A Comparative Study to Evaluate the Diagnostic Accuracy of Bedside Hydrogen Peroxide Test and Light’s Criteria in Differentiating Pleural Effusions as Transudate and Exudate

Introduction

Pleural effusion refers to the accumulation of excess fluid in the pleural cavity.¹ It becomes clinically detectable when the fluid volume exceeds 200-300 mL, and is a significant cause of pulmonary morbidity and mortality.^2,3 In India, the annual incidence of pleural effusions due to various aetiological factors is estimated to be 5.9 lakh per year, with pulmonary tuberculosis being the most prevalent aetiology.⁴

Pleural effusion can be either a transudate or exudate and this categorization of pleural fluid is the first step in evaluating a case of pleural effusion. Exudative effusions result from leakage of pulmonary microvasculature due to inflammatory or traumatic conditions, such as infections, tuberculosis, malignancy, etc. Whereas, transudative effusions are caused by an imbalance in Starling forces, with an intact capillary endothelium, as seen in conditions like chronic liver disease, congestive cardiac failure, etc.⁵

Thus, it is evident that categorizing pleural effusion as a transudate or exudate assists in diagnostic and therapeutic decisions. An exudative effusion warrants further rigorous diagnostic work-up to establish a definitive diagnosis, allowing for the initiation of specific therapy. In contrast, transudative effusions typically resolve with treatment of the underlying systemic condition and usually do not pose significant diagnostic challenges.⁶

Microscopic examination of pleural fluid, biochemical tests, fluid culture, cytology, radiological investigations, and histopathological examination of pleural tissue are commonly used diagnostic methods.⁷ However, these tests have known limitations, as they are often time consuming and tedious to perform. Light et al., developed a landmark set of criteria through a prospective study involving 150 cases of pleural effusion. The criteria include: fluid to serum total protein ratio > 0.5, fluid lactate dehydrogenase (LDH) value > 200 U/L, and fluid to serum LDH ratio > 0.6. The presence of at least one of these three criteria is sufficient to classify an effusion as exudate, with the remaining fluids considered as transudates.⁸ Light’s criteria are the currently accepted gold standard for classification of pleural fluid. However, Light’s method has multiple drawbacks, which have become more evident in the current medical scenario of rapid diagnostic tests. Firstly, it is time-consuming, which delays diagnosis and hinders the treatment protocol. Secondly, the chances of preanalytical errors associated with the transport of pleural fluid sample from the hospital ward to the central laboratory. Moreover, the lack of specificity of Light’s criteria means that about 25% of transudates may be misclassified as exudates.⁹ Due to these limitations, newer and more rapid diagnostic tests are constantly being evaluated. The demand for a single, simple test to classify pleural effusions is yet to be met. 

In this context, the present study was conducted to assess the diagnostic accuracy of a simple bedside hydrogen peroxide (H₂ O₂ ) test, and to compare its performance with Light’s criteria, which remain the current diagnostic standard. In doing so, we analysed the utility of the H₂ O₂ test as a bedside test in terms of simplicity, costeffectiveness, and economy of time.

Materials & Methods

A cross-sectional study was conducted to determine the diagnostic accuracy of a simple bedside H₂O₂ test in differentiating pleural fluid as transudate and exudate among patients with pleural effusion admitted to the tertiary care hospital after obtaining the Institute Ethics Committee clearance (dated 26/03/2019 Ref No. JJMMC/IEC-06-2019). Adult patients of both genders with a clinical diagnosis of pleural effusion willing to give consent for thoracocentesis were included in the study. Patients with blood contaminated pleural fluid or those unwilling to participate were excluded. A total of 100 subjects were enrolled, based on the sample size calculated using sensitivity.

The minimum sample size calculated was 91, which was rounded off to 100.

Around 10 mL of pleural fluid was obtained by thoracocentesis done in the posterior scapular line/mid-axillary line at the point of maximum dullness on percussion. About 2 mL of the specimen was used for H₂ O₂ test, while the remaining 8 mL was used for the estimation of LDH and proteinsin pleural fluid. Additionally, 2 mL of whole blood was drawn under aseptic conditions from the median cubital vein into a sterile, red vacutainer and this was used for the estimation of serum LDH and protein levels, as components of Light’s criteria (Figure 1).^10,11,12 

Classification of pleural fluid as transudate or exudate using Light’s criteria: Pleural fluid protein/serum protein > 0.5, Pleural fluid LDH/serum LDH > 0.6, Pleural fluid LDH > 2/3rd the upper limit of laboratory normal value for serum LDH. Using Light’s Criteria, a diagnosis of exudate is made when there is fulfilment of any one or more of the above criteria, and a diagnosis of transudate is made when there is no fulfilment of any of the above criteria.⁸

In the H₂ O₂ test, a diagnosis of exudate is made when profuse bubbling occurs upon addition of H₂ O₂ to pleural fluid. Conversely, the absence of bubbling within one minute of addition of H₂ O₂ indicates a transudate (Figure 2). SPSS (Statistical Package for Social Sciences) version 20.0 was used for statistical analysis. Qualitative variables are presented as frequencies and percentages. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic accuracy of the H₂ O₂ test were calculated. The level of statistical significance was set at 5%.

Results

A total of 100 cases of clinically and radiologically confirmed pleural effusion were included in the study following approval from the Institutional Ethics Committee and after obtaining written informed consent from all participants. A male predominance was observed, with 58% of the cases being male and 42% female. Pleural effusion was relatively common among urban residents (59%) compared to rural participants (41%). Left-sided pleural effusions were the most common in our study accounting for 42%, followed by bilateral effusions at 31% and right-sided effusions at 27%. Chronic tobacco consumption in the form of smoking and chewing (74%), chronic alcoholism (62%), emerged as risk factors for pleural effusion (Table 1).

