ANALYSIS OF ABNORMAL CONSTITUENTS OF URINE

Sub-competency: BI 11.4- Perform urine analysis to estimate and determine normal and
abnormal constituents

BI11.20 Identify abnormal constituents in urine, interpret the findings and correlate these
with pathological states

Many substances such as glucose, protein, amino acids etc., are present in trace amounts in
normal urine. They escape detection due to the low sensitivity of the tests employed.
Concentrations of these constituents in urine are increased markedly in different pathological
conditions. When they are present in detectable amounts they are termed as abnormal
constituents. Presence of these substances is suggestive of underlying pathological conditions
and of diagnostic importance. Usually the analysis is carried out with a suitably preserved 24
hr. urine specimen. On standing urine undergoes bacterial fermentation. It can be preserved
under refrigeration or using chemicals such as toluene or chloroform.

A. Physical characteristics of urine in pathological condition:

1. Volume: An increase in urinary output (polyuria) occurs in diabetes mellitus and diabetes
insipidus or after administration of drugs like digitalis, salicylate etc.
A diminished urine output (oliguria) occurs in nephritis, fever or diarrhea and vomiting.
A total suppression of urine formation (anuria) may occur during shock, acute nephritis,
incompatible blood transfusion, mercury poisoning or bilateral renal stone formation.
2. Color: The urine appears smoky brown when blood is present, yellow when bilirubin
glucuronide is present, black when melanin is present. Milky appearance may be due to the
presence of pus, bacterial or epithelial cells or lipids.
3. pH: Significantly acidic urine is voided in fever or acidosis. Alkali therapy and urinary
retention make urine alkaline.
4. Specific gravity: Specific gravity is high in acute nephritis and fever and low in diabetes
insipidus.

B. Chemical constituents:

The abnormal constituents which are routinely analyzed in urine are albumin, glucose,
ketone bodies, bile salts, bilirubin (bile pigment) and blood.

Test Observation Inference

Benedict's test for glucose and Light green Presence of reducing sugar
reducing sugars: 5 ml Benedict's or yellow or generally, glucose. Color is
reagent + 8 drops of urine. Boil for 2 brick red suggestive of approximate amount
minutes over a small flame. color of glucose in urine (green 0.5%,
yellow 1%, orange, 1.5%, brick
red >2%).

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 Note: Positive Benedict's test is usually taken for glucose. Glycosuria occurs mainly during
diabetes mellitus. Other reducing sugars in urine can also give positive test. Sugars such as
lactose (during pregnancy/lactation), galactose (in galactosemia), pentoses (in pentosuria)
also give positive test.
Ketone bodies: Rothera’s test for Purple ring Presence of acetone or and
acetone and acetoacetic acid: acetoacetic acid.
5 ml urine + solid ammonium sulphate
a little at a time with mixing to
saturate the solution + 2 or 3 drops of
freshly prepared sodium nitroprusside
solution. Mix gently + 1ml strong
ammonium hydroxide, drop wise
along the side of the test tube. Don't
mix.
Gerhardt’s test for acetoacetic acid: Port wine Presence of acetoacetic acid.
To 3 ml urine, add ferric chloride color is
solution drop by drop. (filter, if a obtained.
precipitate of ferric phosphate is seen).
Note: Acetone, acetoacetic acid and β-hydroxybutyric acid known as ketone bodies, are
found in urine when fat catabolism is excessive as in the case of diabetes mellitus and
starvation.

Test Observation Inference

Hay's test for Bile salts: Sulphur sinks Presence of bile salts. Bile salts
2 ml urine + sprinkle a to the bottom have the property of lowering the
pinch of sulfur powder. surface tension of solution in
Observe without mixing. which they are dissolved.
Fouchet's test for bile pigments:
5 ml of urine solution + 5 ml BaCI2 Green or blue Presence of bile pigments.
solution+ a pinch of MgSO4. Mix color BaCl2 reacts with MgSO4 to
well. BaSO4 is precipitated. After 5 form Barium Sulphate (BaSO4)
minutes filter the solution. Unfold precipitate. This adsorbs any
the filter paper over a dry filter paper. bilirubin present in the urine.
Add a few drops of Fouchet's reagent Fouchet’s reagent oxidises
on the precipitate. bilirubin to green (biliverdin)
and blue (bilicyanin) products.
Note: Presence of bile salts and bile pigments in urine is suggestive of obstructive jaundice
or hepatic jaundice.

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Heat and acetic acid test for albumin: A cloudy Presence of albumin in urine.
10 ml of urine. Hold the tube over flame white
in a slanting position and boil the upper precipitate
half portion of urine. The lower half will be
serves as control. Cool, add few drops observed in
of 1% acetic acid. the heated
portion
Note: The presence of detectable amount of albumin (albuminuria or proteinuria) is
characteristic of kidney diseases such as nephrotic syndrome.

