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ORIGINAL ARTICLE  
Year : 2023  |  Volume : 66  |  Issue : 1  |  Page : 75-80
Body fluids: Comparison of liquid based cytology with conventional cytopreparatory technique


1 Department of Pathology, Government Medical College, Nagpur; Dr. Nemade Nidan Pathology Laboratory and Research Center, Akot, Maharashtra, India
2 Department of Pathology, Government Medical College, Nagpur, Maharashtra, India

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Date of Submission05-Dec-2021
Date of Acceptance01-Jan-2022
Date of Web Publication18-Jan-2023
 

   Abstract 


Introduction: In the diagnosis of malignant tumors, cytological examinations of various body fluids are useful. For the preparation of body fluid, many methods are used till date. The use of liquid-based cytology is new upcoming in the field. Aim: To examine the performance of liquid-based cytology on body cavity fluids as compared to conventional cytopreparatory techniques. Methodology: In the present study, 700 body fluid samples were processed by both liquid- based cytology (BD SurePath) and conventional cytopreparatory technique (Thick & Thin). The performance of both techniques was compared in terms of “smear quality” and “overall diagnostic test performance.” Results: Out of 155 body fluid samples from proven malignancy patients, 32 (20.65%) were reported as Positive for malignancy, 23 (14.84%) as Suspicious of malignancy, and 100 (64.51%) as Negative for malignancy by CS (Thick and Thin). A total of 44 (28.39%) were reported as Positive for malignancy, 12 (7.74%) as Suspicious of malignancy, and 99 (63.87%) as Negative for malignancy by LBC. Conclusion: Liquid-based cytology is advantageous over conventional techniques in cytomorphology of body fluids, but not better in sensitivity and specificity. Also saves cytopathologist's valuable time for screening.

Keywords: Cytomorphology, cytopathology, liquid-based cytology

How to cite this article:
Nemade SH, Kamal MM. Body fluids: Comparison of liquid based cytology with conventional cytopreparatory technique. Indian J Pathol Microbiol 2023;66:75-80

How to cite this URL:
Nemade SH, Kamal MM. Body fluids: Comparison of liquid based cytology with conventional cytopreparatory technique. Indian J Pathol Microbiol [serial online] 2023 [cited 2023 Feb 7];66:75-80. Available from: https://www.ijpmonline.org/text.asp?2023/66/1/75/367938





   Introduction Top


Cytological evaluation of all body fluids like pleural, pericardial, ascetic, cerebrospinal fluid, bronco-alveolar lavage, urine, and fine-needle aspiration fluids are well established in the diagnosis of malignant tumors. Malignant effusions worsen the quality of life in patients with advanced cancer. Malignant serous effusions are usually diagnosed by cytological assessment. Therefore, the ability of cytologists to distinguish between malignant and benign serous effusions is very important for medical management. It is an established fact that the correct interpretation of cells, harvested from serous body cavity fluids, is one of the most difficult problems in diagnostic cytology. Notorious are the reactive mesothelial cells that are a cause of major pitfall for a false-positive diagnosis of malignancy.[1],[2]

For the cyto-preparation of body fluids, various cell concentration techniques used are smearing of the sediment, cytocentrifugation, membrane filtration, cell block preparation, and liquid-based preparations. Each of these methods offers a particular advantage but also suffers from specific shortcomings.[3],[4]

Liquid-based preparation has been basically used for gynecologic cytology. However, during the last decade, liquid-based cytology (LBC) has emerged as an alternative to conventional cytopreparatory methods for nongynecological cytology also.[1] The basic principle of LBC is to collect samples into a liquid fixative solution and then create a monolayer of cells ready for microscopic observation.[2] It also reduces the screening time as cells are concentrated in a small circle.[3]

We decided to investigate the performance of this new technique as compared with conventional cytopreparatory methods.


   Materials and Methods Top


The present study was conducted in the Department of Pathology, Tertiary Care Hospital, Nagpur from July 2016 to July 2018. In the present study, 700 body fluid samples were processed by both, LBC (BD SurePath) and CS (Thick & Thin). The performance of both techniques was compared in terms of “smear quality” and “overall diagnostic test performance.”

Inclusion criteria

I) For assessing the “diagnostic test performance,” cases that were proven malignant either by 1) Clinic-radiologically, 2) histopathologically, 3) patients on chemotherapy and/or radiotherapy treatment were included. There were 155 such cases.