A wide range of aetiological factors was observed among the study subjects, with tuberculosis being the most common cause (35%), followed by congestive heart failure (20%), chronic liver disease (15%), pneumonia (14%), malignancy (8%), peritoneal infections (6%). The least common causes were liver contusion and ruptured ectopic pregnancy, each accounting for 1% of cases (Table 1).

Thoracocentesis was performed under aseptic precautions, tapping about 10 mL of pleural fluid, of which 2 mL was used for a bedside H₂ O₂ test, while the remaining was used for analyzing Light’s criteria parameters. Out of the 100 study subjects, 61% had exudative pleural effusion and 39% had transudative pleural effusion based on Light’s criteria. According to the H₂ O₂ test, 56% were classified as having exudative pleural effusion and 44% as transudative (Figure 3). 

Diagnostic accuracy tests revealed the following details: With H₂ O₂ test and Light’s criteria, 54 samples were categorized as exudates, accounting for true positives and 37 samples as transudates, accounting for true negatives. There were two false positives, categorized as exudates by the H₂ O₂test and transudates by Light’s criteria and seven false negatives, categorized as transudates by H₂ O₂ test but exudates by Light’s criteria (Table 2). 

Discussion

The diagnosis of pleural effusion presents a significant clinical challenge due to the anatomical location of the pleural space, which is an internal cavity with no direct access. Non-invasive diagnostic techniques often have limited utility and are highly dependent on the underlying disease as well as the clinical expertise of the physician.¹³ These factors contribute to delays in establishing a definitive diagnosis, potentially resulting in suboptimal treatment, poor prognosis, and a decline in the patient’s quality of life.

A systematic and structured approach is essential when investigating cases of pleural effusion. A detailed clinical history, physical examination, and radiological investigations should precede thoracocentesis. The categorization of pleural effusion as transudate or exudate is the preliminary step in evaluating a case of pleural effusion. Transudative effusions occur secondary to systemic factors such as congestive heart failure and hypoproteinaemia and do not involve further workup. On the contrary, matrix of exudates is similar to that of plasma and typically results from increase in capillary permeability, either due to inflammation or from lymphatic obstruction.¹⁴

Light’s criteria remain the standard reference for differentiating pleural fluid into transudate or exudate categories. Recent prospective studies evaluating Light’s criteria have demonstrated high sensitivity in identifying exudative pleural effusions, but relatively low specificity.¹⁵ Recently, several studies have been conducted to establish differential diagnoses for transudates and exudates, using parameters such as measurement of pleural fluid cholesterol levels, serum and pleural fluid albumin gradient, and pleural fluid to serum bilirubin ratio. However, these parameters are still under investigation, and their superiority over Light’s criteria is yet to be established.^16,17

Hydrogen peroxide is decomposed into water and oxygen by the enzyme catalase. This catalase activity in pleural fluid forms the basis of H₂ O₂ test to differentiate between transudate or exudate. A unique characteristic of exudative fluid is elevated catalase activity, which is absent in transudates. The resulting oxygen release is easily visualized as a bubbling reaction. Appearance of bubbling within one minute after the addition of H₂ O₂ indicates an exudative effusion, while the absence of bubbling suggests a transudative effusion.¹⁸  

This study was undertaken to evaluate the utility of the H₂ O₂ test. The diagnostic accuracy of the H₂ O₂ test was evaluated by comparing its results with the reference standard, Light’s criteria. In our study of 100 pleural effusion cases, 54 cases were identified as exudates by both Light’s criteria and the H₂ O₂ test (true positives), while 37 cases were categorized as transudates by both methods (true negatives). There were two false positives, diagnosed as exudates by the H₂ O₂ test but classified as transudates by Light’s criteria. Seven false negatives were noted, where the H₂ O₂ test indicated transudates, but Light’s criteria identified them as exudates. 

The H₂ O₂ test showed sensitivity of 88.52%, specificity of 94.87%, positive predictive value of 96.4%, and negative predictive value of 88.4%. These values were found to be slightly lower than those estimated by Sarkar et al., who reported 100% sensitivity and 100% specificity. This is presumably because, their study eliminated the presence of blood in the fluid using benzidine test and excluded samples that tested positive.⁹

Our study findings are comparable to those reported by Taksande et al., who observed a sensitivity of 80.7% and a specificity of 81.8%. However, our results demonstrated much higher sensitivity and specificity. Additionally, the overall diagnostic accuracy of H₂ O₂ test in our study was 91%, which surpasses the 80.8% diagnostic accuracy reported by Taksande et al., Jepson et al.,in their review concluded that the sensitivity and specificity of the H₂ O₂ test are believed to be equivalent to the widely used Light’s criteria, i.e. 98% and 91.3%, respectively. A similar concordance was found in our study, confirming the utility of the H₂ O₂ test in categorizing pleural fluid as a transudate or exudate.^19,20 

Strengths

The sample size used was sufficient to justify our results. A wide range of aetiological factors of pleural effusion were included in the study. Haemorrhagic effusions were excluded, reducing the possibility of false positives.

Limitations

The H₂ O₂ test is not applicable to blood-contaminated pleural fluid samples, necessitating exclusion of such samples from our study. 

Conclusion

We conclude that the simple bedside H₂ O₂ test is an effective method for categorizing pleural fluid as transudate or exudate. Firstly, it significantly reduces the diagnostic turnaround time, enabling earlier initiation of appropriate treatment. Secondly, the test is costeffective and economical, making it particularly suitable for resource-deficient laboratory settings. Given its high statistical and clinical relevance, the H₂ O₂ test represents a practical and viable option for routine clinical practice.

Conflicts of interest

Nil