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Clinical Pathology


By Dayyal Dg.Twitter Profile | Updated: Monday, 08 March 2021 22:00 UTC
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Rothera’s tube test and reagent strip test for ketone bodies in urine
Rothera’s tube test and reagent strip test for ketone bodies in urine

The proportion of ketone bodies in urine in ketosis is variable: β-hydroxybutyric acid 78%, acetoacetic acid 20%, and acetone 2%.

No method for the detection of ketonuria reacts with all three ketone bodies. Rothera’s nitroprusside method and methods based on it detect acetoacetic acid and acetone (the test is 10-20 times more sensitive to acetoacetic acid than acetone). Ferric chloride test detects acetoacetic acid only. β-hydroxybutyric acid is not detected by any of the screening tests.

Methods for detection of ketone bodies in urine are Rothera’s test, Acetest tablet method, ferric chloride test, and reagent strip test.

1. ROTHERA’S’ TEST (Classic Nitroprusside Reaction)

Acetoacetic acid or acetone reacts with nitroprusside in an alkaline solution to form a purple-colored complex (Figure 822.1). Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and 10-25 mg/dl of acetone.

Principles of Rothera Test in Urine
Figure 822.1: Principles of Rothera’s test and reagent strip test for ketone bodies in urine. Ketones are detected as acetoacetic acid and acetone but not β-hydroxybutyric acid


  1. Take 5 ml of urine in a test tube and saturate it with ammonium sulphate.
  2. Add a small crystal of sodium nitroprusside. Mix well.
  3. Slowly run along the side of the test tube liquor ammonia to form a layer.
  4. Immediate formation of a purple permanganate-colored ring at the junction of the two fluids indicates a positive test (Figure 822.2).

A false-positive test can occur in the presence of L-dopa in urine and phenylketonuria.

Rotheras tube test and reagent strip test for ketone bodies in urine
Figure 822.2: Rothera’s tube test and reagent strip test for ketone bodies in urine


This is Rothera’s test in the form of a tablet. The Acetest tablet consists of sodium nitroprusside, glycine, and an alkaline buffer. Purple lavender discoloration of the tablet indicates the presence of acetoacetate or acetone (≥ 5 mg/dl). A rough estimate of the amount of ketone bodies can be obtained by comparison with the color chart provided by the manufacturer.

The test is more sensitive than the reagent strip test for ketones.


The addition of 10% ferric chloride solution to urine causes the solution to become reddish or purplish if acetoacetic acid is present. The test is not specific since certain drugs (salicylate and L-dopa) give a similar reaction. The sensitivity of the test is 25-50 mg/dl.


Reagent strips tests are modifications of the nitroprusside test (Figures 822.1 and 822.2). Their sensitivity is 5-10 mg/dl of acetoacetate. If exposed to moisture, reagent strips often give a false-negative result. The ketone pad on the strip test is especially vulnerable to improper storage and easily gets damaged. Also, readURINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

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