II) For assessing the “quality of the smears,” fluids from cases with proven malignancy that were cytologically positive or suspicious of malignancy were included. Out of 155 cases with proven malignancy, there were 56 such cases that were positive or suspicious of malignancy by cytology.

After taking informed consent (Annexure I), detailed history was taken along with radiological and histopathological findings. All samples (unfixed) received in the cytology section of our pathology department were processed within 1 h. These samples were then centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and the sediment was used to first prepare three conventional smears (Thick and Thin smears). Then, the remaining material was fixed in 12–15 mL of CytoRich-Red solution (provided by the BD SurePath) to lyse RBCs. Conventional smears were stained with Papanicolaou stain, H and E stain, and May–Grünwald–Giemsa stain (MGG) stain. LBC smears were stained with Pap stain in an automated machine.

Both LBC and conventional smears were reported by a single observer. We selected five criteria in order to assess the quality of smears i.e., 1) cellularity, 2) cell distribution, 3) background, 4) cytomorphology, and 5) preservation of architecture. The Mair et al. scoring system was applied to each criterion in order to minimize observation bias. Each of these criteria was given a score of either “0” or “1” or '2” according to the quantitative or qualitative appearance of the material on the slide as shown in [Table 1]. These scores were applied only to fluids that were Positive or Suspicious of malignancy (56 cases) and that also fulfilled the criteria of “proven malignancy.”
Table 1: The Mair et al. scoring system: Criteria and scores to assess quality of conventional smear (CS) and LBC smears

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A cumulative score ranging between 0 and 10 points was calculated for each CS and LBC smears and then categorized into the following three categories:

  • Category 1 (score 0–3): smear unsuitable for diagnosis.
  • Category 2 (score > 3–6): smear adequate for cytological diagnosis.
  • Category 3 (score > 6–10): diagnostically superior smear.


For all practical purposes and following the protocol in our department, the body fluids were reported on cytology either as Positive, Suspicious, or Negative for malignancy using the conventional smears.[5]

We also calculated the diagnostic performance of CS and LBC by comparing with clinico-radiological or histopathological diagnosis (Gold standard).

Statistical analysis

Statistical analysis in the present study was done using OpenEpi Software. Wilson Score was applied for estimation of Sensitivity, Specificity, Positive predictive value, Negative predictive value, and Diagnostic accuracy. True Positivity was calculated for both CS and LBC.

Sample size estimation

The sample size was determined for this diagnostic test design considering sensitivity as the main outcome measure and under the following assumptions-

  1. Sensitivity of Liquid-based cytology = 89%
  2. Sensitivity of conventional method = 75%
  3. Effect size (difference = 14%)
  4. Power of test = 80%
  5. Confidence interval = 95%


Required sample size N = 117

Therefore, total of 155 cases were included in the present study in which both the techniques were applied.


   Results Top


Out of 155 body fluid samples, 32 (20.65%) were reported as Positive for malignancy, 23 (14.84%) as Suspicious of malignancy, and 100 (64.51%) as Negative for malignancy by CS (Thick & Thin). A total of 44 (28.39%) were reported as Positive for malignancy, 12 (7.74%) as Suspicious of malignancy, and 99 (63.87%) as Negative for malignancy by LBC [Table 2].
Table 2: Distribution of cytology results of 155 cases diagnosed by CS and LBC

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The Mair et al. scoring system was applied to five parameters in order to minimize observation bias and to assess the quality of smears. Each of these parameters was given a score of either “0” or “1” or “2” according to the quantitative or qualitative appearance of the material on the slide.

The first criterion was “Cellularity.” When considering the criteria of cellularity, the number of cases having a score of 2 (abundant cells) in LBC was 14 (25%) and in CS was 13 (23.21%) [Table 3]. The proportions of cases with a score of 2 in LBC and CS did not differ significantly (p ≅ 1). However, there was same number of cases having a score 1 (sufficient for diagnosis) in both LBC and in CS. Hence, cellularity in both CS and LBC did not differ significantly.
Table 3: Comparison of criteria of cellularity as seen in CS and LBC smears

