Analysis of the Diagnostic Value of Cytological Smear Method Versus Cell Block Method in Pleural Effusion Fluid of Suspected Lung Cancer Patients
*Ray S,1 Zinnah SA,2 Awal N,3 Hassan I,4 Dewan RK,5 Jeba R,6 Hussain M7
*Dr. Subrata Ray, Assistant Professor, Department of Pathology, Jashore Medical College. email@example.com
- Shahed Ali Zinnah, Associate Professor, Department of Pathology, Dhaka Medical College.
- Dr . Naila Awal , Assistant Professor, Department of Pathology, Greenlife Medical College.
- Imran Hassan, Assistant Professor(CC), Department of Pathology, (Col) Malek Medical College, Manikgonj.
- Rezaul Karim Dewan, Professor and Head, Department of Pathology, Dhaka Medical College
- Dr . Ruksana Jeba, Associate Professor, Department of Pathology, Dhaka Medical College.
- Dr Maleeha Hussain, Professor, OSD, DGHS.
Background: For any kind of lungs pathology accompanying pleural effusion pleural biopsy guided with thoracoscopy, of course, may provide important results. Since biopsy is an invasive procedure, the priority is to do, a cytological examination of pleural fluid obtained with thoracentesis, which is very crucial for the disease staging and treatment strategy. In this purpose, even though conventional cytosmear techniques have been used generally till this day, cell block technique is also being used recently.
Methods: In our study, 100 pleural effusion fluid sample were included. All the fluid specimen was subjected to cytosmear (CS) and cytoblock (CB).
Results: By using conventional CS method, 85 fluid specimens were diagnosed as negative for malignancy (85%), 5 cases were diagnosed as positive for malignancy (5%) and the remaining 10 cases were diagnosed as suspicious for malignancy (10%). On the contrary by using CB, complimented by combined PAS-Alcian blue stain where required, 90 cases were diagnosed as negative for malignancy (90%) and 10 cases were diagnosed as positive for malignancy. Thus, by the CB method, an additional 5 more cases were diagnosed as positive for malignancy, that is, 5% more diagnostic yield for malignancy.
Conclusion: CB technique could be considered as a useful adjunct in evaluating malignant cells in MPE for a final cytodiagnosis, along with the routine CS method.
[Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):99-106]
Key Wards: Cell block , Pleural effusion , Lung cancer
Development of pleural effusion is a common complication in patients with lung cancer either primary or secondary. There may be presence of neoplastic cells in the pleural effusion which is called malignant effusion (MPE) or it may be a para-malignant effusion, which is pleural effusion with absence of cytological evidence of tumor cells in a known setting of malignancy. In UK, 40000 people per year are affected by MPE and it is estimated that up to 50% of the patients with metastatic malignancy develop a pleural effusion – either at the time of diagnosis or during the evaluation of cancer.1,2,3 The common etiologies of MPE are lung cancer, breast cancer, lymphoma, ovarian cancer and gastric cancer in order of decreasing percentage.
For any kind of lung lesion accompanying pleural effusion, pleural biopsy guided with thoracoscopy, of course, may provide important results, but has the risk of being an invasive procedure and it depends on the experience and efficiency of the surgeon. So, in a country with poor resource settings, the priority should be a cytologic examination of pleural fluid obtained by thoracentesis. It not only helps in diagnosis but also can help in disease staging and treatment strategy.4
In conventional cytological smear (CS), discrimination of the reactive mesothelial cells and malignant cells is the most important diagnostic problem. Distinguishing benign from malignant cellular changes may require meticulous screening, careful scrutiny of cellular features and an understanding of the range of reactive changes. Since the introduction of cell block (CB) technique by Bahernburg nearly a century ago, it has been used routinely for processing fluid.5 CB has the advantage of recognition of the histological pattern of disease, possibility of study multiple sections by routine staining, special staining and immunological procedures.
In this study, we assessed the utility of CB and CS techniques and evaluated whether the CB, when it complements conventional CS, can increase the diagnostic yield.
This is an analytic study, carried out at the Department of Pathology, Dhaka Medical College, Dhaka from July 2015 to June 2017, over 100 pleural effusion fluid specimen of suspected lung cancer patients.
10 ml of fresh pleural fluid sample was obtained by thoracentesis from each patient during clinical evaluation. Each sample was divided into two equal parts.
