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Development of a rapid, microplate-based kinetic assay for measuring adenosine deaminase activity in body fluids
Clinica Chimica Acta 413 (2012) 1637–1640
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Development of a rapid, microplate-based kinetic assay for measuring adenosine deaminase activity in body fluids Jun Lu a, David G. Grenache a, b,⁎ a b
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
a r t i c l e
i n f o
Article history: Received 15 February 2012 Received in revised form 3 May 2012 Accepted 4 May 2012 Available online 10 May 2012 Keywords: Adenosine deaminase Tuberculosis Tuberculous pleuritis Tuberculous peritonitis Tuberculous meningitis Reference intervals
a b s t r a c t Background: Adenosine deaminase (ADA) catalyzes the deamination of adenosine to inosine. The activity of ADA in body fluids has clinical utility in the assessment of suspected tuberculosis. Methods: The conversion of adenosine to inosine was monitored in 96-well microplates as a continuous decrease at 265 nm for 15 min at ambient temperature. Analytical precision, sensitivity, linearity, accuracy, and enzyme stability were validated. Reference intervals were established from >120 tuberculosis-negative pleural, peritoneal, and cerebrospinal fluid samples. Results: The molar extinction coefficients of adenosine and inosine at 265 nm were 12,715 and 4,918 l/mol.cm and their difference was used to calculate ADA activity. Maximum within-day imprecision was b 11% and maximum total precision was b19%. Analytical sensitivity was 0.5 U/l and the assay was linear to 40 U/l. ADA recovery was 96–110% over an activity range of 11.7–25.3 U/l. ADA was stable for 1, 7 and 30 days at ~25 °C, 4–8 °C, and − 20 °C storage, respectively. Upper reference limits were 9.4, 7.3, and 1.5 U/l for pleural, peritoneal, and cerebrospinal fluid, respectively. Conclusions: The microplate-based kinetic ADA assay has favorable performance characteristics. This method eliminates the need for assay calibration and allows 96 samples to be tested simultaneously. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Adenosine deaminase (ADA; EC 3.5.4.4) is an enzyme of the purine salvage pathway that irreversibly catalyzes the deamination of adenosine and deoxyadenosine nucleosides into inosine and deoxyinosine, respectively. ADA plays an important role in the proliferation, differentiation, and maturation of lymphocytes and a deficiency of the enzyme leads to impaired lymphoid development and severe combined immunodeficiency disease [1]. Due to the stimulation of T cells by mycobacterial antigens, the measurement of ADA activity in body fluids has clinical utility as rapid, non-invasive test of tuberculous pleuritis, peritonitis, and meningitis [2–4]. Numerous methods for determining ADA activity in biological fluids have been described [5–8]. Many of these rely on determining the concentration of the ammonia produced by the deamination of adenosine and are compromised by the contamination of samples with ammonia. Kinetic methods often use coupled enzyme reactions to generate products that can be measured colorimetrically to avoid the use of ultraviolet wavelengths [9].
Abbreviations: ADA, adenosine deaminase. ⁎ Corresponding author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA. Tel.: + 1 801 583 2787; fax: + 1 801 584 5207. E-mail address: [email protected] (D.G. Grenache). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.05.001
Our laboratory formerly utilized a calibrated, end-point assay to quantify ADA activity in body fluids that was based on the decrease of absorbance at 265 nm as adenosine is deaminated to inosine. This method was constrained by several limitations including the lack of a certified reference material for assay calibration, a 60-min reaction time at 37 °C, and the use of individual quartz cuvettes that limited the number of samples that could be tested simultaneously. The objectives of this study were to develop a more accurate and high-throughput analytical method for measuring ADA activity in body fluids and to establish reference intervals for ADA activity in pleural, peritoneal, and cerebrospinal fluids.
2. Materials and methods Stock solutions of adenosine and inosine (Sigma-Aldrich, St. Louis, MO) of 1.0 mmol/l and ADA (MP Biomedicals, Solon, OH) were prepared in 0.05 mol/l phosphate buffer (pH 7.4). ADA activity was determined by adapting the method originally described by Kaplan et al. [10] in which the conversion of adenosine to inosine is monitored as a continuous absorbance decrease as a function of time at 265 nm. When performed in quartz cuvettes, reactions were initiated by the addition of 20 μl of sample into 980 μl of 0.06 mmol/l adenosine and monitored on a Beckman DU 800 spectrophotometer (Beckman Coulter, Inc., Life Science Division, Indianapolis, IN) for a minimum of 15 min.
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J. Lu, D.G. Grenache / Clinica Chimica Acta 413 (2012) 1637–1640
To increase the number of samples that could be analyzed simultaneously, the use of disposable 96-well, ultraviolet light-transparent microplates (UV-Star®, Greiner Bio-One, Frickenhausen, Germany) was utilized. One hundred twenty microliters of 0.05 mol/l phosphate buffer and 10 μl of sample were added to each well. Reactions were initiated by the addition of 120 μl of 0.12 mmol/l adenosine substrate (0.06 mmol/l final substrate concentration). The plates were covered with an ultraviolet light-transparent sealing film (VWR® PCR Sealing Film, VWR International, Radnor, PA) and absorbance monitored on a SpectraMax® M5 multi-detection plate reader (Molecular Devices, Sunnyvale, CA) for a minimum of 15 min. Because the path length of the reaction volumes in the mircoplates was b1.0 cm, all absorbances were normalized to 1.0 cm using SoftMax® Pro Data Acquisition & Analysis software, ver. 4.8 (Molecular Devices). All reactions were carried out at room temperature. Analytical precision, sensitivity, linearity, accuracy, and enzyme stability were established for pleural, peritoneal, and cerebrospinal fluids using residual samples sent to ARUP Laboratories. For each type of fluid, reference intervals were established from >120 samples that failed to grow any acid fast bacilli when cultured for those organisms. No clinical information was known about the individuals from whom the samples were obtained. The University of Utah Institutional Review Board approved the use of all biological samples.
