ADA (Adenosine Deaminase) Test: Introduction, Principle, Test Requirements, Procedure, Normal Range, and Clinical Significance

Introduction of ADA (Adenosine Deaminase) Test

ADA (Adenosine Deaminase) Test is very useful for suspicious cases of tuberculosis, increased levels of ADA could facilitate diagnosis. ADA is an enzyme that primarily functions in our body in the development and maintenance of the immune system that catalyzes the conversion of adenosine and 2’-deoxyadenosine to inosine and 2’-deoxyinosine and is widely distributed in various tissues and cells. There are two isoforms, ADA1 and ADA2. ADA1 is widely expressed in most cells in the body, particularly in lymphocytes and macrophages. It is present in the cytosol, nucleus, and in the cell membrane. ADA2 was first found in the spleen but is predominantly found in the plasma and serum. Increased serum ADA levels are found in certain infectious diseases such as tuberculosis and various liver diseases such as acute hepatitis, alcoholic hepatic fibrosis, and chronic active hepatitis. ADA is also a marker for T-lymphocyte proliferation.

Principle of ADA (Adenosine Deaminase) Test

ADA (Adenosine Deaminase) Test is based on the enzymatic deamination of adenosine to inosine which is converted to hypoxanthine by purine nucleoside phosphorylase (PNP). Hypoxanthine is then converted to uric acid and hydrogen peroxide (H₂O₂) by xanthine oxidase (XOD). H₂O₂ is further reacted with N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methyl aniline (EHSPT) and 4-amino antipyrine (4-AA) in the presence of peroxidase (POD) to generate quinone dye which is monitored in a kinetic manner.

Test Requirements for ADA Test

• The test kit contains reagent 1, reagent 2, glass cups, and a calibrator.
• Micropiettes and tips
• Spectrophotometer with incubator
• Timer
• Waste bin
• Gloves

Test Procedure of ADA Test

Method: UV kinetic

1. Add reagent first (R1): 360 ul
2. Add sample 10 ul in a cup and finally mix them.
3. Incubate for 3 minutes in an incubator provided by a spectrophotometer.
4. Add reagent second (R2) 180ul.
5. Mix properly and suck to the spectrophotometer.
6. It takes 8 minutes to give the result.

Normal Range of ADA Test

The normal range of ADA depends on the nature of the specimen.

• Seum<15 U/L
• CSF<10 U/L
• Fluid<40 U/L

Clinical Significance of ADA Test

ADA can also be used in the workup of lymphatic pleural effusion or peritoneal ascites, in that such specimens with low ADA levels essentially exclude tuberculosis from consideration. Tuberculosis pleural effusion can now be grown and diagnosed accurately by increased levels of pleural ADA, above 40 U per liter. In the case of tubercular meningitis, the ADA level is high in CSF.

Why this test is important?

To rule out a Mycobacterium tuberculosis infection in pleural fluid in order to assist in the diagnosis of tuberculosis; rarely to detect the infection in other body fluids such as peritoneal fluid or cerebrospinal fluid (CSF) A volume of pleural fluid is collected by a physician using a procedure called thoracentesis; other body fluids are collected using other procedures. Other samples may be CSF, ascitic fluid/peritoneal fluid, synovial fluids (joint fluid), etc. Note: This ADA test requires only 10 microlitre samples so,  0.5 to 1 ml samples are sufficient.

When do we have to do this test?

When a clinician that someone with chest pain. A suspected patient of extrapulmonary tuberculosis and specimen choice is body fluids.

Further Readings

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822331/
2. https://www.researchgate.net/publication/14796714_Kinetic_determination_of_serum_adenosine_deaminase
3. https://www.abcam.com/adenosine-deaminase-ada-activity-assay-kit-colorimetric-ab211093.html
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818597/
5. https://www.diazyme.com/adenosine-deaminase-ada