CHEMICAL EXAMINATION OF

URINE

📌 reference discussion by sir Ulysses (use school email to access :>):

https://drive.google.com/drive/u/0/folders/1a12uhG7PHPuZzGgeVIh5wlIjAEGbPu49

📌 pdf link:
https://drive.google.com/file/d/1Zlyd51V2vwPN1NhrHPqZiLOn9dAOKua0/view

REAGENT STRIP TECHNIQUE

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A. when using the reagent strip you should dip the strip briefly no longer than _____ second
into a well-mixed uncentrifuged urine specimen at _____. 1 second at room temperature

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 B. remove the excess urine by touching the _____ of the strip to the container as the strip is
withdrawn. Edge
C. blot the edge of the strip on a _____. Disposable absorbent pad

D. wait to the specified amount of time for the _____ to occur. Reaction
E. compare the color reaction of the strip pads to the _____ color char in good lighting.
Manufacturer

CARE OF REAGENT STRIPS

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A. store with desiccant in a _____, tightly closed container. Opaque

B. store below _____; do not freeze. 30 deg. Celsius (room
temperature)
C. do not expose to volatile _____. Fumes
D. do not use past the _____ date. Expiration
E. do not use if chemical pads become _____. Discolored
F. remove strips _____ prior to use. Immediately

AUTOMATIC REAGENT STRIP REAGENTS

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the principle of machine used in chemical examination is. Reflectance photometry

Reflectance Photometry
light reflection from the test pads _____ in proportion to the intensity of color produced
by concentration of the test substance. Decrease

the darker the color of the reagent pad, the _____ the ight reflection (vice versa).
Lesser

a monochromatic light is directed towards the reagent pad by placing a filter between
the light source and reflective sources of the pad

uses LED that monochromatic light passes through the test pad and light is reflected to
the detector

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 1. SPECIFIC GRAVITY
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COLORED CHART:

this is a density of solution compared with density of similar volume of distilled water at
a similar temperature. Specific Gravity

the specific gravity are influenced by _____ and _____ of particles in a solution.
Number & Size

Determination of SG
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what are the two methods in the determination of specific gravity?

1. urinometry (urinometer/hydrometer)

2. refractometry (refractometer, Rf/TS meter)

notes to remember:

a. both refractometer (Rf) and urinometer (U) require corrections for glucose and
protein

b. refractometry reading is lower than the urinometer reading by 0.002 (Rf < U by
0.002)

1. Urinometry (Urinometer/Hydrometer)
what are some disadvantages of urinometer?

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 less accurate

requires large volume of urine

not recommended by CLSI

what is the calibration temperature of urinometer? 20 deg. Celsius

for every 3 deg. Celsius BELOW the calibration temperature we ____ it with 0.001.
Substract

for every 3 deg. Celsius ABOVE the calibration temperature we ____ it with 0.001. Add

for every 1g/dL of glucose we should _____ it with 0.004. Substract

for every 1g/dL of protein we should _____ it with 0.003. Substract

how much volume does urinometer requires? 10-15mL

for the calibration of urinometer, we use the _____ solution. Potassium Sulfate
(K2SO4)

you just need to add 20.29g K2SO4 to 1L H2O to get the SG reading of 1.015

when using urinometer, an adequate amount of urine is poured into a proper-size

container and the urinometer is added with a _____. Spinning motion

the scale reading of urinometer is taken at the _____ of the urine meniscus. Bottom

SAMPLE PROBLEM W/ SOLUTION:

2. Refractometry (Refractometer, Rf/TS Meter)

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what are the steps in using refractometer

1. put 1 or 2 drops of sample on the prism

2. close the daylight plate gently

3. sample must spread all over the prism surface

4. look at the scale through the eyepiece

5. read scaled where the boundary line intercepts it

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 6. wipe the sample from the prism clean with tissue and water

what is the formula for the INDIRECT method of refractive index?

since refractive index do NOT need temperature correction, what is the compensated
temperature of RI? 15-38 deg. Celsius

what correction does the refractive index needs?

protein

glucose

the refractometer is calibrated with a reading of, using:

Distilled water 1.000

3% NaCl 1.015 ± 0.001

5% NaCl 1.022 ± 0.001

9% sucrose 1.034 ± 0.001

SAMPLE PROBLEM W/ SOLUTION:

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 Specific Gravity Dilution
specimens with very high SG readings can be diluted and retested.

to obtain the actual SG, multiply the decimal portion of SG by the dilution factor.

