Diazyme adenosine deaminase in the diagnosis of tuberculous pleural effusion: method evaluation and clinical experiences in a New Zealand population.
The analysis of pleural fluid adenosine deaminase (ADA) levels in the diagnosis of tuberculous pleural effusion (TPE) was established in 1978 by Piras et al (1). Adenosine deaminase assay is available on routine biochemistry analysers and meta-analyses of ADA in pleural fluid has shown ADA to have high sensitivity (90-92%) and specificity (90-92%) for tuberculosis (2). Despite its utility as an adjunctive test in the diagnosis of TPE, ADA is infrequently tested in Australasia. This may be due the low prevalence of tuberculosis and the availability of an automated assay.
Clinical cut-offs for ADA levels are often based on published literature, however the various ADA assays are not standardised and results may not be comparable. A meta-analysis of 40 studies published from 1966 to 1999 showed discrepancies in results among the reported studies (2). This can be attributed to the use of different methods of ADA analysis. Thus it is important that new ADA methods are validated by comparison with clinical outcomes.
We report the results of the method evaluation of the Diazyme ADA assay on the Abbott CI8000 auto-analyzer and the clinical experiences of its use over a 6 month period in a New Zealand population.
Materials and methods
An ADA kitset (catalogue number DZ117A-K) manufactured and supplied by Diazyme Laboratories (General Atomics, Ponway, California, US) was evaluated in August 2007 on an Abbott CI8200 automated clinical chemistry and immunoassay analyzer (3).
The reagent kit contains one R1 and one R2 and is ready to use. Parameters for this kitset were obtained after correspondence from the headquarters of Abbott Diagnostics, Brazil. A single level calibrator (catalogue number DZ117A-CAL) and two control levels (catalogue number DZ117A-CON) were also supplied separately by Diazyme. These are lyophilized and stored at -20[degrees]C.
A sample comparison with Queensland Central Laboratories, Royal Brisbane Hospital, Australia was conducted on six samples to externally verify our results. Their ADA method is an in-house non-Giusti method.
Interference studies were investigated at 3 levels of ADA concentrations; low level ADA 6.4 IU/L, medium level 28.4 IU/L and a high level 189.7 IU/L. Samples were spiked with haemolysate for haemolysis studies. Bilirubin standard dissolved in dimethyl sulphoxide (DMSO) representing bilirubin were spiked for icterus studies and 10% intralipid for lipaemia studies. Each sample was matched with a control spiked with equal volumes of 0.9% saline and measured in duplicates. For icterus studies, DMSO was used instead of 0.9% saline.
For intra-batch precision, 15 patient samples were analyzed in duplicate. For inter-batch precision the control levels at 31.9 U/L and 152.9 U/L were analyzed in 10 consecutive batches. Results are presented as coefficient of variation (CV).
The Middlemore Hospital Laboratory analyzes pleural fluid for both Middlemore Hospital and North Shore Hospital which are located in Auckland, New Zealand. They are secondary-level hospitals servicing a population of approximately 475,000 and 500,000 people respectively.
Pleural fluid was analyzed for ADA only if it was classified as a non-transudate. Modified Light's criteria were used to classify pleural fluid as either exudate or transudate (4). Where there was insufficient information to classify a pleural fluid as an exudate or transudate, it was treated as a non-transudate.
Clinical records were used to search for the diagnosis for each pleural fluid ADA tested between 1st of March 2008 to the 8th of September 2008. A diagnosis of tuberculosis required a confirmed culture of Mycobacterium tuberculosis in either sputum, pleural fluid or pleural biopsy. The clinical course was followed for a further 3 to 6 months.
Intra-batch precision was 1.75% while inter-batch precision were 3.4% and 2.7% at the control levels of 31.9 U/L and 152.9 U/L respectively
Good correlation was obtained between our method and that of the Queensland Central Laboratory. However, our results were about 30% lower (Figure 1).
ADA was minimally affected by haemolysis up to 1.8 g/L while ADA results demonstrated a 17% reduction by bilirubin concentrations >300umol/L. Ten percent intralipid reduced ADA levels by 31%. The package insert claims that there is no interference from ascorbic acid up to 4mg/dL However, we did not verify this claim. No other interferences were tested for nor were stated in the package insert.
In the period from 1 March 2008 to 8 September 2008 there were 21 out of 120 patients with ADA results above the cut-off of 30 U/L (note: some patients had more than one pleural fluid where ADA was measured, only the first result was included in the analysis). Of these, 5 were confirmed TB positive, all with ADA results greater than 64 U/L (Figure 3).
