Analysis of CD4, CD8, CD19, CD56-16, CD64, QuantiFERON biomarkers in exudative lymphocyte-dominant pleural effusion

ABSTRACT

Background:

There are two main causes of exudative effusion including malignancy-induced effusion and tuberculosis. Considering that in reactive ejections, such as tuberculosis-induced effusion, the role of B lymphocytes and in the malignant effusion, the role of T lymphocytes are more important, in this study we analyzed the frequency of CD4, CD8, CD19, CD56-16, CD64, QuantiFERON in the pleural and serum samples of patients with exudative lymphocytic-dominant effusion.

Methods:

In total, 73 patients were enrolled in the study by exudative lymphocyte effusion, and finally, 63 patients had definite diagnoses. The patients were sorted into three groups including malignant, tuberculosis, and none. The sample of blood plasma and pleural effusion were collected and CD markers were analyzed using flow cytometry.

Results:

The mean age in the malignancy and tuberculous (TB) groups was 63.16 ± 12 and 52.15 ± 22.62, respectively. There was no significant difference in the frequency of CD8, CD4, and CD16-56 cells in blood samples of patients with tuberculosis and malignancy. Compared to those with tuberculosis, the percentage of CD64 cells was significantly higher in patients with tuberculosis than in malignant subjects. Moreover, a comparison of the frequency of cells with CD8, CD4, CD19, CD64, CD16-56, and CD14 markers in pleural samples showed no significant difference between groups. Other inflammatory factors were also investigated. The erythrocyte sedimentation rate (ESR) value for tuberculosis patients was significantly higher than malignancy. Also, QuantiFERON was positive in 14.3% of malignant patients, and 62.5% of patients with TB, which had a significant difference.

Conclusion:

Considering that there are many confounding variables in the study, such as previous medications, subtypes of Mycobacterium, and race of patients conducting studies in different groups and performing data mining for using a set of parameters can be used to detect the exact diagnosis.

Introduction

The pleura is a serous membrane that covers the surface of the lungs. The pleural space is located between the visceral and peritoneal layers. The mean fluid volume between the pleural space is 4.3 mL ± 8.4. Plurocerebral conduction is through the bronchial artery and drainage is through the pulmonary veins. Parietal pleural blood flow is through the intercostal artery and drainage is through the superior vena cava. Pleural fluid, as a lubricant, is involved in reducing respiratory work.[1]

Surface markers (CDs) are cells that are used to identify and characterize leukocytes, which can be used to detect leukocytes through flow cytometry.[2] CD14 acts as an endotoxin receptor. By connecting to the glycoprotein I (GPI), it connects to the cell surface. It is very positive in monocytes and most tissue macrophages; however, neutrophils and a small proportion of B lymphocytes may strongly express CD14. T cells, dendritic cells, and platelets are CD14-negative.[3,4] CD16 is a molecule found on the surface of natural killer cells, neutrophils, monocytes, and macrophages and is involved in signal transduction.[5] CD64 is a glycoprotein that acts as a restore that binds to IgG.[6] On dendritic cells, stem cells, macrophages, monocytes, and granulocytes. Inherent immune response, adaptation is involved.[7] CD56, which is called neural cell adhesion molecule (NCAM). This molecule is also found in the hematopoietic system.[8] CD19 is present on B cells other than plasma cells and follicular dendritic cells. This protein also acts as a diagnostic biomarker of B lymphocytes. It is used in the diagnosis of leukemia and lymphoma phenotypes.[9]

In vitro quantum biomarkers are used in the differential diagnosis of tuberculous pleural effusion and malignant pleural effusion in areas with a high prevalence of tuberculosis. The sensitivity and specificity of quantiFERONe for the detection of tuberculous pleural effusion (TPE) were 0.93% and 0.60%, respectively. Measurement of gamma interferon release (IGRAs) is a diagnostic tool for latent tuberculosis (LTBI) infection. They are alternative markers of Mycobacterium tuberculosis infection and indicate a cellular immune response to M. tuberculosis in the presence of the latter.[10]

One of the clinical findings in pulmonology is lymphocytic pleural effusions. Important causes of lymphocytic effusions are tuberculosis, primary and metastatic malignancies, lymphoma, collagen vascular diseases, chylothorax, and coronary artery bypass graft (CABG). The main causes of exudative lymphocytic effusion are tuberculosis and malignancy.[11] Pleural effusion is a common complication that affects more than 400 people per 100,000 people. The percentage of patients with TB associated with pleural effusion varies from country to country (more than 25%), about 10% in the United Kingdom, and about 3–5% in the United States. The effusion caused by a mycobacterial infection is usually caused by a delayed sensitivity to Mycobacterium proteins, and in most cases, the amount of bacteria present in the pleural space is reduced to 80 (reduced to 10). A combination of cytology and biopsy increases the detection rate to 73%. Although pleuroscopy can determine the cause of pleural effusion with 95% accuracy, it is invasive and not available in most hospitals.[12,13,14]

The use of inflammatory biomarkers and molecules that detect the level of white blood cells that are invoked in any disease can be used in the differential diagnosis of the causes of effusion.[15] One of these molecules is CD markers, which have different manifestations in different causes of effusion. Therefore, a comparative analysis of the biomarkers of CD4, CD8, CD19, CD56-16, CD64, and QuantiFERON in the pleural and serum samples of patients with exudative lymphocytic effusion adds to the diagnostic value of each of these biomarkers.

