ultraviolet light). 70 Concise Book of Medical Laboratory Technology: Methods and Interpretations A random sample or 24 hours sample may be submitted to the laboratory. Porphobilinogens are always done with porphyrin test. Should a single, fresh-voided specimen

 

color indicates bilirubinuria.

4. Diazo Test

p-nitrobenzene diazonium p-toluene sulfonate is the

active reagent. Place 5 drops of the urine on the mat

provided in the kit. Bilirubin, if present shall be absorbed

onto the mat surface. Place a reagent tablet on it. Let 2 drops

of water flow over the tablet. A positive test is indicated by

the appearance of a blue to purple color within 30 seconds.

Pink/red color is negative. Sensitivity > 0.1 to 0.05 mg% of

bilirubin in urine.

5. Paper Strip Method

After dipping the strip in urine, match with the color chart

provided by the manufacturers.

Causes of Hyperbilirubinuria

1. Moderate to severe hepatocellular damage.

2. Obstruction of bile ducts, extrahepatic or intrahepatic.

In early hepatocellular damage and in hemolysis, urine

bilirubin may be negative.

Urobilinogen and Urobilin

Urobilinogen is colorless, and on standing, it gets oxidized

to urobilin which has a brown color. It is best to perform tests

for urobilinogen on fresh specimens. If delay is inevitable,

collect the sample in a dark bottle, provide a surface layer

of petroleum ether and add sodium carbonate (5 g for

24 hours volume) and refrigerate the sample.

Urobilinogen

Ehrlich’s test: Nitrites and bilirubin interfere with this test.

Sulfonamide and procaine cause yellowish color reactions.

Pyridium, indole, porphobilinogen and PAS yield pink-red

color not different from that produced by urobilinogen.

To 10 mL of fresh sample at room temperature add 1 mL

of Ehrlich’s reagent, invert several times and let stand for

5 minutes. A pink color is normal, cherry or darker red color

indicate abnormal amounts of urobilinogen. Dilutions

may be used. Color reactions are normal in dilutions up to

1:20 (sensitivity > 1.3 mg%).

Urobilin (Schlesinger’s Test)

Convert urobilinogen to urobilin by adding a few drops of

Lugol’s solution. Mix 10 mL of urine with an equal quantity

of saturated alcoholic solution of zinc acetate filter into a

dry test tube.

Abnormal amounts of urobilin give the filtrate a green

fluorescence, which is best seen against a dark background

with a light source from the side, or in sunlight against a

black background.

The filtrate obtained from Harrison’s test for bilirubin

can be used for urobilinogen or urobilin.

Paper strip method (Dipstick tests are available).

Estimated Urobilinogen

Normal values: 0.1–1 Ehrlich unit/dL

Urinary urobilinogen is an important tool in routine

urinalysis since it serves as a guide in detecting and

differentiating liver disease, hemolytic disease, and biliary

obstruction. Sequential determination assists in evaluating

progress of disease and response to treatment. Both

urobilinogen and bilirubin in urine may be regarded as

bile pigments, but the tests provide different information.

Increased values of urobilinogen occur in:

¾ Cirrhosis: Bilirubin in urine may or may not be present

¾ Hemolytic jaundice: Bilirubin does not appear in urine.

A number of drugs produce false positives or negatives.

Urobilinogen (Quantitative)

Normal values

2-hour specimen: 0.1–1.0 Ehrlich units/2 hours

24-hour specimen: 1–4 mg/24 hours.

This is one of the most sensitive tests employed to

determine impaired liver function. Bilirubin, formed from

the metabolism of hemoglobin entering the intestine in

the bile, is transformed through the action of bacteria into

urobilinogen. Part of the urobilinogen formed in intestine

is excreted with the feces; another portion is absorbed into

the portal bloodstream and carried to the liver where it is

metabolized and excreted in bile. Traces of urobilinogen

that escape removal from the blood by the liver are carried

to the kidneys and excreted in the urine.