A
B
3. Results The molar extinction coefficients of adenosine and inosine were determined at several different molar concentrations (Table 1). To maintain the absorbance within reasonable limits, cuvette path lengths of less than 1 cm were used at concentrations above 0.1 mmol/l. The mean coefficients were 12,715 and 4,918 l/mol.cm for adenosine and inosine, respectively. The difference between these two coefficients (7,797 l/mol.cm) was used to calculate ADA activity. One unit of ADA activity was defined as the quantity of enzyme required to catalyze 1 μmol of adenosine to inosine per minute at pH 7.4 and at room temperature (~25 °C). The Michaelis constant (Km) and maximum reaction velocity (Vmax) were determined by measuring reaction velocity over an adenosine concentration range of 0 to 0.14 mmol/l. Woolf–Hanes transformation of these data produced a Km of 0.021 mmol/l and a Vmax of 0.002 ΔA/min (Fig. 1). A substrate concentration that was 3 times the Km (0.06 mmol/l) was used in all other experiments.
Fig. 1. Michaelis–Menten (A) and Hanes-Woolf (B) plots. Linear regression analysis of the Woolf–Hanes transformation produced a slope of 468.6 and a y-intercept of 9.841 that were used to determine the maximum reaction velocity (Vmax = 1/slope) and the Michaelis constant (Km = y-intercept × Vmax).
3.1. Precision Analytical precision was determined by adding ADA to pleural, peritoneal, or cerebrospinal fluid pools to create samples with high and low ADA activity. Within run and total precision were evaluated by testing both pools in two to three replicates once each day for 20 days. For all fluid types, within run and total precision ranged from 3.0% to 10.1% and 7.5% to 18.8%, respectively (Table 2). 3.2. Sensitivity
Table 1 Molar extinction coefficients of adenosine and inosine determined at several different concentrations. The molecular weight of adenosine and inosine is 267.24 and 268.23 g/mol, respectively. Adenosine
Inosine
Concentration (mM)
Path length (cm)
Absorbance at 265 nm
Extinction coefficient at 265 nm (l/mol.cm)
Absorbance at 265 nm
Extinction coefficient at 265 nm (l/mol.cm)
1.0 1.0 0.5 0.5 0.1 0.1 0.1 0.05 0.05 0.05 Mean (SD)
0.2 0.1 0.2 0.1 1.0 1.0 0.1 1.0 1.0 0.1
2.3367 1.2727 1.2954 0.6414 1.2723 1.3013 0.1302 0.6323 0.6528 0.0625
11,684 12,727 12,954 12,828 12,723 13,013 13,020 12,646 13,056 12,500 12,715 (406)
0.9887 0.4884 0.5070 0.2486 0.4850 0.4949 0.0495 0.2383 0.2532 0.0237
4944 4884 5070 4972 4850 4949 4950 4766 5064 4740 4918 (111)
Analytical sensitivity was evaluated as the limit of blank (LOB) and the limit of detection (LOD). For the LOB, ADA activity was determined in 13 replicate measurements in a sample that did not contain the enzyme (reaction substrate alone). The mean (SD) activity was 0.2 (0.09) U/l and the LOB was calculated as the mean activity added to 3 SD of the mean (0.47 U/l). For the LOD, the mean ADA activity was determined in 8 to 10 replicate measurements in pleural, peritoneal, and cerebrospinal fluid samples with an ADA activity just above the LOB. The mean (SD) activities were Table 2 The analytical precision of the ADA assay determined by measuring enzyme activity in 2 (low activity pool) to three (high activity pool) replicates each day for 20 days.
