European Journal of Cardiovascular Medicine

Normally, body fluid are present with their constituents within serous body cavities, in minimal quantities. These body fluids undergo abnormal qualitative and quantitative changes in different pathological conditions and results in effusion. In various inflammatory and neoplastic conditions, cells exfoliate into these body cavity fluids and study of these exfoliated cells help in diagnosis. In cancer patients presence of malignant cells in effusion is an important prognostic indicator ⁽¹⁾.

Cytological examination of effusion samples aids in diagnostic, therapeutic and prognostic implications ⁽²⁾ and by means of conventional smears however carefully prepared leaves behind a large residue that is not further investigated but might contain valuable diagnostic material ⁽³⁾, also at times because of lack of morphological details of the representative cells contributes to difficulties in making diagnosis on conventional smears. The accurate identification of cells as either malignant or reactive mesothelial cells is a diagnostic problem and is a serious pitfall in conventional cytological smears. Due to cellular overlapping, delaying artifact, suboptimal processing, preparatory cytotechniques and useful material that is left behind causes lower diagnostic yield in conventional smear method. This residual material can be used in improving the diagnostic yield by the cell block technique ⁽⁴⁾.

The cell block technique is easy, safe, cheaper and reproducible even in resource limited settings. Cell blocks provided the best material for interpretation with less background staining and it gives results that most closely approximated those found in the surgical pathology literature .They are particularly useful for differentiation of tumors when conventional smears are not sufficient alone. The usefulness of cell block depends on the availability of diagnostic material for further studies like histopathology, histochemistry and immunochemistry for classification of tumor and for identification of etiological agents with microbiological stains ⁽⁵⁾.

According to surgical pathology literature Cell block preparation increases the sensitivity in diagnosing malignancies, has the ability to reduce false positive interpretations, also has better cellularity, architecture, morphological features and an additional yield of malignant cells that increases the sensitivity of cytodiagnosis compared to conventional smear method⁽⁶⁾.

Many techniques are available that vary in scope, type of fixative and embedding technique used, making valid comparison difficult. Method of cell block preparation by using plasma- thromboplastin method has more sensitivity as compared to conventional smear method. This method is very simple and cost effective and requires no extra material compared to other methods⁽⁷⁾.

This study aims to assess the efficacy of cell block preparation for cytodiagnosis of serous effusions. Since cell block technique for fluids has an architectural pattern, it is an effective method in comparison to routine conventional smear cytology. This study will have significant impact mainly in correct pathological diagnosis and hence patient’s management and prognosis.

Aim of our study is to compare cytology of body fluid by using routine conventional smear

method and cell block technique of specimen received in Department of Pathology, M.G.M. Medical college, Indore (M.P.).

A cross-sectional study was conducted in the Department of Pathology, Mahatma Gandhi Memorial Medical College, and M.Y Hospital, Indore Madhya Pradesh, India. Serous effusion samples of pleural and peritoneal cavities obtained from both male and female of all age groups are included. Poorly preserved sample; Inadequate sample (< 10 ml); Body fluid effusion other then pleural and peritoneal fluids are excluded.

Methods of collection of data: Data was collected in a pretested proforma. Clinical findings and radiological data of all case of effusion samples sent to laboratory were noted. These fluids will be analyzed for physical properties like volume, color, appearance and odor. About half amount of the fluid will be processed by routine technique and staining was done using Papanicolaou stain. Remaining fluid was subjected to cell block technique using Plasma- Thromboplastine method and then processed like a histopathology sample.

Samples of fluids received were divided in two halves. One half of specimen was centrifuged at 1500 rpm for 15 minutes. A drop of sediment was put on a glass slide and spread it with another glass slide to make smear. Smears made which were wet fixed and stained with Papanicolaou stain. Remaining half of the specimen was subjected to cell block preparation ⁽⁸⁾.

1. Firstly, centrifuge the sample at 2000 rpm for 10 minutes.
2. Discard the supernatant.
3. Add 2 drops of pooled plasma to the sediment and mix well.
4. Then add 2 drops of thromboplastine (Neoplastine CI Plus by Dignostica Stago S.A.S.) and mix well again.
5. Allow the tube to stand for 5 minutes to form the clot.
6. The clot in the test tube is slid (by gently tapping on the bottom of the tube) onto the Whatman’s No.1 filter paper pre-moistened with formalin fixative.
7. Wrap the sediment securely in the Whatman’s No.1 filter paper and place it into labeled tissue cassette.
8. Put the cassette into a jar containing fixative (at least for 4 hours).
9. Process further as tissue specimen in automated tissue processor.
10. The paraffin embedded cell button (cell block) were prepared by placing the processed cell button in warm liquid paraffin, which forms a firm block after cooling and enables it to be cut on a microtome and identification number is placed adjacent to it.
11. Section of 4-6 micrometer thickness were prepared by microtome and section stained with the hematoxylin and eosin stain.

Method of analysis: A cytological diagnosis was rendered for each case on smears and cell block separately. Each individual slide was objectively analysed for a number of features such as cellularity, nuclear and cytoplasmic preservation, background and the ability to make definite diagnosis on them. Routine smears and cell block were both classified into benign, suspicious for malignancy and malignant.

Benign category includes ⁽⁸⁾ (i) reactive mesothelial proliferation, (ii) acute inflammation, (iii) chronic inflammation, (iv) lymphocytic effusion, and (v) specific infections.

Suspicious for Malignancy represents ⁽⁸⁾ smears that lack morphological features of frank malignancy but are enough to warrant suspicion of malignancy.

Malignant category represents smears that show unequivocal malignant cells ⁽⁸⁾.

