Role of pleural fluid “Cell Block” in malignant pleural effusion: Underutilized, sensitive, and superior over conventional fluid cytology; Does it will decrease need for thoracoscopy guided procedures?

Shital Patil; Sham Toshniwal; Ayachit Rujuta

doi:10.4103/jascp.jascp_23_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 3 | Issue : 3 | Page : 73-79

Role of pleural fluid “Cell Block” in malignant pleural effusion: Underutilized, sensitive, and superior over conventional fluid cytology; Does it will decrease need for thoracoscopy guided procedures?

Shital Patil¹, Sham Toshniwal², Ayachit Rujuta³
¹ Department of Pulmonary Medicine, MIMSR Medical College, Latur, Maharashtra, India
² Department of Internal Medicine, Government Medical College, Latur, Maharashtra, India
³ Department of Pathology, MIMSR Medical College, Latur, Maharashtra, India

Date of Submission	30-Sep-2021
Date of Decision	27-Oct-2021
Date of Acceptance	19-Dec-2021
Date of Web Publication	03-Nov-2022

Correspondence Address:
Shital Patil
Department of Pulmonary Medicine, MIMSR Medical College, Latur, Maharashtra
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jascp.jascp_23_21

Abstract

Background: Malignant pleural effusion missed routinely because of less diagnostic yield of conventional fluid cytology. Materials and Methods: Prospective multicentric study conducted during January 2014 to June 2016 in Venkatesh chest hospital, and Pulmonary Medicine, MIMSR medical college Latur, to find diagnostic yield of conventional pleural fluid cytology and pleural fluid “cell block” in malignant pleural effusion and compare yield of pleural fluid cell block with conventional cytology technique. The study included 200 cases of unexplained, exudative pleural effusion with Adenosine deaminase (ADA) ≤30/IU/l and pleural fluid cytology is either positive for malignant cell with or without cell type differentiation, or cytology suspicious for malignant cell. All cases were subjected to cell block preparation. Statistical analysis was done by using Chi-test. Observation and Analysis: In study of 200 cases, mean age of group was 68 ± 9.5 years and adenocarcinoma was predominant malignancy in 72% cases, mesothelioma in 10% cases, squamous cell carcinoma in 7% cases and 9% cases were having primary tumor outside the thoracic cavity. In study cases, pleural fluid cytology was positive in 42% cases (84/200), and pleural fluid cell block was positive in 96% cases (192/200) in detecting malignant pleural effusion (P < 0.0001). Remaining six and two cases were diagnosed by using image-guided and thoracoscopy-guided pleural biopsies, respectively. Immunohistochemistry (IHC) was done in all pleural fluid cell block preparation for calretinin, cytokeratin, and epidermal growth factor receptor. Conclusion: Pleural fluid cell block is sensitive, superior, cost-effective, and specific diagnostic method over conventional pleural fluid cytology. “Cell block” specimens are enough for primary diagnosis and IHC analysis necessary for cell typing. It will decrease the need for more invasive and costlier diagnostic methods like thoracoscopy and image-guided pleural biopsies. We recommend cell block for every exudative pleural fluid samples with ADA <30 IU/l.

Keywords: Cytology, lung cancer, malignant pleural effusion, pleural fluid cell block

How to cite this article:
Patil S, Toshniwal S, Rujuta A. Role of pleural fluid “Cell Block” in malignant pleural effusion: Underutilized, sensitive, and superior over conventional fluid cytology; Does it will decrease need for thoracoscopy guided procedures?. J Appl Sci Clin Pract 2022;3:73-9

How to cite this URL:
Patil S, Toshniwal S, Rujuta A. Role of pleural fluid “Cell Block” in malignant pleural effusion: Underutilized, sensitive, and superior over conventional fluid cytology; Does it will decrease need for thoracoscopy guided procedures?. J Appl Sci Clin Pract [serial online] 2022 [cited 2023 Mar 27];3:73-9. Available from: http://www.jascp.org/text.asp?2022/3/3/73/360444

Introduction

Lung cancer is a leading cause of cancer-related mortality worldwide, with nonsmall cell lung cancer accounting for around 80%–85% of lung cancers.^[1] Although pleural effusion is one of the clinical signs of malignant disease, its accurate diagnosis is sometimes difficult. Determining the diagnosis of pleural effusion is important in planning the appropriate management and in the prognostication of the malignant disease.^[2],[3],[4] Thoracentesis and/or closed pleural biopsy are generally considered as the first step for diagnosis of pleural effusion because these procedures can be easily performed even in outpatients. Some studies have reported that the diagnostic yield of cytology by thoracentesis was 62% to 90% and that of closed pleural biopsy was 40% to 75%.^[4]