Pleural fluid Peritoneal fluid Cerebrospinal fluid
Mean activity (U/l)
Within run CV (%)
Total CV (%)
8.5 23.8 7.9 22.4 6.4 17.6
5.9 4.4 10.1 4.6 6.1 3.0
18.8 8.4 16.9 8.6 10.1 7.5
J. Lu, D.G. Grenache / Clinica Chimica Acta 413 (2012) 1637–1640
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1.6 (0.36), 2.0 (0.42), and 1.3 (0.18) U/l, respectively. The LOD was calculated as LOB added to 3 SD of the mean of these biological samples and was 1.6, 1.7, and 1.0 U/l, respectively. 3.3. Linearity Assay linearity was determined for each type of body fluid by adding ADA to an aliquot of pleural, peritoneal, or cerebrospinal fluid pools to create a single, high activity sample. This sample was combined in different ratios with aliquots of unaltered fluid pool to prepare a set of 5–6 specimens, each of which were tested in 3 replicates. The assay was linear to 40 U/l in all three types of body fluids (Fig. 2). 3.4. Accuracy Analytical accuracy was determined for each type of body fluid by adding known quantities of ADA to aliquots of pleural, peritoneal, or cerebrospinal fluid pools, testing each specimen in three replicates, and calculating percent recovery. The recovery of ADA ranged from 96% to 110% at activities of 11.7–25.3 U/l (Table 3). 3.5. Enzyme stability The stability of ADA in each type of body fluid over time was determined by storing aliquots of pleural, peritoneal, or cerebrospinal fluid pools at ambient, refrigerated, and frozen temperatures and testing each sample in 2 replicates. The stability of ADA at ~25 °C, 4–8 °C, and −20 °C was 1, 7, and 30 days, respectively (Table 4). 3.6. Reference intervals ADA reference intervals were established from 122, 125, and 127 pleural, peritoneal, and cerebrospinal fluid specimens, respectively, that were negative when cultured for acid-fast bacilli. Samples were stored at −70 °C for up to 9 months prior to testing. Using nonparametric methods, the upper limit of ADA in each body fluid type was 9.4, 7.3, and 1.5 U/l for pleural, peritoneal, and cerebrospinal fluid, respectively. 4. Discussion The bacteria Mycobacterium tuberculosis causes pulmonary tuberculosis. In the US, the incidence is approximately 4/100,000 annually [11]. Global annual incidence is estimated to be nearly 10 million [12]. Disseminated, extrapulmonary tuberculosis may occur in infected people whose immune systems do not successfully contain the primary infection. In the US, disseminated tuberculosis occurs in approximately 20% of those infected [11]. Effusions are common in individuals with disseminated tuberculosis with about 5% and 3.5% developing tuberculous pleuritis or peritonitis, respectively [3,13]. Tuberculous meningitis, the most severe manifestation of tuberculosis, occurs in approximately 1.5% of infected individuals [14]. The use of ADA activity measurements in the differential diagnosis of extrapulmonary tuberculosis has been reported to have high clinical sensitivity. In one study of 2104 patients with pleural effusion (10.5% due to extrapulmonary tuberculosis), a pleural fluid ADA result >35 U/l was 93% sensitive and 90% specific for the diagnosis of tuberculous pleuritis [2]. A systematic review reported that, in peritoneal fluid, an ADA activity >30 U/l had a sensitivity and specificity of >90% for the diagnosis of tuberculous peritonitis [3]. Further, a meta-analysis of ADA in cerebrospinal fluid for the diagnosis of tuberculous meningitis reported a pooled sensitivity and specificity of 79% and 91%, respectively [4]. Several methods for measuring ADA activity have been developed. Many studies that have evaluated the clinical utility of ADA have
Fig. 2. Linearity for each fluid type was determined by diluting a sample with high ADA activity and testing each dilution in triplicate. The error bars represent the 95% confidence interval. The assay was linear to 40 U/l in all three types of body fluid.
Table 3 Analytical accuracy of the ADA assay determined by calculating the recovery of enzyme added to sample pools.
ADA ADA ADA ADA
added (U/l) expected (U/l) measured (U/l) recovered (%)
Pleural fluid (5.8 U/l)
Peritoneal fluid (7.5 U/l)
Cerebrospinal fluid (5.1 U/l)
73.7 12.0 11.7 97.7
73.7 13.5 13.5 100.0
73.7 11.3 10.9 96.2
185.9 22.2 24.3 110.0
185.9 23.7 25.3 106.8
185.9 21.5 22.9 106.3
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Table 4 Stability of ADA over time at ambient (~25 °C), refrigerated (4–8 °C), and frozen (− 20 °C) temperatures. Temperature Time
Pleural fluid
Ambient
U/l 21.1 22.6 20.8 19.8
0 day 1 day 2 days 4 days 7 days Mean difference
21.1 21.7 21.9 21.4 21.6
0 day 7 days 30 days Mean difference Maximum difference allowedb
21.1 0 21.1 0 21.0 − 0.1 − 0.05
0h 2h 6h 24 h Mean difference
Peritoneal fluid
Cerebrospinal fluid
Differencea 0 1.5 − 0.3 − 1.3 − 0.03
U/l 20.2 21.7 19.5 19.5
Difference 0 1.5 − 0.7 − 0.7 0.03
U/l 17.3 17.6 15.4 13.6
Difference 0 0.3 − 1.9 − 3.7 − 1.8
0 0.6 0.8 0.3 0.5 0.6
20.2 22.0 23.1 20.6 19.5
0 1.8 2.9 0.4 − 0.7 1.1
17.3 15.8 16.3 17.1 16.4
0 − 1.5 − 1.0 − 0.2 − 0.9 − 0.9
Refrigerated
Frozen
a b
4.0
20.2 0 19.9 − 0.3 20.2 0 − 0.2 3.9
17.3 0 15.9 − 1.4 13.7 − 3.6 − 2.5 2.6
From time zero. Based on total imprecision.