Table 1: Distribution of study participants in to different Age groups

Table 2: Distribution of study participants according to Gender

Table 3: Distribution of cases as per the type of body fluid received

Table 4: Distribution of cases according to appearance of fluid

Table 5: Distribution of cases according to Cytological Findings in CS method

Table 6: Distribution of cases according to Cytological Finding in CB method

Table 7: Analysis of result by conventional smear method

Table 8: Analysis of result by cell block method

Table 9: Comparison of Results Between Conventional Smear and Cell Block Methods

Table 10: Analysis of discrepancies between CS and CB method.

No. Of suspicious cases (6) on Conventional smear method reduces to 3 on cell block method. Thereby increasing the no. Of proven malignant cases on cell block method.

1. Conventional Smear (CS) Cell Block (CB)

Figure 14: Comparison between CS and CB method. CB method showing better cellular architecture with better preservation of cellular morphology.

1. Conventional Smear (CS) B. Cell Block (CB)

Figure 15: Comparison between CS and CB method . CS showing only inflammatory cells with few atypical cells, whereas CB showing frank malignant cells morphology.

1. Conventional Smear (CS) B. Cell Block (CB)

 Figure 16: Comparison between CS and CB method in case of inflammatory etiology.

1. Conventional Smear (CS) B. Cell Block (CB)

Figure 17: Comparison between CS and CB method in case of malignant etiology.

In the present study of cytodiagnosis of serous effusions by using comparative approach of routine smear method and cell block technique, pleural fluid and ascitic fluid, samples were studied. The study groups vary markedly in various studies done on the subject. Sherwani R et al selected 209 cases, Thapar et al did study of 120 cases, Manisha B et al had a comparable study group to our study with 49 cases of effusion, Jalal R et al studied a very large number of cases i.e. 600 cases^(9,10,5,7). In our study, total cases were 60. 

Various studies have been done on cell block cytology. In a study by Manisha et al out of 38 cases, 23 (60.5%) cases were diagnosed as malignant, another study by Bodele AK et al 39 (30.9%) cases were malignant. Thapar M et al, diagnosed 70 (36.8%) cases as malignant.  In our study of 60 cases, cell block diagnosed 16 (26.6%) cases as malignant. Similar results were obtained by nearly all the studies, increasing the number of positive cases by cell block method. Just as in smears, different studies showed different results on cell block examination, which might be due to sample size, sampling technique and method of cell block preparation.

In a study by Manisha B et al within the group of 38 serous effusion, there was absolute concordance between smear and cell block in 37 cases. In a case of ascitic fluid cytology, smear was negative but cell block was positive. It was an interpretation error in which it was difficult to differentiate between reactive mesothelail cells and adenocarcinoma. More confirmed diagnosis could be rendered on the basis of cell block⁽⁷⁾.Bodele AK et al did a study, out of 126 cases, 39 (26%) fluids were reported as positive for malignant cells by cell block method while by routine method only 29 (23.01%) fluids were reported as positive for malignant cells. On comparing the two techniques, increase in number of positive cases by cell block method was 3%⁽¹²⁾.  In a study by Thapar M et al of the 70 cases which were histologically/ FNAC confirmed of malignancy, 50 cases were reported positive on conventional smear method, while on cell blocks 60 cases were found to be positive for malignancy which came out to be 50 % and 58.3% out of 120 cases. There was increase in number of positive cases on cell block by 8.3%⁽¹⁰⁾. Our study also revealed increase in positive cases by cell block method. Only 11 (18.3%) cases were reported positive for malignant cells by smear method but they were increased to 16 (26.6%) in number on studying cell block. Hence, an increase of 8.3% was observed while reporting by cell block.

In our study, within the group of 60 cases of effusion, there was absolute concordance between smear and cell block in 52(86%) cases. In rest 08(14%) cases, 06(10%) smears were suspicious for malignant cells, while others were negative. But on cell block, 06 (26%) cases were positive for malignant cells and 03 were suspicious for malignancy as shown in Table 8 and 9.Hence, 5(8%) more cases were found to be positive for malignant cells on cell block which were previously given suspicious and negative for malignant cells on smear examination.

Cell block definitely increases the total number of cases being diagnosed as positive when compared to conventional smears. This finding was unanimously observed by all the studies. Increase in percentage was between 3-8 %. Cell block leads to concentration of same cytological material in a small wax section whereas in smears the material is spread on whole of the slide. It is also more convenient to see multiple sections of cell block on fewer slides as compared to viewing large smears on more number of slides to see for malignant cells, hence also reducing the chance of missing malignant cells.

It is suggested that smearing and staining of smears produce more cellular degeneration. Cell block showed better preserved cell morphology, which appears to be due to fixative and better handling of sample when processed as histopathology. And significant difference was observed in retention of architecture between both the techniques if the diagnostic material was more. Cell block sections were diagnostically superior to smears, however if cytological material was abundant, it affected little.

Cell block method of study of body fluid is simple, cost effective and requires minimal additional material. The processing of fluid specimen is done as paraffin embedded cell block by fixing in 10 % buffered formalin.

Apart from increased cellularity, better morphological details were also obtained with cell blocks which includes retention of the architectural patterns like cell balls and papillae and three dimensional clusters, superior nuclear and cytoplasmic preservation, intact cell membrane and fine chromatin details. Yet, conventional smear study is routinely practiced since it is easier to perform and useful for arriving at diagnosis in short period of time. Cell block preparations can be combined with conventional smears whenever it is possible to improve the diagnostic accuracy. Cell block helps in increasing the diagnostic yield compared to the routine methods.

Special stains and other ancillary techniques which can be easily performed on cell block sections even many days after receiving of fluid sections is one of the biggest advantage of cell block. In future, use of cell blocks with these advantages might reduce the need of biopsies from in accessible and difficult sites in body where cell blocks may be used for making the final diagnosis.

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