Adenosine Deaminase Levels in Nontuberculous Lymphocytic Pleural Effusions

Adenosine Deaminase Levels in Nontuberculous Lymphocytic Pleural Effusions* Y. C. Gary Lee, MBChB; Jeffrey T. Rogers, RRT; R. Michael Rodriguez, MD, FCCP; Kent D. Miller MD, PhD; and Richard W. Light, MD, FCCP

Objectives: Adenosine deaminase (ADA) can aid in the diagnosis of tuberculous pleural effusions, but false-positive findings from lymphocytic effusions have been reported. We studied the ADA levels in a variety of nontuberculous lymphocytic effusions and analyzed the relationships between ADA and conventional hematologic and biochemical parameters. Methods: One hundred six lymphocytic pleural fluid samples (lymphocyte count > 50%) were analyzed. These included post-coronary artery bypass grafting (CABG) effusions (n ⴝ 45), malignant effusions (n ⴝ 27), miscellaneous exudative effusions (n ⴝ 10), and transudative effusions (n ⴝ 24). ADA levels were determined using the Giusti method. In 22 randomly selected cases, ADA was measured again on the same sample 6 weeks later. Results: The ADA level reached the diagnostic cutoff for tuberculosis (40 U/L) in only three cases (2.8%): two lymphomas and one complicated parapneumonic effusion. There was no significant correlation between effusion ADA levels and the total leukocyte (r ⴝ 0.08), differential lymphocyte (r ⴝ 0.18) or monocyte (r ⴝ ⴚ 0.18) counts. ADA levels were significantly lower in the transudative effusions (7.2 ⴞ 3.5 U/L) than in post-CABG (16.6 ⴞ 7.2 U/L), malignant (15.3 ⴞ 11.2 U/L), and other exudative (15.4 ⴞ 13.1 U/L) effusions (p < 0.001). ADA measurements were consistent when assayed 6 weeks apart (r ⴝ 0.95; p < 0.00001; coefficient of variation, 14%). Conclusions: ADA levels in nontuberculous lymphocytic effusions seldom exceeded the diagnostic cutoff for TB. Effusion ADA levels cannot be predicted from total or differential leukocyte counts. Post-CABG pleural fluids had ADA levels similar to other nontuberculous lymphocytic effusions. ADA is stable in effusion fluids, and its measurement is reproducible. (CHEST 2001; 120:356 –361) Key words: adenosine deaminase; malignancy; pleural effusion; post-coronary artery bypass; tuberculosis Abbreviations: ADA ⫽ adenosine deaminase; CABG ⫽ coronary artery bypass grafting; LDH ⫽ lactate dehydrogenase; TB ⫽ tuberculosis

pleural effusions are common in L ymphocytic clinical practice. Tuberculosis (TB) is an important cause of lymphocytic effusions, along with malignancies, lymphoma, collagen vascular diseases, chylothorax,1 and post-coronary artery bypass grafting (CABG).2,3 More than 90% of TB effusions show a lymphocytic predominance,4 and hence TB re*From the Department of Pulmonary Medicine, St. Thomas Hospital and Vanderbilt University, Nashville, TN. Support was provided by the Saint Thomas Foundation, Nashville, TN, and a United States-New Zealand Fulbright Graduate Award (Dr. Lee). Manuscript received September 19, 2000; revision accepted February 23, 2001. Correspondence to: Y. C. Gary Lee, MBChB, Department of Pulmonary Medicine, St. Thomas Hospital, 4220 Harding Road, Nashville, TN 37202; e-mail: [email protected] 356

quires exclusion in any lymphocytic exudative effusion where no alternative cause is found. However, the diagnosis of TB pleural effusion remains a common clinical challenge. At least 50% of For editorial comment see page 334 the cases of TB pleural effusion present as primary disease without TB involvement of other organs.5 Tuberculin testing is neither specific nor sensitive.6 The effusion is usually a result of delayed hypersensitivity to proteins of the mycobacteria, and the actual bacterial load in the pleural space is often low. As a result, pleural biopsy and pleural fluid culture findings are often negative.4,6 Adenosine deaminase (ADA) has gained increasClinical Investigations

