The acidity of pleural fluid is a poorly studied parameter in modern literature. Only one study exists that addresses the normal pH of pleural fluid in humans.^[3] It defines normal reference values for pleural fluid pH as ranging between 7.6 and 7.64. The pH of pleural effusions is almost always lower than that of normal pleural fluid and is much closer to the pH of blood. In cases of transudative pleural effusion, there is a significant increase in pleural pH (7.45–7.55) compared to exudative pleural effusions, where it typically ranges from 7.30 to 7.45.^[4]

When pleural pH falls below 7.3, the condition is known as pleural acidosis.^[5] Values below 7.20 are associated with severe forms of pneumonia, pleural carcinomatosis, esophageal rupture, tuberculosis, rheumatoid diseases, and are even considered an indication for urgent thoracentesis, as persistently low pH dramatically increases the risk of developing pleural empyema.^[6] Several authors have studied the mechanism of pleural acidosis^[7–10], linking it to active inflammatory processes, activation of immune cells (particularly leukocytes), and increased acid production.^[11–13] Accumulation of carbon dioxide and lactate further deepens the acidosis.^{[14, 15]}

Carbon dioxide buildup is linked to both increased production and reduced transport across pleural membranes, as well as increased leukocyte glycolysis.^{[16, 17]} Lactate is formed as a byproduct of neutrophil glycolysis and pathogen metabolism in bacterial hydrothorax.^[18]

Detailed literature analysis even reveals cases of pleural alkalosis, where pleural fluid pH reaches 7.8.^[19] This is most commonly associated with the presence of a chylous effusion, also known as chylothorax.

In addition to distinguishing between exudates and transudates, one of the most practical uses of pleural pH measurement is its prognostic value in predicting the success of pleurodesis. Pleural pH remains the sole independent predictor for successful pleurodesis, with the likelihood of obliterating the pleural space progressively decreasing with lower pleural pH. When pH falls below 7.15, the positive predictive value drops to around 45.7%.^[20]

The specific gravity of pleural fluid is the most precise indirect method for measuring its viscosity. Tavana’s comprehensive study^[21] aimed to determine the sensitivity and specificity of specific gravity in differentiating exudative from transudative pleural effusions.

Samples were collected from 100 patients undergoing thoracentesis. Based on Light’s criteria, punctates were classified, and specific gravity was measured using refractometry. Among the samples, 70% were classified as exudative effusions and 30% as transudative. In the exudative group, the mean specific gravity was 1033.6, while it was 1021.4 in the transudative group.

The optimal sensitivity and specificity were achieved at a threshold of 1024, with sensitivity, specificity, positive predictive value, and negative predictive value of 91.4%, 66.7%, 86.5%, and 76.9%, respectively.

Quantitative determination of glucose in pleural fluid is an important step in the biochemical analysis of pleural effusions. Values can vary significantly, ranging from 0.00 to 29.36 mmol/L, with a threshold value of 3.30 mmol/L considered significant.^[22]

Several studies have examined the correlation between glucose and pleural fluid acidity.^[22–26] Many authors adopt the hypothesis that there is a direct proportional relationship, where a decrease in pH corresponds to glucose depletion. However, an in-depth study by Fitzgerald et al.^[27], involving data from multiple centers, demonstrates high variability in predictive intervals, suggesting the two parameters should be interpreted independently.

For example, while linear dependence is observed in many non-infectious effusions, tuberculous effusions often exhibit drastically low glucose levels unrelated to pleural fluid pH.^[28] Glucose reduction is attributed to the presence of bacteria, polymorphonuclear cells, and malignant cells.

Normal pleural fluid is relatively low in proteins. Protein presence and composition are critical for distinguishing effusion types, as emphasized in Light’s criteria.

Samanta et al.^[29] conducted a case-control study, identifying elevated serum albumin and pleural fluid albumin levels in tuberculosis-associated effusions compared to malignant effusions. Tuberculosis effusions demonstrated significantly higher serum effusion albumin gradient (SEAG) than malignant effusions. The critical threshold for total protein in pleural fluid is 6.2 g/dL, providing 92% sensitivity and 100% specificity in distinguishing tuberculosis effusions from malignancies.

LDH is a cytoplasmic enzyme found in all organ cells that plays an essential role in anaerobic metabolism. It is an indicator of inflammation or cellular damage, such as ischemia, dehydration, or bacterial and chemical injury.

In pleural effusions, LDH values above 1000 U/L are significant, often pointing to infectious causes, rheumatoid serositis, tuberculosis, or malignancy.^[30] LDH levels are also incorporated into Light’s criteria for distinguishing exudative from transudative effusions.

