Comparative study of manual liquid-based cytology (MLBC) technique and direct smear technique (conventional) on fine-needle cytology/fine-needle aspiration cytology samples.

BACKGROUND: Liquid-based cytology technique enables cells to be suspended in a liquid medium and spread in a monolayer, making better morphological assessment. Automated techniques have been widely used, but limited due to cost and availability. AIM: The aim was to establish manual liquid-based cytology (MLBC) technique on fine-needle aspiration cytology (FNAC) material and compare its results with conventional technique.
MATERIALS AND METHODS: In this study, we examined cells trapped in needles hub used for the collection of FNAC samples. 50 cases were examined by the MLBC technique and compared with the conventional FNAC technique. By centrifugation, sediment was obtained and imprint was taken on defined area. Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) staining was done. Direct smears and MLBC smears were compared for cellularity, background, cellular preservation, and nuclear preservation. Slides were diagnosed independently by two cytologists with more than 5 years' experience. Standard error of proportion was used for statistical analysis.
RESULTS: Cellularity was low in MLBC as compared with conventional smears, which is expected as remnant material in the needle hub was used. Nuclei overlap to a lesser extent and hemorrhage and necrosis was reduced, so cell morphology can be better studied in the MLBC technique. P value obtained was <0.05.
CONCLUSION: This MLBC technique gives results comparable to the conventional technique with better morphology. In a set up where aspirators are learners, this technique will ensure adequacy due to remnant in needle hub getting processed.

Introduction

Liquid-based cytology

Liquid-based cytology is a technique that enables cells to be suspended in liquid medium and spread in monolayer and thus better morphological assessment is possible. It includes the preparation and evaluation of cells collected in a liquid fixative. It is being introduced in developed countries to improve the sensitivity of the Pap test.[1] Liquid-based preparations are increasingly being used both for gynecologic (gyn) and nongynecologic (nongyn) cytology, including fine-needle aspirations.[2] Two technologies-ThinPrep (Hologic, Marlborough, MA, U.S.A.) and BD SurePath (BD Diagnostics-TriPath, Burlington, NC, U.S.A.) have been more widely used.[1]

The advantages of liquid-based cytology include improved sensitivity and specificity since fixation is better and nuclear details are well-preserved. Abnormal cells are not obscured or diluted by other epithelial or inflammatory cells. There is, therefore, a lower rate of unsatisfactory cervical cytology samples. The residual cell suspension can be used to make further cytological preparations or used for other tests like detection of human papilloma virus DNA. Other ancillary techniques like immunocytochemistry can also be performed on the residual sample.[13]

The more widely used technologies for liquid-based cytology require expensive equipment.[1]

The aims and objectives of the study were to prepare cytology smears using the manual method of liquid-based cytology on material remaining in the needle hub and syringes after fine-needle cytology (FNC)/fine-needle aspiration cytology (FNAC),[4] observe morphological features, compare results of direct smear examination with manual liquid-based smears and correlate with histopathological findings in those cases in which a biopsy was also done.

Materials and Methods

Patients attending cytology outpatient department for FNC/FNAC in a rural tertiary health care center were selected for study. All cases where definitive diagnosis can be made on conventional technique were included in the study, while cases where material was inadequate for diagnosis by either method were excluded from the study. Fifty cases were enrolled in this study approved by the Ethics Committee of the Institute. In this study, we used needle hub material because using entire material would necessitate second prick. This being first study for the establishment of the technique; for ethical reasons second prick was avoided.

Informed consent from all cases was taken. Conventional FNC/FNAC of the patients was carried out. One smear was wet fixed in 95% ethyl alcohol and Pap stained. Air dried smear was kept for May-Grünwald Giemsa (MGG) staining. Material remaining in the needle hub and the syringe was processed immediately for manual liquid-based cytology (MLBC) technique. Whenever, there was delay, samples were preserved at 4°C and processed subsequently for MLBC technique. For this purpose, we prepared the tubes in which molten paraffin wax was added at the base. Over the flat surface of the wax filter paper equal to the size of the bore of the tube was inserted carefully. The needles were removed from the syringes after the smears were made by the conventional method. 2 mL of phosphate buffer at the pH 7.4 was then aspirated into the syringe and the needle was attached to the syringe. The phosphate buffer was then expelled through the needle forcing the cells to be released with the buffer. Glacial acetic acid was added to the hemorrhagic aspirate. Our final tubes contained paraffin wax at the base, filter paper and material obtained in phosphate buffer at the top [Figure 1]. The tubes were centrifuged for about 8 min at 1200 rpm and the supernatant removed. Paper was withdrawn carefully from the tube and imprint was taken on a slide. Wet fixed smears were Pap stained. Air dried smears were MGG stained [Figure 2].