ing popularity as a diagnostic test for tuberculous pleuritis since 1978, especially in countries where the prevalence of TB is high. It carries a high sensitivity (90 to 100%)7–10 and is inexpensive and easy to measure.6 ADA is an enzyme involved in purine catabolism and is present in abundance in lymphocytes.11 ADA is related to lymphocytic differentiation and proliferation, and ADA activity increases during antigenic response of lymphocytes.12 Although previous reports have demonstrated the usefulness of ADA in pleural effusions, false-positive findings were present in most studies. These studies included pleural fluids of various cell types, including neutrophilic effusions. Most of the false-positive findings reported9,10,13–15 were from parapneumonic effusions or empyemas, which are usually neutrophilic predominant. Little is known about the usefulness of ADA measurements in lymphocytic pleural fluids. This is important because in clinical practice, TB is usually suspected only if the pleural fluid is lymphocytic in nature. We believe that false-positive diagnoses of TB effusion by ADA can be significantly reduced if ADA measurement is limited to lymphocytic pleural fluids. This would exclude most, if not all, of the empyema fluids, and better resemble everyday clinical practice. We therefore hypothesize that elevated ADA levels are uncommon in nontuberculous lymphocytic pleural effusions. The purpose of this study is to assess the ADA levels in nontuberculous lymphocytic pleural effusions of different etiologies, including post-CABG effusions. We also studied the correlation between the ADA levels and usual hematologic and biochemical parameters in the pleural fluids. In addition, we examined the stability and reproducibility of pleural fluid ADA measurements by comparing the results on the same samples 6 weeks apart.

Materials and Methods This study was approved by the Institutional Review Board of Saint Thomas Hospital, and all patients signed an informed consent. Lymphocytic effusions were defined as effusions with a lymphocyte count ⬎ 50% of the total WBC count, as conventionally defined.16 –20 One hundred six pleural fluid samples were randomly selected from all nontuberculous lymphocytic pleural fluids collected from patients who underwent thoracentesis in our hospital between September 1, 1997, and July 1, 1999. The diagnoses of the pleural fluid samples are listed in Table 1. The clinical diagnoses of the pleural effusions were independently verified by at least one qualified pulmonologist (Y.C.L., R.M.R., or R.W.L.). The definitions for the diagnosis of the effusions were the same as previously published.21,22 A postCABG effusion was one that developed within the first 3 months after coronary artery bypass surgery with or without heart valve replacement with no other identifiable causes (eg, congestive heart failure, chylothorax, or infection). A pleural effusion was

Table 1—Diagnoses of the Pleural Fluid Samples Studied Diagnoses

Patients, No.

Post-CABG pleural effusions Malignant pleural effusions Lung Breast Lymphoma Others Miscellaneous exudates Parapneumonic Chylothorax Rheumatoid arthritis Constrictive pericarditis Chronic idiopathic pleuritis Superior vena caval obstruction Undetermined/post-lung resection Transudates Congestive heart failure Hepatic cirrhosis Renal failure

