone
 Detectable only when glycine is
present

 Reagent
 Sodium nitroprusside reaction
 Sequential because???
 Acetoacetic acid in alkaline medium reacts
 Making use of 2 enzymes and the reactions
with sodium nitroprusside to yield a purple
are sequential (sunodsunod)
color
 The product of the 1st reaction
eventually becomes the substrate of
OTHER REAGENT STRIP BRANDS WITH THEIR
the 2nd reaction
 st
1 enzyme = GOX SENSITIVITIES
 2nd enzyme = PEROXIDASE (hydrolyzes H2O2)

OTHER REAGENT STRIP BRANDS WITH THEIR

CHROMOGENS

BLOOD

KETONES

 Read at 60s
 Blood in the urine sample is an indication of a
 Ketones, or ketone bodies are formed during the
possible bleeding episode in the renal tract.
catabolism of fatty acids.
 Red urine = urine sample contains blood
 Primary Source of Energy – CHO
 may mean 3 different conditions:
 But with CHO Depletion –METABOLISM OF
(hematuria, hemoglobinuria
FAT FOR ENERGY > KETONE BODIES
myoglobinuria)
(byproduct)
 Hematuria (presence of intact rbcs that has not been
destroyed; speckling pattern) versus
 Ketone bodies:
Hemoglobinuria (presence of hemoglobin in urine,
 Acetoacetic acid (diacetic acid)
rbcs have been destroyed, lysed; color is
 PARENT!!
homogenous)
 from this you can produce the other
2 KBs:  Myoglobinuria
 Brownish-red colored urine
 decarboxylation = acetone
 Behaves just like myoglobin
 spontaneously = B-HA
 Muscles
 **most of the reagent strips, they
only detect AA
 Reagent Strip Reaction:
 B-hydroxybutyric acid
 Pseudoperoxidase activity of Hemoglobin
  Activity is similar to peroxidase
 Heme > hydrolyzes hydrogen peroxide >
forming h20 and 02
OTHER REAGENT STRIP BRANDS WITH THEIR
CHROMOGENS
BILIRUBIN
 Detected at 30s
 Bilirubin is a breakdown product of hemoglobin that
is formed in the reticuloendothelial cells of the
spleen, liver, and bone marrow
 Reflects liver disease
 B1 versus B2
 B1 –unconjugated, water insoluble
 B2 –conjugated, water soluble
 Only detected
 Reagent Strip Reaction:
 Diazo Reaction/Azo-coupling Reaction
 Bilirubin reacts with 2,4-dichloroaniline-
diazonium salt (Multistix) or 2,6-
dichlorobenzene-diazonium-
tetrafluoroborate in an acidic medium to
produce an azo dye (tan/pink-violet)
 Fresh urine
 If not fresh the B2 will be converted to free
haemoglobin (not detected)
 Avoid exposure to sunlight
 So that the bilirubin wwill not be converted
back to biliverdin (not detected)
*miscellaneous test to help confirm if there is bilirubin in your
sample
 Yellow brown to reddish brown
 Yellow foam that does not disintegrate (shake)
 *major interfering factor =presence of
PHENAZOPYRIDINE
 History taking is important
UROBILINOGEN
 Detected at 60s
 Urobilinogen represents a group of closely related
tetrapyrrole compounds formed from reduction of
bilirubin
 Reagent Strip Reaction:
 Ehrlich Aldehyde Reaction
 PDAB
 Diazo Reaction/Azo-coupling
(MULTISTIX)
 Both produce pink to red colors if positive
 Urobilinogen is normally present in the urine but in
concentrations of 1 Ehrlich unit or less per 100 mL
of urine
 Strip can still detect this level, lower than
this, not anymore
 (REPORT AS NORMAL AND NOT
NEGATIVE)
 Urobilinogen instability is a problem
 Fresh specimen
 When it is exposed to air, it will be converted
to urobilin (not detected)
 When is the best time to collect urine for
urobilinogen testing?
 Between 2pm to 4pm : peak
NITRITE
 Read at 60s
 The nitrite test is a rapid, indirect method for the
early detection of significant and asymptomatic
bacteriuria
  Not directly isolating and cultivating bacteria
 If (+) =there is presence of bacteria
 Detection of UTI
 Mostly caused by Enterobactericiae –
enzyme nitrate reductase =able to convert
nitrate to nitrate
 Normally, urine has nitrate
  glucose, blood, bilirubin, nitrite, and
 Reagent Strip Reaction:
 Greiss reaction
 Nitrate at an acidic pH reacts with an
aromatic amine (para—arsanilic acid or
sulfanilamide) to form a diazonium
compound that then reacts with
tetrahydrobenzoquinolin compounds to
produce a pink colored azodye.
 Any degree of uniform pink color should be
interpreted as a positive nitrite test
 suggesting the presence of 105 or more
organisms per milliliter .
 First morning urine is the specimen of choice
 Usually takes 4 hrs to convert nitrate to
nitrite
LEUKOCYTE ESTERASE
 Read after 120s
 Detects the presence of esterase in the granulocytic
white blood cells
 BEN
 Esterases also are present in Trichomonas and
histiocytes (interferences)
 Help diagnose UTI
 Reagent Strip Reaction:
 Action of LE to catalyze the hydrolysis of an
acid ester
 (PURPLE)
 A positive LE test result is most frequently
accompanied by:
 the presence of bacteria
 which may or may not produce a
positive nitrite reaction
SPECIFIC GRAVITY
 The addition of a specific gravity testing area to
reagent strips has eliminated a time-consuming
step in routine urinalysis and has provided a
convenient method for routine screening.
 Reagent Strip Reaction:
 Change in pKa (dissociation constant) of
a polyelectrolyte in an alkaline medium
 HIGHER SOLUTE = MORE HYDROGEN
RELEASE = ACID = YELLOW
Additional Reagent Strip Parameter
ASCORBIC ACID
 Ascorbic acid may inhibit several reagent strip
reactions
leukocyte esterase
 BBLNG
 REDUCING AGENT!
 It can neutralize the R.S. which is oxidative
in nature
 CAN ALSO COUNTERACT THE ACTIVITY OF
AZO DYE
 Reagent Strip Reaction:
 10sec reading time
 Other reagent strip brands include:
 Calcium, creatinine, and microalbumin
CONFIRMATORY TESTING
 Using different reagents or methodologies to detect
the same substance as detected by the reagent strip
with some or greater sensitivity and specificity
 Tablets and liquid chemicals
 But not often used today due to increased sensitivity
and specificity of reagent test strips
SULFOSALICYLIC ACID TEST FOR PROTEIN:
 Sulfosalicylic acid testing
 Add 3mL of 3% SSA to 3mL of centrifuged urine
 Mix by inversion and observe for cloudiness
 Grade the turbidity
OTHER CONVENTIONAL CHEMICAL TESTS
 Heller’s Ring Test for Protein
 Benedict’s Test for Glucose
 Rothera’s Test for Ketone bodies
  Gerhardt’s Test for Ketone Bodies
 Gmelin’s Test for Bile Pigments
 Smith Iodine Test for Bile Pigments

TABLE-BASED TESTS
 Clinitest -glucose
 Acetest –ketone bodies
 Advantageous, you can use other body
fluids
 Ictotest -bilirubin
 Better than R.S.

dm us @REVIEWERMODIHA for corrections.