Figure 1

The steps involved in the manual liquid based cytology technique

Figure 2

The final steps involved in the manual liquid based cytology technique

Standardization of the method

Various variables studied were, speed of centrifugation -tried 800, 1000, 1200, and 1500 rpm, and finally set at 1200 rpm. Time-samples centrifuged for 8, 10, and 12 min and finally set at 8 min. Tube — tried conical tubes, small size tubes and finally, selected the present tubes. Addition of fixative with albumenized slides was tried but it gave pinkish background so it was not used.

Parameters studied

Conventional smears and MLBC smears were compared under the heading of cellularity, background, cellular preservation and nuclear preservation.

Slides were diagnosed and parameters to be studied were graded independently by two cytologists with more than 5 years’ experience. Final diagnosis on both slides was given. Analysis of 50 cases was done. Standard error of the proportion was used for background study analysis. Histopathological correlation was done wherever possible.

Results

In our study, 50 cases were studied. All cases were having same diagnosis on conventional technique and MLBC technique. Histopathological correlation was done in 14 cases [Table 1]. Cellularity was low in (MLBC) as compared with conventional smears as we are using only material remained in the needle hub, but nuclei overlap to the lesser extent, so better cell morphology can be studied in the MLBC technique [Figure 3]. For background study, we applied standard error of proportion statistics to the data obtained.[5] In MLBC technique hemorrhage and necrosis was reduced as compared with the conventional technique, P value was obtained <0.05, which is highly significant [Figures 4, 5 and Table 2]. Myxoid stroma, colloid and mucin was retained [Table 2].

Table 1

Distribution of the cases, diagnosis on conventional and MLBC techniques and correlation with histopathology

Figure 3

Comparison of nuclear characteristics in conventional and manual liquid-based cytology techniques

Figure 4

Comparison of necrotic background in case of Squamous cell carcinoma respectively in conventional and manual liquid-based cytology technique

Figure 5

Comparison of hemorrhagic background in case of advanced breast malignancy respectively in conventional and manual liquid-based cytology technique

Table 2

Background studies

Discussion

This study was conducted in a resource poor setting in a setup where FNAC is done by residents who rotate every 6 months. This is true for large number of hospitals attached to medical colleges. In this case, diagnostically important material remains in the needle hub and is wasted. This study fulfilled various needs in this scenario. There are no ethical issues in this study as the cellular material remaining in needle hub was used as an adjunct to routinely processed material. The patients in our setting are often diagnosed with advanced stage of disease, for example, caseating lymph nodes, large necrotic metastasizing malignancies. However, even in necrotic inflammatory or hemorrhagic smears malignancy can be underdiagnosed as demonstrated by early findings of RODEO study team.[6] Liquid based cytology is gaining popularity in non gynec FNAC, due to advantages of a cleaner background and reproducibility, better cellular morphology and ability to gain sample for ancillary techniques.[7] Compared to cell block MLBC technique has the advantage of viewing cell morphology comparable to conventional smear without presence of necrosis, hemorrhage. In thyroid FNAC study Saleh et al.[8] found ThinPrep (liquid-based cytology [LBC]) preparation superior to cell block technique to detect atypical/neoplastic thyroid lesions. We have attempted this manual technique considering limitations of our setup. All materials used in this technique are readily available in a modest pathology laboratory and the technique can be easily mastered. We can use better quality filter papers if available. In resource poor settings MLBC is viable approved alternative to automated LBC techniques.[9]

This method was found to retain diagnostically important material such as myxoid stroma, colloid, mucin and a larger study is needed to assess diagnostic accuracy and limitations in specific organs. Though this is the first study using this technique, other MLBC protocols have been tried successfully. Kavatkar et al.[1] found MLBC as cost-effective alternative to automated methods. Godwin et al.[4] studied MLBC on needle hub remaining material in breast malignancy cases and found it diagnostically useful. Alves et al.[10] compared manual and automated methods of liquid-based cytology and found MLBC as effective as in preserving cellular details as automated techniques. The choice of method will depend on price, availability and procedures involved. Maksem et al.[11] found manual method makes liquid-based cytology inexpensive and does not require specialized preparative devices.

Conclusion

This simple technique using centrifugation, sediment cytology Preparation and layering due to flat surface on paraffin wax can be used as an adjunct in necrotic or large masses or as a diagnostic method after larger studies.