45 27 12 8 3 4 10 3 2 1 1 1 1 1 24 21 2 1

categorized as malignant if pleural fluid cytology or pleural biopsy findings were positive for malignancy, or if the patient had known metastatic malignancy with no other explanation for the effusion. All other exudative effusions were included in the miscellaneous exudate group. Exudates and transudates were classified according to the criteria of Light et al.23 At the time of the thoracentesis, pleural fluid was collected in a citrated tube for ADA quantification, in an ethylenediaminetetra-acetic acid tube for measurement of total and differential WBC counts, and in a plain tube for protein and lactate dehydrogenase (LDH) analysis. For ADA quantification, the pleural fluid aspirates were immediately centrifuged at 3,000 revolutions per minute for 20 min at 4°C. The supernatant was stored at ⫺ 70°C until analysis. RBC and WBC counts were obtained by manual microscopy. The differential leukocyte count was obtained by manually counting 100 cells on a Wright’sstained smear after the cells had been concentrated by cytocentrifugation at 2,000 revolutions per minute for 10 min. Protein and LDH concentrations were measured using a Vitro Model 950 automated analyzer (Johnson & Johnson; New York, NY). The upper limit of normal serum LDH level is 620 U/L in our laboratory. Total ADA was measured by the colorimetric method of Giusti,24 employing reagents optimized for ammonia measurement by Kaplan.25 The laboratory cutoff for tuberculous pleural effusion is 40 U/L. To confirm the reproducibility of the ADA measurements, 22 samples (21%) were randomly selected from the 106 samples originally assayed and measured again using the same method 6 weeks after the initial ADA quantification. Statistical Analysis All data were analyzed with statistical software (SigmaStat V2.03; Jandel Scientific; San Rafael, CA). Results were expressed as mean ⫾ SD unless otherwise stated. One-way analysis of variance was used to compare the levels among subgroups, and Tukey’s test was used to perform multiple comparison procedures. If the log-transformed data did not satisfy the normality and equal variance tests, Kruskal-Wallis one-way analysis of variance on ranks was used to compare the levels among subgroups and Dunn’s method was used to perform multiple CHEST / 120 / 2 / AUGUST, 2001

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effusion ADA levels and the total leukocyte counts, lymphocyte counts, or monocyte counts (Table 3). Twenty-two samples were reassayed again with the same method 6 weeks after the first ADA quantification. The results of the two separate measurements were remarkably consistent (r ⫽ 0.95; p ⬍ 0.00001; coefficient of variation, 13.9%; Fig 2).

Discussion

Figure 1. ADA levels in different diagnostic groups. Bar indicates the median value of the group.

comparison procedures. Correlation was analyzed with Pearson’s correlation test. p ⬍ 0.05 was considered significant.

Results The ADA levels in the post-CABG effusions (16.6 ⫾ 7.2 U/L), malignant effusions (15.3 ⫾ 11.2 U/L), and miscellaneous exudative effusions (15.4 ⫾ 13.1 U/L) were all significantly higher when compared with the transudative group (7.2 ⫾ 3.5 U/L; p ⬍ 0.001; Fig 1). The ADA level reached the diagnostic cutoff for TB (40 U/L) in only 3 of the 106 cases (2.8%; Fig 1). Two patients had lymphomas, and one patient had a complicated parapneumonic effusion. The hematologic and biochemical analyses of the pleural fluid samples were listed in Table 2. There were no strong correlation between the ADA levels and the various hematologic and biochemical parameters, whether they were analyzed in the entire cohort or within each diagnostic subgroup. When compared with the biochemical parameters, the effusion ADA level was weakly correlated with the protein and LDH concentrations in the pleural fluids (Table 3). Of note, no correlation was found between

Our study showed that ADA levels in nontuberculous lymphocytic pleural effusions seldom exceeded the cutoff set for tuberculous effusions. The pleural fluid ADA levels were significantly higher in different types of exudative effusions, including postCABG effusions, than in transudates. The ADA levels cannot be predicted from any of the conventional hematologic or biochemical parameters. We also showed that ADA measurements were accurately reproducible when assayed 6 weeks apart. The global incidence of TB continues to rise,26 and it remains an important cause of exudative pleural effusions.1 While TB pleuritis often resolves spontaneously, up to 65% of untreated patients will eventually develop active TB.27 Hence, the correct diagnosis for patients with TB pleuritis followed by appropriate antimicrobial therapy is important. When investigating an effusion of unknown cause, the initial questions are (1) whether it is an exudate or transudate, and (2) what are the cell count differentials. Most TB effusions are lymphocyte predominant and ⬎ 50% of total WBC count is conventionally accepted as the practical cutoff for lymphocytic effusion.16 –20 Previous studies19,20 have demonstrated that ⬎ 90% of TB effusions have a lymphocyte count that exceeds this cutoff. Lymphocytic effusions are common in clinical practice, and TB should be excluded from other common causes of exudative lymphocytic effusions, such as malignancy, connective tissue disorders, chylothorax, and atypical infections. However, the diagnosis of TB pleural effusion is often difficult. In