Materials and Methods

Study design

The present study was done as a cross-sectional descriptive cohort study from June 2018 to 2019 July for 1 year. All patients referred to Masih Daneshvari Hospital of Shahid Beheshti University of Medical Sciences who presented with shortness of breath or other respiratory symptoms and were diagnosed with pleural effusion in diagnostic tests including lung and chest x ray (CXR), which were diagnostically taped. After obtaining consent, they consciously entered the study. Then, two plasma and pleural effusion samples were sent to the laboratory at the same time. Patients with predominant exudative pleural effusion of lymphocytes were included in the study. The criterion of exudativeness of the sample was the presence of one of the following: pleural fluid to serum protein ratio greater than 50, pleural fluid to serum lactate dehydrogenase (LDH) ratio greater than 0.6, or LDH ratio greater than two-thirds of the highest normal serum LDH level. Lymphocyte predominant effusions known as more than 50 percent of total cell count as lymphocytes. The use of other immunosuppressive drugs, no chronic underlying lung disease, no chemotherapy drugs, no TB, and criterion oncology: death before diagnosis, concomitant tuberculosis and malignancy at diagnosis, no complete diagnostic measures including biopsy and culture and smear of samples obtained from the patient, inadequate biopsy and diagnostic doubt in the confirmation of tuberculosis or malignancy were determined. After diagnosis, patients were divided into one of the three groups of TB, malignant, and unspecified tumors.

Study patients

A total of 73 patients were enrolled in one of the three groups: definitive diagnosis of TB with confirmation of sputum culture, positive sampling and smear (n = 20), definitive diagnosis of malignancy with confirmation of pathology diagnosis (n = 30), and patients without a diagnosis after culture and smears were diagnostic molecular tests for tuberculosis and pathology (n = 13). A number of 10 patients were excluded from the study due to incomplete diagnoses such as incomplete data. From each patient, 2 mL of pleural effusion sample and 10 whole blood of the patient were sent to the laboratory for flow analysis by cell cytometry. Other diagnostic tests such as cytology, chest CT scan, chest X-ray, culture and smear for mycobacteriology, and biopsy were performed as needed.

Laboratory measurements

To measure the CD marker, the flow cytometry method was used, which is a fast method for identifying cells and evaluating their properties. In this method, the scattering properties of light by cells and fluorescence emission were used. The signals were detected by the device or from light sources at 635 nm and 488 nm. The following antibodies were used: Cytognas LOT # 1910200 CD F/CD 8, eBioscience LOT # E13993-105 CD19/APC, Cytognas LOT # 526231 CD 56, eBioscience LOT # 4161538 CD 16 F, eBioscience LOT # E11825-1468 CD F. Antigen concentration at the surface or inside the cell was appropriate for the concentration of antibody used. In this method, appropriate positive and negative controls were used in cell analysis.[12,16,17,18,19,20,21,22]

Statistical analysis

Categoric variables are reported as percentages, and continuous variables are reported as the mean ± standard deviation or median (interquartile range) as appropriate, attending to normal distribution. The assumption of normality was evaluated using the Shapiro–Wilk or Kolmogorov–Smirnov test. Demographic data, ESR, and CD marker were compared using analysis of variance (ANOVA) and Tukey’s post-hoc tests. CD markers were analyzed by receiver operating characteristic (ROC) curve analysis for sensitivity and specificity. Data were analyzed using GraphPad prism® version 8.3.0 software and SPSS version 18.

Results

According to the results, 20 patients showed a definite diagnosis of TB (with culture and positive smear), 30 patients with a definitive diagnosis of malignancy (pathological), 13 patients without diagnosis (after culture and smear, molecular diagnostic tests for tuberculosis and pathology), and 10 patients showed diagnosis process remained incomplete (incomplete data) [Table 1].

Table 1.

Final diagnosis of pleural samples and their relations with Gender

Disease	Malignancy (n=30)	Tuberculosis (n=20)	Non TB or Malignancy (n=13)	Total (n=63)
Percentage	47.6%	31.7%	20.6
Gender	56.7% (n=17)	35% (n=7)	30.8% (n=4)	44.4% (n=28)

There was no significant difference in the level of CD4 in the blood and pleura in patients with tuberculosis and malignancy; however, in patients who had both diagnoses rejected, the percentage in the blood was higher than in the pleura (P = 0.005).

The percentage of CD8 in the blood was significantly lower in patients with malignancy and unclear diagnosis. Blood CD8 was also significantly different in malignancy and indeterminate diagnosis.

The percentage of CD19 in blood or pleural fluid in patients in all three groups was not significantly different from each other.

