|Year : 2017 | Volume
| Issue : 4 | Page : 245-249
Comparison of efficacy of cell block versus conventional smear study in exudative fluids
Sandeep S Matreja1, Kamal Malukani1, Shirish S Nandedkar2, Amit V Varma1, Anjali Saxena1, Arpita Ajmera1
1 Department of Pathology, Sri Aurobindo Medical College and PG Institute, Indore, India
2 Department of Pathology, R.D. Gardi Medical College, Ujjain, Madhya Pradesh, India
|Date of Web Publication||18-Jan-2018|
Dr. Kamal Malukani
Department of Pathology, Sri Aurobindo Medical College and PG Institute, Indore, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: Cytological examination of serous effusions helps in staging, prognostication and management of patients with malignancy. The method has disadvantage of lower sensitivity in differentiating reactive atypical mesothelial cells from malignant cells. Aim: The aim of this study is to compare the cytological features of pleural and peritoneal exudative fluids by conventional smear (CS) method and cell block (CB) method and also to assess the utility of a combined approach for cytodiagnosis of these effusions. Materials and Methods: One hundred and fifty-three pleural and peritoneal exudative fluid samples were subjected to evaluation by both CS and CB methods over a period of 2 years. Cellularity, architecture patterns, morphological features and yield for malignancy were compared, using the two methods. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for diagnosing malignancy were calculated by both methods, using histology as a gold standard. Results: CB method provided higher cellularity, better architectural patterns and additional yield for malignancy as compared to CS method (P < 0.005). Sensitivity, specificity, PPV, NPV, and accuracy by CS method were 69.2%, 95%, 56.25%, 97.08% and 92.8%, while by CB method were 92.30%, 99.2%, 92.30%, 99.28% and 98.6%. Conclusion: The present study shows that it is advisable to routinely make CBs before discarding specimens that are suspicious for malignancy by smear examination.
Keywords: Cellblock, cytological smear, exudative fluids
|How to cite this article:|
Matreja SS, Malukani K, Nandedkar SS, Varma AV, Saxena A, Ajmera A. Comparison of efficacy of cell block versus conventional smear study in exudative fluids. Niger Postgrad Med J 2017;24:245-9
|How to cite this URL:|
Matreja SS, Malukani K, Nandedkar SS, Varma AV, Saxena A, Ajmera A. Comparison of efficacy of cell block versus conventional smear study in exudative fluids. Niger Postgrad Med J [serial online] 2017 [cited 2020 Jul 14];24:245-9. Available from: http://www.npmj.org/text.asp?2017/24/4/245/223465
| Introduction|| |
Accumulation of fluid in a body cavity is referred to as serous effusion and is classified into two types-transudate and exudate. All effusions are pathological regardless of their cellular constituents. Cytological examination of serous fluids is one of the commonly performed investigations as it reveals information about inflammatory and malignant lesions of serous membranes. Accurate identification of malignant or reactive mesothelial cells is a diagnostic problem in conventional cytological smears. Cytodiagnosis by conventional smears (CSs) have got lower sensitivity due to overcrowding of cells, cell loss and different laboratory processing methods. The cellblock technique of examining the fluids, along with concomitant use of smears has shown an added advantage in such cases. The main advantages of cellblock technique are preservation of tissue architecture and to obtain multiple sections from the same material for special stains.
Hence, the present study was undertaken to evaluate the utility of cellblock preparation in increasing the sensitivity of cytodiagnosis of serous effusions received in our laboratory. Cell block preparation with conventional techniques such as agar gel or formol-alcohol is laborious and time-consuming. Therefore, in the present study, plasma-thromboplastin cell block technique (CB) was performed. This technique is simple, cost-effective and readily adaptable in routine hospital laboratories.
The aim of this study is to compare the cytomorphological features of pleural and ascitic exudative fluids by cytological smear method and cellblock method and also to assess the utility of combined approach for cytodiagnosis of these effusions.
| Materials and Methods|| |
The present study was conducted in the Department of Pathology of a tertiary hospital over a period of 2 years from August 2014 to July 2016 and included 153 body fluid samples.
All the body fluids (pleural and ascitic) which were exudative in nature and having protein level of >3.0 gm/dl (determined by Vitros 5,1 J and J dry chemistry analyser).
- All other fluids except pleural and ascitic
- Fluids which were transudative in nature, i.e., having protein level of <3.0 g/dl.