Table 2—Hematologic and Biochemical Analysis of Pleural Fluids in Diagnoses Subgroups* Variables

Post-CABG (n ⫽ 45)

Malignant (n ⫽ 27)

Miscellaneous Exudates (n ⫽ 10)

Transudates (n ⫽ 24)

Protein, g/dL LDH, U/L WBC, ␮L Neutrophil, % Lymphocyte, % Monocyte, %

4.2 ⫾ 0.9† 811 ⫾ 689† 3,436 ⫾ 10,354† 6.5 ⫾ 8.0 72.2 ⫾ 12.4 17.1 ⫾ 11.9

3.7 ⫾ 0.9† 602 ⫾ 500 1,955 ⫾ 1,750† 5.8 ⫾ 6.1 71.8 ⫾ 13.8 19.7 ⫾ 12.6

3.4 ⫾ 1.0† 773 ⫾ 722 3,377 ⫾ 3,580† 8.6 ⫾ 8.7 78.0 ⫾ 13.7 12.7 ⫾ 10.1

2.4 ⫾ 0.7 487 ⫾ 503 771 ⫾ 532 8.9 ⫾ 8.0 66.3 ⫾ 11.0 22.5 ⫾ 10.5

*Data are presented as mean ⫾ SD. †p ⬍ 0.05 when compared with the transudate group. 358

Clinical Investigations

Table 3—Correlation r Values Between ADA Levels and Other Hematologic and Biochemical Markers in the Pleural Fluid Variables

ADA

Protein LDH RBC count Total leukocyte cell count Total neutrophil count Percentage neutrophil Total lymphocyte count Percentage lymphocyte Total monocyte count Percentage monocyte

0.43* 0.32† 0.04 0.08 0.13 ⫺ 0.08 0.07 0.18 0.03 ⫺ 0.18

*p ⬍ 0.00001. †p ⬍ 0.001.

more than half of the patients, the pleura is the only site of infection, with no other manifestation of TB elsewhere to aid the diagnosis.5 There is no serum marker, and tuberculin skin test is nonspecific and findings can be negative in one third of cases.6 As the mycobacterial load in the pleura is often low,4 pleural fluid culture findings are positive in ⬍ 20% of TB pleural effusions,16 and culture takes several weeks. As a result, more invasive procedures, eg, pleural biopsy or thoracoscopy, are often sought. Pleural fluid ADA now forms part of the routine diagnostic workup for tuberculous effusions in many countries where TB is endemic. Numerous reports7–9 have attested to its high sensitivity (⬎ 95% in most series). Unlike interferon-␥ or polymerase chain reaction, ADA is inexpensive, quick, and technically easy to measure (in the United States, the cost of measuring ADA is approximately $15 per sample, with a turnaround time of 2 h). False-positive findings were frequently reported when ADA was used to diagnose TB pleural effusions7,8,10,15,28,29; careful examination of those studies revealed that pleural fluids of any cell type predominance were included. Also, most of the false-positive findings were from pleural fluids of empyemas, which are neutrophilic in nature. Although elevated ADA levels in pleural fluids of nontuberculous lymphocytic effusions have been reported, they were far less common. To reduce the number of false-positive findings, various strategies including the measurement of ADA isoforms (ADA-1 and ADA-2) had been tried. ADA-2 is produced by monocytes and predominates over ADA-1 in TB effusions. Hence, a high ADA-2 or a low ADA-1:total ADA level would be useful to help exclude neutrophilic effusions such as empyemas. However, in TB effusions, ADA-2 accounts for 88% of the total ADA,30 such that the measurement

