Role of pleural fluid adenosine deaminase activity and lymphocytosis in the etiological diagnosis

Correspondence: Dr Kricha Pande, MBBS, MD Department of Pathology, Nepal Medical College and Teaching Hospital and Green city Hospital, Kathmandu,Nepal Email: krichapande@gmail.com Background: Pleural effusion is a common medical condition with many possible underlying etiologies. However, Tuberculosis is the most common cause of pleural effusion especially in countries like Nepal. Pleural fluid lymphocytosis is seen in tuberculosis, malignancy and few auto-immune diseases. Adenosine Deaminase activity (ADA) level in tubercular pleural effusion is markedly increased compared to non-tubercular effusions. ADA estimation being a simple colorimetric method is suitable for the rapid diagnosis of tubercular effusion. This study aims to correlate the diagnostic efficacy of ADA with the value of differential count (lymphocytosis) in establishing different etiology of pleural effusion.

The analysis of pleural fluid includes total and differential count, biochemical examination as well as cytology and microbiology depending upon the type of case.Neutrophilic predominant exudative effusions are due to acute processes whereas lymphocytic effusions have a much longer list of differential diagnoses.However, in areas with high incidence of tuberculosis, tuberculosis and malignancy are the most likely cause of lymphocytic pleural effusion. 1Pleural fluid lymphocytosis can be seen in malignancy, collagen vascular disease, sarcoidosis and up to third of all transudates.Para pneumonic and emphysematous effusions are characterized by neutrophils-predominant, exudative effusions. 2enosine Deaminase activity (ADA) analysis in effusion as well as in serum are in practice as this is the cheapest test with high diagnostic value.Adenosine Deaminase is an enzyme in the purine salvage pathway required for converting adenosine to inosine.Its level is ten times higher in lymphocytes than in erythrocytes and particularly in T lymphocytes. 3ADA level in tubercular pleural effusion is markedly increased compared to non-tubercular effusions.ADA estimation being a simple colorimetric method is imminently suitable for the rapid diagnosis of tubercular effusion. 4ADA levels in the pleural fluid are estimated by the spectrophotometric method described by Guisti. 3ere has been a need to study the diagnostic efficacy of ADA and the value of differential count (lymphocytosis) in establishing the etiological diagnosis (tubercular versus non tubercular) pleural effusion, in countries like Nepal where the cost effectiveness of each test counts.

MATERIALS AND METHODS
Study Design and samples: A cross sectional study was conducted in the department of pathology, Green city hospital for the duration of twenty-one months from October 2014 to July 2016.Clearance from the institutional review committee was obtained.The study comprised of 50 selected patients with pleural effusion (in-patients as well as OPD based).A thorough history, examination and confirmation of effusion by chest x-ray or ultrasound was done in all patients.Pleural tapping was done by clinicians and radiologists under full aseptic precaution.Both ultrasound guided as well as blind tapping were included.
Pleural fluid analysis: The pleural fluid sent to the laboratory was analyzed for gross findings, biochemical findings, total and differential count of pleural fluid, ADA level in pleural effusion.The volume, color and transparency were noted in physical examination.Total count was obtained manually via neubaur chamber whereas Wright's stained slides were prepared for differential count.Lymphocytic effusions were defined as effusions with a lymphocyte count >50% of the total nucleated cells 5 .
The pleural fluid was analyzed for protein, albumin, sugar, lactate dehydrogenase (LDH) and ADA levels.Centrifuge preparation and cytology (Papaniculaou and Giemsa stain) for malignant cells were also done as and when required.Special stain (AFB-TB) and gram stain: Ziehl Neelsen stain (AFB-TB) and Gram stain was done for the suspected patients of tuberculosis, pneumonia and lung abscess.
Presence of first or more than one of the following criteria was adopted to label a case as tuberculosis: (1) Bacteriological confirmation of Mycobacterium tuberculosis (identification of the bacillus in the pleural fluid or biopsy specimen by stain or by culture); (2) Histopathology finding of caseating granulomas; (3) Radiological findings consistent with TB; (4) Clinical presentation consistent with TB with positive tuberculin test (>20 mm indurations) with exclusion of other clinical considerations; (5) Definite clinical and radiological improvement in 2 months of administration of exclusive anti-tubercular treatment.Effusion was called malignant when pleural fluid cytology or pleural biopsy showed evidence of malignancy or if the patient had proved metastatic malignancy with no other detectable cause of effusion. 2,6termination of ADA level: Adenosine Deaminase was estimated by Giusti and Galanti colorimetric method (spectrophotometric method). 3The ADA value 30-40 Unit/liter (U/L) was kept as suspected whereas 40-60 U/L was taken as strong suspect and more than 60 U/L was considered as positive for tuberculosis.All cases within the range 30-40 U/L were considered negative if clinical features did not correlate.Since the most widely accepted cut-off level of ADA for the diagnosis of tubercular pleural effusion is 40 U/L, in this present study we took ADA ≥ 40 U/L as in agreement with other studies. 6So, strong suspects were also considered as positive if other clinical features were suggestive of Tuberculosis.

Statistical analysis
The results were subjected to manual analysis.Mean, percentage, Chi squared tests (with Fischer's exact correction), sensitivity, specificity, positive and negative predictive value were calculated.2.

Both
Table 3 shows that Lymphocytosis was more in tubercular pleural effusion (87%) as well as in non-tubercular effusion (68%).Table 4 and 5 shows the correlation between ADA activity and Lymphocytosis.
The sensitivity and specificity of ADA alone to diagnose tubercular pleural effusion was 92% each and when lymphocyte alone was considered sensitivity was 85% with specificity of 32% whereas the combined effect of ADA in effusion with pleural fluid lymphocytosis was 100% (sensitivity) and 87% (specificity), 83% (positive predictive value) and 100% (negative predictive value) respectively with statistically significant p value (<0.05).