Cytologic techniques have been universally recognized as the most important diagnostic tool in the recognition of malignant tumors in effusions.^[5] Accurate identification of the exact nature of cells (benign/malignant/reactive) is often a practical problem in conventional cytology smears (CS), due to overcrowding of cells, cell loss, and different laboratory processing methods.^[6]

On the other hand, cell block is also a useful method to evaluate pleural effusion by enabling observation of tissue architecture and providing additional sections that are easily available for special stains and immunochemistry.^[7],[8]

Quincke in 1882, first published detailed description of cancer cells in abdominal and pleural fluids using cell films from sediment^[9] while Bahrenburg first introduced cell block technique or paraffin embedding of sediments in 1896.^[10] Many techniques for CB are described like the plasma thromboplastin method,^[11] bacterial agar method,^[5],[11] simplified cell block technique,^[11],[12] compact cell block technique,^[13] histogel technique,^[14] and fixed sediment method.^[11]

In this study, we assessed diagnostic yield of pleural fluid cell block in comparison to conventional fluid cytology. We also assessed utilization of cell block specimens for immunohistochemistry (IHC) analysis.

Materials and Methods

Prospective multicentric study conducted in Venkatesh chest hospital, and Pulmonary Medicine, MIMSR medical college Latur during January 2014 to June 2016, to find diagnostic yield of conventional pleural fluid cytology in malignant pleural effusion and its comparison with pleural fluid cell block specimens. We also analyzed IHC analysis of cell block specimens. Total 200 cases of unexplained, exudative pleural effusion were enrolled in study after Institutional Review Board approval and written informed consent of patient.

Inclusion criteria

Unexplained, exudative pleural effusion
Exudative pleural effusion with pleural fluid ADA ≤30/IU/l
Hemorrhagic or reddish colored pleural effusion with ADA ≤30/IU/l
Pleural fluid cytology is either positive for malignant cell with or without cell type differentiation, or cytology suspicious for malignant cell
Clinical and radiological feature suggestive of malignant pleural effusion.

(Radiological features of malignant pleural effusion-massive pleural effusion, pleural effusion with fixed mediastinum or central mediastinum).

Exclusion criteria

Transudate pleural effusion
Exudative pleural effusion with ADA >44 IU/l and high index of suspicion for tuberculosis.
Bilateral pleural effusion with co-morbidity like heart failure, kidney disease, or hypoproteinemia, anemia
Cases not willing to participate in study or not willing for pleural fluid aspiration.

Methodology

Cases attending outdoor unit after scrutinizing inclusion and exclusion criteria with high index of suspicion of malignancy on clinical and radiological criteria were enrolled in study. All 200 study cases undergone pleural fluid aspiration and at least 100 ml pleural fluid is aspirated as per standard guidelines for thoracentesis. Pleural fluid aspiration was done under ultrasound guidance and aspirated fluid was divided into two aliquots, one sent for cytology and second for cell block preparation. All study samples were evaluated by two different cytopathologists and oncopathologists having expertise in field of thoracic oncology. Those cases not diagnosed by fluid cytology or cell block were undergone image-guided pleural biopsy and thoracoscopy-guided pleural samplings to confirm the diagnosis.

Procedure of pleural fluid cytology

Twenty milliliter pleural fluid was were centrifuged at 2500 rpm for 10 min. A minimum of 3 smears were prepared from the sediment. One smear was prepared after air drying and it was stained with the May-Grunewald-Giemsa stain. The other two smears were immediately fixed in 95% alcohol and were stained with hematoxylin-eosin stain.

Cytology results were categorized as:

Cytology suspicious for malignant cells or malignant cells with undifferentiated morphological type
Cytology showing clear morphological malignant cells differentiation
Cytology negative for malignant cell or showing benign cellularity.

Procedure of “pleural fluid cell block”

Cell block processing for serous effusion-Modified Thromboplastin method is used. After centrifugation at 2500 rpm for 10 min, drain the supernatant or pipette out the supernatant cell and residual sediment was formed. Excess supernatant was blotted out, 2 drops of plasma added to the tube, and then 4 drops of thromboplastin added and allowed to clot for 20 min. Then inert the tube and collect the cell block on filter paper. The cell blocks were embedded in paraffin and sectioned at 4 μm thickness (process clot as any tiny biopsy specimen).

Finally, paraffin blocks were cut into 3-μm sections for hematoxylin-eosin stain. All cell block specimens were send for IHC analysis.