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The second criterion was “Cell distribution.” When considering the criteria of cell distribution, the number of cases having a score of 2 (even distribution) in LBC was 26 (46.43%) and in CS was 14 (25%). On the contrary, there was a greater number of cases having a score of 1 (uneven distribution) i.e., 35 (62.50%) in CS as compared to 30 (53.57%) in LBC [Table 4]. The proportion of cases with scores 2 and scores 1 in CS and LBC differed significantly with significant P value, (P = 0.0071).
Table 4: Comparison of criteria of cell distribution as seen in CS and LBC smears

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The third criterion was “Smear background.” When considering the criteria of background, the number of cases having a score of 2 (diagnosis easy) in LBC were 20 (35.71%) and in CS were only 4 (7.14%), however, there was a greater number of cases having a score of 1 (diagnosis possible) i.e., 44 (78.57) in CS as compared to 36 (64.29) in LBC [Table 5]. The proportionof cases with scores 2 and scores 1 in CS and LBC differed significantly, P = 0.0004.
Table 5: Comparison of criteria of background as seen in CS and LBC smears

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The fourth criterion was “cytomorphology.” When considering the criteria of cytomorphology, the number of cases having a score of 2 (good cytomorphology) in LBC was more i.e., 41 (73.21%) as compared to 26 (46.43%) in CS. This difference was statistically significant, P = 0.005. The reverse was true for a score of 1 (fair cytomorphology). There was a greater number of cases having a score of 1 in CS 30 (53.57%) as compared to 15 (26.79%) in LBC [Table 6].{Table 5}

The fifth criterion was “preservation of architecture.” When considering the criteria of architecture, the number of cases having a score of 2 (architecture preserved) in LBC was 13 (23.21%) and in CS was 11 (19.64%) [Table 7]. The proportion of cases with score 2 and score 1 in CS and LBC did not differ significantly, P = 0.6474.
Table 7: Comparison of criteria of architecture as seen in CS and LBC smears

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The cumulative score of the five smear quality parameters (ranging between 0 and 10 points) was calculated for CS and LBC smears and then categorized into the following three categories:

  • Category 1 (score 0–3): smear unsuitable for diagnosis.
  • Category 2 (score > 3–6): smear adequate for cytological diagnosis.
  • Category 3 (score > 6–10): diagnostically superior smear.


In category 3 (diagnostically superior smear), there were more numbers of cases of LBC (35) as compared to CS (19). However, in category 2 (smear adequate for cytological diagnosis), there were more numbers of cases of CS (33) as compared to LBC (21) [Table 8].
Table 8: Category wise distribution of 56 cases

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The true positivity of LBC (36.13%) was greater than that of CS (35.48%). Hence, LBC could pick up more positive cases compared to CS [Table 9].
Table 9: Comparison of true positivity of CS and LBC with Gold standard (Clinico-Radiological or histopathological features)

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   Discussion Top


Cytopreparation of any specimen is as important as cytodiagnosis. The use of cell concentration has improved the diagnostic accuracy, especially for small volumes of the fluid specimen such as body fluid, urine, etc. After the conventional sediment method, many techniques have come into use like the membrane filtration technique which utilizes the use of filter paper with very small pores to trap the cellular material in a limited diameter. However, it is becoming obsolete because of several reasons, it is very labor intensive, filter paper is clogged by noncellular elements, and at times, the cellular morphology is compromised.[6]

LBC has emerged as alternatives to all the above conventional and semiautomated cytopreparatory methods.[7] The improved quality of the resulting liquid-based cytopreparation is because of excellent specimen preservation and cell prefixation. This also enhances the cytomorphologic details. Later, the cells are concentrated or allowed to settle in a small circle onto the glass slide, with this automated system. The automation in monolayer cell preparation provides ease in interpretation of nonoverlapping cells and thereby enhances the ability of the cytopathologists to detect precancerous changes, malignancies, and infectious diseases with high accuracy.