For conventional smear technique, 5 ml fluid specimen was centrifuged at 2500 rmp for 10 min. A minimum two smears were prepared from the sediment. They were immediately fixed in 95% alcohol and stained with the Papanicoloau and Haematoxylin- Eosin stain.
For cell block, we used AAF (95% ethyl alcohol + acetic acid + 10% formalin). After centrifuge at 2500 rmp for 10 min, cell sediments were mixed with thrice the volume of AAF fixative and again centrifuged at 2000 rmp for 10 min. The centrifuged tube was aside undisturbed for six hours. The cell button was scraped out and wrapped in filter paper and processed in automatic tissue processor for routine histopathological sections. The cell blocks were embedded in paraffin and sectioned at 4 micro meter thickness.
The samples were studied in detail taking into account the available clinical and radiological data and various investigation reports. Each individual slide was objectively analyzed for cellularity, background blood, nuclear and cytoplasmic details and arrangement of the cells using the point scoring system described by Mair et al., (1989).6 All these criteria were put together and each cases was categorized as Benign, Suspicious for malignancy and Malignant effusion. Special stain of cell blocks, including combined PAS-Alcian blue stain was done whenever needed. In cases, where pleural biopsy of the same patients was available, used to confirm the diagnosis.
In our study, 100 pleural effusion fluid sample were included. The age-rang from 26 to 82 years. Most of the patients are between the age group 51-60 years (30%). The male female ratio was 1.8:1. All the fluid specimen was subjected to CS and CB. By using connectional CS method, 85 fluid specimens were diagnosed as negative for malignancy (85%), 5 causes were diagnosed as positive for malignancy (5%) and the remaining 10 causes were diagnosed as suspicious for malignancy (10%).On the contrary by using CB, complimented by combined PAS-Alcian blue stain where required , 90 cases were diagnosed as negative for malignancy (90%) and 10 cases were diagnosed as positive for malignancy. Thus by the CB method, an additional 5 more cases were diagnosed as positive for malignancy, that is, 5% more diagnostic yield for malignancy .
Among the 10 malignant cases, diagnosed by CB, 9 cases were subcategorized as adeno carcinoma (90%) and 1 cases was diagnosed as lymphoma.
The malignant effusion was more common in male (70%) than female (30%). The male female ratio was 7:3.
When the cytological smear and cell block techniques were studied for their quality using the point scoring system of Mair et al, (1989).6 it was noticed that 5% of the CS and 6% of the CB was inadequate and unsuitable for diagnosis. 50% of the CB and 32% of the CS showed highest cellularity. 41% of the CB and only 11% of the CS yielded highest morphological details. Wilcoxon Signed Rank test was done to measure the level of significant, which was highly significant in CB compared to CS regarding cellularity and morphological details.
Lung cancer, whether primary or secondary, is invariably accompanied by pleural effusion. The cell population in sediment of pleural fluid represent much larger surface area than obtained by needle biopsy. Malignant cells first involve the visceral pleura and tend to be focal in the parietal pleura.7 This explains why, pleural fluid cytology is a more sensitive diagnostic test than closed percutaneous pleural biopsy.8,9
Thoracentesis followed by cytological examination is the first investigation performed in a pleural effusion fluid of a suspected lung cancer patients and has been accepted as a routine laboratory procedure. The differentiation of a malignant effusion from a para-malignant effusion is extremely important, not only in the diagnosis of a malignant lesion, but also in staging, prediction of prognosis and to reform a proper treatment protocol.10
In our study, 100 pleural effusion fluid specimen of radiologically and clinically suspected lung cancer patients were examined by using conventional CS and CB techniques. In CS, of 85 patients, the fluids were diagnosed as negative for malignancy (85%).
All the 85 negative cytosmear cases were confirmed by CB. In conventional CS method 10 cases were diagnosed as suspicious for malignancy, of which 5 cases turned to be negative and another 5 cases proved to be positive for malignancy in CB. In conventional CS method, reactive mesothelial cells, an abundance of inflammatory cell and paucity of representative cells contributed to the considerable difficulties in making conclusive diagnosis of malignancy.
