| Abstract|| |
Background: The role of fine needle aspiration cytology (FNAC) in the diagnosis of malignant lesions is well documented. Conventionally obtained fine needle aspiration (FNA) smears frequently contain well preserved viable intact tissue fragments (microbiopsies). Aims: The present study was aimed at evaluating the microbiopsies and carrying out further cyto-histopathologic correlation in order to assess what additional information they could provide. Materials and Methods: A total of 116 FNA smears from clinically suspected malignant lesions were examined, of which 81 smears (70.0%) contained representative tissue fragments of the tumors. Histopathological details were available in 75 cases. Immunocytochemistry (ICC) was applied in selected smears as and when required. Results: Tumors in which microbiopsies aided in diagnosis chiefly included soft tissue malignancies (12 cases), typed into malignant peripheral nerve sheath tumor, fibrosarcoma, spindle cell sarcoma and malignant melanoma; lung tumors (14 cases) classified as small cell carcinoma, large cell carcinoma, squamous carcinoma and adenocarcinoma; breast tumors (08 cases), typed into ductal, medullary and lobular carcinoma; and lymph node metastasis (19 cases) from primaries in lung, larynx and thyroid. The cyto-histopathologic concordance was raised from 81.2% in the absence of microbiopsies to 93.2% in their presence. Conclusions: FNA smears containing microbiopsies are of ample help in establishing firm diagnosis, tumor typing, and predicting possible primary sites in metastatic tumors which were not possible by cytology alone. Hence, this technique can be utilized to enhance the diagnostic accuracy of FNAC, if put into practice in evaluation of routine cytology smears, without increasing any financial burden on patients.
Keywords: FNAC, histopathology, ICC, microbiopsy, malignancy
|How to cite this article:|
Sherwani RK, Akhtar K, Ray PS, Basha M. The significance of microbiopsies in cytological smears
. Indian J Pathol Microbiol 2013;56:200-3
| Introduction|| |
FNAC is one of the first line investigations in the diagnosis of clinically suspected malignant lesions. In modern day oncology practice, it is an indispensible tool, both as a modality of pre-therapeutic investigation and also in detecting recurrences. Yet it is not always possible to reach a definitive diagnosis on the basis of FNAC alone.
Orell et al., are of the opinion that, "If the treatment involves neoadjuvant, preoperative therapy, then cytodiagnosis must be equivalent to a histological tissue diagnosis, i.e. providing malignancy grade and histogenetic tumor type." With this background in mind, the main focus of this study was on the assessment of microbiopsies, which are defined as well preserved viable tissue fragments obtained on cytology smears. 
The objective of the present study was to evaluate FNA smears containing tissue fragments (microbiopsy) obtained from malignant lesions and to carry out further cyto-histopathologic correlation. Our study also aimed at exploration of further findings of significance.
| Materials and Methods|| |
This study was conducted on a total of 116 FNA smears obtained from clinically suspected malignant lesions over a period of 6 months.
The tumors were aspirated using 22-23 gauge disposable needles and 10 ml disposable syringes except in cases of 3 bone lesions where 18 gauge needles were used. Deep seated lesions were aspirated under computed tomography (CT) or ultrasonography (US) guidance. The smears prepared were fixed in 95% ethyl alcohol, stained with hematoxylin and eosin (H and E) and papanicolaou stains, and examined microscopically for the presence of well preserved viable tissue fragments, disregarding the loose tumor cells in the background. Special stains like Periodic acid Schiff and immunocytochemistry (ICC) were used in selected number of cases, as and when required. For ICC, smears were dipped in 3 changes of 100% absolute alcohol for 5 min each, followed by washing in running tap water for 5 min. Next, the smears were subjected to endogenous peroxidase for 30 min (0.3% H 2 O 2 in absolute methanol). This is followed by washing under running tap water, antigen retrieval (low heat with microwave) and 3 changes of wash with 10 mM Tris Buffer solution (TBS of pH 6.0) for ~ 5 min. Subsequently, blocking serum was applied, followed by incubation with primary antibody (diluted 1:40; Biogenex, San Ramon, CA-94583,USA) for ~1 h and TBS washing. Further, secondary antibody (Biogenex, San Ramon, CA) was applied for 30 min, followed by washing with TBS and application of avidin - biotin complex(ABC). Afterwards, TBS washing was carried out with subsequent staining with Diamino Benzidine reagent (DAB- Biogenex). Harris' hematoxylin was used as counter stain. Finally, the smears were dehydrated with absolute alcohol, cleared with xylene and mounted with DPX. The cases were accompanied with appropriate controls.
Subsequent histopathological correlation with the results of FNAC was made, wherever available. The histopathological diagnosis was taken as the gold standard against which the cytological diagnosis was compared.
| Results|| |
The study population was distributed over an age group of 13 years (youngest patient) to 90 years (oldest patient) with a mean age of 50.1 years. Majority of the cases, 35 (30.2%) were clustered in the sixth decade. There was a male preponderance with a male-female ratio of 1.9:1.
Out of the total of 116 FNA smears, 81 smears (70.0%) contained well preserved representative tissue fragments (microbiopsies) of the tumors which aided in correct diagnosis in almost all cases. The organs with highest yield of microbiopsies were lymph nodes, 23 cases (85.2%) followed by soft tissue tumors,12 cases (75.0%), lungs, 14 cases (73.8%) and breast, 8 cases (72.7%), bones, 6 cases (66.7%), thyroid, 5 cases (62.5%), liver, gallbladder and parotid, 3 cases each (42.8%), prostate, 2 cases (50.0%) and a single case each (100%) of ovary and rectum.
