Blood Safety and Clinical Technology

Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Qualitative Tests
 
     Appearance

Normal urine colour varies from light yellow to deep amber. Urine colour sometimes may vary
depending upon the drink, if any, consumed by the patient. The colour of urine is sometimes related
to a pigment called "urochrome". The degree of colour also depends on whether the specimen is
concentrated or dilute.

Normal urine is usually clear. If the pH is alkaline, turbidity may be observed due to the precipitation
of phosphates. Such urine should be centrifuged before analysis. Turbidity due to the presence
of chyle (chylomicrons) cannot be centrifuged, but requires filtration using a special cellulose filter
having <0.1 m m diameter.

     pH

Use a narrow range pH paper or a pH meter.

In some clinical situations, measurements of approximate pH within + 0.5 pH units using a narrow-

range pH paper or exact pH using a pH meter may be very helpful.

      Procedure
 
     Using pH paper

Put a drop of urine on a portion of pH indicator paper. The colour obtained is compared with a

standard chart. For checking the reliability of the pH paper cross check the pH of buffer
solutions of known pH values having acidic and alkaline pH ranges.

     Using a pH meter

Calibrate the pH meter using standard buffers, one having an acidic pH and the other an
alkaline pH, preferably less than 9.0. Pour about 40-50ml of urine into a 100ml beaker,
calibrate the pH meter once again using the buffer of pH 7.0 and measure the pH of the urine.

     Result

Normal urine pH ranges from 4.5 to 8.0.

The urine pH values are reported for example as 6.0 if pH paper is used or as 6.1 if a pH meter is
used.

     Interpretation and quality control

Urine pH is usually acidic in normal people, especially non-vegetarians, and is usually alkaline in
vegetarians.

An early morning urine pH <5.5 indicates that renal tubular acidification mechanism is intact.

As a quality control measure, use certified reference buffers (commercial source), one in acidic
range, say, pH 4.0 and the other in alkaline range, preferably pH 9.2 to check the reliability of the
pH paper used or to assess the performance of the pH meter.

Always use a pH indicator paper before the date of expiry. Do not use outdated pH papers. Always
 close the bottle containing the pH paper tightly.

The pH meter must be calibrated against a correct calibration buffer.

     Ketone Bodies - Rothera's test

The three main ketone bodies are acetone , acetoacetic acid (diacetic acid) and beta-

hydroxybutyric acid. Testing for ketone bodies should be done on fresh urine or the specimen kept at
4 0C.

     Principle

Acetone and acetoacetic acid react with sodium nitropruside in the presence of alkali to produce a

purple colour.

      Reagents
 
     Rothera's Reagent : Dry mixture

Pulve210rize 7.5g sodium nitropruside with 200g ammonium sulphate. Store in a clean amber

bottle at 250-350C. Stable for 6 months.

     Ammonia concentrated, specific gravity 0.91

 
 
     Procedure

To about 5ml of urine taken in an 18 x 150mm glass tube, add about one teaspoon of the mixture,
mix well, then add 0.5 to 1.0 ml of concentrated ammonia down to the side of the tube so that it
layers on top of the urine. Observe for any colour change within 30-60 seconds.

     Result

If acetone and diacetic acid are present, then a purple (permanganate calomel red) colour will form
at the junction of the two layers within 30-60 seconds. The result can be graded from trace to 3+
based on the intensity of the colour formed, as detailed below.

No change in colour - Negative

Pinkish ring - +
Red ring - ++
Deep purple ring - +++

     Interpretation and quality control

Ketone bodies are intermediary products of fat metabolism and their presence in the blood and
then in the urine are indications that the metabolism is disordered or incomplete. This is associated
with metabolic acidosis. This occurs in poorly controlled diabetes mellitus and also in starvation.

Normal urine does not contain methyl ketone. Weak false positive reactions may occur if the urine
contains L-dopa and phenyl pyruvic acid.

If there is suspicion of a false positive test, heat the urine in a test tube in a bunsen burner flame
for one minute, allow to cool and repeat the Rothera's test. Heated urine will not give a
positiveRothera's due to ketone bodies.

As a quality control measure, the reagent should be checked frequently using a positive control (1-
2 drops of acetone is added to 5ml of urine). The use of distilled water in place of urine for
 negative control isrecommended.

     Urobilinogen – Erhlich’s test
 
     Principle

Erhlich's reagent in conc HCI. reacts with urobilinogen to form a pink coloured aldehyde complex in

chloroform.

      Reagents
 
     Erhlich's reagent

Dissolve 2 g of P-dimethyl aminobenzaldehyde in 100ml of 20% HCI. Store at 25-350C in an

amber coloured bottle. Stable for 3 months.

     10g/dl Barium chloride

Dissolve 10g barium chloride in 100ml of distilled water. Store at 25-350C. Stable for 6 months.

     Saturated ammonium sulphate
     Chloroform AR or GR quality
 
      Procedure

To 12 ml of urine taken in a 25ml or 50 ml measuring cylinder add 3 ml of barium chloride followed

by 3 drops of saturated ammonium sulphate. Mix well. Transfer a portion of it into a 15 x 120mm
glass tube. Centrifuge for 5 minutes at 3500 rpm. Transfer about 5ml of the supernatant into an 18
x 150 mm glass tube and add 0.5 ml of Erhlich's reagent. Mix well. Then add 3ml chloroform and
shake well. Allow to stand one minute. Observe for any colour change in the chloroform layer
(bottom layer).

     Result

Urobilinogen

Colourless : Not detected
Faint red colour : Normal
Red or bright red : Positive or highly positive depending 
upon the intensity of colour.

     Interpretation and quality control

Urobilinogen is normally excreted in trace and a normal urine will always show a faint red colour in
the chloroform layer. It is always a good practice to run a normal urine as control whenever
an urobilinogen test is done. Excess urobilinogen is seen in urine in haemolytic jaundice, viral
hepatitis and cirrhosis and is absent in obstructive jaundice.

     Bilirubin - (Harison spot test) Fouchet's test

 
     Principle

When ferric chloride in acid solution is added to a precipitate (Ref : Urobilinogen procedure) of

urine containing bilirubin, a green colour is produced as the bilirubin in the urine is oxidized
tobiliverdin.

      Reagent
 
      Fouchet's reagent.

Dissolve 25g of trichloroacetic acid in about 50ml of distilled water, then add 1g ferric chloride,

mix to dissolve and then make up to 100ml with distilled water. Store at 25 – 35 0C. Stable for 6
months.

     Procedure

To 12 ml of urine taken in a 25 ml or 50 ml measuring cylinder add 3 ml of barium chloride

followed by 3 drops of saturated ammonium sulphate. Mix well. Transfer a portion of it into a 15 x
120mm glass tube. Centrifuge for 5 minutes at 3500 rpm. Decant the supernatant and add 1or 2
drops of Fouchet's reagent to the precipitate. Examine for any colour change.

     Result

No colour change in the precipitate : Negative

Appearance of a green or blue colour : Positive

     Interpretation and quality control

Bilirubin isnot present in normal urine. For a positive control, a few drops of either
a bilirubin standard or an icteric serum are added to a normal urine sample and the specimen
is analysed for the presence of bilirubin. Any bilirubin present in the urine is conjugated and
indicates excess in the serum due to cholestasis.