Esbach Test- Definition, Principle, Procedure, Result, Uses

The Esbach test is a biochemical test that can detect and measure the amount of protein, especially albumin, in a urine sample. Albumin is a type of protein that is normally found in the blood but can leak into the urine due to various conditions such as kidney disease, diabetes, hypertension, or infection. The presence and level of albumin in the urine can indicate the severity and progression of these conditions and help in diagnosis and treatment.

The Esbach test was developed by Georges Hubert Esbach, a German physician and chemist, in 1874. It is one of the oldest and simplest methods of protein estimation in urine. The test involves mixing a urine sample with a reagent containing picric acid and citric acid. These acids react with the protein molecules in the urine and form a yellow precipitate. The amount of precipitate can be measured by using a special glass tube called an albuminometer, which has graduations that indicate the grams of albumin per 1000 ml of urine.

The Esbach test is also known as the picric acid test or the Esbach albuminometer test. It is a qualitative and quantitative test that can provide a quick and easy estimation of proteinuria (the presence of excess protein in the urine). However, it also has some limitations and sources of error that need to be considered. In this article, we will discuss the principle, procedure, result, uses, and limitations of the Esbach test in detail.

The main objective of Esbach test is to detect the presence and amount of proteins in a sample, especially urine. Proteins are essential molecules for the structure and function of living cells, but they are not normally found in urine. The presence of proteins in urine, also known as proteinuria, can indicate various diseases or disorders of the kidneys, liver, or other organs. Therefore, Esbach test can be used as a simple and quick screening tool to diagnose and monitor these conditions.

Another objective of Esbach test is to specifically detect the presence and amount of albumin in urine. Albumin is the most abundant protein in blood plasma, and it plays a vital role in maintaining the fluid balance and transporting various substances in the body. Albuminuria, or the loss of albumin in urine, can be a sign of kidney damage or dysfunction, as well as other diseases such as diabetes, hypertension, or heart failure. Albuminuria can also increase the risk of cardiovascular complications and mortality. Therefore, Esbach test can be used as a modified method to measure albumin levels in urine and assess the severity and prognosis of kidney and cardiovascular diseases.

The test is based on the principle of precipitation of albumin in the presence of organic acids. The organic acids (citric acid and picric acid) added to the sample exist as negative ions in the solution form. Similarly, the proteins in the urine sample exist as cations when the isoelectric pH of the protein is on the acidic side. Thus, it is essential that the urine sample to be tested has to be acidic enough for the dissociation to take place. When the organic acids are added to the sample, the positively charged ions of proteins combine with the negatively charged ions of the organic acids. As a result, a salt of protein is formed with results in the formation of a precipitate. For the quantitative estimation of albumin, the precipitation formed in the tube is measured by the means of graduations present on the albuminometer tube used for the test. In order to estimate just albumin, a modified version of the Esbach test is developed. As per the modified method, urine is reacted with 10% potassium ferrocyanide, and acetic acid in a specially designed graduated conical flask. After about five minutes of centrifugation, it is said that every 0.1 ml of precipitate represents 1% albumin.

To perform the Esbach test, the following requirements are needed:

• Reagent: Esbach’s reagent is a solution of picric acid and citric acid in distilled water. It is used to precipitate albumin and other proteins in the urine sample. A fresh reagent is preferred for a more accurate result.
• Urine sample: The urine sample should be collected over a 24-hour period and filtered to remove any cells or other particles. If the urine is not acidic enough, few drops of 10% acetic acid should be added to the urine sample. The urine should also have a specific gravity of less than 1.010, otherwise it should be diluted with distilled water.
• Materials required: The materials required for the test include Esbach’s albuminometer, pipettes, test tubes, rubber plugs, and a centrifuge (for the modified version of the test). Esbach’s albuminometer is a glass tube with markings that indicate the levels of urine and reagent to be added. It also has graduations from 0 to 12 grams that represent the amount of albumin per 1000 ml of urine. The pipettes are used to transfer the urine and reagent to the albuminometer tube. The test tubes are used for the qualitative test. The rubber plugs are used to seal the albuminometer tube after mixing the contents. The centrifuge is used to spin the mixture of urine and potassium ferrocyanide and acetic acid in the modified version of the test.

The procedure of Esbach test can be divided into two parts: qualitative test and quantitative test. The qualitative test is used to detect the presence or absence of albumin in the urine sample, while the quantitative test is used to measure the amount of albumin in the urine sample.