Technical considerations for cell block preparation as we specifically recommend are:

Pooled plasma remains well in a freezer up to 1 month
Thromboplastin is to be kept in the refrigerator
Reagents should be brought to room temperature before processing.

Cell block preparation results were categorized as:

Histology showing malignant cell undifferentiated type
Histology showing malignant cells with exact differentiation
Histology negative for malignant cell or showing benign cellularity.

Cell block specimens after primary evaluation and confirmation as malignancy was send for IHC analysis for reanalysis of primary diagnosis by cell block method and mutation analysis in tumor cells to avail exact treatment to have excellent treatment outcome. In IHC analysis, we specifically recommend for epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), calretinin, carcinoembryonic antigen, and ALK analysis.

Cell block IHC specimen's results were categorized as:

Confirmatory and sample sufficient
Confirmatory and sample insufficient.

The statistical analysis was done using Chi-squared test (three methods of Chi-squared test such as independence, goodness of fit, and proportion test). Significant values of Chi-square were seen from probability table for different degree of freedom required. P value was considered significant if it was below 0.05 and highly significant in case if it was <0.001.

Observation and analysis

Total 200 patients between age group 31–90 years, with mean age 68 ± 9.5 years, male population constitutes 66% and females 34% of total. In study cases, only 9% cases were smoker. Commoner symptoms were shortness of breath in (91.33%), cough in (54.00%) and chest pain (46.66%) cases, and massive pleural effusion 42%, mass with effusion 28%, effusion with fixed mediastinum in 21%, and bilateral pleural effusion 9% were more common radiological abnormalities.

In study of 200 cases with malignant pleural effusion, 84 cases were diagnosed by conventional cell cytology; out of which only 48 cases were diagnosed with clear histological type.

Sensitivity of conventional cell cytology in detecting malignant pleural effusion is 42% [Table 1].

Table 1: Yield of pleural fluid cytology in study cases (n=200)

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In study of 200 cases with malignant pleural effusion, 192 cases were diagnosed by cell block histology technique, out of which 158 cases were diagnosed with exact histological type.

Sensitivity of “cell block” in detecting malignant pleural effusion is 96% [Table 2].

Table 2: Yield of pleural fluid “cell block” in study cases

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In study of 200 cases with malignant pleural effusion, 192 cases were diagnosed by pleural fluid cell block, while only 84 cases were diagnosed by pleural fluid cytology. Pleural fluid cell block has very significant yield as compared to conventional pleural fluid cytology (P < 0.00001) [Table 3].

Table 3: Comparison of pleural fluid cytology and “cell block” in confirmed cases by these techniques in study cohort (n=192/200)

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Pleural fluid cell block has 96% (192/200) diagnostic yield as compared to conventional cytology having 42% (84/200) diagnostic yield. Pleural fluid cell block has 2.28 times more detection rate than cytology (P < 0.00001) [Table 4].

Table 4: Comparison of overall yield of pleural fluid cytology and “cell block” in study cohort

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IHC analysis in pleural fluid cell block specimens was confirmatory, and sample was sufficient for diagnosis in 93.75% cases [Table 5].

Table 5: Immunohistochemistry analysis on pleural fluid “cell block” specimens

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Discussion

Yield of pleural fluid cytology in study cases

In the present study of 200 cases with malignant pleural effusion, 84 cases were diagnosed by conventional cell cytology, out of which only 48 cases were diagnosed with clear histological type. Sensitivity of conventional cell cytology in detecting malignant pleural effusion is 42%. Studies by Rivera et al.,^[15] McGrath and Anderson,^[16] Gupta et al.,^[17] and Hooper et al.^[18] documented that in malignant pleural effusion, cell cytology from pleural fluid provides a diagnostic rate of 60%, ranging from 40% to 87%.

Various studies by Köksal et al.,^[19] Jing et al.,^[20] Ugurluoglu et al.,^[21] and Bhanvadia et al.^[22] observed that improper smear, fixation, and staining techniques in pleural fluid cell cytology can cause cell overlapping or overcrowding, cell loss, artifacts, and poor background staining, while these are less frequent in cell block.

Studies by Köksal et al.,^[19] Ugurluoglu et al.,^[21] Bhanvadia et al.,^[22] Dekker and Bupp,^[23] and Shivakumarswamy et al.^[24] mentioned that discrimination of reactive mesothelial cells and malignant cells is a major challenge in cytology smear, as reactive mesothelial cells may express large irregular nucleoli, coarse chromatin, and enlarged nuclei, mimicking malignancy.

