Medically reviewed and approved by a board-certified member
Clinical Pathology

Proteinuria: Purpose, Test Procedure and Results

By Dayyal Dg.Twitter Profile | Updated: Friday, 08 December 2023 18:03 UTC
Login to get unlimited free access
Evaluation of proteinuria
Evaluation of proteinuria

A urine protein test checks the amount of protein in urine. Normally, healthy people don't have much protein in their urine. But if the kidneys aren't working well or if there are high levels of certain proteins in the bloodstream, protein might show up in the urine.

Qualitative Estimation of Proteins

Method 1: Heat and Acetic Acid Test (Boiling Test)

This test operates on the principle that when proteins are heated in an acidic solution, they undergo precipitation.


Ensure clarity in urine; if not, either filter it or utilize the supernatant from a centrifuged sample.

Confirm urine acidity (verify with litmus paper); if not acidic, gradually add 10% acetic acid until the blue litmus paper transitions to red.

Take a test tube and fill it two-thirds with urine. Incline the tube at an angle and heat the upper portion over a flame. (Specifically, only heat the upper portion to avoid disrupting the precipitate with convection currents generated by heat, allowing for a clear comparison between the upper and lower segments). Compare the heated part with the lower part. The presence of cloudiness or turbidity indicates the existence of either phosphates or proteins (refer to Figure 1). Introduce a few drops of 10% acetic acid, then boil the upper portion again. Turbidity originating from phosphates disappears, whereas that arising from proteins persists.

Principle of heat test for proteins
Figure 1: Principle of heat test for proteins.

This test may yield a false-positive outcome in the presence of tolbutamide and elevated levels of penicillins.

Method 2: Reagent Strip Test

The reagent section of the strip is covered with an indicator and buffered to an acidic pH, displaying a color change in the presence of proteins (refer to Figures 821.2 and 821.3). This phenomenon is referred to as the "protein error of indicators" principle.

Principle of reagent strip test for proteins
Figure 2: Principle of reagent strip test for proteins. The principle is called as ‘protein error of indicators’ meaning that one color appears if protein is present and another color if protein is absent. Sensitivity is 5-10 mg/dl. The test does not detect Bence Jones proteins, hemoglobin, and myoglobin.

The reagent region is saturated with bromophenol blue indicator buffered to pH 3.0 using citrate. As the dye adheres to proteins, there is a shift in ionization (and consequently, pH) of the indicator, resulting in a color change. The intensity of the produced color is directly proportional to the protein concentration. This test is semi-quantitative in nature.

Grading of proteinuria with reagent strip test
Figure 3: Grading of proteinuria with reagent strip test (above) and sulphosalicylic acid test (below).

The reagent strip test primarily reacts to albumin but may yield false-negative results in the presence of Bence Jones proteins, myoglobin, and hemoglobin. If solely relying on the reagent strip method, there is a risk of overlooking overload (Bence Jones) proteinuria and tubular proteinuria. To ensure accuracy, it is advisable to follow this test with the sulphosalicylic acid test, which serves as a confirmatory measure. It's important to note that highly alkaline urine, significant hematuria, and contamination with vaginal secretions can potentially cause false-positive reactions in this test.

Method 3: Sulphosalicylic Acid Test

Introduction of sulphosalicylic acid into urine leads to the precipitation of a white substance if proteins are present. Organic acids denature proteins, causing them to precipitate out of the solution.

To perform the test, take 2 ml of clear urine in a test tube. If the urine's pH is neutral or alkaline, introduce a drop of glacial acetic acid. Following this, add 2-3 drops of sulphosalicylic acid (3 to 5%) and observe for turbidity against a dark background (refer to Figure 3). This method proves to be more sensitive and dependable compared to the boiling test.

However, it's crucial to note that false-positive results may arise due to various factors such as gross hematuria, highly concentrated urine, radiographic contrast media, excess uric acid, tolbutamide, sulphonamides, salicylates, and penicillins. On the contrary, a false-negative outcome may occur when dealing with very dilute urine.

This test is proficient in detecting a range of proteins, including albumin, hemoglobin, myoglobin, and Bence Jones proteins. For a comprehensive understanding, Table 1 illustrates a comparison between the reagent strip test and the sulphosalicylic acid test.

Table 1: Comparison of two tests for proteinuria.
ParameterReagent strip testSulphosalicylic acid test
Principle Colorimetric Acid precipitation
Proteins detected Albumin All (albumin, Bence Jones proteins, hemoglobin, myoglobin)
Sensitivity 5-10 mg/dl 20 mg/dl
Indicator Color change Turbidity
Type of test Screening Confirmatory

Quantitative Estimation of Proteins

The indications for quantifying protein levels in urine encompass several key aspects within the realm of clinical diagnosis and therapeutic monitoring. These include:

  1. Diagnosis of Nephrotic Syndrome: Determining the presence of nephrotic syndrome stands as a primary motive for conducting quantitative estimations of proteins in urine. This serves as a crucial diagnostic parameter, aiding in the identification and characterization of renal conditions associated with abnormal protein excretion.
  2. Detection of Microalbuminuria or Early Diabetic Nephropathy: Another pivotal application is the identification of microalbuminuria, a subtle yet significant indication of early-stage diabetic nephropathy. This underscores the proactive role of protein quantification in recognizing incipient renal complications in diabetic patients, facilitating timely intervention.
  3. Monitoring Response to Therapy in Renal Disease: Quantitative assessment of proteins in urine plays a vital role in monitoring the efficacy of therapeutic interventions in renal diseases. This ongoing evaluation provides valuable insights into the patient's response to treatment, guiding adjustments and refinements in the therapeutic approach as needed.