DISCUSSION
Tuberculosis is a common cause of pleural effusion 7 especially in Nepal.The incidence of tuberculosis is increasing worldwide. 8Although tubercular pleural effusion can resolve spontaneously but up to 65% untreated tubercular pleural effusion can develop active tuberculosis. 9o, rapid and accurate diagnosis and prompt treatment is necessary for tubercular pleural effusion.Whenever a patient of pleural effusion presents, the usual investigation is in the form of gross, microscopic and biochemical parameters including ADA level.Although lymphocytic predominant fluid is usually seen in tubercular pleural effusion but all lymphocytic predominant fluid can't be tubercular, it could be malignant. 10Piras et al were one of the earliest workers who studied ADA activity in pleural effusion of different etiology and concluded that the tubercular pleural fluid ADA activity was significantly higher when compared to Pande K et al. different non tubercular cases 11 which is similar to this study.
The immediate cause of the effusion in TB is a delayed hypersensitivity response to mycobacterial antigen in the pleural space.For this reason microbiological analyses are often negative and limited by the lengthy delay in obtaining results.In areas with high TB prevalence, pleural fluid ADA levels greater than 40 U/l argue strongly for TB; in contrast, low levels of pleural ADA have high negative predictive value in low-prevalence countries. 12The presence of ADA in pleural fluid reflects cellular immune response in pleural cavity and in particular, the activation of T lymphocytes. 13DA has gained increasing attention as a diagnostic tool, especially in countries where the prevalence of TB is high.It achieves a high sensitivity (90 to 100%) and is inexpensive and easy to measure. 14 this present study, tuberculosis accounted for 26% of all cases.ADA level was raised (≥40U/L) in 92% of tubercular pleural effusion which correlates well with the conclusion made by Castro et al, in which they stated that adenosine Deaminase level < 40 IU/L virtually excludes tuberculosis in lymphocytic pleural effusions. 15Non-tubercular causes of pleural effusion were COPD, miscellaneous causes (which included cholelithiasis, anemia, fever-PUO, laparotomy, UGI bleeding, renal diseases), idiopathic cases, parapneunomic effusion, road traffic accident, malignancy and auto-immune (rheumatoid arthritis).
The sensitivity and specificity of ADA alone was 92% each in this study which correlates well with other studies 7,16 whereas the sensitivity improved to 100% when the value of lymphocytosis was combined with pleural fluid ADA levels.The results correlated well with other researchers. 7,15evkota et al studied 100 cases of pleural effusions in which they found out that mean ADA level was 105.8 + 67.23 in TB which was much higher than other etiologies.The sensitivity of ADA was 90.8% with specificity of 82.8% when the cut off value was 42.9%. 16The sensitivity and specificity of ADA alone to diagnose tubercular pleural effusion was 92% each and when lymphocyte alone was considered sensitivity was 85% with specificity of 32% whereas the combined effect of ADA in effusion with pleural fluid lymphocytosis was 100% (sensitivity) and 87% (specificity), 83% (positive predictive value) and 100% (negative predictive value) respectively with statistically significant p value (<0.05).
From this data, it can be concluded that the combination of pleural fluid differential count (lymphocytosis >50%) and ADA level (≥40U/L) provides us with much more positive result than each component alone.) similar to the present case.They also stated that with the decline in the prevalence of tuberculous pleural effusion in some areas, the positive predictive value of pleural fluid ADA also declines but the negative predictive value remains high. 15Besides, lymphocyte count and ADA level in pleural fluid, even other parameters like protein, sugar and red blood cell counts will also help to build up a conclusion towards etiology as stated by Liam et al.This study showed that a higher proportion of patients with tuberculosis had lymphocyte predominant effusion and tuberculous effusion had higher lymphocyte percentage, lower red cell count and higher protein content.However, there was considerable overlap of these characteristics in maliganant and tubercular effusions. 17wer school of thoughts has been provided by a study done by Mohan et al suggesting that the combined use of adenosine Deaminase activity along with lymphocyte neutrophils ratio would provide a more efficient means for diagnosing tuberculosis pleuritis than the use of ADA alone. 18Measuring the isoenzymes of ADA particularly estimation of activity of both ADA-1 and ADA-2 isoenzymes and 2'-deoxyadenosine/adenosine activity ratio also improvises the diagnostic efficacy according to Desai et al 19

Table 1 .
Our study showed 92% of the tubercular pleural effusion had ADA level of more than or equal to 40 U/L and most of the non-tubercular origin of effusion had ADA lower than the cut off value which was expected, illustrated numerically on Table with non tuberculosis pleural effusion (n= 37) was 56. 2 years (Range 16-87); there were 24 males.Causes of non tubercular pleural effusions were Para pneumonic effusions, COPD, Trauma, miscellaneous, Idiopathic, Neoplastic and autoimmune causes.Etiology, age and gender distribution are depicted in

Pleural Fluid Adenosine Deaminase activity and LymphocytosisTable 5 : Comparison of ADA activity, Lymphocytosis in pleural fluid and combined value showing sensitivity, specificity, Positive predictive value (PPV) and negative predictive value (NPV) Criteria used to diagnose Tuberculosis ADA level Pleural fluid Lymphocytosis
20d Safianowska et al.20CONCLUSIONIn conclusion, this can be stated that the measurement of ADA level in pleural fluid in combination with the differential count of pleural fluid will give best results to categorize and to rule in the diagnosis of tubercular pleural effusion.The ADA is relatively inexpensive, easy to perform and less time consuming test whereas lymphocytosis in pleural fluid being an easily available test, these tests are