Yield of pleural fluid cell block in study cases

In study of 200 cases with malignant pleural effusion, 192 cases were diagnosed by cell block histology technique; out of which 158 cases were diagnosed with exact histological type. Sensitivity of “cell block” in detecting malignant pleural effusion is 96%.

Pleural effusion cell block is a useful alternative because collection is easy and better morphologic preservation of the architectural pattern may be obtained, compared with conventional cytology.^[24] In previous studies by Nathan et al.,^[7] Kern and Haber^[25] Axe et al.,^[26] Wojcik and Selvaggi,^[27] Leung and Bedard,^[28] and Norimatsu et al.^[29] documented sensitivity of cell block varied widely from 60% to 89.4%, probably because of differences in sampling type, size, type of specimens, and aspiration techniques. Thapar et al.^[6] observed 65.7% positivity for malignancy on cell block while in Nathan et al.,^[7] study cell block confirmed malignancy in 92.7% of cases. Miyoshi et al.^[30] showed that pleural biopsy using flex-rigid pleuroscopy had a significantly higher diagnostic yield (94.2%) than pleural effusion cell block (71.4%) for malignant pleural disease.

Cellularity is higher by cell block compared with fluid cytology and is concentrated in one small area that can be evaluated at a glance, with all cells lying in the same focal plane of the microscope.^[23],[24] In addition, cell block provides better cellular morphological details, such as better nuclear and cytoplasmic preservation, intact cell membrane, and crisp chromatin; there is also less difficulty in microscopic observation, in spite of the presence of excess blood in the background.^[24]

Comparison of cytology and cell block in study cases

In study of 200 cases with malignant pleural effusion, 192 cases were diagnosed by pleural fluid cell block, while only 84 cases were diagnosed by pleural fluid cytology. Pleural fluid cell block has very significant yield as compared to conventional pleural fluid cytology (P < 0.00001). Some studies by Udasimath et al.^[24] and Thapar et al.^[6] have shown additional cases of malignancy on CB by increasing the diagnostic yield by 9% and 20%, respectively. Various studies by authors Thapar et al.,^[6] Köksal et al.,^[19] Ugurluoglu et al.,^[21] Bhanvadia et al.^[22] Shivakumarswamy U et al.^[24] showed an additional diagnostic rate of CB to CS around 10%–15% in MPE. They also documented that in a group that was considered benign with the Conventional smear cytology technique, there were malignant cases proven with the cell block technique. Besides, they also detected that it was possible to type cancer with cell block technique in the cases considered malignant with the conventional smear cytology technique.

In a study by Dekker and Bupp,^[23] the rate of recovery of tumor cells by cell block preparation was double that obtained by smear alone. By using cell block method, tumors were subsequently demonstrated in 38% of the patient who had negative or atypical cytological reports. Thapar et al.,^[6] showed a diagnostic yield of 20% by cell block preparations. In a study done by Khan et al.,^[31] additional findings were diagnostic in 16% of malignant cases. Khan et al.^[31] in another study titled as “usefulness of cell block verses smears in malignant effusion cases” reported that the recovery rate for malignant lesions by cell block preparation was 20% greater than that obtained for specimen examined in smear only.

Similar findings were also observed in studies by Dekker and Bupp,^[23] Takagi,^[9] Chapman and Whalen,^[32] Velios and Griffin,^[33] and Ugurluoglu C et al^[21] When conventional smears were compared with cellblock preparation for morphological preservation, the cell block sections showed clearly recognizable cells with minimal shrinkage and aberrations. The cytomorphologic features were well maintained with minimal shrinkage and aberration.

Contradictory to our observation studies by Shafigh and Siadaty^[34] and Nathan et al.^[7] documented sensitivity of smears and cell blocks tended to be similar whereas the cell blocks proved to be superior in our material. Kung et al.^[35] especially mentioned in their study that cell block entails a risk of losing material during preparation, especially in the case of fixation technique that might explain negative cell block results but positive conventional smear cytology results in some cases.

In this study, pleural fluid cell block has 96% (192/200) diagnostic yield as compared to conventional cytology having 42% (84/200) diagnostic yield. Pleural fluid cell block has 2.28 times more detection rate than cytology (P < 0.00001). In a study by Dekker and Bupp,^[23] the rate of recovery of tumor cells by cellblock preparation was double that obtained by smear alone.

The advantages of the cellblock preparation as we observed in our study are:

Recognition of histologic patterns of diseases that sometimes cannot be identified reliably in smears
Possible processing of the multiple sections of the same material for routine staining, special staining, and immunologic procedure
Less cellular dispersion, which permits easier microscopic observation than do the conventional smears
Less difficulty in spite of background showing excess blood on microscopic observation
Lower cost than the biopsies and need for thoracoscopy-guided procedure
Possibilities of storing slides for retrospective studies. Storage of the conventional smear is a practical problem.

