ADA
Adenosine Deaminase
Colorimetric - Kinetic
Quantitative determination of Adenosine Deaminase ADDITIONAL EQUIPMENT
(ADA) in serum and plasma samples - Spectrophotometer or colorimeter measuring at 540/550 nm.
IVD - Thermostatic bath at 37ºC ( 0.1ºC)
- Matched cuvettes 1.0 cm light path.
Store at 2-8ºC - General laboratory equipment.
PRINCIPLE OF THE METHOD SAMPLES
The ADA assay is based on the enzymatic deamination of adenosine Fresh serum and non-hemolyzed serum or plasma.
to inosine which is converted to hypoxanthine by purine nucleoside Stability: 7 days at 2-8ºC.
phosphorylase (PNP). Hypoxanthine is then converted to uric acid
and hydrogen peroxide (H2O2) by xanthine oxidase (XOD). H2O2 is PROCEDURE
further reacted with N-Ethyl-N-(2-hydroxy-3-sulphopropyl)-3- 1. Assay conditions:
methylaniline (EHSPT) and 4-aminoantipyrine (4-AA) in the presence Wavelength (main/sub): . . . . . . . . . . . .550 nm
of peroxidase (POD) to generate quinone dye which is monitored in a Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . 1 cm light path
kinetic manner. The entire enzymatic reaction scheme is shown Constant temperature . . . . . . . . . . . . …37ºC
below. 1. Mix 5 μl sample with 180 μl R1 and incubate at 37ºC for 3 minutes.
ADA 2. Add 90 μl R2 into cuvette, mix and wait for 5 minute.
Adenosine + H2O Inosine + NH3 3. Read initial absorbance and start timer simultaneously, read again
PNP after 3 minutes.
Inosine + Pi Hypoxanthine + Ribose 1-phosphate 4. Calculate absorbance change per minute ( A/min)
XOD
Hypoxanthine + 2H2O + 2O2 Uric acid + 2H2O2 CALCULATIONS
POD Asample /min
2H2O2 + 4-AA + EHSPT 4H2O + Quinone dye ADA (U/L) = × Calibrator value
(max 556nm) Acalibrator /min
One unit of ADA is defined as the amount of ADA that generates one Units: One international unit (IU) is the amount of enzyme that transforms 1 mol
μmole of inosine from adenosine per min at 37ºC. of substrate per minute, in standard conditions. The concentration is expressed
in units per litre of sample (U/L).
CLINICAL SIGNIFICANCE
ADA is an enzyme catalyzing the deamination reaction from QUALITY CONTROL
adenosine to inosine. The enzyme is widely distributed in human Control sera are recommended to monitor the performance of assay
tissues, especially high in T lymphocytes. Elevated serum ADA procedures. ADA Control (2 levels).
activity has been observed in patients with acute hepatitis, alcoholic If control values are found outside the defined range, check the instrument,
hepatic fibrosis, chronic active hepatitis, liver cirrhosis, viral hepatitis reagents and technique for problems.
and hepatoma
1,2
. Increased ADA activity was also observed in Each laboratory should establish its own Quality Control scheme and
3
patients with tuberculous effusions . Determination of ADA activity in corrective actions if controls do not meet the acceptable tolerances.
patient serum may add unique values to the diagnosis of liver REFERENCE VALUES
diseases in combination with ALT or γ-GT (GGT) tests. ADA assay 0-15 U/L
3
may also be useful in the diagnostics of tuberculous pleuritis . These values are for orientation purpose; each laboratory should establish
REAGENTS its own reference range.
Tris-HCl pH 8.0 50 mM PERFORMANCE CHARACTERISTICS
4-AA 2 mM Linearity: The assay is linear up to ADA concentration of 200 U/L.
R1
PNP 0.1 U/mL If the results obtained were greater than linearity limit, dilute the sample 1/2
XOD 0.2 U/mL with NaCl 9 g/L and multiply the result by 2.
Peroxidase 0.6 U/mL Precision: In the study, two serum specimens containing 11 and 30 U/L
Tris-HCl pH 4.0 50mM ADA were tested with 2 runs per day with duplicates over 15 working days:
R2
Adenosine 10 mM
EHSPT 2 mM Within Run (N=30) Run to Run (N=30)
ADA CAL 11 U/L 30 U/L 11 U/L 30 U/L
Mean (U/L) 11.11 30.74 9.63 29.62
PREPARATION SD 0.16 0.45 0.47 0.59
Reagents are ready to use. ADA Calibrator and Control are in CV (%) 1.47 1.45 4.90 2.00
lyophilized form, and need to be recontituted with 1.0 mL of distilled
Sensitivity: The minimum detectable concentration of ADA with an
water before use.
acceptable level of precision was determined as 0 U/L.
PRECAUTIONS The results of the performance characteristics depend on the analyzer used.
R1 is light-sensitive and should be stored in a dark place.
Solution R1 and CAL contain Sodium Azide. Avoid ingestion or INTERFERENCES
contact with skin or mucous membranes. In case of skin contact, flush Bilirubin (up to 30 mg/dL), Hemoglobin (up to 200 mg/dL), Triglycerides (up
affected area with copious amounts of water. In case of contact with to 750 mg/dL) and Ascorbic acid (up to 4 mg/dL) do not interfere.
eyes or if ingested, seek immediate medical attention. NOTES
All specimens used in this t est should be considered potentially Audit Diagnostics has instruction sheets for several automatic
infectious. analyzers. Instructions for many of them are available on request.
CALIBRATION BIBLIOGRAPHY
Recommend that this as say should be calibrated using the ADA 1. Kobayashi F, Ikeda T, Marumo F, Sato C: Adenosine deaminase isoenzymes in
Calibrator. liver disease. Am. J. Gastroenterol. 88: 266-271 (1993)
2. Kallkan A., Bult V., Erel O., Avci S., and Bingol N. K. : Adenosine deaminase
STORAGE AND STABILITY and guanosine deaminase activities in sera of patients with viral hepatitis. Mem
All the components of the kit are stable until the expiration date on the Inst. Oswaldo Cruz 94(3) 383-386 (1999)
label when stored tightly closed at 2-8ºC, protected from light and 3. Burgess LJ, Maritz FJ, Le Roux I, et al. Use of adenosine deaminase as a
contaminations prevented during their use. diagnositic tool for tuberculous pleurisy. Thorax 50: 672-674 (1995)
Do not use reagents over the expiration date.
Stability: 1 month at 2-8ºC after opening, if contamination avoided and
vials recapped immediately after use.
Signs of reagent deterioration:
- Presence of particles and turbidity.
