Urine specimens collected over a 24-hour period should be
pooled in a clean detergent free container and refrigerated
at 2–8°C. Thimerosal (0.01%) or sodium azide (0.1%) are
recommended as urine preservatives.
Materials Provided with the Kit
Antihuman albumin reagent, albumin latex reagent,
positive control reactive with antihuman albumin reagent,
negative control non-reactive with antihuman albumin
Glass slide with six reaction circles, pipettes for dispensing
urine specimen, mixing sticks and rubber teats.
A high intensity direct light source and stopwatch.
Bring all reagents and samples to room temperature before
1. Place one drop of clear urine under test on the glass
slide using disposable pipettes provided with the kit.
2. Add one drop of antihuman albumin reagent to the
drop of urine under test on the slide. Deliver the drop
3. Using a mixing stick, mix the antihuman albumin
reagent and urine over the circle for 30 seconds.
4. Add one drop of well mixed albumin latex reagent to
the mixture. Mix uniformly over the entire circle.
5. Immediately start the stopwatch, rock the slide
gently back and forth observing for agglutination
macroscopically at three minutes.
Measure and record the total volume of patient urine
collected over a 24-hour period. Centrifuge an aliquot
of the 24 hours urine specimen. Using isotonic saline,
prepare progressive dilutions from the centrifuged urine
specimen. Perform the qualitative test procedure using
Agglutination is a negative test result indicating the absence
of detectable levels of albumin in urine signifying absence
of microalbuminuria. No agglutination is a positive test
result indicating the presence of albumin in concentrations
above 25 mg/L in urine signifying microalbuminuria.
No agglutination in the highest urine dilution corresponds
to the titer of microalbumin per liter of the specimen.
To calculate the concentration of microalbumin in the
specimen uses the following formula:
S = Sensitivity of the test, i.e. 25 mg/L
D = Highest dilution of urine showing no agglutination.
1. Microalbuminuria also occurs in response to acute
inflammatory conditions such as ischemia, trauma and
thermal injury, surgery, pancreatitis and inflammatory
bowel disease. In many of these conditions, the albumin
excretion increases within minutes or hours of the
initiating stimulus and only lasts for 24–72 hours.
2. Use only urine as test specimen. Do not use serum.
3. Albumin excretion is increased after physical activity.
It is, therefore, recommended to use urine sample that
has been produced at rest whenever random urine
4. As albumin excretion is subject to physiological
fluctuations, it is necessary to take two measurements
in consecutive days; in case of contradictory results,
three measurements on different days must be done
5. Liquid intake of the patient must be in the normal
6. To diagnose incipient nephropathy microalbuminuria
must be present in at least 2 out of 3 specimens over a
7. It is recommended that results of the tests should be
correlated with clinical findings to arrive at the final
8. It is recommended that reagents should be tested with
positive and negative controls periodically to validate
9. The agglutination pattern in negative urines will vary
from sample to sample since it is affected by the salt
concentration and pH of the urine under test.
24-hour specimen: 100 mg/24 hours
Renal threshold level: 180 mg/dL.
Benedict’s Qualitative (Semiquantitative) Glucose
In this method, the cupric ion is reduced to Cu2O
(cuprous oxide). If only 0.1% or less of glucose is present,
the precipitate may not appear until cooling. To 5 mL of
Benedict’s qualitative reagent, add 8 drops of urine (0.5 mL).
Heat to boiling and set in a boiling water bath for 5 minutes
or else boil it over a flame for 2 minutes.
Yellow-green = + (< 0.5% glucose)
Greenish yellow = ++ (0.5-1% glucose)
Orange to brick red = ++++ (over 2% glucose)
Sensitivity of this test is 50 mg% or more.
Glucose oxidase methods: Glucose oxidase reacts with
glucose to yield gluconic acid and hydrogen peroxide.
Hydrogen peroxide and orthotolidine yield a blue color.
This is a specific test. The reagents may be impregnated
on paper strips (as mentioned above) and dipping them
in urine provide the result in lesser time as compared to
Benedict’s method (sensitivity = 0.1%).
Benedict’s Quantitative Glucose Test
Place a small quantity of powdered pumice, 10 g of
anhydrous sodium carbonate and 25 mL of quantitative
Benedict’s reagent in a 250 mL container and heat. While
the mixture is boiling, add urine rapidly from a buret until
the blue color begins to fade, then add urine drop by drop
until all blue color is gone and only a gray color remains. At
this point, all cupric ions originally in solution is reduced.
The amount of urine used contains 0.05 gm of glucose. To
calculate grams of glucose per 100 mL of urine, divide 5 by
the number of mL of urine used.
Urinary sugar can also be detected by routine biochemical
Sugar Tests in Urine (Table 5.3)
Significance of Sugars in Urine
Other endocrine disorders: Acromegaly, Cushing’s syndrome, hyperthyroidism, pheochromocytoma.
Pancreatic disease: Cystic fibrosis—advanced stage,
hemochromatosis, severe chronic pancreatitis, carcinoma.
CNS dysfunction: Asphyxia, tumors or hemorrhage,
Massive metabolic derangement: Severe burns, uremia,
advanced liver disease, sepsis, cardiogenic shock.
Drug induced: Corticosteroids and ACTH, thiazides, oral
Glycosuria without Hyperglycemia
¾ Pregnancy (differentiate from gestational diabetes).
Galactose: Detecting galactosemia in newborn period
may prevent irreversible liver and CNS damage. Galactose
spills into urine only if milk is being taken.
Fructose: Essential fructosuria (rare).
Pentose: Very high fruit intake may cause pentosuria in
a. Pregnancy and lactation may cause a false positive
in enzymatic tests. About 70% of women show a
temporary glucosuria that appears to be of no clinical
b. Ascorbic acid, creatinine in concentrated urine,
streptomycin and homogentisic acid may cause a false
positive reduction test (Benedict’s), usually it will only
c. Stress excitement, testing after a heavy meal, and
following the administration of IV glucose may cause
false positives of all tests. Usually, it is a trace reaction.
d. Ascorbic acid in large amounts may cause a false
e. False negatives may be obtained if deteriorated
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