Relevant information regarding age, sex, clinical and radiological findings were obtained from the patient records. Efforts were made in this study to immediately process the fluids, but in 5.88% of samples, when there was delay these specimens were stored for 2–6 h in refrigerator at 2°C to 8°C and then processed. Routine examination of all samples was done and then processed further by the conventional technique and also by CB technique before microscopic examination.
Processing of fluids
All exudative ascitic and pleural fluids were divided into two equal parts. One part was kept for conventional cytology smear preparation (centrifuged smear– CS) while the other part was used for CB preparation.
Specimens were centrifuged at 1500 rpm for 15 min and four smears were made from the sediment in each case. Two smears were made by the cytospin method  entailing centrifugation of specimens at 1100 rpm for 7 min. Half of the smears were fixed by 95% ethanol for Pap staining, and the remaining were air dried for Giemsa staining.
Cell block preparation
Plasma-thromboplastin method  for the CB preparation: the fluids were centrifuged at 2,500 rpm for 15 min. The supernatant was poured off, and 2 drops of thromboplastin and 2 drops of pooled plasma were added to the sediment and centrifuged again at 2500 rpm for 3 min for proper aggregation and condensation of sediments, then cell button sediments were put in a filter paper, wrapped and placed in 10% formalin for 8 h for fixation. Same were processed in histokinette as part of routine paraffin-embedded section preparation. Paraffin-embedded CB sections were stained by haematoxylin and eosin method. Sections were mounted with DPX mountant and examined after drying. Slides were studied under the microscope taking available clinical data into account.
Quality of smears and CBs was assessed according to Miar's criteria which include:,
- Volume of blood/clot obscuring background (large: 0, moderate: 1, minimal: 2)
- Amount of diagnostic cellular material present.(minimal: 0, moderate: 1, abundant: 2)
- Degree of cellular degeneration and cellular trauma.(marked: 0, moderate: 1, minimal: 2)
- Retained architecture/cellular arrangement.(minimal: 0, moderate: 1, excellent: 2).
0–2 point score was given to individual smear/CB based on each of the above criteria, and the final score was calculated by adding the scores of four criteria. Qualitative grouping of smears and CBs was done into three categories:
- Diagnostically unsuitable– (0 score)
- Diagnostically adequate– (1–4 score)
- Diagnostically superior– (4–8 score).
The cytological smears and block sections were examined separately for cellularity, architectural patterns and morphology (cytoplasmic and nuclear details) to render a cytological diagnosis for each case, and the findings were compared. Cytomorphological features were studied to identify the malignancy and the most probable primary site. Yield for malignancy was identified by both the methods. Subsequent histologic study (biopsy from relevant site) was done in all malignant cases and most of the benign cases. Diagnostic accuracy of both CS and CB method for diagnosing malignancy was calculated.
| Results|| |
As shown in [Table 1], 153 fluids were processed, 77 (50.3%) of which were from males while 76 (49.7%) were from females. The peak age was in the 21–30 year age group, accounting for 26.1% (40/153) of cases and those older than 71 years constituted the least frequency (3/153). Pleural fluid constituted 89 (58.2%) of cases while peritoneal fluid constituted the remaining 64 (41.8%) cases. There was no association between type of fluid drained and gender (P = 0.168) [Table 2]. When grouped according to Miar's criteria, 62 (40.52%) cytological smears were diagnostically superior while 91 (59.48%) were diagnostically adequate. On the other hand, 85 (55.56%) CBs studied were diagnostically superior and 68 (44.44%) were diagnostically adequate. No case was found to be diagnostically unsuitable on CS or CB study. There were 66 (43.14%) cases which showed mild cellularity on cytological smear whereas moderate and marked cellularity were seen in 59 (38.56%) and 28 (18.3%) of cases, respectively. Similarly, there were 36 (23.53%) mildly cellular CBs while moderately and markedly cellular CBs were seen in 91 (59.47%) and 26 (17%) cases, respectively. Architectural patterns, such as cell balls, sheets, cell clusters, glands and papillae were better observed in CBs as compared to CSs (P< 0.005). Acute inflammation was observed in 12 (18.8%) of cases, chronic inflammation in 43 (67.2%) of cases, 3 (4.7%) were suspicious for malignancy, and 6 cases (9.4%) were malignant on CS examination of peritoneal fluids. Examination of CBs of peritoneal fluids showed acute inflammation in 12 (18.8%) cases, chronic inflammation in 44 (68.8%) of cases and malignancy in 8 cases (12.5%).