utilized methods that rely on the measurement of the ammonia that is liberated from the deamination of adenosine. While these methods do not require the use of ultraviolet wavelengths they are influenced by sample contamination with ammonia [15]. Other methods are based on the measurement of the inosine product [9,16]. Our laboratory previously utilized an ADA assay that directly determined enzyme activity by measuring the decrease in absorbance at 265 nm as adenosine was deaminated to inosine. While simple to perform, this method had several limitations. First, it was an end-point assay that required the use of a calibrator. Because there is no certified reference material for ADA, a commercially available ADA preparation was used as the standard yet we lacked an independent method of validating the activity value assigned to the material. Second, the reaction required a 60-min incubation at 37 °C and that, combined with the mechanical configuration of a spectrophotometer that can accommodate only six cuvettes, limited the number of samples that could be analyzed simultaneously. In an effort to increase assay accuracy and efficiency, we sought to develop a more accurate and high-throughput analytical method for measuring ADA activity. To accomplish this, we abandoned the use of a calibrated assay and opted to determine ADA activity from a molar extinction coefficient. However, both adenosine and inosine strongly absorb light at 265 nm although inosine is approximately 40% lower than that of adenosine (Table 1). Therefore, we utilized the difference between the two molar extinction coefficients to calculate ADA activity. Because this difference is a constant, the accuracy of the assay was improved, as it is no longer dependent on the activity assignment of a commercially available ADA preparation. Assay throughput was considerably improved due to the use of a 96-well microplate. Whereas the previous assay could test 6 samples simultaneously, the assay described here can simultaneously determine
ADA activity in 16 times that number of samples. The reaction time of the revised assay was reduced from 60 to 15 min and was validated at ambient temperatures which eliminated the need to determine enzyme activity at 37 °C. Perhaps most importantly, we established upper reference limits of ADA activity in pleural, peritoneal, and cerebrospinal fluid samples that were negative when cultured for acid-fast bacilli. Because otherwise healthy individuals do not usually have these types of body fluids collected, the reference limits may not be representative of a “healthy” population. However, because ADA activity in body fluids is used as a surrogate marker for disseminated tuberculosis, the population used is the most appropriate. That is, we utilized samples from individuals in whom tuberculous pleuritis, peritonitis, or meningitis was suspected but for whom the disease was ruled out. In summary, we have developed and validated a rapid, microplatebased kinetic assay for determining ADA activity in pleural, peritoneal, and cerebrospinal fluids. The assay is precise, accurate, and linear to 40 U/l and upper reference limits were determined to be 9.4, 7.3, and 1.5 U/l, respectively. The kinetic method described here eliminates the need for calibration, is performed at ambient temperatures, and is 75% faster as compared to the original method.
Acknowledgements This work was supported by the ARUP Institute for Clinical and Experimental Pathology.
References [1] Fischer A. Severe combined immunodeficiencies (SCID). Clin Exp Immunol Nov. 1 2000;122(2):143–9. [2] Porcel JM, Esquerda A, Bielsa S. Diagnostic performance of adenosine deaminase activity in pleural fluid: a single-center experience with over 2100 consecutive patients. Eur J Intern Med Oct. 2010;21(5):419–23. [3] Sanai FM, Bzeizi KI. Systematic review: tuberculous peritonitis—presenting features, diagnostic strategies and treatment. Aliment Pharmacol Ther Oct. 15 2005;22(8): 685–700. [4] Xu H-B, Jiang R-H, Li L, Sha W, Xiao H-P. Diagnostic value of adenosine deaminase in cerebrospinal fluid for tuberculous meningitis: a meta-analysis. Int J Tuberc Lung Dis Nov. 2010;14(11):1382–7. [5] Giusti G, Galanti B. Adenosine deaminase: colorimetric method. In: Bergmeyer HU, editor. Methods of enzymatic analysis, 3rd ed., vol. 4. Verlag Chemie; 1984. p. 315–23. [6] Kaplan NO. Specific adenosine deaminase from intestine. Methods Enzymol 1955;2: 473–5. [7] Ellis G, Goldberg DM. A reduced nicotinamide adenine dinucleotide-linked kinetic assay for adenosine deaminase activity. J Clin Lab Med 1970;76:507–17. [8] Hopkinson DA, Cook PJL, Harris H. Further data on the adenosine deaminase (ADA) polymorphism and a report of a new phenotype. Ann Hum Genet 1969;32:361–7. [9] Oosthuizen HM, Ungerer JP, Bissbort SH. Kinetic determination of serum adenosine deaminase. Clin Chem Oct. 1 1993;39(10):2182–5. [10] Kaplan NO, Colowick SP, Ciotti MM. Enzymatic deamination of adenosine derivatives. J Biol Chem Feb. 1952;194(2):579–91. [11] National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention (U.S.). Division of Tuberculosis Elimination. In: Miramontes R, editor. Reported tuberculosis in the United States, 2010; 2010. p. 134. CDC. [12] WHO. WHO global tuberculosis control report 2010. Cent Eur J Public Health Dec. 2010;237. [13] Kataria YP, Khurshid I. Adenosine deaminase in the diagnosis of tuberculous pleural effusion. Chest Aug. 2001;120(2):334–6. [14] Phypers M, Harris T, Power C. CNS tuberculosis: a longitudinal analysis of epidemiological and clinical features. Int J Tuberc Lung Dis Jan. 2006;10(1):99–103. [15] Giusti G, Gakis C. Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues. Enzyme 1971;12(4):417–25. [16] Delacour H, Sauvanet C, Burnat P. Analytical performances of the Diazyme ADA assay on the Cobas(®) 6000 system. Clin Biochem 2010;43(18):1468–71.