Rishu Mittal et al. (2016)[8] in their study observed that out of 75 cases examined by liquid-based cytology, 12 (16%) were malignant, 2 (2.67%) were Suspicious of malignancy, 53 (70.67%) were rendered inflammatory or benign, and 8 (10.66%) were rendered inconclusive. In our study out of the 700 LBC preparations from the body fluids, 104 (14.86%) were reported as Positive for malignancy, 43 (6.14%) as Suspicious of malignancy, and 553 (79%) as Negative for malignancy which was comparable with Rishu Mittal et al.[8] [Table 2]. Out of the conventional smears in the study by Rishu Mittal et al. (2016) 7 were (9.33%) malignant, 5 (6.67%) were Suspicious of malignancy, 45 (60%) were inflammatory or benign, and 18 (24%) were inconclusive. In our study, out of 700 CS preparations, 93 (13.29%) were reported as Positive for malignancy, 62 (8.86%) as Suspicious of malignancy, and 545 (77.86%) as Negative for malignancy [Table 2]. These values were comparable with Rishu Mittal et al. (2016).[8]

In the study by Rishu Mittal et al. (2016),[8] four out of five cases reported as Suspicious of malignancy by CS were rendered as Positive for malignancy by LBC. In our study, also 11 cases that were diagnosed as Suspicious by CS were confirmed as Positive for malignancy by LBC. Our results are comparable with that of Rishu Mittal et al. (2016).[8]

Charlotte Gabriel et al. (2004)[9] observed that liquid-based preparation gives excellent morphology. Nasar Yousuf Alwahaibi et al. (2014)[7] also observed that the cytomorphology by ThinPrep method was better preserved (61%) than with conventional method (41%). Hoda RS et al. (2007),[10] Priya Singh et al. (2016)[11] and Seung-Myoung Son et al. (2012)[12] in their studies observed better cytmorphology with LBC preparations. In another study conducted by Ann T. Moriarty et al. in 2008,[13] the authors observed that overall ThinPrep performed slightly better and specifically in terms of cytomorphology. But in their study, only pelvic washings and peritoneal fluids demonstrated statistically significant improved performance with ThinPrep. We also had similar observations. In our study, the score of 2 for cytomorphology (meaning that cells showed good nuclear and cytoplasmic details) was seen in 73.21% with LBC and 46.43% with CS.

Aasma Nalwa et al. (2018)[14] using bronchial wash specimens observed the sensitivity and the specificity of LBC as 76.7% and 100%, respectively and that of CS (Cytospin) were 79.5% and 100%, respectively. Abha Thakur et al. (2017)[15] in their retrospective analysis of bronchial washings/bronco-alveolar lavage (BAL) observed that the sensitivity and specificity were 55.8% and 100%, respectively with LBC. Seung-Myoung Son et al. (2012)[12] also observed sensitivity and specificity for CellprepPlus were 50% and 100%, respectively. In our study, the sensitivity and specificity of the LBC technique were 70% and 56.25%, respectively [Table 10]. The sensitivity observed in our study was comparable with other studies. However, the observed Specificity is low in our study as compared to other studies. The reason may be that fluid specimens even from cases of proven malignancy need not always be positive for malignancy. This is either because of early stage of malignancy or response to ongoing therapy.[16]
Table 10: Comparing sensitivity and specificity of LBC with CS

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   Conclusion Top


  • For 700 body fluid samples, positivity rate of LBC was more than that of CS.
  • Cell distribution with the LBC technique as compared to CS (Thick & Thin) technique, was even and in a monolayer without overlapping or overcrowding of cells. The representative cell population in monolayer aids in making an accurate diagnosis in the LBC technique.
  • The background was free of blood and an inflammatory cell as in LBC smears, whereas in CS blood, inflammatory cells and mucus obscured cytological details.
  • Cytomorphology was observed better in LBC than CS.
  • There was increased sensitivity of the LBC technique as compared to the CS technique. The true positivity of LBC was also greater than that of CS. Hence, LBC could pick up more cases that were positive for malignancy as compared to CS.


Research quality and ethics statement

The authors of this study declare that this scientific work complies with reporting quality, formatting, and reproducibility guidelines set forth by the EQUATOR Network. The authors also attest that this clinical investigation study was approved by the institutional ethics committee (IEC), and the corresponding approval number is [935 EC/Pharmac/GMC/NGP-01/12/2016].

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.


   Annexure I Top


Written Consent

I, the undersigned…………………………………… - have been explained by my doctor in my language that a study is being conducted at this hospital – ”Body Fluids: A Comparison of Liquid-Based Cytology with Conventional Cytopreparatory Technique.” I have been informed to my satisfaction the nature, purpose, and risk of the research work.

I have been explained that information given by me and my records will be kept confidential and will be used only for study purpose.

