Efficacy of liquid based cytology versus conventional smears

Liquid based cytology versus conventional smear; Journal of cytology:2015; pap smear comparison

1. Efficacy of liquid-based cytology versus conventional smears in FNA samples Journal of Cytology / January 2015 / Volume 32 / Issue 1 Tripathy , et al Department of pathology, Shrirama Chandra Bhanja Medical College, Cuttack, Odisha , India

2. What does 'Liquid Based Cytology‘ mean ? It means cytology (study of cells )through a liquid medium . Liquid-based cytology (LBC) as a technique was introduced and tried on Pap smears . It received the Food and Drug Association (FDA) approval in 1996.[1] Thereafter, this technique was performed for non-gynecological cytology, including conventional fine needle aspiration cytology (FNAC), guided FNAC and fluid cytology. INTRODUCTION

3. Cells are collected from cervix or any other site and are placed directly into liquid preservative, rather than transferred to slide. Sample is processed and resultant thin smear is easy to screen . The thin-prep (TP) smears were consistently devoid of obscuring elements and the cells were adequately preserved and evenly dispersed.

4. FDA approved Thin Prep: Filtration and collection of vacuum packed cells on a membrane & transferring to glass slide Sure Path: Centrifugation & sedimentation through a density gradient Not FDA approved Cytoscreen Turbitac Cellslide Papspin Centrifugation and sedimentation by manual techniques. LIQUID BASED CYTOLOGY TECHNIQUES

5. Thin prep Sure Path

6. 1. Dispersion 2. Cell Collection 3. Cell Transfer Principle of thin prep

7. The prepared slide with the new thin prep

8. Relatively inexpensive method Equipments required- vortexer and Laboratory centrifuge Manual membrane based method of LBC

9. Continuing improvement to the Cervical screening Programme Limitations of conventional cytology because there is suboptimal preservation of cells and cellular obscuring by unwanted material, leading to a high proportion of cases reported as inadequate or unsatisfactory for assessment Modernisation of the technique Extra tests – HPV, chlamydia, Neiseria , Gonorrhoea . Why LBC (Liquid based cytology) has been introduced ?

10. Simplifies the collection process for the smear taken Reduced inadequate rate Cells better preserved and not obscured by blood, mucus , or inflammatory cells Infectious organisms retained and better preserved Quicker to screen and report Multiple slides can be produced allowing further testing Residual material in the vial can be made available for further tests eg ; HPV tests Facilitates computer assisted screening which can be available in future(automated cytology) Advantages of LBC

11. More costly than conventional pap smears Preparation is more labor intensive than conventional Loss of background material Some differences in architecture and morphology Requires training for both the screeners and the smear takers Disadvantages of LBC

12. Overcoming the Inherent Limitations of the Conventional Pap Smear Conventional Pap Smear - Majority of cells not captured - Non-representative transfer of cells - Clumping and overlapping of cells - Obscuring material LBC - Virtually all cells of sample are collected - Randomized , representative transfer of cells - Even distribution of cells - Minimizes obscuring material

13. Although studies on LBC are documented for breast, non- gynecologicalspecimen , thyroid, salivary gland and soft tissues, the authors supplemented bone lesions to all the above in their study.

14. This study was conducted in a 2000-bedded hospital. The aim of study was to determine the efficacy of LBC technology over conventional smear methods in FNAC samples. The study was performed on patients attending the Cytology Outpatient Department (OPD) of the hospital over a period of 2 years . Dry tap samples were not included in the study. Samples lost during processing were also rejected. A total of 110 cases were studied. MATERIALS AND METHODS

15. For LBC, samples were processed using the LiquiprepTM processing kit. Processing was performed manually in three steps, i.e. collection , concentration and cellular encapsulation. The LB cytology processing kit contains a preservative solution, a cleaning solution and a cellular base solution. Using the LBC processing kit, the first step is collection of samples. Here , the samples were pushed into a preservative solution, approximately three-times the volume of the sample, and left as such for at least 1 hour.

16. This is followed by the next step, i.e. concentration, where the preserved sample is centrifuged at 3000 rpm for 30 min. For cystic fluids, the supernatant fluid is discarded and, after adding the cleaning solution , it is again centrifuged for 15 min to increase its concentration . A pellet is produced, the supernatant fluid is discarded and a base solution of about 50 microlitre is added to the pellet to make a thin, homogenous suspension

17. S uspension was placed on an ethanol-cleaned glass slide T wo circular smears of 1 cm diameter were made. The second option , which uses a cellular base solution, was used in certain cases , particularly if the sample contains necrotic substance or mucin . About 4 mL of cleansing solution was used in selected cases , along with the preservative solution, and the processing was performed in a similar fashion. This study used both the options.