Cholesterol, a key sterol in the human body, enters pleural fluid through increased vascular permeability or as a product of degenerating cells. A clinical-laboratory threshold of >45 mg/dL is used for differentiating exudative from transudative effusions.^[31] In chylous effusions, cholesterol levels are <5.18 mmol/L (<200 mg/dL), while pleural triglycerides are >1.24 mmol/L (>110 mg/dL).^[32]

A good amount of evidence suggests a correlation between total protein and albumin, as a major component in its composition. This justifies considering both parameters together.

High total protein content in pleural effusion increases the likelihood that it is malignant. This was proven by Hsieh et al.^[33], when the fluid from 27 patients, 14 of whom were cytologically verified as having malignant hydrothorax, was subjected to two-dimensional gel electrophoresis. Using ELISA and Western blot methods, the protein profiles of each effusion were identified.

Their results clearly showed that total protein content was significantly elevated in the malignant group.

The albumin gradient, as a function of albumin content in pleural fluid, provides much more accurate information compared to isolated albumin testing. It can effectively differentiate exudative from transudative pleural effusions. This concept is supported by recent observations from a study conducted by Benin Ceyhan, who recommends using an upper limit of the albumin gradient at 1.2 g/dl, which provides 76% sensitivity and 100% specificity for detecting exudative pleural effusions.^[34] Additionally, Roth et al.^[35] demonstrated that a low albumin gradient successfully predicts the malignant etiology of pleural effusion.

To reinforce the benefits of studying the albumin gradient (serum-effusion albumin gradient), we will present a prospective observational study conducted by Sandeesha et al.^[36], where pleural punctates from 66 patients were examined and then classified into two groups based on the etiology of the pleural effusions. The data show that of the 22 transudative effusions, 20 were correctly classified using the albumin gradient. Of the 44 exudative effusions, 41 fell into the correct group. When compared to Light’s criteria, it was found that testing the albumin gradient provided diagnostic value for distinguishing exudative from transudative pleural effusions at 92.42%, compared to 87.87% for Light’s criteria.

By conducting a comparative analysis between the two groups in our study, we found the following. Using Kolmogorov-Smirnov test values, total protein (p=0.013) and albumin gradient (p=0.009) show non-normal distribution. When applying the Mann-Whitney U test, with p=0.002 and p<0.001 for total protein and albumin gradient respectively, we see that these values show promising levels of statistical significance in distinguishing malignant from benign pleural effusions. The analysis of the results also leads us to the hypothesis that elevated protein content is associated with the severity of pleural damage, whether malignant or inflammatory.

The relationship between triglyceride levels and secondary malignant diseases of the pleura remains controversial. Research on triglycerides in pleural fluid does not provide significant advantages for diagnosing malignant pleural effusions based on the sources used. There is a noticeable lack of adequate studies examining their values in secondary malignant diseases of the pleura. According to the results we obtained from the Kolmogorov-Smirnov test, the triglyceride values in the examined patients with pleural effusions do not follow a normal distribution with p<0.001. On the other hand, Independent Samples tests show conflicting results, with the Equality of Variances test giving p=0.178, and the Equality of Means test showing p=0.315.

Additionally, when analyzing the Mann-Whitney U data, triglycerides show good statistical significance with p=0.013. Therefore, triglyceride values in pleural punctates should be interpreted cautiously and in the context of the overall analysis of the expanded biochemical panel.

An extensive review of available literature shows that increased levels of cholesterol in pleural fluid are associated with an exudative nature of hydrothorax. In a study conducted by Shen et al.^[37], pleural cholesterol was linked to high sensitivity (88%) and specificity (96%) in detecting malignant pleural effusions. On the other hand, Leers et al.^[38] reports sensitivity, specificity, PPV, and NPV values for the method as 75.7%, 98%, 99.1%, and 59.2%, respectively. In an effort to determine reference values for cholesterol in pleural fluid, Guleria et al.^[39] suggests an upper limit of 60 mg/dl, which, according to their study, provides a sensitivity of 88.2% and specificity of 100%. For comparison, in the same study, using Light’s criteria, sensitivity of 98% and specificity of 80% were achieved.^[39]

In addition, Hamm et al. demonstrates that malignant pleural effusions register significantly elevated levels of pleural cholesterol, independent of serum cholesterol with an average of 94 mg/dl, followed by inflammatory pleural effusions (76 mg/dl) and non-malignant effusions (30 mg/dl). He also suggests an upper limit of 60 mg/dl, which differentiates exudative from transudative pleural effusions with only a 5% error.^[40]

Comparing these data with our results, we can observe several intersections. From the Kolmogorov-Smirnov test, with p=0.009, we can conclude that the values do not follow a normal distribution. From the Equality of Variances test, there is an equal variation in the data as p=0.165. However, the Equality of Means test gives a difference between the means with p=0.011.