Acute inflammation was observed in 20 (22.4%) of cases, chronic inflammation in 62 (69.7%) of cases, 3 cases (3.4%) were suspicious for malignancy and 4 cases (4.5%) were malignant on CS examination of pleural fluids. Examination of CBs of pleural fluids, on the other hand, showed acute inflammation in 19 (21.4%) cases, chronic inflammation in 65 (73%) of cases and malignancy in 5 cases (5.6%). [Figure 1] show photomicrographs of acute and chronic inflammation in conventional smear and cell block.
|Figure 1: (a) Photomicrograph showing acute inflammation in conventional smear (Pap, ×400), (b) Photomicrograph showing acute inflammation in cell block (H and E, ×400), (c) Photomicrograph showing chronic inflammation in conventional smear (Giemsa, ×400), (d) Photomicrograph showing chronic inflammation in conventional smear (Pap, ×400)|
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Most common cause of benign/reactive effusion was tuberculosis (33/140; %) followed by trauma (23/140; 16.43%), pneumonia (25/140; %), bacterial and viral peritonitis (22/140; %), chronic obstructive lung diseases (9/140; 6.43%), renal diseases (8/140; %), liver cirrhosis (10/140;), pancreatitis (6/140; %) and malignancies (4/140; 2.85%). [Table 3] shows comparison of cytological diagnosis on CS and CB study. [Table 4] shows comparison of cytological diagnosis with clinicoradiological and histological diagnosis. [Figure 2] show photomicrographs of adenocarcinoma in conventional smear and cell block.
|Figure 2: (a) Photomicrograph showing adenocarcinoma in conventional smear (Giemsa, ×400), (b) Photomicrograph showing adenocarcinoma in cell block (H and E, ×400), (c) Photomicrograph showing papillary adenocarcinoma in conventional smear (Giemsa, ×400), (d) Photomicrograph showing papillary adenocarcinoma in cell block (H and E, ×400)|
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|Table 3: Comparison of cytological diagnosis on conventional smear and cell block study|
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|Table 4: Comparison of cytological diagnosis with clinico-radiological and histological diagnosis|
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Thirteen cases were found to be malignant on examination of CBs and on histology. Of these, 8 (5.22%) were adenocarcinoma and 5 (3.2%) were papillary carcinoma. Out of the eight cases which were diagnosed as malignant peritoneal effusions, primary malignancy was from ovary in 5 (62.5%) of cases and from pancreas and stomach in one (25%) of case each. The primary was unknown in the remaining 1 (12.5%) case. Out of the five cases which were diagnosed as malignant pleural effusions, primary malignancy was in the lungs in 3 (60%) of cases and from the breast in one (20%) of cases. In the remaining one case (20%), the primary was unknown.
Out of total 153 cases, 9 were true positive, 133 were true negative, 7 were false positive and 4 were false negative for malignancy on cytology smear examination when compared to histology. Sensitivity of cytology smears for diagnosing malignancy was 69.2% and specificity was 95%. Positive and negative predictive values (NPV) were 56.25% and 97.08%, respectively while accuracy was 92.81%.
Out of total 153 cases, 12 were true positive, 139 were true negative and one each was false positive and false negative on CB examination as compared to histopathology. The sensitivity of CB for diagnosing malignancy was 92.30% and specificity was 99.2%. Positive and NPVs of CB for diagnosing malignancy were 92.30% and 99.28% respectively. Accuracy of cytological CB for diagnosing malignancy was 98.69%.
| Discussion|| |
In the index study, most cases were in the age group 21–30 years. This contrasts with findings by Bansode et al. and Padmavathi et al., who have reported modal number of cases in the age group 41–60 years as 54% and 69.3%, respectively. Greater numbers of inflammatory effusions and relatively fewer malignant effusions were found in our study as compared to those cited,, where more cases of malignant effusions were reported may explain this difference. This is corroborated by our finding that mean age of cases of malignant effusion was 49.6 years.
Even though no significant difference was observed in gender-wise distribution of cases based on our almost equal M:F ratio of 1:0.98. Padmavathi et al. have reported M:F ratio of 1.4:1 while Bansode et al. have reported M: F ratio of 2.1:1. However, similar to our study, others , have also found greater frequency of peritoneal aspirates from females and conversely greater number of pleural aspirates from males, reflecting pattern of pathologies from organs in these respective cavities.
