Heat and Acetic Acid Test for Proteinuria – Principle, Procedure, Result, Uses

What is Heat and Acetic Acid Test?

Heat and acetic acid test (heat coagulation test) is a simple qualitative and semi quantitative test which is used for detection of excess protein in urine sample. Mainly albumin is detected but globulin may be present also. It is done as a screening test for proteinuria.

Principle of this test is based on protein denaturation on heating. When urine is heated the protein molecules are unfolded due to breaking of weak bonds and they are coagulated to form white turbidity or precipitate. The reaction is seen in heated portion as cloudiness.

In this test clear urine is taken in a test tube and it is filled partially. Only upper one third portion is boiled so that lower unheated part acts as control. If protein is present white cloudiness is produced in upper heated portion. But turbidity may be due to phosphates and carbonates also so acetic acid is added to confirm.

Few drops of dilute acetic acid is added from side of test tube after boiling. Acetic acid dissolves phosphate and carbonate precipitate but proteins remains insoluble (pH is brought near isoelectric point). If turbidity disappears after adding acetic acid it is due to salts. If turbidity persists or increases and again heating is done then test is positive for protein.

Result is graded by visual observation from trace to 4+ based on density of turbidity and coagulum formation. This test is used in kidney diseases urinary tract infection and preeclampsia in pregnant women. It is low cost and minimal equipment is required so it is useful in resource limited settings.

Principle of heat and acetic acid test

Principle of heat and acetic acid test is based on denaturation and precipitation of proteins by heat in acidic medium. When urine sample is heated the increased kinetic energy breaks the weak non covalent bonds which maintain the three dimensional structure of protein. Due to this protein is unfolded and hydrophobic groups are exposed. These exposed regions are aggregated and a visible white turbidity or coagulum is formed.

In this test acetic acid is added after heating and it has two main function. First it adjusts pH of urine near isoelectric point of protein (about pH 4.5–5.0) so the charge on protein is neutralized and solubility is decreased therefore precipitation is increased. Second it acts as differentiating agent because heating can precipitate phosphate and carbonate also and it gives false turbidity. Acetic acid dissolves these salts but true protein coagulum remains intact.

Therefore if the turbidity persists or increases after heating and adding acetic acid then presence of protein is confirmed. If turbidity disappears then it is due to inorganic salts and protein is absent.

Requirements for heat and acetic acid test

• Urine specimen– Fresh clear urine sample is taken (first morning urine is preferred because it is more concentrated). If urine is turbid it is filtered or centrifuged to make it clear.
• Acetic acid– Dilute acetic acid solution is required (3% or 5% or 6% or 10% acetic acid is used).
• Test tube– Clear glass test tube (about 5–10 ml capacity or standard size) is taken.
• Heat source– Bunsen burner or spirit lamp or water bath is required for boiling urine.
• Test tube holder– Holder or tong is used for holding tube during heating.
• Dropper– Dropper is used to add acetic acid drop by drop.
• Syringe or measuring device– 5 ml syringe is used for taking and transferring urine.
• Test tube rack– It is used for keeping test tube safely.
• Lighting material– Match stick is required for igniting burner or lamp.
• Safety items– Gloves soap and towel is used for hygiene and infection control.
• Labelling material– Label tape and marker pen is used to label the sample and to avoid interchange.

Procedure of heat and acetic acid tests

1. Step 1– Clear urine sample is taken in a clean dry test tube. The tube is filled about two third to three fourth. If urine is cloudy then it is filtered or centrifuged first.
2. Step 2– Test tube is held with holder and it is kept in slanting position. Only upper one third portion of urine is heated over flame till boiling is occured. Lower unheated portion is kept as control.
3. Step 3– Heated portion is observed against dark background. If cloudiness or turbidity is formed it is noted (it may be due to protein or phosphate or carbonate).
4. Step 4– 2–5 drops of dilute acetic acid (6% or 10%) is added carefully from side of test tube.
5. Step 5– The upper portion is boiled again for second time.
6. Step 6– Observation is taken again. If turbidity persists or increases then test is positive for protein. If turbidity disappears completely then it is due to phosphates or carbonates and test is negative for protein.