Clinical Relevance

Increase in Urinary Urobilinogen

This occurs in any condition that causes an increase

in the production of bilirubin and by any disease that

Urine Analysis 69

prevents the liver from normally removing the reabsorbed

urobilinogen from the portal circulation.

a. Increased urobilinogen is found whenever there is

excessive destruction of RBCs as in:

• Hemolytic anemias

• Pernicious anemia

• Malaria.

b. Values above normal also occur in:

• Infectious and toxic hepatitis

• Pulmonary infarction

• Biliary disease

• Cholangitis

• Hemolytic jaundice and anemia

• Chemical injury to liver due to chloroform and

carbon tetrachloride poisoning

• Cirrhosis

• Congestive heart failure

• Infectious mononucleosis.

c. An increased urobilinogen level is one of the earliest

signs of acute liver cell damage.

Decrease in Urinary Urobilinogen

This occurs when normal amounts of bilirubin are not

excreted into the intestinal tract. It usually indicates partial

or complete obstruction of the bile ducts. As occurs in:

¾ Cholelithiasis

¾ Severe inflammatory disease

¾ Cancer of head of pancreas

¾ During antibiotic therapy. Suppression of normal

gut flora may prevent breakdown of bilirubin to

urobilinogen, leading to its absence in urine

¾ Decreased values are also associated with:

• Severe diarrhea

• Renal insufficiency.

Interfering Factors

a. Drugs and foods that may cause urobilinogen to be

increased include:

• Para-aminosalicylic acid (PAS)

• Antipyrine

• BSP

• Cascara

• Phenothiazines

• Sulfonamides

• Drugs causing hemolysis of RBCs

• Bananas

• Phenazopyridine.

b. Drugs that may cause decreased urobilinogen include

those that cause cholestasis and those that reduce

bacterial flora in the GI tract (e.g. antibiotics).

c. Peak excretion is said to occur from noon to 16:00

hours. The urinary urobilinogen is subject to diurnal

variation.

d. Strongly alkaline urine will show higher value and

strongly acid urine will show a lower level.

Porphyrins

Perform Ehrlich’s test for urobilinogen by mixing equal

parts of urine and Ehrlich’s reagent. Add 2 parts of saturated

sodium acetate solution and mix. If turbid, filter. Shake with

a small quantity of chloroform. Urobilinogen is soluble in

chloroform, while porphobilinogen is not. If after several

extractions with chloroform the aqueous phase is still

pink, the test is positive for porphobilinogen.

Causes

Conditions producing increased levels of any of the heme

precursors are called porphyrias. The two rare major

categories of genetically determined porphyria and

erythropoietic porphyrias, in which the major diagnostic

abnormalities occur in red cell chemistry, and hepatic

porphyrias, in which heme precursors are found in urine

or feces. In acquired disorders, precursors accumulate

more in urine and feces than in red cells.

Normal Values

Porphobilinogens : 2 mg/24 h or negative

Porphyrins : 50–300 mg/24 h

DAL or ALA : 1–710 mg/24 h

Fluorescent : Negative

Porphyrins are cyclic compounds formed from deltaaminolevulinic acid (DAL or ALA), which is important in

the formation of hemoglobin and other hemoproteins that

function as carriers of oxygen in the blood and tissues. In

health, insignificant amounts of porphyrin are excreted

in the urine. However, in conditions like porphyria

(disturbance in metabolism of porphyrin), liver disease,

lead poisoning, and pellagra, there is an increased level of

porphyrins as well as DAL and ALA in the urine. Disorders

of porphyrin metabolism also result in porphobilinogen.

In acute attacks of porphyria, the patient may suffer

skin lesions, abdominal pain, neuropathy, and mental

disturbances. The urine of patients with this disease

usually has a pinkish to reddish-black tinge and will

become darker upon standing. In the laboratory, the urine

is tested for the presence of porphyrins, porphobilinogen,

and DAL or ALA. It is also given the black light screening

test (porphyrins fluorescence when exposed to black or

ultraviolet light).

70 Concise Book of Medical Laboratory Technology: Methods and Interpretations A random sample or 24 hours sample may be submitted

to the laboratory. Porphobilinogens are always done with

porphyrin test. Should a single, fresh-voided specimen

be ordered, only a porphobilinogen will be done. Protect

specimen from light. The test must be performed within

60 minutes. Random sample should be obtained between

10:00 and 14:00 hours. Observe and record the color of

urine. If porphyrins are present, the urine may have a

grossly recognisable amber red or burgundy color. It may

vary from pale pink to almost black. Some patients will

excrete urine of normal color that turns dark after standing

in the light.