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Development of a rapid, microplate-based kinetic assay for measuring adenosine deaminase activity in body fluids Jun Lu a, David G. Grenache a, b,⁎ a b
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
a r t i c l e
i n f o
Article history: Received 15 February 2012 Received in revised form 3 May 2012 Accepted 4 May 2012 Available online 10 May 2012 Keywords: Adenosine deaminase Tuberculosis Tuberculous pleuritis Tuberculous peritonitis Tuberculous meningitis Reference intervals
a b s t r a c t Background: Adenosine deaminase (ADA) catalyzes the deamination of adenosine to inosine. The activity of ADA in body fluids has clinical utility in the assessment of suspected tuberculosis. Methods: The conversion of adenosine to inosine was monitored in 96-well microplates as a continuous decrease at 265 nm for 15 min at ambient temperature. Analytical precision, sensitivity, linearity, accuracy, and enzyme stability were validated. Reference intervals were established from >120 tuberculosis-negative pleural, peritoneal, and cerebrospinal fluid samples. Results: The molar extinction coefficients of adenosine and inosine at 265 nm were 12,715 and 4,918 l/mol.cm and their difference was used to calculate ADA activity. Maximum within-day imprecision was b 11% and maximum total precision was b19%. Analytical sensitivity was 0.5 U/l and the assay was linear to 40 U/l. ADA recovery was 96–110% over an activity range of 11.7–25.3 U/l. ADA was stable for 1, 7 and 30 days at ~25 °C, 4–8 °C, and − 20 °C storage, respectively. Upper reference limits were 9.4, 7.3, and 1.5 U/l for pleural, peritoneal, and cerebrospinal fluid, respectively. Conclusions: The microplate-based kinetic ADA assay has favorable performance characteristics. This method eliminates the need for assay calibration and allows 96 samples to be tested simultaneously. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Adenosine deaminase (ADA; EC 3.5.4.4) is an enzyme of the purine salvage pathway that irreversibly catalyzes the deamination of adenosine and deoxyadenosine nucleosides into inosine and deoxyinosine, respectively. ADA plays an important role in the proliferation, differentiation, and maturation of lymphocytes and a deficiency of the enzyme leads to impaired lymphoid development and severe combined immunodeficiency disease [1]. Due to the stimulation of T cells by mycobacterial antigens, the measurement of ADA activity in body fluids has clinical utility as rapid, non-invasive test of tuberculous pleuritis, peritonitis, and meningitis [2–4]. Numerous methods for determining ADA activity in biological fluids have been described [5–8]. Many of these rely on determining the concentration of the ammonia produced by the deamination of adenosine and are compromised by the contamination of samples with ammonia. Kinetic methods often use coupled enzyme reactions to generate products that can be measured colorimetrically to avoid the use of ultraviolet wavelengths [9].
Abbreviations: ADA, adenosine deaminase. ⁎ Corresponding author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA. Tel.: + 1 801 583 2787; fax: + 1 801 584 5207. E-mail address: [email protected] (D.G. Grenache). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.05.001
Our laboratory formerly utilized a calibrated, end-point assay to quantify ADA activity in body fluids that was based on the decrease of absorbance at 265 nm as adenosine is deaminated to inosine. This method was constrained by several limitations including the lack of a certified reference material for assay calibration, a 60-min reaction time at 37 °C, and the use of individual quartz cuvettes that limited the number of samples that could be tested simultaneously. The objectives of this study were to develop a more accurate and high-throughput analytical method for measuring ADA activity in body fluids and to establish reference intervals for ADA activity in pleural, peritoneal, and cerebrospinal fluids.
2. Materials and methods Stock solutions of adenosine and inosine (Sigma-Aldrich, St. Louis, MO) of 1.0 mmol/l and ADA (MP Biomedicals, Solon, OH) were prepared in 0.05 mol/l phosphate buffer (pH 7.4). ADA activity was determined by adapting the method originally described by Kaplan et al. [10] in which the conversion of adenosine to inosine is monitored as a continuous absorbance decrease as a function of time at 265 nm. When performed in quartz cuvettes, reactions were initiated by the addition of 20 μl of sample into 980 μl of 0.06 mmol/l adenosine and monitored on a Beckman DU 800 spectrophotometer (Beckman Coulter, Inc., Life Science Division, Indianapolis, IN) for a minimum of 15 min.
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To increase the number of samples that could be analyzed simultaneously, the use of disposable 96-well, ultraviolet light-transparent microplates (UV-Star®, Greiner Bio-One, Frickenhausen, Germany) was utilized. One hundred twenty microliters of 0.05 mol/l phosphate buffer and 10 μl of sample were added to each well. Reactions were initiated by the addition of 120 μl of 0.12 mmol/l adenosine substrate (0.06 mmol/l final substrate concentration). The plates were covered with an ultraviolet light-transparent sealing film (VWR® PCR Sealing Film, VWR International, Radnor, PA) and absorbance monitored on a SpectraMax® M5 multi-detection plate reader (Molecular Devices, Sunnyvale, CA) for a minimum of 15 min. Because the path length of the reaction volumes in the mircoplates was b1.0 cm, all absorbances were normalized to 1.0 cm using SoftMax® Pro Data Acquisition & Analysis software, ver. 4.8 (Molecular Devices). All reactions were carried out at room temperature. Analytical precision, sensitivity, linearity, accuracy, and enzyme stability were established for pleural, peritoneal, and cerebrospinal fluids using residual samples sent to ARUP Laboratories. For each type of fluid, reference intervals were established from >120 samples that failed to grow any acid fast bacilli when cultured for those organisms. No clinical information was known about the individuals from whom the samples were obtained. The University of Utah Institutional Review Board approved the use of all biological samples.