Results of heat and acetic acid tests

• Negative– Urine remains clear and no turbidity is seen (normal protein level is less than 10 mg/dL). If any initial cloudiness disappears after adding acetic acid then test is negative for protein. If it dissolves cleanly it indicates phosphate. If it dissolves with gas bubble then it indicates carbonate.
• Trace– Faint haziness or slight opalescence is seen (mostly visible against dark background). It indicates about 10–20 mg/dL of protein.
• 1+– Definite cloudiness is present but no granules or flocculation is seen. It indicates about 30–50 mg/dL of protein.
• 2+– Heavy granular cloudiness is present but distinct flocculation is not seen. It indicates about 50–200 mg/dL of protein.
• 3+– Dense opaque turbidity is present with marked flocculation or clumping. It indicates about 200–500 mg/dL of protein.
• 4+– Thick curdy or solid coagulum is formed with large flocculi and it may become solid. It indicates more than 500 mg/dL of protein (heavy proteinuria).

Uses of Heat and Acetic Acid Test

• Screening for proteinuria– It is used as qualitative and semi quantitative test for detection of excess protein (mainly albumin) in urine.
• Pregnancy monitoring– It is routinely used in pregnant women for screening of preeclampsia and eclampsia (proteinuria with high blood pressure).
• Kidney disorders– It helps in detection evaluation and monitoring of renal damage and renal function (nephrotic syndrome glomerulonephritis etc).
• Urinary tract infection– It is used as an aid for screening of UTI also.
• Bence Jones protein– By variation of this test (slow heating and cooling) Bence Jones protein can be detected and it helps in diagnosis of multiple myeloma and Waldenström macroglobulinemia.
• Confirmatory test– It is used for confirmation of abnormal dipstick result because acetic acid removes false positive turbidity (alkaline urine or disinfectant).
• Resource limited diagnosis– It is useful in rural and developing areas because it requires simple material (heat source and acetic acid) and it gives specific result.

Advantages of Heat and Acetic Acid Test

• Cost effective– It is very cheap test and minimal equipment is required so it is accessible in primary care and resource limited areas.
• Broad protein detection– It can detect other proteins also (globulin paraprotein and Bence Jones protein) not only albumin like dipstick.
• High specificity– It gives low false positive result compared to SSA test Heller nitric acid test and dipstick. Acetic acid dissolves inorganic salts and removes false turbidity due to alkaline urine or cleaning agent.
• Simple method– Procedure is easy and result is observed visually so minimal training is required and it can be done by health workers also.
• Adaptable– If acetic acid is not available then other mild acids (lemon juice) can be used for similar result in extreme condition.
• Screening and confirmatory– It is useful as screening test and also confirmatory test for doubtful dipstick result and it correlates with protein creatinine index in spot urine.

Limitations of Heat and Acetic Acid Test

• False negative– In dilute urine (low specific gravity) protein concentration becomes low so test may show negative.
• Excess acetic acid– If too much acetic acid is added then coagulated protein may be dissolved and false negative is produced.
• Highly alkaline urine– In very alkaline urine proteins may not coagulate properly on heating if sample is not acidified first.
• Old urine sample– Improperly stored urine allows bacterial growth it increases pH and proteins may be degraded so result becomes inaccurate.
• Low molecular weight protein– It may fail to detect some low molecular weight proteins which are excreted in renal tubular disease.
• False positive– Some drugs (penicillin sulfonamides tolbutamide tolmetin) may precipitate and gives turbidity like protein.
• Contrast media– Iodine containing radiographic contrast may produce false positive reaction.
• Other turbidity– High urates or mucin may cause turbidity and it looks like protein.
• Semi quantitative– It gives only approximate range not exact value so it is not fully quantitative.
• Type of protein– It cannot identify specific type of protein causing proteinuria.
• Clear urine needed– Urine should be clear for proper observation otherwise it is filtered or centrifuged before test.
• Equipment needed– Glass test tube and heat source (Bunsen burner or spirit lamp) is required so it is not as simple like dipstick.
• Technical error– If whole test tube is heated then control part is lost and interpretation becomes difficult.

Frequently Asked Questions

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