Quantitative determination of Adenosine Deaminase
(ADA) in serum and plasma samples
IVD
Store at 2-8ºC
PRINCIPLE OF THE METHOD
The ADA assay 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-
sulphopropyl)-3-methylaniline (EHSPT) and 4-aminoantipyrine (4-AA) in the
presence of peroxidase (POD) to generate quinone dye which is monitored in a
kinetic manner. The entire enzymatic reaction scheme is shown below.
ADA
Adenosine + H2O Inosine + NH3
PNP
Inosine + Pi Hypoxanthine + Ribose 1-phosphate
XOD
Hypoxanthine + 2H2O + 2O2 Uric acid + 2H2O2
POD
2H2O2 + 4-AA + EHSPT 4H2O + Quinone dye
(max 556nm)
One unit of ADA is defined as the amount of ADA that generates one μmole of
inosine from adenosine per min at 37ºC.
CLINICAL SIGNIFICANCE
ADA is an enzyme catalyzing the deamination reaction from adenosine to inosine.
The enzyme is widely distributed in human tissues, especially high in T
lymphocytes. Elevated serum ADA activity has been observed in patients with
acute hepatitis, alcoholic hepatic fibrosis, chronic active hepatitis, liver cirrhosis,
viral hepatitis and hepatoma 1,2. Increased ADA activity was also observed in
patients with tuberculous effusions 3
. Determination of ADA activity in patient
serum may add unique values to the diagnosis of liver diseases in combination
with ALT or γ-GT (GGT) tests. ADA assay may also be useful in the diagnostics
of tuberculous pleuritis 3.
PRECAUTIONS
R1 is light-sensitive and should be stored in a dark place.
The reagents contain 0,1% Sodium Azide. Avoid ingestion or contact with skin
or mucous membranes. In case of skin contact, flush affected area with copious
amounts of water. In case of contact with eyes or if ingested, seek immediate
medical attention.
All specimens used in this test should be considered potentially infectious.
CALIBRATION
Recommend that this assay should be calibrated using the ADA Calibrator
ref.1002230 included in the kit.
STORAGE AND STABILITY
All the components of the kit are stable until the expiration date on the label when
stored tightly closed at 2-8ºC, protected from light and contaminations prevented
during their use.
Do not use reagents over the expiration date.
Signs of reagent deterioration:
- Presence of particles and turbidity.
ADDITIONAL EQUIPMENT
- Spectrophotometer or colorimeter measuring at 540/550 nm.
- Thermostatic bath at 37ºC ( 0,1ºC)
- Matched cuvettes 1,0 cm light path.
- General laboratory equipment.
SAMPLES
Serum, heparinized plasma, pleural fluid, or cerebrospinal fluid may be assayed.
Ideally, venous blood should be collected and handled anaerobically. Do not use
citrate or oxalate as anticoagulant.
Plasma and serum, after prompt separation from cells or clot, should be kept
tightly stoppered. ADA content of blood is stable for 1 week when stored at 2–
8°C. Pleural fluid should be collected in a sterile or heparinized tube and
processed within 2 hours at room temperature or stored at 2-8°C or -20°C for 2
days and up to 2,5 years at -80°C.7,8,9 Cerebrospinal fluid (CSF) should be clear
and collected in a sterile tube without anticoagulant. ADA is stable in CSF for 24
hours at 25ºC, 7 days at 2-8ºC and 3 months at -20ºC.10
PROCEDURE
1. Assay conditions:
Wavelength (main/sub): . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550 nm
Cuvette: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 cm light path
Constant temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37ºC
1. Mix 5 μl sample with 180 μl R1 and incubate at 37ºC for 3 minutes.
2. Add 90 μl R2 into cuvette, mix and wait for 5 minute.
3. Read initial absorbance and start timer simultaneously, read again after 3 minutes.
4. Calculate absorbance change per minute (A/min)
CALCULATIONS
Asample /min
ADA (U/L) = × Calibrator value
Acalibrator /min
Units: One international unit (IU) is the amount of enzyme that transforms 1 mol of
substrate per minute, in standard conditions. The concentration is expressed in units per
litre of sample (U/L).
