Sulphosalicylic Acid Test for Proteinuria – Principle, Procedure, Result, Uses

Sulfosalicylic acid (SSA) test is a simple screening test which is used to detect excessive proteins in urine. It is a rapid and low cost method and it is mainly used for checking proteinuria which indicates kidney impairment. The test is done on clear urine sample after centrifugation so that turbidity is observed clearly.

It is based on turbidimetry. When SSA reagent is added to clear centrifuged urine the strong acidic nature causes protein to be denatured and precipitated out from solution. Due to precipitation a cloudy appearance is produced in urine and the degree of turbidity is proportional to concentration of protein present in the sample. The turbidity is graded visually from negative(clear) to 4+ or 5+(dense turbidity or solid mass) depending on amount of precipitate formed.

A major advantage is that it detects broad range of proteins. Dipstick mainly detects albumin but SSA test can detect albumin globulins glycoproteins and Bence-Jones proteins(immunoglobulin light chains). So it is used for confirmation of dipstick result and it is used for screening of preeclampsia in pregnant women and it is helpful for detecting non albumin proteinuria in conditions like multiple myeloma.

Principle of Sulphosalicylic Acid Test

It is based on turbidimetric assay. In this test sulphosalicylic acid(SSA) reagent is added to urine sample and pH of fluid is lowered due to acidic nature of reagent. When pH is dropped below the isoelectric point of proteins the protein molecules gain net positive charge and this step helps in precipitation of protein.

The negatively charged sulfosalicylate anions binds with positively charged protein molecules by electrostatic interaction. Due to ionic binding and acidic environment the solubility of proteins is altered and the proteins is denatured and unfolded. These unfolded proteins are aggregated to form large insoluble complexes and turbidity(cloudiness) is produced in previously clear urine. The degree of turbidity formed is directly proportional to total protein concentration present in sample and it is used for visual estimation of protein level.

Requirements for Sulphosalicylic Acid Test

The requirements for sulphosalicylic acid(SSA) test are–

• Fresh urine specimen is required. First morning urine sample is preferred.
• Centrifuge is required for centrifugation of urine(about 2000–3000 rpm for 3–5 minutes) so that clear supernatant is obtained.
• Sulphosalicylic acid(SSA) reagent is required for denaturation and precipitation of proteins. It is commonly prepared as 3% 5% 20% or 25% solution.
• Litmus paper is required to check initial pH of urine sample.
• Dilute acetic acid(3%–10%) is required to acidify urine to pH 5.0–6.0 when sample is highly alkaline. Alkaline urine can neutralize SSA and false negative result is obtained.
• Test tubes(clear glass test tubes like 10 × 75 mm) is required for holding and mixing urine supernatant with SSA reagent.
• Visual aid is required such as dark background or standardized card behind the test tube for grading turbidity.

Procedure of Sulphosalicylic Acid Test

The procedure of sulphosalicylic acid(SSA) test are–

1. Urine sample is collected and it is mixed properly. First morning urine sample is preferred.
2. The urine sample is centrifuged at about 2000–3000 rpm for 3–5 minutes so that clear supernatant is obtained. Debris and cells are removed because it can give false turbidity.
3. The pH of clear supernatant is checked. When urine is highly alkaline(pH more than 7.0) it can neutralize SSA reagent and false negative result is obtained.
4. Few drops of dilute acetic acid(3%–10%) is added to alkaline urine and pH is adjusted to about 5.0–6.0.
5. The clear pH adjusted urine supernatant is taken in test tube.
6. SSA reagent is added to urine in test tube. Equal volume is mixed such as 2 mL urine + 2 mL of 3% SSA. Some method uses 1 part urine and 3 part SSA.
7. The test tube is covered and it is mixed gently by inverting for few times.
8. The tube is kept undisturbed for about 10 minutes so that protein-anion complex is formed and aggregation occurs.
9. Turbidity is observed visually against dark background or with print card.
10. The result is graded from negative(clear) to 4+ or 5+(dense turbidity with solid or flocculent precipitate) based on obscuring of background text/lines.