Given our results and the available information, we can conclude that pleural cholesterol is one of the best diagnostic tools to aid the diagnosis of malignant pleural effusions.

Several studies link about one-third of malignant pleural effusions with a pleural fluid pH<7.30 at the initial manifestation of hydrothorax.^{[41, 42]} This lowered pH is associated with glucose levels in the pleural effusion <60 mg/dL.^[43] These results are connected to the degree of tumor invasion in the pleural space. Malignant pleural effusions with low pH and glucose are more often positive for tumor cells and are diagnosed already in the cytopathological exami- nation.^{[44, 45]}

According to our data, the pH of the punctate has a non-normal distribution of the data in the Kolmogorov-Smirnov test with p<0.001. In the Independent Samples tests, particularly the Equality of Variances test, we get p=0.306, while the Equality of Means test gives p=−1.007.

Additionally, the Mann-Whitney U test shows that there is no statistically significant difference between the malignant and benign groups with p=0.117. Therefore, the acidity of pleural effusion in malignant pleural diseases is not as informative as, for example, in inflammatory diseases.

Regarding glucose values, we see that according to our results, there is a non-normal distribution of the obtained values, p<0.001 in the Kolmogorov-Smirnov test. Expanding this analysis, in the Independent Samples tests, the Equality of Variances test gives p=0.763, and the Equality of Means test gives p=0.874. The statistical significance assessment from the Mann-Whitney U test confirms the hypothesis that glucose does not have statistical significance in differentiating between malignant and benign pleural effusions with p=0.871.

The study of specific gravity in pleural fluid to differentiate malignant from benign pleural effusions is sparsely studied in the available literature. However, there are studies that examine its potential use in distinguishing between exudative and transudative pleural effusions. A study by Abdollahi and Nozarian^[46] shows that using an upper limit of 1022, measuring specific gravity can achieve sensitivity, specificity, PPV, and NPV values of 92.1%, 68.1%, 88.1%, and 78.3%, respectively, in differentiating exudates from transudates. They conducted a prospective cross-sectional study for two years, collecting 268 samples, 125 of which were pleural punctates and 143 peritoneal fluids. From the pleural group, based on Light’s criteria, 61 cases were classified as exudates and 64 as transudates. Analyzing the sensitivity and specificity of relative weight in the two groups, he concluded that measuring specific gravity through refractometry is an acceptable method for differentiation between exudative and transudative pleural effusions.

According to our data, the values of relative weight in pleural punctates from the examined patients show a non-normal distribution according to the Kolmogorov-Smirnov test (p<0.001). This is also true in the Independent Samples t-test. Although the parameters show equal variation in the data in the Equality of Variances test p=0.391, the Equality of Means test gives p=0.005. When applying the Mann-Whitney U test, we see that p=0.004, indicating a statistically significant difference between the values in the two groups.

Several studies highlight the importance of lactate dehydrogenase in the diagnosis of malignant pleural effusions and its significance in differentiating between exudative and transudative pleural effusions.^[47]

According to Vergnon et al.^[48], the increased amount of LDH isoenzyme LDH-5 is a good marker for detecting malignant pleural effusions, specifically hydrothorax caused by primary small-cell lung carcinoma and lymphoma. They also note that this isoenzyme and its values in pleural punctates are dependable for monitoring such patients. LDH values are also related to the prognosis for survival in patients with malignant pleural effusions. This is best studied in patients with secondary pleural involvement from lung adenocarcinoma, as shown by a retrospective cohort study by Verma et al.^[49]

From the data obtained from the Kolmogorov-Smirnov test, we see that LDH does not demonstrate a normal distribution of the data with p<0.001. When applying the Independent Samples tests, it is evident that the parameters show equal variation in the data in the equality of variances test with p=0.801. In the Equality of Means test, p=0.345. The Mann-Whitney U test reveals a statistically significant difference for LDH between the malignant and benign groups with p=0.003.

As with the total protein levels in the punctate, we hypothesize that LDH levels are indicative of the severity of pleural damage in both inflammatory and malignant pleural effusions, with malignant effusions possibly being a predictor of the degree of pleural carcinomatosis and a poor prognostic factor.^[49]

From all examined parameters of the extended panel of biochemical markers, the tests performed, and the correlations drawn, the most dependable remain the examination of total protein and albumin gradient, lactate dehydrogenase levels. However, the diagnostics can be enhanced by strengthening their application and including cholesterol levels, as well as investigating the total leukocyte count and their subpopulations.