Example:

1. Urine specimen diluted 1:4 has a reading of 1.014. What is the actual SG reading?

0.014 x 4 = 0.056 + 1 = 1.056, because:

a. decimal portion of 1.014 is 0.014

b. you multiply it with the dilution factor of 4 which is equal to 0.056

c. then add the whole number from SG which is 1, then the final SG is 1.056

Reagent Strip Reaction for SG

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reagent strip reaction for SG is read at? 45seconds

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 Harmonic Oscillation Densitometry
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it is based on frequency of soundwave entering a solution changes in proportion to the

density of solution.

it is originally used in early automated urinalysis instrument, the addition of reagent

strip analysis forces SG to replace densitometry into an automated system

what is the example of hormonic oscillation densitometry?

yellow IRIS (International Remote Imaging System) which models 300 & 500
workstations

IRIS diagnostics requires how much urine volume? 6mL

the 4mL (of 6mL) is for IRIS slide less microscope

the 2mL (of 6mL) is for IRIS mass gravity meter (for SG determination by using
harmonic oscillation)

2. pH
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COLORED CHART:

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 it is important in the identification of crystals and determination of unsatisfactory
specimens

it is determined by the concentration of the free hydrogen ion. pH

what is the normal pH of the urine?

random: 4.5-8.0

1st morning: 5.0-6.0 (acidic)

protein diet: 4.5-6.5

what is the pH of unpreserved urine? 9.0

what are the clinical significance of pH?

1. respiratory or metabolic acidosis/alkalosis

2. renal tubular acidosis

3. identification of crystals

4. determination of unsatisfactory specimens

how do you measure the pH of the urine?

pH meter w/ glass electrode

litmus or nitrazine paper

reagent strip

this occurs after meals due to withdrawal of H+ ions for the purpose of secretion of HCl.
Alkaline tide

Reagent Strips Reaction for pH

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 reagent strip reaction for pH is read at? 60seconds

when pH is decreased, your _____ ion is increased. Hydrogen

relationship is inversely proportional

3. PROTEIN
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this is the most indicative of renal disease. Protein

what is the normal value of protein in the urine? 150mg/24hrs

what are the proteins that are normally found in urine?

albumin

tamm-horsefall protein

serum/tubular microglobulins

proteins from prostatic/vaginal secretions

protein produces ______ foam in urine when shaken. White

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 presence of increased protein sin urine >30mg/dL or 300mg/L is called. Clinical
Proteinuria

clinical proteinuria is categorized into three (3) groups and these are:

1. pre-renal

2. renal

3. postrenal

1. Pre-Renal (Before)/Overflow Proteinuria

this is caused by conditions that affect the plasma prior to its reaching the kidney:

a. intravascular hemolysis - hemoglobin

b. muscle injury - myoglobin

c. severe infection & inflammation - increase APRs

d. multiple myeloma - proliferation of Ig-producing plasma cells (Bence Jones

Protein)

NOT and indicative of actual renal diseases and NOT detected by reagent strip. Pre-
renal proteinuria

increased quantities of low molecular weight plasma proteins in the blood results in
an overflow of these proteins in the urine.

what are the examples of pre-renal proteinuria?

1. acute phase reactants

2. hemoglobin

3. mygobn

4. bence-jones protein

Bence Jones Protein (BJP)

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 it is an immunoglobulin light chain. BJP

what tests are performed in BJP?

serum electrophoresis

immunofixation electrophoresis

what is the urine reading of BJP?

precipitation at 40-60 deg. Celsius (cloudy) and dissolves at 100 deg. Celsius
(clear)

interference due to the other precipitated proteins can be removed by filtering the
specimen at _____ and observing the specimen for turbidity as it cools to between 40
deg. Celsius and 60 deg. Celsius. 100 deg. Celsius

2. Renal Proteinuria (True Renal Disease)

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this is associated with TRUE renal disease. Renal proteinuria

Glomerular Proteinuria
this is the most common and most serious type of renal proteinuria. Glomerular
protenuria

what are the examples of renal proteinuria?