18. For each FNAC, two passes were performed, the first pass was for CP and the second pass yielded material for the TP preparation , which was processed by the TP kit marketed by Cytcec Corporation. Different stains were used for staining the CP and LP smears, like HE, Pap and Diff Quik . The representative CS and LB preparations were compared by a semiquantitative scoring system using several criteria [Table 1 ].

19. The concentration method used in the procedure was useful in the diagnosis of cystic lesions like aneurysmal bone cysts, Warthin’s tumor of salivary glands and metastatic carcinoma Statistical analysis was performed using the Wilcoxon signed rank test on the SPSS program (Chicago, IL, USA). Every cytological diagnosis was recorded and tabulated and compiled to yield the P -value. P -value is the probability of occurrence of the research finding. P < 0.005 is significant. The lesser the P -value, the more accurate is the result.

20. A total of 110 cases were studied, which were distributed among 30 cases of breast (10 fibroadenoma , 8 mastitis and 12 ductal carcinoma), 40 cases of lymph node (20 reactive hyperplasia, 12 granulomatous lymphadenitis, 3 lymphoma and 5 metastatic carcinoma ), 10 cases of salivary gland ( 2 sialadenitis , 5 mixed tumor , 1 Warthin’s tumor and 2 mucoepidermoid carcinoma ), 18 cases of thyroid (10 colloid goiter, 4 thyroiditis and 4 carcinoma cases) 12 cases of soft tissue and bone lesions (5 benign and 7 malignant ). RESULT

21. Pitfalls noticed as cytomorphologic alterations in LBCs are small-sized cell clusters, with more single cells than sheets. The cells are generally smaller, occasionally show spindling chromatin detail is attenuated, nucleoli are more prominent , intranuclear inclusion is difficult to visualize, decrease of extracellular particles is seen, there is a decrease in the number of small mononuclear cells/red blood cells/ myoepithelial cells and the background matrix is altered.

22. Comparison between cytologicalfeatures in LBC and CS was carried out using the Wilcoxon signed rank test separately for each organ . In this study , in two cases of pleomorphic adenoma, one case of Warthin’s tumor and two cases of colloid goiter, a support of CS was needed due to pitfalls in LBC described above . All the bony lesions were perfectly diagnosed in LBC.

23. For breast cases, fibroadenoma , mastitis and ductal carcinoma were better diagnosed on LBC preparations due to clarity of the nuclear features [Figure 1]. In fibroadenoma breasts, although stromal fragments were lost, LBC proved to be useful in the diagnosis based on visualization of ductal aggregates and bipolar cells. For duct carcinomas, the same criteria were useful Comparison data for lymph nodes suggested that the RS cells were more easily diagnosed on LBC smears due to monolayering . Immature lymphoid cells and foreign cells were also better visualized because of similar reasons.

24. Liquiprep cytosmear of the breast showing pleomorphic-looking ductal epithelial cells in a cluster and acinar pattern in a clear background

25. In case of salivary gland lesions, the mixed salivary tumor was better visualized in the conventional smear because of preservation of the stromal component. Cystic neoplasms of the salivary glands like Warthin’s tumor, mucoepidermoid and adenoid cystic carcinoma were better appreciated in LBC smears because of concentration. In sialoadenitis , the LBC preparations were superior to the CP smears .

26. In the thyroid gland, LBCs were not very useful in goiter and infectious lesions, but they proved very useful in neoplastic lesions especially anaplastic and medullary carcinoma. In lytic bone lesions like aneurismal bone cyst (ABC), giant cell tumor (GCT) and metastasis, better results were obtained by LBC preparations because of background clearing and concentration phenomenon In soft tissue lesions, LBC showed good results because of lack of background material.

27. Liquiprep cytosmear showing papillary carcinoma of the thyroid in a clear background with nuclear grooving

28. Because of the pitfalls of LBC, as described above, in some selected cases of granulomatous lymphadenitis ( lacking epitheloid cells), mucinous tumors (lack of mucin ), colloid goiter (thin colloid fragmented) and pleomorphic adenomas of the salivary gland ( myxoid background poor and present in droplets ), a support on CS may prove useful. As observed by the authors, LBC of bony lesions do not require support of CS.

29. TP preparations are superior to CPs with regard to clear background , monolayer cell preparation and cell preservation . It is easier and less time consuming to screen and interpret TP preparations because the cells are limited to smaller areas on clear backgrounds, with excellent cellular preservation . CONCLUSION

30. However , TP preparations are more expensive than CP and require some experience for interpretation. Familiarity with artifacts is essential to avoid misdiagnosis. LBC is strongly advocated in the best interest of public health, by improving the quality of the sample and reducing the likelihood of false negative cytology results.