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Clinical Pathology

Test for Detection of Urobilinogen in Urine

By Dayyal Dg.Twitter Profile | Updated: Wednesday, 06 December 2023 16:48 UTC
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In the analysis of urinary urobilinogen, it is imperative to utilize a freshly collected urine sample. This crucial choice is rooted in the fact that, over time, urobilinogen undergoes conversion into urobilin, rendering it undetectable through routine tests. For a more nuanced examination, particularly when timing is a factor, a timed sample, such as a 2-hour postprandial collection, proves to be a valuable alternative for urobilinogen testing.

To quantify increased levels of urobilinogen in urine, two notable methods come to the forefront: the Ehrlich’s aldehyde test and the reagent strip test. These methodologies serve as reliable tools in the diagnostic arsenal, offering distinct advantages in discerning elevated urobilinogen concentrations.

Ehrlich's aldehyde test stands as a venerable technique, capitalizing on the reaction between urobilinogen and Ehrlich's reagent. This chemical interplay results in a characteristic color change, enabling precise quantification of urobilinogen levels. This method, steeped in scientific tradition, provides a meticulous approach for discerning subtle variations in urobilinogen concentration.

Complementing this, the reagent strip test emerges as a modern and efficient means of urobilinogen detection. This method capitalizes on advanced chemical indicators incorporated into specialized strips. These strips undergo specific reactions with urobilinogen, yielding color changes that can be quantified to gauge the concentration accurately. The reagent strip test, with its rapid and user-friendly nature, aligns with contemporary demands in clinical settings.

Ehrlich’s Aldehyde Test

Ehrlich’s reagent, also known as pdimethylaminobenzaldehyde, engages in a distinctive reaction with urobilinogen within urine, culminating in the manifestation of a discernible pink color. The intensity of this coloration serves as a direct correlate to the concentration of urobilinogen present in the specimen. It is crucial to note that the presence of bilirubin can impede this reaction. Consequently, in cases where bilirubin is detected, it becomes imperative to eliminate its interference.

To address the interference posed by bilirubin, a meticulous process is employed. Equal volumes of the urine specimen and a 10% solution of barium chloride are thoroughly amalgamated and subsequently subjected to filtration. The ensuing filtrate serves as the medium for conducting the urobilinogen test. This strategic filtration step aids in the removal of bilirubin, ensuring the fidelity of the urobilinogen reaction.

However, it is essential to exercise caution and discernment in the interpretation of results, as a parallel reaction can be induced by porphobilinogen. Porphobilinogen, a substance excreted in the urine of individuals afflicted by porphyria, shares a similar reactivity with Ehrlich's reagent, leading to analogous color changes. Thus, in the clinical assessment of urobilinogen, it becomes imperative to distinguish between the contributions of urobilinogen and porphobilinogen to the observed reactions.

Ehrlichs aldehyde test for urobilinogen
Figure 1: Ehrlich’s aldehyde test for urobilinogen

Methodology for Urobilinogen Assessment

In the pursuit of accurate urobilinogen analysis, a meticulous method is prescribed to ensure precision and reliability. Commencing with a 5 ml aliquot of freshly collected urine, the protocol unfolds by introducing 0.5 ml of Ehrlich’s aldehyde reagent into a designated test tube. This reagent formulation comprises hydrochloric acid (20 ml), distilled water (80 ml), and paradimethylaminobenzaldehyde (2 gm). Following this amalgamation, the concoction is allowed to stand at room temperature for a judicious period of 5 minutes.

The interpretive nuances lie in the observed color changes. The emergence of a pink hue signifies a normal concentration of urobilinogen, offering a visual cue to the discerning analyst. Conversely, a transition into a dark red coloration serves as a pivotal indicator of an elevated urobilinogen presence, as elucidated in Figure 1.

Given the potential for overlapping reactions between urobilinogen and porphobilinogen, a supplementary evaluation becomes imperative. Enter the Watson-Schwartz test, a discriminating assay designed to delineate between these chemically akin entities. With the introduction of 1-2 ml of chloroform and a subsequent two-minute agitation period, the solution undergoes a distinctive phase separation. A telltale pink coloration in the chloroform layer signifies the exclusive presence of urobilinogen, while a corresponding manifestation in the aqueous portion signals the dominance of porphobilinogen. This critical differentiation is pivotal for accurate diagnosis and is depicted in Figure 2.

To fortify the diagnostic accuracy, the pink layer is meticulously decanted and subjected to further scrutiny through interaction with butanol. A persistent pink color in the aqueous layer unequivocally indicates the presence of porphobilinogen, thus offering a conclusive resolution to the diagnostic conundrum.

Interpretation of Watson Schwartz test
Figure 2: Interpretation of Watson-Schwartz test

Potential Factors Influencing False-Negative Reactions in Urobilinogen Testing

It is crucial to be cognizant of potential confounding factors that may lead to false-negative reactions. Two notable circumstances warrant consideration in this context.

Firstly, the presence of a urinary tract infection (UTI) introduces a dynamic that can obscure the accuracy of urobilinogen assessments. Specifically, the oxidation of urobilinogen to urobilin can occur in the presence of nitrites, a phenomenon commonly associated with UTIs. This chemical transformation during the testing process may yield a false-negative outcome, necessitating a judicious interpretation of results in the context of suspected or known UTIs.

Secondly, the impact of antibiotic therapy on urobilinogen testing integrity is noteworthy. Antibiotics, while instrumental in combatting infections, possess the capacity to disrupt the delicate balance of gut bacteria responsible for urobilinogen production. Consequently, the destruction of these bacteria can lead to a reduction in urobilinogen levels, potentially resulting in false-negative reactions during testing.

Urobilinogen Assessment via Reagent Strip Method

In the pursuit of urobilinogen quantification, the Reagent Strip Method emerges as a targeted and specialized approach. This method intricately focuses on the specific detection of urobilinogen, underscoring its precision in elucidating this particular component within urine samples.

The method hinges on the strategic impregnation of the test area with either p-dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate. These meticulously chosen reagents serve as discriminating agents, interacting selectively with urobilinogen to yield discernible reactions. The deliberate incorporation of these compounds into the test area ensures a high level of specificity, facilitating the accurate identification and quantification of urobilinogen amidst the complex milieu of urinary components.

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