1. post-streptococcal glomerulonephritis

2. orthostatic proteinuria

3. nephrotic syndrome

this is a decreased glomerular filtration (defective) and may lead to renal failure.
Diabetic Nephropathy

what is the indicator of the disease diabetic nephropathy? Microalbuminuria

this microalbuminuria is a proteinuria undetectable by routine reagent strip

this also signifies onset of renal complications of DM

Significant if: 30 to 300 mg/24 hours

Albumin Excretion Rate: 20 to 20 ug/min

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 what is the albumin excretion rate (AER)? ug/min or mg/24hrs

what is the normal AER? 0-20 ug/min

what is the AER when you have the indicator microalbuminuria? 20-200 ug/min (30-
300 mg/24hr)

what is the AER of clinical albuminuria? >200 ug/min

Micral Test
this is a test for microalbuminuria. Micral test

since undetectable man ang microalbuminuria sa routine reagent strip, then we

need to use this test

this is a strip employing antibody-enzyme conjugate that binds albumin. Micral test

the principle of mircral test is the. Enzyme immunoassay (Immunochemical method)

what are the reagents used in micral test?

gold-labeled antibody

b-galactoside

chlorophenol red galactoside

what is the sensitivity value of the micral test? 0-10 mg/dL

what are some interference for micral test? It is false negative to diluted urine

Immunodip
what is the principle of immunodip? Immunochromographics

what is the sensitivity for this test? 1.2-8.0 mg/dL

what are the reagents used for immunodip? Antibody coated blue latex particles

what are some interference in immunodip? False negative to diluted urine

Albumin: Creatine Ration-Clinitest Microalbumin Strips/Multistix-Pro

what is the principle for this?

sensitive albumin tests related to creatine concentration to correct for patient

hydration

what are the reagents used?

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 albumin: diiodo-dihydroxydinitrophenyl tetrabromosulfonpthalein

creatine: copper sulfate, tetramethylbenzidine, diisopropylbenzenedihydroperoxide

what is the sensitivity of albumin and creatine?

albumin: 10-15 mg/dL

creatine: 10-300 mg/dL (0.9-26.5 mmol/L)

what are the interferences for this test?

visibly bloody/abnormally colored urine

creatine: cimetidine can cause false-positive

1. Orthostatic/Cadet/Postural Proteinuria
it is a proteinuria when standing due to increased pressure to renal veins.
Othostatic/Cadet/Postural proteinuria

Difference between Orthostatic and Clinical Protenuria:

2. Tubular Proteinuria
it is an filtered albumin that can no longer be reabsorbed. Tubular proteinuria

such as:

1. fanconi’s syndrome

2. toxic agents/heavy metals

3. severe viral infections

3. Post-Renal Proteinuria (After)

urine includes proteins produced by the urinary tract

1. lower UTI/inflammation

2. injury/trauma

3. menstrual contamination

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 4. prostatic fluid/spermatozoa

5. vaginal secretions

Reagents Strip Reaction for Protein

reagent strip reaction for protein is read at? 60seconds

Sulfosalicylic Acid (SSA) Precipitation Test

this is a cold precipitation test that reacts equally with all forms of protein. SSA
precipitation test

what is the volume needed for SSA? 3mL

what is the reagent used in SSA? 3% SSA

SSA has a sensitivity of? 5-10mg/dL

what is the procedure for SSA precipitation test?

3mL of 3% SSA (using Extons’s reagent) + 3mL centrifuged urine, then the
positive result should be presence of cloudiness

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 4. GLUCOSE (Dextrose)
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COLORED CHART:

this is the most frequently performed chemical examination of urine. Glucose

under normal consumption test almost all glucose are filtered by the glomerulus
and is actively reabsorbed in the proximal convoluted tube (PCT)

reagent strip reaction for glucose is read at? 30seconds

what is the tubular reabsorption of glucose? Active transport

this is a plasma concentration of a substance at which tubular reabsorption stops.

Renal threshold

what is the renal threshold for glucose? 160-180 mg/dL

what is the clinical significance of glucose reagent strip? Detection and monitoring of
diabetes mellitus

presence of detectable levels of glucose in urine is called. Glycosuria/Glucosuria

what is the normal value of glucose in the urine? 15mg/dL

fasting: 2-20mg/dL per 100mL of urine

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 what are the other sugars found in the urine that are identified by TLC?

1. fructose (levulose)

2. galactose

3. lactose (glucose + galactose)

4. pentose

5. sucrose (glucose + fructose

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 NOTE: False (+): similar to the test principle, while False (-): opposite to the test principle

Clinitest Procedure
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what is needed in the clinitest procedure?