Figure 2. ADA levels measured 6 weeks apart.

of either one gives similar results. Valdes et al,9 in a study of 350 effusion samples, found no significant differences in the usefulness of ADA-2 over total ADA, and thus concluded that “the extra labor involved in the estimation of ADA2 would not be justified in clinical practice.” To our knowledge, our study is the first that applied ADA measurement to only lymphocytic effusions. We believe that such practice resembles clinical decision making where TB is most commonly suspected only in lymphocytic effusions. By applying this simple practice, one can easily exclude most of the empyema and parapneumonic pleural fluids. As a result, we found that false-positive findings were rare (⬍ 3%) if we applied ADA to lymphocytic effusions only. Given its high sensitivity, ADA is a valuable screening tool, whereby a negative result helps to exclude TB effusion. A high ADA level in a lymphocytic effusion strongly favors TB. Our study demonstrated that false-positive findings are rare when nontuberculosis lymphocytic effusions were studied on the whole. There were no false-positive findings in any of the post-CABG effusions or transudative (mainly congestive cardiac failure) effusions studied. False-positive findings were rare in the malignant effusion group in general. However, two of the three lymphoma samples registered levels above the diagnostic cutoff for TB effusions. In clinical practice, effusions from lymphoma can be distinguished from TB effusions with the use of flow cytometry.31 Pleural effusions after CABG are common,2 and their incidence continues to increase as more CABGs are performed each year. Pleural effusions ⬎ 1 month after CABG are often characterized by very high lymphocyte counts, but the origin of the lymphocytes remains unknown.2,3 While uncommon, TB effusions developing shortly after CABG have been described.32 To our knowledge, our study is the CHEST / 120 / 2 / AUGUST, 2001

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first to assess the ADA levels in post-CABG effusions, and found that the mean level was similar to other exudative nontuberculous lymphocytic effusions, but was significantly higher than that of transudative lymphocytic effusions. Why some lymphocytic effusions have higher ADA levels than others remains an intriguing question. Our study showed no correlation between the lymphocyte or monocyte counts and ADA activity. Other studies33,34 also failed to demonstrate any close correlation between monocytes, total lymphocytes, or T-cell subtypes with either total ADA or its isoenzyme levels. The standard ADA determination measures its activity, rather than the amount of enzyme present. One unit of ADA is the enzymatic activity required to produce 1 mol/L of ammonia per minute from adenosine at standard assay conditions. One possible explanation would be that while the quantity of ADA is related to the amount of T lymphocytes present, the ADA activity varies dependent on different pathologic conditions, such that the ADA activity is highest with TB, and also lymphoma. This is supported by the in vitro observation that ADA activity is higher in activated T lymphocytes, eg, following mitogenic and antigenic challenges. Another possible explanation is that the ADA activity depends on how rapidly the T lymphocytes proliferate. Some authors believe that mycobacterial antigenic stimulation leads to the increased production of ADA, which is essential to the accelerated T-cell blastogenesis and accumulation of CD4 subpopulation, commonly seen in tuberculous effusions. Given the nonspecific presentation of TB pleuritis, the diagnosis is often unsuspected during the initial investigations. Our study demonstrated that the ADA measurement was consistent and reproducible when assayed several weeks apart. This result is of clinical value such that if the initial workup of the lymphocytic effusion failed to yield a definite diagnosis, the pleural fluid sample, if stored properly, can still be used for ADA determination, thus avoiding the need of a repeat thoracentesis. Our findings further supported the use of ADA in the diagnosis of tuberculous pleural effusions, as we showed that false-positive findings were uncommon in nontuberculous lymphocytic pleural effusions of various etiologies. Also, the ADA measurements in pleural fluids were accurately reproducible.

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