A
B
3. Results The molar extinction coefficients of adenosine and inosine were determined at several different molar concentrations (Table 1). To maintain the absorbance within reasonable limits, cuvette path lengths of less than 1 cm were used at concentrations above 0.1 mmol/l. The mean coefficients were 12,715 and 4,918 l/mol.cm for adenosine and inosine, respectively. The difference between these two coefficients (7,797 l/mol.cm) was used to calculate ADA activity. One unit of ADA activity was defined as the quantity of enzyme required to catalyze 1 μmol of adenosine to inosine per minute at pH 7.4 and at room temperature (~25 °C). The Michaelis constant (Km) and maximum reaction velocity (Vmax) were determined by measuring reaction velocity over an adenosine concentration range of 0 to 0.14 mmol/l. Woolf–Hanes transformation of these data produced a Km of 0.021 mmol/l and a Vmax of 0.002 ΔA/min (Fig. 1). A substrate concentration that was 3 times the Km (0.06 mmol/l) was used in all other experiments.
Fig. 1. Michaelis–Menten (A) and Hanes-Woolf (B) plots. Linear regression analysis of the Woolf–Hanes transformation produced a slope of 468.6 and a y-intercept of 9.841 that were used to determine the maximum reaction velocity (Vmax = 1/slope) and the Michaelis constant (Km = y-intercept × Vmax).
3.1. Precision Analytical precision was determined by adding ADA to pleural, peritoneal, or cerebrospinal fluid pools to create samples with high and low ADA activity. Within run and total precision were evaluated by testing both pools in two to three replicates once each day for 20 days. For all fluid types, within run and total precision ranged from 3.0% to 10.1% and 7.5% to 18.8%, respectively (Table 2). 3.2. Sensitivity
Table 1 Molar extinction coefficients of adenosine and inosine determined at several different concentrations. The molecular weight of adenosine and inosine is 267.24 and 268.23 g/mol, respectively. Adenosine
Inosine
Concentration (mM)
Path length (cm)
Absorbance at 265 nm
Extinction coefficient at 265 nm (l/mol.cm)
Absorbance at 265 nm
Extinction coefficient at 265 nm (l/mol.cm)
1.0 1.0 0.5 0.5 0.1 0.1 0.1 0.05 0.05 0.05 Mean (SD)
0.2 0.1 0.2 0.1 1.0 1.0 0.1 1.0 1.0 0.1
2.3367 1.2727 1.2954 0.6414 1.2723 1.3013 0.1302 0.6323 0.6528 0.0625
11,684 12,727 12,954 12,828 12,723 13,013 13,020 12,646 13,056 12,500 12,715 (406)
0.9887 0.4884 0.5070 0.2486 0.4850 0.4949 0.0495 0.2383 0.2532 0.0237
4944 4884 5070 4972 4850 4949 4950 4766 5064 4740 4918 (111)
Analytical sensitivity was evaluated as the limit of blank (LOB) and the limit of detection (LOD). For the LOB, ADA activity was determined in 13 replicate measurements in a sample that did not contain the enzyme (reaction substrate alone). The mean (SD) activity was 0.2 (0.09) U/l and the LOB was calculated as the mean activity added to 3 SD of the mean (0.47 U/l). For the LOD, the mean ADA activity was determined in 8 to 10 replicate measurements in pleural, peritoneal, and cerebrospinal fluid samples with an ADA activity just above the LOB. The mean (SD) activities were Table 2 The analytical precision of the ADA assay determined by measuring enzyme activity in 2 (low activity pool) to three (high activity pool) replicates each day for 20 days.