5 gtts(drop) urine + 10 gtts(drop) H2O + clinitest tablet

the clinitest tablet contains:a

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 a. CuSO4: main reacting agent

b. NaCO3: eliminates interfering O2

c. Na citrate: for heat production

this phenomenon occurs when >g/dL sugar is present. Pass-through phenomenon

blue>green>yellow>brick red >>>>> blue or green-brown

to prevent pass through, use 2gtts urine (use separate color chart to interpret the
reaction)

5. KETONES
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COLORED CHART:

this is a result from increased fat metabolism due to inability to metabolize

carbohydrates. Ketones

reagent strip reaction for ketones is read at? 40seconds

ketones are seen in:

type 1 DM

vomiting

starvation

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 malabsorption

the tablet acetest contains:

sodium nitroprusside

disodium phosphate

glycine

lactose

6. BLOOD
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COLORED CHART:

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 reagent strip reaction for blood is read at? 60seconds

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 7. BILIRUBIN
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COLORED CHART:

this is an early indication of liver disease. Bilirubin

reagent strip reaction for bilirubin is read at? 30seconds

this type of bilirubin is water soluble. Conjugated bilirubin

what is the color of urine if there is a presence of bilirubin? Tea-colored/Amber (w/

yellow foam)

the clinical significance of bilirubin is. Identification of liver disorders

hepatitis, cirrhosis

bilary obstruction (gallstones, carcinoma)

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 this tablet is more sensitive than strip with less interference. Ictotest tablet

ictotest tablet contains:

p-nitrobenzene-diazonium p-toluenesulfonate

SSA

sodium carbonate

boric acid

(+) blue to purple color

8. UROBILINOGEN
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COLORED CHART:

this is a bile pigment that result from hemoglobin degradation. Urobilinogen

reagent strip reaction for urobilinogen is read at? 60seconds

the preferred specimen of urobilinogen is:

afternoon urine (2-4PM)

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 what is the normal value of urobilinogen? <1 mg/dL or Ehrlich unit

PRINCIPLES OF WATSON-SCHWARTZ TEST:

if the test is soluble to chloroform and butanol then it is. Urobilinogen

if the test is insoluble to chloroform and butanol then it is. Porphobilinogen

if its soluble only to butanol then it is. Other ERC

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 During PRE-HEPATIC stage:

1. the lifespan of RBC under normal condition is 120 days, at which time they are
destroyed by the liver and the spleen (macrophages) by phagocytic cell of the
reticuloendothelial system

2. the liberated hemoglobin is broken down into heme and globin

3. globin will go to the amino acid pool

4. heme is divided into iron and protophorphyrin IX

5. the body uses iron in the bone marrow

6. and converts protophorphyrin IX into unconjugated bilirubin (UB)

7. the unconjugated bilirubin (UB) is released in the circulation which binds to albumin
and is transported to the liver

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 During HEPATIC stage:

1. when UB together with albumin is transported to the liver, the liver will convert the
UB to conjugated blirubin (CB) by UDPGT (Uridine Diphosphate Glucoronyl
Transferase)

During POST-HEPATIC stage:

1. CB does not appear in the urine because it is passed directly from the liver into the
bile duct and intestine

2. the intestinal bacteria will then convert bilirubin to urobilinogen

3. which is then excreted in the feces as fecal urobilinogen in the form of

stercobilinogen and urobilin in the form of stercobilin

4. some urobilinogen is absorbed in the intestine into the blood and the blood
circulates to the liver, and excreted back to bile duct and intestine

5. the stercobilinogen cannot be reabsorbed and it will remain in the intestine and will
be oxidized to stercobilin

6. the recirculated urobilinogen reaches to the intestine and will be oxidized urobilin

both urobilin and stercobilin are excreted in the feces and these responsible for
the brown pigment of poop (stercobilin)

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 7. the urobilinogen appears in the urine because as it circulate back to the liver, it
passes sad in the kidney and are filtered by glomerulus

8. small amount of urobilinogen (<1mg/dL) is normally found in the urine and becomes
urobilin

9. NITRITE
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COLORED CHART:

this is a rapid screening test for UTI of bacteriuria. Nitrite

reagent strip reaction for nitrite is read at? 60seconds

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 10. LEUKOCYTES
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COLORED CHART:

reagent strip reaction for leukocyte is read at? 2minutes

the clinical significance of leukocyte is for:

UTI/Inflammation

screening of culture specimen

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 11. ASCORBIC ACID
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COLORED CHART:

this causes false negative reactions on blood, bilirubin, leukocytes, nitrite, and glucose.
Ascorbic acid

what is the 11th reagent pad? Ascorbic acid

ascorbic acid (≥5 mg/dL) + phosphomolybdate = (+) molybdeum blue

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