Table 1 shows the ADA results >30 U/L and the diagnosis. Table 2 compares ADA data from Middlemore Hospital with published studies and shows that our sensitivity (100%) and specificity (86%) compared well with published studies. Positive predictive value (PPV) was 24% and the negative predictive value (NPV) was 100% for an ADA cut-off of >30 U/L. The PPV increased to 45% and NPV was 99% at an ADA cut-off of >70 U/L. However, the sample size of tuberculous pleural effusions were small and calculations based on this maybe subject to variation.
The diagnosis of TPE is usually determined on a combination of clinical, radiological and laboratory findings. In most series of patients with TPE, stains for acid fast bacilli are detected in less than 10% of cases (5). Pleural fluid cultures for TB are more sensitive with a yield ranging from 12% to 70% (5), however, turnaround time for cultures is usually 3 to 4 weeks. Polymerase chain reaction (PCR) has excellent specificity (78%-100%) but there are significant differences in sensitivity amongst methods used (20%-90%) owing to the presence of amplification inhibitors, type of primer used, genomic sequence amplified and the number of mycobacteria (2). Pleural biopsy is the gold standard for diagnosis of TPE but this is an invasive procedure and there are risks of complications. A biopsy involves a stain, culture and histology. In patients with TPE, pleural biopsy culture is positive in 39%-80% of cases and histological examination reveals granulomatous inflammation in 50%-97% of cases (2).
Adenosine deaminase (ADA) is an enzyme in the purine salvage pathway that catalyses the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine with the release of ammonia. ADA is found in most cells but plays an important role in the differentiation of lymphoid cells (in particular, active T-lymphocytes). Elevated pleural fluid ADA levels are not specific for TPE and it may be seen in diseases such as lymphoma, empyema, malignancy, pneumonia and rheumatoid-associated pleural effusions (rheumatoid arthritis or systemic lupus erythrematosus).
The methods for measuring ADA can be grouped into broadly two groups: Giusti and non-Giusti. One of the first methods for ADA was described by Giusti and Galanti (5). In this reaction the first step, where adenosine plus water is converted to inosine and ammonia by ADA, is the same. Ammonia then reacts with sodium hypochlorite and phenol in an alkaline solution (with sodium nitroprusside as the catalyst) to form an intensely blue indophenol compound. Non-Giusti methods includes the reduced nicotinamide adenine dinucleotide-linked kinetic method, ADA deaminases adenosine to inosine. The ammonium released is then used by glutamate dehydrogenase to convert 2-oxoglutarate to L-glutamate, with the concomitant oxidation of NADH to NAD+, allowing the reaction to be monitored by following the decrease in absorbance at 340 nm (6). The Diazyme ADA is a non-Giusti method, based on the enzymatic deamination of adenosine to inosine. Inosine is converted to hypoxanthine by purine nucleoside phosphorylase. Hypoxanthine is converted to uric acid and hydrogen peroxide by xanthine oxidase. Hydrogen peroxide is further reacted with two other compounds to generate a quinone dye. This is a rate up reaction measured at 548nm.
At Middlemore Hospital samples for ADA estimation are analysed three times per week. Our studies have shown that ADA in pleural aspirates are stable for up to six hours at room temperature and up to 1 month at 2[degrees]C-8[degrees]C (unpublished studies). However, we advise transportation at 4[degrees]C. Miller et al looked at using preservatives, such as a mixture of glycerol and ethylene glycol or a mixture of glycerol and sodium sulphate, to enable samples to be shipped at ambient temperatures. However, this may be more beneficial in regions where refrigerated transport of samples is problematic (7).
We noted significant differences in ADA levels between different methods as illustrated in Figure 1. In meta-analyses optimum cutoff for ADA were different depending on the method utilised. There are no international standardisation programs for adenosine deaminase assays and published cut-off should therefore be viewed with caution. Determination of adenosine deaminase cutoffs should be method specific and use local populations where possible.
The sensitivity and specificity of ADA increases when the cut-off of >70 U/L is used, however, ADA cannot be used alone to diagnose TPE. Its value as an early marker of TPE was highlighted by one of our cases, where a high ADA result was obtained 55 days before a sample from this patient grew Mycobacterium tuberculosis. Cultures will still be required to identify mycobacterial drug resistance.
In the New Zealand population an elevated ADA level >70 U/L has a low positive predictive value (24%) for tuberculosis. However, in practice the causes of false positive results, such as empyema, are often identified promptly. The negative predictive value is very high (100%) and in cases where there is a low pre-test probability of TPE, a low ADA result can rule out the need for invasive procedures such as pleural biopsy. Most of the literature cites 40 U/L as the cut-off levels for ADA. The meta-analysis by Goto el al (including Giusti, non-Giusti and unknown methodologies) featured studies that used a cut-off levels between 30 U/L and 71 U/L.
