Interfering Factors 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

 

For Semiquantitative Method

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

Reagents

Antihuman albumin reagent, albumin latex reagent,

positive control reactive with antihuman albumin reagent,

negative control non-reactive with antihuman albumin

reagent.

Accessories

Glass slide with six reaction circles, pipettes for dispensing

urine specimen, mixing sticks and rubber teats.

Additional Materials Required

A high intensity direct light source and stopwatch.

Test Procedure

Bring all reagents and samples to room temperature before

use.

Qualitative Method

1. Place one drop of clear urine under test on the glass

slide using disposable pipettes provided with the kit.

Deliver the drop vertically.

2. Add one drop of antihuman albumin reagent to the

drop of urine under test on the slide. Deliver the drop

vertically.

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.

Semiquantitative Method

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

each dilution as specimen.

Interpretation of Results

Qualitative Method

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.

Semiquantitative Method

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:

Microalbumin (mg/L) = S × D

where

S = Sensitivity of the test, i.e. 25 mg/L

D = Highest dilution of urine showing no agglutination.

Urine Analysis 65

Remarks

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

specimen is used.

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

preferably within a week.

5. Liquid intake of the patient must be in the normal

range, i.e. 1.5–2 liters/day.

6. To diagnose incipient nephropathy microalbuminuria

must be present in at least 2 out of 3 specimens over a

3–6 months period.

7. It is recommended that results of the tests should be

correlated with clinical findings to arrive at the final

diagnosis.

8. It is recommended that reagents should be tested with

positive and negative controls periodically to validate

their performance.

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.

Tests for Glucose

Normal values

Random : Negative

24-hour specimen: 100 mg/24 hours

Renal threshold level: 180 mg/dL.

Benedict’s Qualitative (Semiquantitative) Glucose

Test

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.

Read as follows:

Blue to cloudy

Green color = Negative, 0

Yellow-green = + (< 0.5% glucose)

Greenish yellow = ++ (0.5-1% glucose)

Yellow = +++ (1-2% 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.

Quantitative Method

Urinary sugar can also be detected by routine biochemical

kits too.

Sugar Tests in Urine (Table 5.3)

Significance of Sugars in Urine

Glycosuria with Hyperglycemia

Diabetes mellitus

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,

especially of hypothalamus.

Massive metabolic derangement: Severe burns, uremia,

advanced liver disease, sepsis, cardiogenic shock.

Drug induced: Corticosteroids and ACTH, thiazides, oral

contraceptives.

Glycosuria without Hyperglycemia

¾ Renal tubular dysfunction

¾ Pregnancy (differentiate from gestational diabetes).

66 Concise Book of Medical Laboratory Technology: Methods and Interpretations Nonglucose Sugars in Urine

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

normal persons.

Interfering Factors

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

value.

b. Ascorbic acid, creatinine in concentrated urine,

streptomycin and homogentisic acid may cause a false

positive reduction test (Benedict’s), usually it will only

be a trace reaction.

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

negative in the enzyme tests.

e. False negatives may be obtained if deteriorated

reagents strips have been used, or directions not

followed exactly.