Pleural fluid Peritoneal fluid Cerebrospinal fluid
Mean activity (U/l)
Within run CV (%)
Total CV (%)
8.5 23.8 7.9 22.4 6.4 17.6
5.9 4.4 10.1 4.6 6.1 3.0
18.8 8.4 16.9 8.6 10.1 7.5
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1.6 (0.36), 2.0 (0.42), and 1.3 (0.18) U/l, respectively. The LOD was calculated as LOB added to 3 SD of the mean of these biological samples and was 1.6, 1.7, and 1.0 U/l, respectively. 3.3. Linearity Assay linearity was determined for each type of body fluid by adding ADA to an aliquot of pleural, peritoneal, or cerebrospinal fluid pools to create a single, high activity sample. This sample was combined in different ratios with aliquots of unaltered fluid pool to prepare a set of 5–6 specimens, each of which were tested in 3 replicates. The assay was linear to 40 U/l in all three types of body fluids (Fig. 2). 3.4. Accuracy Analytical accuracy was determined for each type of body fluid by adding known quantities of ADA to aliquots of pleural, peritoneal, or cerebrospinal fluid pools, testing each specimen in three replicates, and calculating percent recovery. The recovery of ADA ranged from 96% to 110% at activities of 11.7–25.3 U/l (Table 3). 3.5. Enzyme stability The stability of ADA in each type of body fluid over time was determined by storing aliquots of pleural, peritoneal, or cerebrospinal fluid pools at ambient, refrigerated, and frozen temperatures and testing each sample in 2 replicates. The stability of ADA at ~25 °C, 4–8 °C, and −20 °C was 1, 7, and 30 days, respectively (Table 4). 3.6. Reference intervals ADA reference intervals were established from 122, 125, and 127 pleural, peritoneal, and cerebrospinal fluid specimens, respectively, that were negative when cultured for acid-fast bacilli. Samples were stored at −70 °C for up to 9 months prior to testing. Using nonparametric methods, the upper limit of ADA in each body fluid type was 9.4, 7.3, and 1.5 U/l for pleural, peritoneal, and cerebrospinal fluid, respectively. 4. Discussion The bacteria Mycobacterium tuberculosis causes pulmonary tuberculosis. In the US, the incidence is approximately 4/100,000 annually [11]. Global annual incidence is estimated to be nearly 10 million [12]. Disseminated, extrapulmonary tuberculosis may occur in infected people whose immune systems do not successfully contain the primary infection. In the US, disseminated tuberculosis occurs in approximately 20% of those infected [11]. Effusions are common in individuals with disseminated tuberculosis with about 5% and 3.5% developing tuberculous pleuritis or peritonitis, respectively [3,13]. Tuberculous meningitis, the most severe manifestation of tuberculosis, occurs in approximately 1.5% of infected individuals [14]. The use of ADA activity measurements in the differential diagnosis of extrapulmonary tuberculosis has been reported to have high clinical sensitivity. In one study of 2104 patients with pleural effusion (10.5% due to extrapulmonary tuberculosis), a pleural fluid ADA result >35 U/l was 93% sensitive and 90% specific for the diagnosis of tuberculous pleuritis [2]. A systematic review reported that, in peritoneal fluid, an ADA activity >30 U/l had a sensitivity and specificity of >90% for the diagnosis of tuberculous peritonitis [3]. Further, a meta-analysis of ADA in cerebrospinal fluid for the diagnosis of tuberculous meningitis reported a pooled sensitivity and specificity of 79% and 91%, respectively [4]. Several methods for measuring ADA activity have been developed. Many studies that have evaluated the clinical utility of ADA have
Fig. 2. Linearity for each fluid type was determined by diluting a sample with high ADA activity and testing each dilution in triplicate. The error bars represent the 95% confidence interval. The assay was linear to 40 U/l in all three types of body fluid.
Table 3 Analytical accuracy of the ADA assay determined by calculating the recovery of enzyme added to sample pools.
ADA ADA ADA ADA
added (U/l) expected (U/l) measured (U/l) recovered (%)
Pleural fluid (5.8 U/l)
Peritoneal fluid (7.5 U/l)
Cerebrospinal fluid (5.1 U/l)
73.7 12.0 11.7 97.7
73.7 13.5 13.5 100.0
73.7 11.3 10.9 96.2
185.9 22.2 24.3 110.0
185.9 23.7 25.3 106.8
185.9 21.5 22.9 106.3
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Table 4 Stability of ADA over time at ambient (~25 °C), refrigerated (4–8 °C), and frozen (− 20 °C) temperatures. Temperature Time
Pleural fluid
Ambient
U/l 21.1 22.6 20.8 19.8
0 day 1 day 2 days 4 days 7 days Mean difference
21.1 21.7 21.9 21.4 21.6
0 day 7 days 30 days Mean difference Maximum difference allowedb
21.1 0 21.1 0 21.0 − 0.1 − 0.05
0h 2h 6h 24 h Mean difference
Peritoneal fluid
Cerebrospinal fluid
Differencea 0 1.5 − 0.3 − 1.3 − 0.03
U/l 20.2 21.7 19.5 19.5
Difference 0 1.5 − 0.7 − 0.7 0.03
U/l 17.3 17.6 15.4 13.6
Difference 0 0.3 − 1.9 − 3.7 − 1.8
0 0.6 0.8 0.3 0.5 0.6
20.2 22.0 23.1 20.6 19.5
0 1.8 2.9 0.4 − 0.7 1.1
17.3 15.8 16.3 17.1 16.4
0 − 1.5 − 1.0 − 0.2 − 0.9 − 0.9
Refrigerated
Frozen
a b
4.0
20.2 0 19.9 − 0.3 20.2 0 − 0.2 3.9
17.3 0 15.9 − 1.4 13.7 − 3.6 − 2.5 2.6
From time zero. Based on total imprecision.