At the initiation of the ADA assay we provided reference interval and interpretative comments based on the literature and the comparison of ADA results with an external laboratory. In view of the 30% lower ADA levels as compared to an external laboratory, we conservatively stated a low cut-off value of 30 U/L to maintain the assay's negative predictive value. A clinical grey-zone was disclosed, to acknowledge the uncertainty of the optimum cut-off for this assay in the New Zealand population. Comments were as follows:
A. <30 U/L: Not strongly suggestive of tuberculous effusion. There is a high negative predictive value when the pretest probability is low. However, if clinical details suggest a high pre-test probability, adenosine deaminase results should be interpreted with caution.
B. >30- 70 U/L: Borderline range, value of indeterminate significance. Specificity for tuberculosis is significantly improved if the effusion is predominately lymphocytic and empyema is excluded.
C. >70 U/L: Highly suggestive of tuberculosis. However, high levels can be seen in empyema, lymphoma and rheumatoid pleural effusions.
A limitation is that the study design was a retrospective audit and as such suffers from deficiencies inherent with this methodology; diagnoses other than tuberculosis were based on judgement from clinicians and did not have strict inclusion and exclusion criteria. There were only five confirmed cases of tuberculous pleural effusion in this cohort, therefore caution is advised when forming conclusions.
In conclusion, ADA is an economical enzymatic assay able to be performed on automated commercial analyzers. It is a useful adjunctive test in the diagnosis of tuberculous pleural effusions, especially in view of its short turnaround time. It identifies those patients who are high risk for tuberculosis and guiding investigations which are required to confirm the diagnosis. In low prevalence populations its excellent negative predictive value may save costs and unnecessary invasive procedures.
We would like to thank Ameeta Chand, Section Head of TB, Microbiology; Dr Susan Taylor, Clinical Microbiologist; and Dr Conroy Wong, Respiratory Physician; all at Middlemore Hospital.
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(3.) Guisti G, Galanti B. Methods in Enzymatic Analysis. Academic Press, New York, NY; 1974: 414-9.
(4.) Ellis G, Goldberg DM. A reduced nicotinamide adenine dinucleotide-linked kinetic assay for adenosine deaminase activity. J Lab Clin Med 1970; 76: 507-17.
(5.) Adenosine deaminase assay kit package insert. Diazyme Laboratories, catalogue number: DZ117A-K.
(6.) Light RW. Clinical practice. Pleural effusion. N Engl J Med 2002; 346: 1971-7.
(7.) Miller KD, Barnette R, Light RW. Stability of adenosine deaminase during transportation. Chest 2004; 126: 19337.
(8.) Zaric B, Kuruc V, Milovancev A, Markovic M, Sarcev T, Canak V, et al. Differential diagnosis of tuberculous and malignant pleural effusions: what is the role of adenosine deaminase? Lung 2008; 186: 233-40.
(9.) Jiang J, Shi H-Z, Liang Q-L, Qin S-M, Qin X-J. Diagnostic value of interferon-/ in tuberculous pleurisy: a metaanalysis. Chest 2007; 131: 1133-41.
David Song was the primary author, carried out the method evaluation and clinical correlations. Andrea Lund liaised with Diazyme Laboratories for the ADA assay and was involved with the method evaluation. Weldon Chiu advised on method evaluation, was involved with the reference intervals and contributed to writing of the article.
David Song, MB ChB, Chemical Pathology Registrar
Andrea R Lun, BMLS, Section Head General Chemistry
Weldon Chiu, FRACP FRCPA, Chemical Pathologist
Middlemore Hospital, Auckland
Address for correspondence
Dr David Song, Diabetes Centre, Level, Greenlane Clinical Centre, Greenlane West, Auckland. Email: Dsong@adhb.govt.nz
Table 1. Diagnosis of pleural effusion ADA results >30 U/L Diagnosis Prevalence (n) Tuberculosis 24% (5) Neoplasm 29% (6) Empyema 24% (5) Para-pneumonic effusion 19% (4) SLE pleuritis 5% (1) Table 2. Middlemore Hospital ADA data compared with published data Study # Patients Cut- Sensitivity or studies off (U/L) MH 120 patients 30 100% Zaric et al (8) 121 patients 49 89.2% Liang et al (9) 63 studies 92% Goto et al (2) 40 studies 30-50 92.2% Study Specificity PPV or NPV or LR * LR * MH 85% 23% 100% Zaric et al (8) 70.4% 84.4% 78.4% Liang et al (9) 90% 9.03 * 0.1 * Goto et al (2) 92.2% MH = Middlemore Hospital * LR = likelihood ratio