The percentage of CD16-56 in blood or pleura in patients of all three groups was not significantly different from each other. However, in the uncertain diagnosis group, the percentage of CD16-56 pleura increased significantly with blood.

The percentage of CD64 in the blood in patients of all three groups was significantly higher than its ratio to pleural fluid. Blood CD64 percentage in tuberculosis was significantly higher than that in malignancy.

The percentage of CD14 in the blood in malignancy was significantly lower than that in the unknown diagnosis. The percentage of CD14 in the blood in patients with malignancy and the indeterminate diagnosis was significantly lower than that in the pleura.

QuantiFERON was positive in 14.3% of malignant patients. Also, 62.5% of cases were positive in patients with tuberculosis, which was a significant difference. Therefore, positive quantiferous is a risk factor in the patient [Table 2].

Table 2.

Relation between CD markers in blood with diagnosis of Tuberculosis and Malignancy

	n	Mean	SD	SE	95% confidence Interval for Mean		Min	Max
CD_4 blood
Malignancy	23	41.30	8.73	1.82	37.52	45.7	25.0	64.0
TBM	11	44.27	9.77	2.95	37.70	50.83	28.0	68.0
NOT TB	14	38.77	10.20	2.73	32.88	44.66	19.0	57.0
CD_8 blood
Malignancy	23	19.92	6.05	1.26	17.30	22.54	8.0	32.0
TBM	10	26.14	5.47	1.73	22.22	30.05	21.0	37.0
NOT TB	14	21.85	3.86	1.03	19.62	24.08	17.0	32.0
CD 19_blood
Malignancy	23	9.36	4.76	0.99	7.30	11.42	3.0	19.0
TBM	10	7.62	3.46	1.09	5.14	10.09	2.0	14.0
NOT TB	14	8.41	2.61	0.70	6.70	9.91	3.5	13.1
CD_16-56_blood
Malignancy	23	4.28	4.59	0.96	2.30	6.27	1.0	22.5
TBM	10	3.66	1.29	0.40	2.75	4.58	1.5	6.5
NOT TB	14	6.20	4.92	1.31	3.35	9.03	2.3	22.5
CD 64_blood
Malignancy TB	23	73.35	13.17	2.75	67.66	79.04	45.0	94

Discussion

Numerous studies have been performed in the past on the differentiation of malignant pleural effusions from tuberculosis. Different biomarkers and cytokines have been used, which produced different responses. In some studies, the age difference was significant; however, in our study, it was not different. Some studies had different lymphocyte percentages; however, our study did not differ between groups. The results of our biomarkers were consistent with some studies and did not agree with some.

This study showed that the use of inflammatory biomarkers and different populations of immune system cells can help in the differential diagnosis of tuberculosis from malignancy. In this study, it was found that the use of CD markers in serum is more involved in differential diagnosis than pleural markers. Serum CD64 was significantly higher in tuberculosis than in malignancy.

The use of a set of biomarkers seems to increase the positive predictive value of cultures and by increasing the specificity and diagnostic sensitivity, the need to spend time to obtain the results of microbial culture or the cost of invasive tests such assampling Reduced [Figure 1].

Figure 1.

Sensitivity and specificity of ESR and CD markers in differentiating of Tuberculosis and malignant effusions

CD14 levels did not differ between the two groups. There was no significant difference in our study. Acid glycoprotein (AGP), adenosine deaminase (ADA), and immunosuppressive acidic protein (IAP) in tuberculosis effusion were significantly higher than in malignant effusion, carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), and CA19-9 in patients with malignant effusion were significantly higher than in tuberculosis. In general, studies of cytokine biomarkers along with studies of CD biomarkers can increase the accuracy of diagnosis.

In a 2012 study: The percentage of lymphocytes in tuberculosis infection was higher than in malignancies. CD3 + T in tuberculosis-induced effusion were higher than malignant effusion, CD4 + CD25 + were also higher in malignancy in tuberculosis. The levels of interleukin-16 and interferon gamma in the cells were higher than the malignant effusion. In our study, interferon was high.

A similar study in 2015 showed that CD8+ and CD4+ markers that produce interleukin-27 were significantly increased in cell effusion. This study showed that in addition to interferon-gamma, ADA, the study of IL-27 has high sensitivity and specificity for the detection of tuberculous effusion tuberculosis.

Conclusion

The use of different and combined parameters seems to have a cumulative effect on the diagnosis of tuberculosis from malignancy and with the scoring system for different biomarkers to create a higher positive predictive value and reduce the probability of false positives and negatives in the diagnosis. Cytokine biomarkers along with studies of CD biomarkers can increase diagnostic accuracy. CD64, which is higher in the blood of tuberculosis patients than in malignant patients, is a marker of dendritic tuberculosis and it seems that the amount of inflammation and cell recruitment in tuberculosis infection is higher than in malignancy.

Limitation of the study

Patients had to enter the scheme with the health information system (HIS) system, which in some cases was lost due to the inconsistency of the samples.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.