When more than trace amounts of protein are found in urine, this is termed proteinuria. Detection of proteinuria is an important indicator of renal disease because protein has a very low maximal tubular rate of reabsorption. The following methods are used to test for proteinuria:

• Qualitative Tests:
  Heat and acetic acid test
  Sulphosalicyclic acid test
  Hellers nitric acid test
• Quantitative Tests:
  Esbach’s method
  Aufrecht’s method
• Other tests:
  Protein reagent strip test
  Biuret test
  Urine protein electrophoresis

Sulphosalicylic Acid Test

Principle

Proteins are precipitated by 5-sulphosalicylic acid. Any resulting turbidity will give an estimation of the amount of protein present in the urine which can be subjectively quantitated visually or more precisely quantitated using photometry. Cells and casts in the urine must be removed by centrifuging before carrying out the test. The test can detect albumin, hemoglobin, myoglobin, and Bence Jones proteins.

Requirements

• Specimen:
  Random urine
• Apparatus:
  Centrifuge, general purpose
  Graduated pipettes/micropipettes
  Measuring Cylinder
  Test Tubes
  pH paper/ pH meter
• Reagents:
  5-sulphosalicylic acid solution-3%
  Glacial acetic acid-10%

Procedure

1. Check the pH of a portion of urine, if it is alkaline or neutral, add 10% acetic acid solution, drop by drop, until it is just acidic (about pH 6).
2. If the urine is cloudy, filter or centrifuge the urine (5 minutes, 2000-3000 rpm).
3. Take 2ml clear urine in a test tube.
4. Add 2 ml 5-sulphosalicylic acid solution and mix. Do not shake. Examine for turbidity against a dark background.

Result and Interpretation

Grade the turbidity as follows:

Negative : No cloudiness
Trace: Faint turbidity.
1+ : definite turbidity
2+ : Heavy turbidity but no flocculation
3+ : Heavy turbidity with light flocculation.
4+ : Heavy turbidity with heavy flocculation.

Note:

Normal urine doesn’t contain detectable protein by this method. A false positive result may be obtained if the patient is receiving tolbutamide, penicillin and some other drugs. High concentration of urates in the urine may cause a false positive result due to precipitation of urate in an acidic urine.

References

1. World Health Organization, 1986. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies. In Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies.
2. Ridley J.W. (2018) Procedures for Complete Urinalysis/Confirmation Testing. In: Fundamentals of the Study of Urine and Body Fluids. Springer, Cham. https://doi.org/10.1007/978-3-319-78417-5_10
3. Cheesbrough, M., 1981. Medical laboratory manual for tropical countries (Vol. 1). M. Cheesbrough, 14 Bevills Close, Doddington, Cambridgeshire, PE15 OTT..

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When more than trace amounts of protein are found in urine, this is termed proteinuria. Detection of proteinuria is an important indicator of renal disease because protein has a very low maximal tubular rate of reabsorption. The following methods are used to test for proteinuria:

Qualitative Tests:

1. Heat and acetic acid test
2. Sulphosalicyclic acid test
3. Hellers nitric acid test

Quantitative Tests:

1. Esbach’s method
2. Aufrecht’s method

Other tests:

1. Protein reagent strip test
2. Biuret test
3. Urine protein electrophoresis

Heat and acetic acid test

Principle:

This test is based on the principle that proteins get precipitated when boiled in an acidic medium.

Procedure:

1. Take 5-10ml clear urine in a test tube.
2. Boil the upper portion over a flame.
3. Compare the heated part with the lower part. Cloudiness or turbidity indicates the presence of either proteins or phosphates/carbonates.
4. Add 2-4 drops of 10% glacial acetic acid and boil the upper portion again.
5. If turbidity is still present, protein is present in urine. If turbidity disappears, that is due to phosphates or carbonates present in urine.

Result and Interpretation:

Grade the turbidity as follows:

• Negative : No cloudiness
• Trace: Barely visible cloudiness.
• 1+ : definite cloud without granular flocculation
• 2+ : heavy and granular cloud without granular flocculation
• 3+ : densed cloud with marked flocculation.
• 4+ : thick curdy precipitation and coagulation

References

1. Sood R. Concise book of Medical Laboratory Technology. Jaypee Brothers Pvt. Limited; 2015.
2. Cheesbrough M. Medical laboratory manual for tropical countries. M. Cheesbrough, 14 Bevills Close, Doddington, Cambridgeshire, PE15 OTT.; 1981.

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When the rate of production exceeds, excess ketone bodies are eliminated in urine and the condition is known as ketonuria. Two conditions most commonly associated are starvation and Diabetes mellitus. Ketonuria is also seen in case of prolonged vomiting, severe diarrhoea, anesthesia, liver damage, high fat intake and low carbohydrate intake.

Various methods are available for detecting ketones in urine:

• Rothera’s test
• Gerhardt’s test
• Lang’s test
• Lindeman’s test
• Han’s test
• Tablet test

The commonly used methods for ketonuria are based on the principle of Rothera’s nitroprusside test. However, other tests with different principle can also be used. Gerhardt’s Test is not very sensitive test because it can only detect about 25 to 50 mg/dl of acetoacetic acid.

Principle of Gerhardt’s test

Gerhardt’s test is based on the reaction of ferric chloride with acetoacetic acid to form a port wine or Bordeaux red color. This test detects acetoacetic acid only, Acetone and beta-hydroxybutyrate can’t be detected by this method. It also detects salicylates in urine.

Requirements

1. Urine specimen
2. Test tubes
3. 10% Ferric chloride:
   (10ml of ferric chloride in 100 ml of distilled water)

Procedure of Gerhardt’s test

1. Transfer about 3-5 ml of urine to a test tube.
2. Add 5ml of ferric chloride drop by drop to the urine. If phosphates are present, they get precipitated as ferric phosphates.
3. On addition of a slight excess of ferric chloride, if diacetic acid is present, Bordeaux red color will develop.
4. To confirm the presence of acetoacetic acid, boil the test solution for 5 minutes.
5. Observe the color change.

Observations and Results

• If the color disappears, then acetoacetic acid is present. The acetoacetic acid on boiling loses carbon dioxide and is converted to acetone. Acetone doesn’t react with ferric chloride.
• If the color persists, acetoacetic acid is absent. Previous color is due to salicylates.

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Hyaline Casts

Hyaline casts are the most commonly occurring casts in urine. They are composed almost entirely of Tamm-Horsfall mucoprotein. They have smooth texture and a very low refractive index, similar to that of urine in which they are suspended. They appear colorless and almost invisible under bright field microscope and have parallel sides and rounded ends. Occasional granular inclusions may be present in the matrix.

Clinical Significance

The presence of 0-2 hyaline casts per LPF is considered normal. Some physiological and pathological conditions are associated with increased amount of hyaline casts in urine.

Physiological Causes

Increased amounts of hyaline casts are usually associated with:

• Physical exercise
• Physiologic dehydration
• Exposure to heat
• Emotional stress

Pathological Causes

Pathologically, increased amount of hyaline casts are usually associated with

• Acute glomerulonephritis
• Pyelonephritis
• Chronic renal disease
• Congestive heart failure
• Meningitis
• Diabetic nephropathy

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