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 Specific Gravity (Mass Density)
 Urinometer method
 Principle
Specific gravity is a function of the number, density
and weight of the solute particles present and is used as a measure of the
concentrating power of the kidney. The specific gravity of urine is its
density compared with the density of distilled water that is conveniently
fixed as 1.000 at 200C.
It ismeasured using a weighted cylinder called
"urinometer", which floats in the urine
and which is calibrated against distilled water at 20° C. Check the working
of the urinometer by floating it in distilled water
to see if the reading is 1.000. As the specific gravity varies with
temperature, apply temperature correction before reporting.
Procedure
 Pour about 40 - 50ml of urine into a 100ml
glass measuring cylinder.
lower the urinometer gently into the urine, rotate and release
(avoid frothing).
Wait for the urinometer
to settle (make sure that the urinometer does not
come into contact with the sides or bottom of the cylinder).
Read the specific gravity given on the scale
at the surface of the urine (use the lower point of the meniscus for
reading).
Observe the temperature of the urine.
Result
Check the temperature at which the urinometer
is calibrated. It is usually at 200C. For every 30C
that the urine temperature is above the calibration temperature, add 0.001 to
the measured specific gravity and for every 30C that the urine
temperature is below the calibration temperature, subtract 0.001 from the measured
specific gravity.
Example
Urinometer is calibrated at 200C
Urine temperature 230C
The measured specific gravity 1.023
The temperature of urine is 30C higher than the calibration
temperature.
\ Specific gravity to be added to the measured specific gravity =
3/3 x 0.001 = 0.003/3 = 0.001.
\ The actual specific gravity = 1.023+0.001=1.024.
Interpretation and quality control
The normal urine-specific gravity is 1.010 – 1.030
The presence of an increased amount of protein affects
the specific gravity by 0.001 for every 0.4g/dl protein level in urine. As a
quality control measure, the functioning of the urinometer
must also be checked by floating in other liquids whose densities are greater
than distilled water. There is also an adjustment for glucose Subtract 0.001 for every 270 mg/dl glucose in the urine.
Proteins – Heat and acetic acid method
Principle
Proteins in urine are coagulated by heat and the degree
of coagulation isdirectly proportional to the amount of proteins
present. Coagulation can be further enhanced when drops of acetic acid are
added.
Procedure
Pour 2-3 ml of urine into a 13 x 100mm glass tube and
hold it using a tube holder. Check the urine pH; if it is >pH 7 or <3,
adjust to between 4-5 using 3% acetic acid. Heat the
upper half of the column of urine in a flame until it boils. Look for the
appearance of cloudiness in the heated portion and contrast it with the lower
portion of the tube. Appearance of cloudiness in the upper portion indicates
the presence of proteins. Add 2-3 drops of 3% acetic acid to the precipitate
and observe. If the precipitate disappears, it indicates the presence of
phosphates and carbonate (later produces effervescence when the precipitate
disappears). Persistence of the precipitate shows the presence of albumin. On
adding 2-3 drops of conc. HN03 ifthe precipitate
disappears, the presence of mucin or nucleoprotein
is suggested.
Result
This test may be used as semi-quantitative, as follows;
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Colour change
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Result
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No cloudiness
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Negative
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Faint cloudiness
(may be observed only if the tube
is held against a black background).
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Trace
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Definite nongranular cloud
without flocculation
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1+
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Heavy and granular cloud without flocculation
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2+
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Dense cloud with marked flocculation
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3+
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Thick curdy flocculation
& coagulation
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4+
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Interpretation
and quality control
This test is sensitive enough to detect protein down to
a concentration of 2-3 mg%.
For quality control, dilute 22g% of human albumin
solution to get a concentration of 5 mg/dl. Use this as a test and check the
reliability and sensitivity of this method.
Note: If an alkaline urine is boiled,
the protein may be converted into the so- called "alkaline metaprotein", which is not coagulated by heat.
Therefore it is always better to acidify the urine before doing this test.