Test Observation Inference

Benzidine test for Blood: Presence of blood.
Mix 2-3 drops of benzidine solution and Blue or green Heme of hemoglobin decomposes
2 drops of hydrogen peroxide. Add 2 color forms H2O2 to oxygen. The liberated
drops of this mixture to 2 ml of urine which is stable oxygen oxidizes benzidine to green
solution taken in a second tube. Observe only for a few or blue colored products which are
the change in color as you add. seconds and quite unstable.
later forms a
stable brown
color
Note: Hematuria (presence of RBC in urine) occurs due to bleeding in the urinary tract or due
to trauma caused by introduction of catheter through urethra. Renal cancer or stones in urinary
tract may also cause bleeding. When hemolyzed blood is found in urine the condition is
known as hemoglobinuria. This occurs in severe burns, chemical poisoning and incompatible
blood transfusion.

Observations

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Sub-competency: BI 11.4- Perform urine analysis to estimate and determine normal and
abnormal constituents
BI11.20Identify abnormal constituents in urine, interpret the findings and correlate these
with pathological states

Attempt 1 Attempt 2 Attempt 3

B M E B M E B M E

Heat and acetic acid test

Benedict’s test

Rothera’s test

Gerhadt’s test

Hay’s test

Fouchet’s test

Benzidine test

Grade

Signature of the assessor

with date

57
 DIPSTICKS FOR URINE ANALYSIS

Sub-competency:

BI 11.4- Perform urine analysis to estimate and determine normal and abnormal constituents

BI11.20 Identify abnormal constituents in urine, interpret the findings and correlate these
with pathological states

Dipsticks (urine analysis reagent strips) are used for both qualitative and semi-quantitative
urine analysis. These contain invitro reagent for diagnostics. (Dry chemistry tests). The urine
parameters that can be tested for using dipsticks are: Leukocytes, nitrite, urobilinogen, protein,
pH, blood, specific gravity, ketone bodies (acetoacetic acid), glucose & bilirubin. The results
of the specific test parameters on the strips can be read visually as well as using instruments.

Principle of each parameter tested using dipstick

Glucose: This test is based on a double sequential enzyme reaction. One enzyme, glucose
oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation
of glucose. A second enzyme, peroxidase, catalyzes the reaction of hydrogen peroxide with
potassium iodide chromogen to oxidize the chromogen to colors ranging from blue-green to
greenish-brown through brown and dark brown. High concentration of ketones may give a
false negative result.

pH: This test is based on the well-known double pH indicator method, where bromothymol
blue and methyl red give distinguishable colors over the pH range of 5-9. The colors range
from red-orange to yellow and yellow-green to blue-green

Note: A highly alkaline urine may give false positive results

Protein: pH indicator which is anionic reacts with the cationic protein, which produces a color
change. Colors range from yellow for a "Negative" reaction to yellow-green and green to blue-
green for a "Positive" reaction.

Ketones: This test is based on the reaction of acetoacetic acid with sodium nitroprusside in
a strongly basic medium. The colors range from beige or buff-pink color for a "Negative"
reading to pink and pink-purple for a "Positive" reading. Highly pigmented urine or levo dopa
in urine may give false positive results.

Blood: This test is based on the pseudoperoxidase action of hemoglobin and erythrocytes
which catalyzes the reaction of 3, 3', 5, 5' - tetramethyl-benzidine and buffered organic
peroxide. The resulting colors range from orange to yellow-green and dark green. Very high
blood concentration may cause the color development to continue to dark blue.

Note: Certain oxidizing contaminants like hypochlorite or microbial peroxidase (in case of UTI) may
lead to false positive results.

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 WARNINGS AND PRECAUTIONS

Urine Reagent Strips are for in vitro diagnostic use. Do not touch test areas of Urine Reagent strips.

TEST PROCEDURE

1. Remove from the bottle only enough strips for immediate use and replace cap tightly.
2. Completely immerse reagent areas of the strip in fresh, well-mixed urine. Remove the strip
immediately to avoid dissolving out the reagent areas.
3. While removing, touch the side of the strip against the rim of the urine container to remove
excess urine. Blot the lengthwise edge of the strip on an absorbent paper towel to further
remove excess urine and avoid running over (contamination from adjacent reagent pads).
4. Compare each reagent area to its corresponding color blocks on the Color chart and read
at the times specified. Proper read time is critical for optimal results.
5. Obtain results by direct color chart comparison.

Note: All reagent areas except Leukocytes may be read between 1-2 minutes for screening
positive urine from negative urine. Changes in color after 2 minutes are of no diagnostic value.

Observation

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 Sub-competency: BI 11.4- Perform urine analysis to estimate and determine normal and
abnormal constituents
BI11.20Identify abnormal constituents in urine, interpret the findings and correlate these with
pathological states

Attempt 1 Attempt 2 Attempt 3 Attempt 4

B M E B M E B M E B M E
Grade
Signature
of the
assessor
with date

60