Qualitative test

• Take about 3 ml of Esbach’s reagent in a clean test tube.
• Add 2 ml of a filtered urine sample to the test tube drop by drop.
• Observe the test tube for any immediate precipitation.

Quantitative test

• Filter the urine sample to remove any cells or other particles that might interfere with the test.
• If the urine sample is not acidic enough, add a few drops of 10% acetic acid to make it acidic. This is important because the precipitation only occurs under acidic conditions.
• Fill the albuminometer tube with the urine sample up to the ‘U’ mark. The albuminometer tube is a special glass tube with markings and graduations that help to measure the amount of precipitate formed.
• Fill the albuminometer tube with Esbach’s reagent up to the ‘R’ mark.
• Close the tube with a rubber plug and mix the contents by gently inverting the tube several times.
• Leave the tube at rest in an upright position for about 24 hours. This allows the precipitate to settle at the bottom of the tube.
• Read the scale on the tube to determine the amount of precipitate. The scale shows the grams of albumin per 1000 ml of urine sample. For example, if the precipitate is level with the 4 graduation on the tube, this means that the urine sample contains 4 grams of albumin per 1000 ml.

Qualitative test

• Positive result: A positive result of the Esbach test is represented by the formation of yellow precipitate as soon as the reagent and urine sample come in contact with one another. This indicates the presence of albumin or other proteins in the urine sample.
• Negative result: A negative result of the Esbach test is represented by the absence of yellow precipitate or the formation of precipitate after some time. This indicates the absence or low concentration of albumin or other proteins in the urine sample.

Quantitative test

• Results of the quantitative test can be read directly from the albuminometer tube. The height of the precipitate corresponds to the concentration of albumin in the urine sample. For example, if the precipitate is level with the 4 graduations on the tube, this indicates that the urine contains 4 grams of albumin per 1000 ml urine sample.
• The results should be interpreted with caution as they may not reflect the true amount of albumin in the urine. Some factors that may affect the accuracy of the test are:
  • The presence of other substances that may precipitate along with albumin, such as peptones, mucin, and proteoses.
  • The specific gravity and acidity of the urine sample. High specific gravity may prevent proper settling of the precipitate, while low acidity may reduce the precipitation reaction.
  • The age and quality of the reagent. Fresh reagent is preferred for a more accurate result.
  • The duration and temperature of incubation. The tube should be left undisturbed for about 24 hours at room temperature for optimal precipitation.

• Esbach test is used as a clinical test for the quantitative estimation of albumin in the urine. Albumin is a type of protein that is normally present in the blood but not in the urine. The presence of albumin in the urine can indicate kidney damage or disease, as well as other conditions such as diabetes, hypertension, or heart failure. Therefore, measuring the amount of albumin in the urine can help diagnose and monitor these disorders.
• Besides, Esbach test can also be used for the detection of other proteins in urine as well as other samples. For example, Esbach test can be used to detect globulins, peptones, mucin, and proteoses in urine. These proteins can also indicate various pathological conditions such as inflammation, infection, or malignancy. Esbach test can also be applied to other fluids such as cerebrospinal fluid, ascitic fluid, or pleural fluid to detect abnormal protein levels. However, Esbach test is not very specific or sensitive for these proteins and may give false-positive or false-negative results. Therefore, other methods such as electrophoresis or immunological assays are preferred for these purposes.

• Esbach test is not very accurate as it precipitates not only albumin but also other proteins in urine such as peptones, mucin, and proteoses. This can lead to overestimation of albumin concentration in the sample.
• Esbach test requires a 24-hour urine collection to obtain a representative sample. This can be inconvenient and impractical for some patients and may affect the quality of the sample.
• Esbach test is sensitive to the specific gravity and pH of the urine sample. If the specific gravity is too high, the urine sample should be diluted to avoid interference with the precipitation. If the pH is too low or too high, the urine sample should be acidified or alkalinized to ensure optimal conditions for the reaction.
• Esbach test has a limited range of detection. It cannot detect albumin concentrations below 0.5 g/L or above 12 g/L in the urine sample. Therefore, it is not suitable for monitoring mild or severe proteinuria.
• Esbach test is affected by the presence of other substances in urine such as glucose, urea, bilirubin, and drugs. These substances can interfere with the precipitation or the reading of the result.
• Esbach test is time-consuming and labor-intensive. It requires a special tube (Esbach`s albuminometer) and a reagent (Esbach`s reagent) that are not readily available in most laboratories. It also requires 24 hours of incubation and careful measurement of the precipitate.

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