QUALITY CONTROL
Control sera are recommended to monitor the performance of assay procedures: ADA
Control ref. 1002232 (2 levels).
If control values are found outside the defined range, check the instrument, reagents and
technique for problems.
Each laboratory should establish its own Quality Control scheme and corrective actions
if controls do not meet the acceptable tolerances.
REFERENCE VALUES
Serum samples: 0-15 U/L1-4
; Pleural fluid: 0-30 U/L; CSF: 0-9 U/L 4,6
These values are for orientation purpose; each laboratory should establish its own
reference range.
PERFORMANCE CHARACTERISTICS
Linearity: The assay is linear up to ADA concentration of 200 U/L.
If the results obtained were greater than linearity limit, dilute the sample 1/2 with NaCl 9
g/L and multiply the result by 2.
Precision: In the study, two serum specimens containing 11 and 30 U/L ADA were
tested with 2 runs per day with duplicates over 15 working days:
Within Run (N=30) Run to Run (N=30)
11 U/L 30 U/L 11 U/L 30 U/L
Mean (U/L) 11,11 30,74 9,63 29,62
SD 0,16 0,45 0,47 0,59
CV (%) 1,47 1,45 4,90 2,00
Sensitivity: The minimum detectable concentration of ADA with an acceptable level of
precision was determined as 0 U/L.
The results of the performance characteristics depend on the analyzer used.
INTERFERENCES
Bilirubin (up to 30 mg/dL), Hemoglobin (up to 200 mg/dL), Triglycerides (up to 750
mg/dL) and Ascorbic acid (up to 4 mg/dL) do not interfere.
NOTES
SPINREACT has instruction sheets for several automatic analyzers. Instructions
for many of them are available on request.
BIBLIOGRAPHY
1. Kobayashi F, Ikeda T, Marumo F, Sato C: Adenosine deaminase isoenzymes in
liver disease. Am. J. Gastroenterol. 88: 266-271 (1993)
2. Kallkan A., Bult V., Erel O., Avci S., and Bingol N. K. : Adenosine deaminase and
guanosine deaminase activities in sera of patients with viral hepatitis. Mem Inst.
Oswaldo Cruz 94(3) 383-386 (1999)
3. Burgess LJ, Maritz FJ, Le Roux I, et al. Use of adenosine deaminase as a
diagnositic tool for tuberculous pleurisy. Thorax 50: 672-674 (1995)
4. Boonyagars L., Kiertiburanakul S.: Use of Adenosine Deaminase for the
Diagnosis of Tuberculosis: A Review. J. Infect. Dis Antimicrob Agents 2010;
27:111-8
5. Delacour H., Sauvanet C., Ceppa F., Burnat P.: Analytical perfor-mances of the
Diazyme ADA assay on the Cobas 6000 system. Clini-cal Biochemistry 43 (2010)
1468-1471.
6. Feres MC, De Martino MC, Maldijian S, et al.: Laboratorial valida-tion of an
automated assay for the determination of adenosine deami-nase activity in pleural
fluid and cerebrospinal fluid. J Bras Pneumol. 2008; 34(12): 1033-1039.
7. Porcel, JM.: Handling Pleural Fluid Samples for Routine Analyses. Derleme. June
2013; 19-22.
8. Al-Shammary FJ.: Adenosine Deaminase Activity in Serum and Pleural Effusions
of Tuberculous and Non-Tuberculous Patients. Bi-ochemistry and Molecular
Biology International 43(4) 763-779 (1997)
9. Bielsa S., Esquerda A., Palma RM, et al.: Influence of Storage Time on Pleural
Fluid Adenosine Deaminase Activity. Clin.Lab. 2014; 60: 501-504.
10. Gupta BK, Parul G, Haren B, et al.: Cerebrospinal fluid Adenosine deaminase: its
evaluation as a marker for diagnosing tu-berculous meningitis in paediatric
patients. IOSR-JDMS Jan.-Feb. 2013; 4(1): 21-24.