In the context of proteinuria levels, specific thresholds offer diagnostic distinctions:

  • Proteinuria exceeding 1500 mg in a 24-hour period signals the presence of glomerular disease.
  • When proteinuria surpasses 3500 mg in 24 hours, it is categorized as nephrotic range proteinuria. Conversely, in tubular, hemodynamic, and post-renal diseases, proteinuria typically falls below 1500 mg in a 24-hour timeframe.

The grading of albuminuria, a subset of proteinuria, is systematically presented in Table 2. This classification aids in stratifying the severity of albumin excretion, providing a standardized framework for clinical interpretation.

Two distinct methodologies are employed for the quantitative assessment of proteins in urine:

  1. Estimation of Proteins in a 24-hour Urine Sample: This method involves collecting urine over a complete day to analyze the total protein excreted during that period.
  2. Estimation of Protein/Creatinine Ratio in a Random Urine Sample: An alternative approach involves determining the ratio of protein to creatinine in a random urine sample. This method offers a more immediate assessment, providing valuable information without the need for a full-day urine collection.
Table 2: Grading of albuminuria
Conditionmg/24 hrmg/Lmg/g creatinineμg/minμg/mg creatinineg/mol creatinine
Normal < 30 < 20 < 20 < 20 < 30 < 2.5
Microalbuminuria 30-300 20-200 20-300 20-200 30-300 2.5-25
Overt albuminuria > 300 > 200 > 300 > 200 > 300 > 25

Quantifying Proteins in a 24-Hour Urine Sample: Accurate assessment of protein levels begins with the collection of a 24-hour urine sample, a procedure outlined earlier. The reliability of this sample is ensured by verifying its adequacy through the calculation of the expected 24-hour urine creatinine excretion. The daily output of urinary creatinine, a marker influenced by muscle mass, tends to remain relatively stable within an individual. For adult males, the range of creatinine excretion falls between 14-26 mg/kg/24 hours, while in females, it spans from 11-20 mg/kg/24 hours. Diverse methods are available for the precise quantification of proteins, encompassing Esbach’s albuminometer method, turbidimetric methods, the biuret reaction, and immunologic methods.

Assessing Protein/Creatinine Ratio in a Random Urine Sample: Recognizing the challenges associated with incomplete 24-hour urine collections, many laboratories opt for measuring the protein/creatinine ratio in random urine samples. A normal protein/creatinine ratio is < 0.2. In cases of low-grade proteinuria, the ratio ranges from 0.2 to 1.0; in moderate cases, it falls between 1.0 and 3.5; whereas in instances of nephrotic-range proteinuria, it surpasses 3.5. This method offers a pragmatic alternative, addressing the practical limitations of the 24-hour collection process and providing valuable insights into protein excretion levels.


This is characterized by the urinary excretion of 30 to 300 mg/24 hours (equivalent to 2-20 mg/dl) of albumin in the urine.

Significance of Microalbuminuria

  1. Microalbuminuria stands as the initial indicator of renal impairment in diabetes mellitus, specifically in the context of diabetic nephropathy. This phenomenon signifies an elevation in capillary permeability to albumin, marking the onset of microvascular disease. Notably, microalbuminuria precedes the onset of diabetic nephropathy by several years. Rigorous management involving aggressive control of blood glucose levels and hypertension during this stage can significantly impede or forestall the progression toward irreversible renal damage and eventual renal failure.
  2. Microalbuminuria emerges as an autonomous risk factor for cardiovascular disease in individuals with diabetes mellitus.

Detection of Microalbuminuria

Microalbuminuria eludes detection through routine proteinuria tests. Various methods employed for its identification include:

  • Assessing the albumin-creatinine ratio in a random urine sample
  • Measuring albumin levels in an early morning or random urine sample
  • Determining albumin levels in a 24-hour urine sample

Commercially available test strips are designed to screen for microalbuminuria. For precise quantification, immunologic assays such as radioimmunoassay or enzyme-linked immunosorbent assay (ELISA) can be employed.

Bence Jones Proteinuria

Bence Jones proteins, integral to the realm of plasma cell dyscrasias like multiple myeloma and primary amyloidosis, represent monoclonal immunoglobulin light chains—either κ or λ—synthesized by neoplastic plasma cells. The heightened production of these light chains leads to their excess presence, resulting in overflow proteinuria as they are excreted in urine.

These proteins exhibit a distinctive thermal behavior. Upon heating, Bence Jones proteins precipitate within the temperature range of 40°C to 60°C— a phenomenon setting them apart, as other proteins typically precipitate between 60-70°C. Notably, the precipitate vanishes upon further heating at 85-100°C, a behavior not shared by other proteins. Upon cooling within the range of 60-85°C, the precipitate of Bence Jones proteins reappears. It is crucial to note that while this test provides insights, it lacks specificity for Bence Jones proteins, and both false-positive and -negative results can arise. Consequently, the contemporary standard involves protein electrophoresis of a concentrated urine sample (see Figure 4).

Urine protein electrophoresis showing heavy Bence Jones proteinuria
Figure 4: Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine.

Figure 5 illustrates the subsequent assessment of enduring overt proteinuria.

Evaluation of proteinuria
Figure 5: Evaluation of proteinuria.

It is important to note that quantifying proteins and assessing creatinine clearance are conducted for all patients experiencing persistent proteinuria.

Was this page helpful?
(1 Vote)
End of the article