Result and Interpretation

The result and interpretation of sulphosalicylic acid(SSA) test are–

• Negative(0)– Solution remains clear and no turbidity or precipitate is seen. It indicates no clinically significant protein is present(about less than 5–10 mg/dL).
• Trace– Faint white precipitate or slight opalescence is seen and it is usually seen against dark background(about 10–20 mg/dL).
• 1+– Distinct turbidity is present but printed text and background lines can be read through the test tube(about 15–30 mg/dL).
• 2+– Moderate turbidity is seen. Printed text behind specimen becomes blurred or illegible but background lines are still visible(about 40–100 mg/dL).
• 3+– Heavy turbidity with fine granules are formed and it is not possible to see through the specimen(about 150–400 mg/dL).
• 4+(and 5+)– Dense turbidity is formed with large flocculent precipitates or solid gelled mass is formed(about more than 500 mg/dL).
• False positive(overestimation)– Turbidity can be increased due to uncentrifuged cloudy urine radiographic contrast agents high concentration of urates or by drugs like penicillins cephalosporins sulfonamides and tolbutamide.
• False negative(underestimation)– The proteins may not be precipitated when urine is highly dilute or highly alkaline. Alkaline urine can neutralize acid reagent and false negative is obtained.
• Diagnostic interpretation– SSA test detects broad range of proteins. If SSA test shows significant turbidity but urine dipstick(which mainly detects albumin) is negative or trace then it suggests non albumin protein such as Bence-Jones proteins(immunoglobulin light chains) and it is seen in multiple myeloma.

Uses of Sulphosalicylic Acid Test

The uses of sulphosalicylic acid(SSA) test are–

• It is used for screening of proteinuria in urine and it is a sensitive test for wide range of proteins.
• It is used to confirm standard urine dipstick result. It helps in detecting false negative or verifying false positive because dipstick mainly detects albumin.
• It is used for detecting non albumin proteins such as Bence-Jones proteins(immunoglobulin light chains) and it is important in diagnosis of plasma cell dyscrasias like multiple myeloma.
• It is used for screening of preeclampsia and pregnancy induced hypertension in pregnant women. It is rapid and low cost method in primary care and resource limited settings.
• It is used in monitoring chronic kidney disease(CKD). Quantitative SSA method can be used for protein to creatinine ratio and for tracking disease progression.
• It is used for evaluation of cerebrospinal fluid(CSF) for presence of proteins in nervous system diagnosis.
• It is used for cleansing sample for biochemical assays by precipitating and removing interfering proteins for accurate spectrophotometric reading.
• It is used for preparing samples in molecular biology for electrophoresis(SDS-PAGE) and chromatography by isolating specific molecules and removing unwanted proteins.
• It is used as educational tool in biology and medical laboratory to show protein denaturation and precipitation.
• It is used in pharmacological quality control during drug formulation and stability testing for detecting minute protein impurities in injectable and parenteral drugs.

Advantages of Sulphosalicylic Acid Test

The advantages of sulphosalicylic acid(SSA) test are–

• It detects broad spectrum of proteins. Dipstick mainly detects albumin but SSA test can detect albumin globulins glycoproteins and Bence-Jones proteins(immunoglobulin light chains).
• It is a good confirmatory test. It is used for confirming dipstick result and for detecting non albumin proteinuria which is missed by routine test.
• It is highly sensitive. Very low concentration of protein can be detected and detection limit is about 5–10 mg/dL.
• It is cost effective and inexpensive test. It is suitable for screening in public sector and resource limited environment.
• It is rapid and simple to perform. Immediate result is obtained and minimal equipment is required. It can be performed by personnel with minimal training and inter observer variability is less.
• It shows high specificity for confirming clinically significant proteinuria in pregnant women with hypertension and it helps in rapid intervention for preeclampsia.
• It is versatile test. It can be used for other biological fluids like cerebrospinal fluid(CSF) and it is also used in biochemical assay for precipitating and removing interfering proteins.

Limitations of Sulphosalicylic Acid Test

The limitations of sulphosalicylic acid(SSA) test are–

• False positive result is obtained due to interfering factors. High dose of drugs like penicillins cephalosporins sulfonamides tolbutamide and para aminosalicylic acid can produce turbidity.
• Radiographic contrast agents(iodinated contrast media) used in imaging procedure like IV pyelogram can cause dense precipitation and result becomes falsely high.
• Cloudy uncentrifuged urine containing cellular debris pus(leukocytes) or blood(gross hematuria) gives factitious high turbidity.
• Highly concentrated urine or high concentration of urates can be precipitated in acidic environment and false positive is obtained.
• False negative result is obtained in some conditions. Strongly buffered alkaline urine neutralizes SSA reagent and required pH drop is not produced so proteins is not precipitated.
• Highly dilute urine(low specific gravity) dilutes protein concentration below detection limit(about 5–10 mg/dL) and existing proteins may not be detected.
• Interpretation is subjective because turbidity is assessed visually and inter observer variability can occur.
• It is semi quantitative test. It gives estimated grade(negative to 4+) and exact quantitative value of protein concentration is not obtained by this visual method alone.

FAQ

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