utilized methods that rely on the measurement of the ammonia that is liberated from the deamination of adenosine. While these methods do not require the use of ultraviolet wavelengths they are influenced by sample contamination with ammonia [15]. Other methods are based on the measurement of the inosine product [9,16]. Our laboratory previously utilized an ADA assay that directly determined enzyme activity by measuring the decrease in absorbance at 265 nm as adenosine was deaminated to inosine. While simple to perform, this method had several limitations. First, it was an end-point assay that required the use of a calibrator. Because there is no certified reference material for ADA, a commercially available ADA preparation was used as the standard yet we lacked an independent method of validating the activity value assigned to the material. Second, the reaction required a 60-min incubation at 37 °C and that, combined with the mechanical configuration of a spectrophotometer that can accommodate only six cuvettes, limited the number of samples that could be analyzed simultaneously. In an effort to increase assay accuracy and efficiency, we sought to develop a more accurate and high-throughput analytical method for measuring ADA activity. To accomplish this, we abandoned the use of a calibrated assay and opted to determine ADA activity from a molar extinction coefficient. However, both adenosine and inosine strongly absorb light at 265 nm although inosine is approximately 40% lower than that of adenosine (Table 1). Therefore, we utilized the difference between the two molar extinction coefficients to calculate ADA activity. Because this difference is a constant, the accuracy of the assay was improved, as it is no longer dependent on the activity assignment of a commercially available ADA preparation. Assay throughput was considerably improved due to the use of a 96-well microplate. Whereas the previous assay could test 6 samples simultaneously, the assay described here can simultaneously determine
ADA activity in 16 times that number of samples. The reaction time of the revised assay was reduced from 60 to 15 min and was validated at ambient temperatures which eliminated the need to determine enzyme activity at 37 °C. Perhaps most importantly, we established upper reference limits of ADA activity in pleural, peritoneal, and cerebrospinal fluid samples that were negative when cultured for acid-fast bacilli. Because otherwise healthy individuals do not usually have these types of body fluids collected, the reference limits may not be representative of a “healthy” population. However, because ADA activity in body fluids is used as a surrogate marker for disseminated tuberculosis, the population used is the most appropriate. That is, we utilized samples from individuals in whom tuberculous pleuritis, peritonitis, or meningitis was suspected but for whom the disease was ruled out. In summary, we have developed and validated a rapid, microplatebased kinetic assay for determining ADA activity in pleural, peritoneal, and cerebrospinal fluids. The assay is precise, accurate, and linear to 40 U/l and upper reference limits were determined to be 9.4, 7.3, and 1.5 U/l, respectively. The kinetic method described here eliminates the need for calibration, is performed at ambient temperatures, and is 75% faster as compared to the original method.
Acknowledgements This work was supported by the ARUP Institute for Clinical and Experimental Pathology.
References [1] Fischer A. Severe combined immunodeficiencies (SCID). Clin Exp Immunol Nov. 1 2000;122(2):143–9. [2] Porcel JM, Esquerda A, Bielsa S. Diagnostic performance of adenosine deaminase activity in pleural fluid: a single-center experience with over 2100 consecutive patients. Eur J Intern Med Oct. 2010;21(5):419–23. [3] Sanai FM, Bzeizi KI. Systematic review: tuberculous peritonitis—presenting features, diagnostic strategies and treatment. Aliment Pharmacol Ther Oct. 15 2005;22(8): 685–700. [4] Xu H-B, Jiang R-H, Li L, Sha W, Xiao H-P. Diagnostic value of adenosine deaminase in cerebrospinal fluid for tuberculous meningitis: a meta-analysis. Int J Tuberc Lung Dis Nov. 2010;14(11):1382–7. [5] Giusti G, Galanti B. Adenosine deaminase: colorimetric method. In: Bergmeyer HU, editor. Methods of enzymatic analysis, 3rd ed., vol. 4. Verlag Chemie; 1984. p. 315–23. [6] Kaplan NO. Specific adenosine deaminase from intestine. Methods Enzymol 1955;2: 473–5. [7] Ellis G, Goldberg DM. A reduced nicotinamide adenine dinucleotide-linked kinetic assay for adenosine deaminase activity. J Clin Lab Med 1970;76:507–17. [8] Hopkinson DA, Cook PJL, Harris H. Further data on the adenosine deaminase (ADA) polymorphism and a report of a new phenotype. Ann Hum Genet 1969;32:361–7. [9] Oosthuizen HM, Ungerer JP, Bissbort SH. Kinetic determination of serum adenosine deaminase. Clin Chem Oct. 1 1993;39(10):2182–5. [10] Kaplan NO, Colowick SP, Ciotti MM. Enzymatic deamination of adenosine derivatives. J Biol Chem Feb. 1952;194(2):579–91. [11] National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention (U.S.). Division of Tuberculosis Elimination. In: Miramontes R, editor. Reported tuberculosis in the United States, 2010; 2010. p. 134. CDC. [12] WHO. WHO global tuberculosis control report 2010. Cent Eur J Public Health Dec. 2010;237. [13] Kataria YP, Khurshid I. Adenosine deaminase in the diagnosis of tuberculous pleural effusion. Chest Aug. 2001;120(2):334–6. [14] Phypers M, Harris T, Power C. CNS tuberculosis: a longitudinal analysis of epidemiological and clinical features. Int J Tuberc Lung Dis Jan. 2006;10(1):99–103. [15] Giusti G, Gakis C. Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues. Enzyme 1971;12(4):417–25. [16] Delacour H, Sauvanet C, Burnat P. Analytical performances of the Diazyme ADA assay on the Cobas(®) 6000 system. Clin Biochem 2010;43(18):1468–71.