Immunohistochemistry analysis in study cases

In our study, we documented that, IHC analysis in pleural fluid cell block specimens was confirmatory and sample was sufficient for diagnosis in 93.75% cases. Analysis was done for EGFR, ROS, calretinin, carcinoembryonic antigen, and ALK mutation. Authors Kung IT et al^[35] Zhou et al.,^[37] mentioned that, cell block preparations are commonly used for diagnostic pathology and gene analysis by Fluorescence in situ hybridization and IHC. A few reports Zhou et al.^[37] and Liu et al.^[38] reported that compared with tumor tissue, pleural effusion cell block had 81.8% sensitivity and 80% specificity for detection of EGFR mutation and 62.5% to 100% sensitivity and 100% specificity for ALK detection. Ensani et al.^[39] and Ikeda et al.^[40] observed that with cell block method, cellularity is increased, the morphological details are better observed, special histochemical stainings and immunohistochemical studies can be done and sensitivity in diagnosis increases.

In our study, we documented that, IHC analysis in pleural fluid cell block specimens was confirmatory and sample was insufficient for diagnosis in 6.25% cases. For successful detection of the targeted gene, sufficient amount of malignant cells are needed; however, in some cases, observed by Miyoshi et al.,^[30] the amount of malignant cells in cell block samples are few.

Studies by authors, Esteban et al.,^[41] Mason et al.,^[42] Doglioni et al.,^[43] Ko EC et al^[44] and Cibas et al.,^[45] documented that distinction of reactive mesothelial cells from malignant cells is always a diagnostic concern in cytodiagnosis of serous fluids. In such situations, IHC may be helpful.

Other important observations in study

Does volume of pleural fluid makes difference in yield?

In this study, we observed that pleural fluid cell block has 96% (192/200) diagnostic yield as compared to conventional cytology having 42% (84/200) diagnostic yield with 100 ml pleural fluid sent for analysis. Baumann^[46] observed that malignant cells are considered to be present heterogeneously within the pleural effusion and can be precipitated by gravity. Position of the patient's body and the site of puncture may affect the diagnostic yield of cytology or cell block from thoracentesis. Miyoshi et al.^[30] documented that heterogeneity of pleural fluid cells probably did not affect the diagnostic yield of the cell block preparations because pleural fluid was collected under direct vision by flex-rigid pleuroscope. However, scarcity of free malignant cells in pleural fluid samples remains a serious challenge. A prospective study by Swiderek et al.^[47] demonstrated that at least 150 mL of pleural fluid is needed for analysis with both cytology and cell block. Miyoshi et al.^[30] in their study analyzed 150 mL pleural was routinely collected in all patients; and they also mentioned that, the volume of pleural fluid can be increased if assessment by cell blocks requires more cell volume.

Other diagnostic modalities used in study

In this study, 192 cases were diagnosed by pleural fluid cell block technique. Remaining six and two cases were diagnosed by using image-guided and thoracoscopy-guided pleural biopsies, respectively. Rational for low yield of diagnostic malignant cells on cell block may be due to hypocellularity and bleeding in the cell block preparations. The amount of malignant cells was few, as seen in conventional cytology, and this may have contributed to the negative cell block results. Norimatsu et al.,^[29] Sweeney et al.,^[48] documented that cellularity of biological fluid samples with excessive blood decrease the quality and compromise accurate assessment of specimens, and treatment with a hemolytic agent is recommended to improve the cellularity in pleural fluid samples with excessive blood.

We have used thoracoscopy in two cases and reached diagnosis in them, approximately yield is 100%. Boutin and Rey^[49] and Tassi et al.^[50] documented diagnostic yield of thoracoscopic biopsy has been reported to be >90%.

Conclusion

Pleural fluid cell block is more sensitive, superior, cost-effective, and specific diagnostic method over conventional pleural fluid cytology in malignant pleural effusion. “Cell block” specimens are enough for primary diagnosis and IHC analysis necessary for cell typing.

Results of cell block are comparable to more invasive and costlier diagnostic methods like thoracoscopy and image-guided pleural biopsies. In addition, it will decrease need for thoracoscopy guided techniques, especially in resource-limited setting like India where availability and cost factor make more difference.

We recommend cell block for every exudative pleural fluid samples with ADA <30 IU/l to have early diagnosis. More emphasis should be given to pleural fluid cell block analysis training.

Compliance with ethical standards

Research involving human participants and/or animals

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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