If too much acetic acid is added, the protein may be
converted to the so-called "acid metaprotein",
which is also not coagulated by heat. Therefore the urine should be only
mildly acidic.
Protein – Sulphosalicylic
acid method
Principle
Urine proteins are precipitated by sulphosalicylic
acid, which gives a white precipitate, and the degree of the precipitate is
proportional to the protein level.
Reagent
3g % sulphosalicylic acid
(SSA)
Weigh 7.5 g of sulphosalicylic
acid and dissolve it in about 200ml of distilled water and then make up to
250 ml with distilled water. Store at 25 - 350C. Stable for 6
months.
Procedure
To 2ml of urine taken in a 13 x 100mm glass tube, add 2
ml of 3g% SSA. Mix gently. Leave for 5 minutes at room temperature. Compare
the degree of the precipitate with 4ml of SSA taken in a similar test tube.
Result
Same as given on page 99 "(c) Result"
Interpretation
and quality control
The sulphosalicylic acid
method will not detect protein in a normal urine,
but will be sensitive enough to detect protein present down to 20mg%. As a
quality control measure, a 22g/dl albumin solution can be diluted
appropriately with 0.9 g/dl sodium chloride to get standards containing 20,
50, 200, 500 and 2500 mg/dl proteins. These standards are stable for one month
when stored at 2-80C. When they are subjected to the same
procedure as urine, the results can be interpreted as follows:
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Concentration of proteins
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Reported as
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20 mg/dl
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Trace
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50 mg/dl
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1+
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200 mg/dl
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2+
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500 mg/dl
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3+
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2500 mg/dl
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4+
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Sugar: Benedict's test
Principle
Urinary sugars when boiled in Benedict's reagent reduce
copper sulphate to a reddish cuprous oxide
precipitate in hot alkaline medium, the intensity of which is proportional to
the amount of sugar present in the urine. The results are reported as I+,2+, etc. depending upon the colour
and intensity of the cuprous oxide precipitate.
Reagent
Dissolve 17.3g of crystalline copper sulphate
in about 800ml of distilled water, then add 100 g of
sodium carbonate, mix to dissolve and finally add 175g of sodium citrate. Mix
well to dissolve and then make up to one litre with
distilled water. Store in an amber coloured bottle
at 25-350C. Stable for one year.
Procedure
To 5 ml of Benedict's reagent taken in an 18 x 150mm
glass tube, add 8 drops (0.5 ml) of urine, mix well
and boil for 2.3 minutes, preferably in a boiling waterbath.
Cool the tube and observe for any colour change.
Result
The results are reported as follows:
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Observation
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Inference
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No change in the original colour
of Benedict’s solution
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Negative
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Solution appears pale green
and slightly cloudy
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Trace
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Definite cloudy green
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1+
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Yellow to orange precipitate
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2+ (1 g/dl)
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Orange to red precipitate
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3+ (2 g/dl)
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Brick red precipitate & clear supernatant
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4+ (>2 g/dl)
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Interpretation
and quality control
Normal urine does not contain any reducing sugar. If
protein is present in large amounts, it may interfere with the precipitation
of the cuprous oxide.
To overcome this problem, precipitate the proteins using
3% SSA filter using a Whatman filter paper and use
the filtrate to test the amount of sugar present.
As a quality control measure, standards containing known
amounts of glucose are prepared in saturated benzoic acid and one of the
standards is used every day to check the reliability of the patient’s
results. The standard results may be transformed in the following
semi-quantitative way.
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100mg/dl
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Trace
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250mg/dl
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1+
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500mg/dl
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2+
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750mg/dl
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3+
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2 g/dl
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4+
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False positive reactions are known to occur due to
the presence of non- carbohydrate substances like ascorbic acid, homogentisic acid, creatinine
and uric acid. Reducing sugars like lactose, galactose,
fructose and pentoses will also give a positive
reaction.
The dipstick technique is specific for glucose and
eliminates the false positive reaction due to the substances mentioned above.
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