The false positive diagnosis in CS was secondary to the marked atypia of the mesothelial cell which may be due to the microbiological, chemical, physical, immunological or metabolic insult to the serous membrane or due to the subtle cytomorphological features of some malignant neoplasm, particularly well differentiated adenocarcinoma.11 The problem was compounded by artifact caused by poor fixation, preparation and staining technique. In our settings, fixation and transportation are contributory factors. Generally the reactive mesothelial cells appears rounded and have single centered or eccentric nucleus. Some of the mesothelial cells form cell ball, clusters and takes a signet-ring cells appearance, thus closely mimicking malignancy. The malignant cells have irregular nuclear membrane, nuclear molding and prominent nucleoli with absence of windows. However, these differentiating features are sometimes difficult to identify.
By using CB method, 5 more malignant cases were diagnosed, which were diagnosed as suspicious for malignancy by conventional CS, thus increasing the diagnostic yield by 5% (5/10). Further special stain (PAS-Alcian blue) supported the diagnosis of malignancy.
Another 5 cases were diagnosed as negative for malignancy by CB, which were diagnosed as suspicious for malignancy by conventional CS. The benign cells did not take PAS-Alcian blue stain.
In a parallel study on effusion fluid, Shivakumaraswamy et al, (2012)5 found 15% more diagnostic yield for malignancy on CB. Bhanvadia et al., (2014)12 in another study observed 10% more diagnostic yield in CB. Thapar et al, (2009)4 also showed 13% more diagnostic yield by CB.
In our study, after the final diagnosis as benign or malignant effusion, a critical evaluation was made for diagnostic yield taking a consideration on cellularity, morphology, architecture and background blood. The conventional CS and CB technique were studied for their quality by using point scoring system of Mair et al, (1989).6 In CB, 41% ensured highest morphological details where as in CS, highest morphological detail was acquired in only 11% cases. The comparative result of our point scoring system was analyzed by Wilcoxson Signed Ranks test which was highly significant in respect of cellularity and cellular morphology.
Reactive mesothelial cells have in the past been responsible for simulating malignancy in CS, largely due to the formation of rosettes, pseudo acini or acini, with or without the presence of prominent nucleoli. The CB effectively puts both the features in their proper prospective. That is, the nucleoli does not appear as prominent in CS and the pseudoaciner or acinar strictures can be better appreciated when present in the CB. Similar findings were noticed in the Dekker and Bapp (1978)(13) study. More important is, this CB is a valuable tool in the evaluation of well differentiated adenocarcinoma, where the presence of true acini is seen in the CB with mucin. Positive stain for mucin in CB indicated malignancy.
In our study, we noticed a significant number of macrophages admixed with malignant cells having large pleomorphic nuclei simulating malignant cells. Multiple large PAS positive granules were identified by special stain that were diagnosed as suspicious for malignancy by CS. We also noticed pericellular lacunae in many of the cases of adenocarcinoma specially of mucin secreting type, characterized by cell clusters and Bull’s eye (Target) inclusion like finding was seen in one case of metastatic carcinoma.
There had not been yet any standard method for the preparation of CB from effusion fluid. Our study was done by using 10% alcohol formalin fixative. In our paraffin embedded CB section our attention was drawn by the presence of large clusters of cells either malignant or reactive mesothelial cells in a separate peripheral layer leaving a thick amorphous proteinaceous material in the center. These helped in reducing background artifact and ensured more nuclear details for the distinction between reactive mesothelial cells and malignant cells. Few inflammatory cells were noticed in a less peripheral separate layers admixed with other cells in malignant cases. The other advantage of CB was concentration of cellular materials in one small area that can be evaluated at a glance with all cells lying in the same focal plane of microscope. It bridges the gap between cytology and histology
The cell block made by using 10% formalin-alcohol as a fixative, is a simple, inexpensive method, and does not require any special training or instrument. Multiple sections could be obtained required, for special stain. Therefore, CB technique could be considered as a useful adjunct in evaluating malignant cells in MPE for a final cytodiagnosis, along with the routine CS method.
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- Maskell N, Gleeson F & Davies R. Standard pleural biopsy versus CT-guided cutting-needle biopsy for diagnosis of malignant disease in pleural effusions: a randomised controlled trial. The Lancet, 2003;361(9366):1326-30.
- Thapar M et al. Critical analysis of cell block versus smear examination in effusions. Journal of Cytology, 2009;26(2):60–64.
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- Price BA, Ehya H & Lee JH, 1992. Significance of pericellular lacunae in cell blocks of effusions. Acta Cytologica, 1992;36(3):333-337.
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