Examination of urine

Preliminary and Confirmatory Tests for Urine

Urine stains can be important biological evidence in forensic investigations. Various preliminary (presumptive) and confirmatory tests are used to identify urine in questioned samples such as clothing, bedding, or other materials.

Visual Examination

• Visual examination is based on the characteristic yellow color of urine and the distinctive odor of urine stains.

• Under alternative light sources, urine stains emit a fluorescent light, which helps in locating urine stains on clothing and bedding.

Presumptive Tests

The following tests are commonly used as presumptive tests for urine detection:

1. DMAC Assay

2. Urease Test

3. Jaffe’s Test

4. Murexide Test

5. Urea Nitrate Crystal Test

6. Urea Oxalate Crystal Test

Para-Dimethylaminocinnamaldehyde (DMAC) Assay (Detects Urea)

The DMAC assay can be performed using two different methods:

• Colorimetric method

• Fluorometric method

A. Colorimetric Method

1. A portion of a stain (~1 cm²) is cut and extracted with 1 mL of distilled water.

2. The extraction is transferred onto a piece of filter paper and allowed to dry.

3. One drop of 0.1% DMAC solution is added to the filter paper. DMAC reacts specifically with urea, producing a pink or magenta-colored product if present.

4. The appearance of a pink color within 30 minutes after applying the DMAC reagent is considered a positive reaction.

B. Fluorometric Method

This method is useful for locating urine stains on large pieces of evidence such as clothing and bedding.

1. The evidence to be examined, such as a garment, is covered with a sheet of filter paper pre-absorbed with DMAC solution.

2. The evidence and filter paper are wrapped together in aluminum foil and left overnight in a press to ensure close contact.

3. Alternatively, the layers may be heated for 30 seconds using an iron.

4. The fluoresced stain is best observed with a 549 nm filter.

Note: Colored fabrics may interfere with the assay because dyes and pigments can inhibit fluorescence.

Urea Test (Urease Test)

• Urea present in urine is hydrolyzed by the enzyme urease into ammonia and carbon dioxide.

• The released ammonia increases the pH, causing a color change in the pH indicator.

Reaction

Urea + Water → (Urease) → Ammonia + Carbon dioxide

Procedure

1. Take a small quantity of the suspected urine sample in a test tube.

2. Add urease enzyme solution.

3. Add a pH indicator (phenolphthalein or bromothymol blue).

4. Incubate at room temperature for a few minutes.

5. Observe the color change.

Positive Result

• Indicator turns pink or blue due to alkaline pH.

Creatinine Test (Jaffe’s Test)

Creatinine reacts with alkaline picrate to form an orange-red colored complex. Creatinine is a characteristic constituent of urine.

Procedure

1. Take the suspected sample in a test tube.

2. Add saturated picric acid solution.

3. Add sodium hydroxide to make the solution alkaline.

4. Mix well and allow to stand for 2–5 minutes.

5. Observe the color development.

Positive Result

• Formation of orange to reddish color.

Uric Acid Test (Murexide Test)

• Uric acid is oxidized by nitric acid to form alloxan and alloxantin, which on exposure to ammonia produce murexide, giving a purple color.

Procedure

1. Place a drop of urine sample in a porcelain dish.

2. Add a few drops of concentrated nitric acid.

3. Evaporate gently to dryness.

4. Expose the residue to ammonia vapor or add dilute ammonia.

5. Observe the color change.

Positive Result

• Development of purple or mauve color.

Urea Nitrate Crystal Test

• Urea reacts with concentrated nitric acid to form urea nitrate, which crystallizes in a characteristic pattern.

Reaction

Urea + Nitric acid → Urea nitrate (crystals)

Procedure

1. Place a small drop of the suspected urine sample on a clean glass slide.

2. Add one drop of concentrated nitric acid.

3. Allow the reaction to occur at room temperature.

4. Cover with a coverslip.

5. Observe under a microscope (low to medium power).

Positive Result

• Formation of colorless rhombic, hexagonal, or plate-like crystals, often arranged in clusters.

Urea Oxalate Crystal Test

• Urea reacts with oxalic acid to form urea oxalate, which produces distinctive needle-shaped crystals.

Procedure

1. Place a drop of urine sample on a glass slide.

2. Add a drop of saturated oxalic acid solution.

3. Allow the mixture to stand for a few minutes.

4. Cover with a coverslip.

5. Examine under a microscope.

Positive Result

• Formation of fine needle-shaped crystals, usually arranged in bundles or rosettes.

Confirmatory Assays

Confirmatory tests provide specific evidence for the presence of urine.

Identification of Tamm–Horsfall Protein

• Tamm–Horsfall protein (THP), also known as uromodulin, is the most abundant protein in urine, accounting for about 40% of urinary proteins.

• THP is exclusively synthesized in the epithelial cells of Henle’s loop.

• An adult excretes approximately 20–100 mg of THP daily in urine.

• THP acts as a urine-specific biomarker for forensic urine identification.

• It can be detected using:

  • Enzyme-linked immunosorbent assay (ELISA)

  • Immunochromatographic assay (RSID-Urine)

• The RSID-Urine assay uses a polyclonal rabbit antibody specific to THP.

Identification of 17-Ketosteroids

• Urine contains derivatives of 17-ketosteroids, including:

  • Androsterone

  • Dehydroepiandrosterone (DHEA)

  • Etiocholanolone

• Androsterone is a steroid hormone with weak testosterone-like activity.

• DHEA acts as a metabolic intermediate in the biosynthesis of gonadal steroids.

Major 17-Ketosteroid Conjugates in Human Urine

1. Androsterone glucuronide

2. Androsterone sulfate

3. DHEA sulfate

4. Etiocholanolone glucuronide

5. Etiocholanolone sulfate

• These five conjugates can be identified using liquid chromatography–mass spectrometry (LC-MS).

• Some of these conjugates may also be present in serum, therefore all five conjugated 17-ketosteroids must be detected to confirm a urine stain.

• The profiles of 17-ketosteroid conjugates are human-specific, allowing differentiation between human and animal urine.

Read more

Introduction to Urine, Nephron & its structure and Formation of urine - click here

Composition of urine, its properties and Forensic Significance - Click here