I understand that my participation is voluntary, and that I may refuse to participate or may withdraw from the study at any time, without penalty or loss of benefits to which I am otherwise entitled.

I also understand that the information thus obtained will help in the advancement of medical sciences and serve the humanity.

I hereby voluntarily give my consent to be a part of the study. I will cooperate with the investigator's requests and directions with respect to the study.

Name and Signature of the Patient:

I confirm that I have explained the nature, purpose, and risks of the above research work to -……………………………………………

Name and Signature of the Investigator:



 
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Bae YS, Cheong JW, Chang WS, Kim S, Oh EJ, Kim SH. Diagnostic accuracy of cerebrospinal fluid (CSF) cytology in metastatic tumors: An analysis of consecutive CSF samples. Korean J Pathol 2013;47:563-8.  Back to cited text no. 4
    
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Qamar I, Rehman S, Mehdi G, Maheshwari V, Ansari HA, Chauhan S. Utility of cytospin and cell block technology in evaluation of body fluids and urine samples: A comparative study. J Cytol 2018;35:22-32.  Back to cited text no. 5
    
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Baloch ZW, Lee A, Cobbs C, Roberts S, LiVolsi VA, Gupta PK. Millipore filter cell block preparation: An alternative to cell block in nongynecologic specimens of limited cellularity. Diagn Cytopathol 1999;20:389-92.  Back to cited text no. 6
    
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Alwahaibi NY, Alnoumani NS, Bai UR. Comparison of ThinPrep and conventional preparations for peritoneal and pleural cytology smears. Annu Res Rev Biol 2014;4:3139-49.  Back to cited text no. 7
    
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Mittal R, Ramesh K, Harpal S, Gupta D, Kaur B, Sandhu HS. Study of ascitic fluid by liquid based cytology and its comparision with conventional cytosmears and cell block prepration. Ann Int Med Dent Res 2016;2:16-22.  Back to cited text no. 8
    
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Gabriel C, Achten R, Drijkoningen M. Use of liquid-based cytology in serous fluids: A comparison with conventional cytopreparatory techniques. Acta Cytol 2004;48:825-35.  Back to cited text no. 9
    
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Hoda RS. Non-gynecologic cytology on liquid-based preparations: A morphologic review of facts and artifacts. Diagn Cytopathol 2007;35:621-34.  Back to cited text no. 10
    
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Singh P, Rohilla M, Dey P. Comparison of liquid-based preparation and conventional smear of fine-needle aspiration cytology of lymph node. J Cytol 2016;33:187-91.  Back to cited text no. 11
[PUBMED]  [Full text]  
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Son SM, Koo JH, Choi SY, Lee HC, Lee YM, Song HG, et al. Evaluation of urine cytology in urothelial carcinoma patients: A comparison of CellprepPlus liquid-based cytology and conventional smear. Korean J Pathol 2012;46:68–74.  Back to cited text no. 12
    
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Moriarty AT, Schwartz MR, Ducatman BS, Booth CN, Haja J, Chakraborty S, et al. A liquid concept—Do classic preparations of body cavity fluid perform differently than ThinPrep cases? Observations from the College of American Pathologists Interlaboratory Comparison program in nongynecologic cytology. Arch Pathol Lab Med 2008;132:1716–8.  Back to cited text no. 13
    
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Nalwa A, Walia R, Singh V, Madan K, Mathur S, Iyer V, et al. Comparison of conventional smear and liquidbased cytology preparation in diagnosis of lung cancer by Bronchial Wash and transbronchial needle aspiration. J Cytol 2018;35:94-8.  Back to cited text no. 14
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Thakur A, Bakshi P, Kaur G, Verma K. Liquid-based and conventional cytology for bronchial washings/bronchoalveolar lavages in the diagnosis of malignancy-An institutional experience. J Cytol 2017;34:127–32.  Back to cited text no. 15
    
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Lee YM, Hwang JY, Son SM, Choi SY, Lee HC, Kim EJ, et al. Comparison of diagnostic accuracy between cellprepplus and thinprep liquid-based preparations in effusion cytology. Diagn Cytopathol 2014;42:384-90.  Back to cited text no. 16
    

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Correspondence Address:
Shyam H Nemade
Dr. Nemade Nidan Pathology Laboratory and Research Center, First Floor, Shital Plaza, Bus Stand Road, Akot – 444101, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_1182_21

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]



 

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