In the index study, sensitivity, specificity, NPV and positive predictive value (PPV) of cytological smear for diagnosing malignancy were reported as 69.2%, 95%, 56.25% and 97.1%, respectively. Bansode et al. have reported sensitivity, specificity, NPV and PPV of cytological smear for diagnosing malignancy as 79%, 100%, 100% and 93%, respectively. Padmavathi et al. have reported sensitivity, specificity, NPV and PPV of cytological smear for diagnosing malignancy as 91.3%, 100%, 100% and 98.3%, respectively. Nair and Manjula have reported have reported sensitivity, specificity, NPV and PPV of cytological smear for diagnosing malignancy as 32.3%, 100%, 14.48% and 85.5%, respectively.
On the other hand, sensitivity, specificity, PPV and NPV of CB were 92.3%, 99.2%, 92.3% and 99.28%, respectively in the index study, which were greater than that for CS. Bansode et al. have reported sensitivity, specificity, PPV and NPV of CBs as 88%, 100%, 100% and 97%, respectively. Padmavathi et al. have reported sensitivity, specificity, PPV and NPV of CB for diagnosing malignancy as 47.8%, 99.1%, 91.7% and 90.6%, respectively. Nair and Manjula have reported sensitivity, specificity, PPV and NPV of CB for diagnosing malignancy as 67.14%, 98.63%, 33.56% and 66.43%, respectively.
In the present study, diagnostic yield for malignancy was 6.53% on CS examination which was increased to 8.5% by CB technique. Hence, additional yield of malignancy was reported as 1.97% which was confirmed by histology. Similarly, in a study by Bansode et al., 15% yield for malignancy on CS examination was increased to 18% on CB study. Similar additional diagnostic yield for malignancy was also noted when CS technique was supplemented by CB method in studies by Khan et al. (20%), Shivakumarswamy et al. (15%), Shukla et al. (15%), Udasimath et al. (13.63%), Thapar et al. (13%), Bodele et al. (7%), Poorana (5%), Sujathan et al.(2.35%), and Padmavathi et al.(1.47%).
Similar to our study, Nair and Manjula  have reported most common primary neoplasm causing pleural effusion as carcinoma of the lung, followed by carcinoma of the breast. Whereas most common primary malignancy in cases of malignant ascitic effusions were adenocarcinoma of gastrointestinal tract (GIT) followed by carcinoma of the ovary, in their study. Sears and Hajdu  have reported most common primary neoplasm causing pleural effusions as carcinoma of the breast (24%) in their study, followed by carcinoma of the lung (19%), and malignancies of lymphoreticular system (16%). Whereas common primary malignancies in cases of malignant ascitic effusions were carcinoma of the ovary (32%), carcinoma of the breast (15%), and lymphoreticular malignancies (7%) in their study. In 15% of cases primary site was unknown. Spieler and Gloor  stated that common primary lesions identified in their study were in breast, ovary, lung and GIT. Shivakumarswamy et al. have reported that common primary lesions in their study were in lung and then in GIT.
In the present study, accuracy of cytological smears for diagnosing malignancy was 92.8% while that of CB was 98.6%. Increased accuracy of CBs over cytological smears in diagnosing malignancy was also noted by previous authors. Thapar et al. found accuracy of cytological smears as 71.42% and that of CBs as 85.72%, Bansode et al. have reported accuracy of cytological smears as 85%–90% and that of CBs as 97%.
In the CS method, reactive mesothelial cells, paucity of representative cells, abundance of inflammatory cells obscuring the morphology of atypical cells and subtle morphological features of some malignant neoplasms contribute to the difficulties which are faced in making a diagnosis. In many nonmalignant conditions, mesothelial cells may show reactive changes such as cytomegaly, high nucleocytoplasmic ratio, multinucleation and mitotic figures. Conventional smear method has low sensitivity due to overcrowding of cells, cell loss and lack of tissue architecture. The CBs which are prepared from the residual fluids can be particularly useful for the identification of tumors that cause diagnostic difficulties in CSs.
| Conclusion|| |
The CB method provides high cellularity, better architectural patterns, better morphological features, additional yield of malignant cells and increased sensitivity for cytodiagnosis of malignant lesions as compared to the CS method.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
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