When assorted according to the type of malignancy diagnosed on the basis of microbiopsies [Table 1], adenocarcinoma was found to be the most common diagnosis, 17 cases (20.9%), followed by squamous cell carcinoma, 11 cases (13.6%), spindle cell sarcoma, 08 cases (9.9%), and ductal carcinoma breast, 05 cases (6.2%). A single aspirate from an ovarian mass taken under US guidance showed tissue fragment with multilayered finger like papillae composed of aggregates of malignant glandular cells and hobnailing with psamomma bodies [Figure 1]. The FNA diagnosis of papillary serous cystadenocarcinoma of ovary was found to be concordant with the histological diagnosis. A case of a gallbladder mass aspirate in a 55 year old lady showed tissue fragments composed of cuboidal cells having pleomorphic nuclei with nuclear faceting [Figure 2] and a cytologic diagnosis of adenocarcinoma was made. Subsequent histopathology showed nests of malignant cuboidal epithelial cells arranged in well-formed glands and thus affirmed with the FNA diagnosis. Another aspirate from a thigh swelling in a 42 year old male patient showed cellular fragment with thin wavy nuclei and palisading in a fascicular growth pattern [Figure 3]. A cytological diagnosis of malignant peripheral nerve sheath tumor (MPNST) was made which was confirmed by histopathology.
|Figure 1: Papillary Serous Cystadenocarcinoma Ovary: Tissue fragment shows multilayered finger like papillae composed of aggregates of malignant glandular cells and hobnailing with psamomma bodies. Periodic acid Schiff stain ×40|
Click here to view
|Figure 2: Adenocarcinoma Gallbladder: Tissue fragments composed of cuboidal cells having pleomorphic nuclei with nuclear faceting. Papanicolaou stain ×40|
Click here to view
|Figure 3: Malignant peripheral nerve sheath tumor (MPNST): Cellular fragment with thin wavy nuclei and palisading in a fascicular growth pattern. H and E stain ×40|
Click here to view
|Table 1: Distribution of cases according to the type of malignancy diagnosed on the basis of microbiopsies|
Click here to view
A comparative study was done between primary and metastatic tumors diagnosed on the basis of microbiopsies [Table 2]. Primary tumors comprised 58 cases (71.6%) while 23 cases (28.4%) were metastatic from various sites. Lungs were the most common primary site, 14 cases (17.3%) while lymph nodes were the most common secondary site, 19 cases (23.5%). Since all specimens from lung tumors were obtained under image guidance in our study, the cytological yield was better with more tissue fragments.
|Table 2: Comparison between primary and metastatic tumors diagnosed on microbiopsies|
Click here to view
Histopathological details were available in 75 cases. Of these, 59 cases corresponded to those which had microbiopsies on FNA smears and remaining 16 were from lesions in which microbiopsies were absent on FNA smears. When compared, the histological diagnosis was same as the cytological diagnosis in 55 cases in the former group (concordance 93.2%) and in 13 cases in the latter group (concordance 81.2%).
ICC was applied in a few selected cases where morphology alone could not give a conclusive diagnosis. One such case of growth in lower end of femur in an 18 year old boy, showed tissue fragment composed of monolayered sheets of small blue round cells with ill defined rosettes on FNA smear. A suspicion of Ewing's sarcoma/primitive neuroectodermal tumor (PNET) was given on cytology, with histology revealing similar features. CD99 positivity confirmed the diagnosis.
| Discussion|| |
The scope of FNAC as a diagnostic tool of malignant lesions is ever increasing. Certain pitfalls, however, do exist in conventionally assessed FNA smears. Frequently, they contain very small quantity of tissue material and the relative absence of recognizable tissue architecture in FNA smears often makes diagnosis very difficult, especially in lesions arising from soft tissues. A commonly faced problem is whether an undifferentiated tumor is a carcinoma, sarcoma, melanoma, or lymphoma. In these cases, cytology can provide a differential diagnosis rather than a conclusive diagnosis. Application of ancillary techniques like ICC helps in arriving at the definitive diagnosis in such cases. However, this is time consuming and expensive. Some tumors may even express inappropriate antigens where immunostaining can give misleading results.  Hence, in this study, we tried to overcome these shortcomings by focusing on the microbiopsies in order to see what additional information they could provide.
Microbiopsies are often disregarded while evaluating cytology smears in favor of other areas where cells are thinly dispersed and diagnostic. Yet careful examination of the periphery of these fragments, where they are often thinner, can provide invaluable clue to the microhistology of the lesions.  Some authors have advocated the use of cell block technique for evaluating microbiopsies obtained on FNA. , But the processing of cell blocks increases the reporting time as well as the cost in an economically poor country like ours, where there is huge patient load. Takenaka et al., obtained ample amounts of microbiopsies while using 21 gauge cutting FNA needles while Buscarini et al., has stressed that larger caliber cutting needles are associated with greater number of complications. We used 22-23 gauge disposable needles and obtained well preserved representative tissue fragments (microbiopsies) of tumors in 81 cases (70.0%).
Different authors have described various techniques of processing and evaluation of microbiopsies from cytology smears. ,, But we applied our own procedure, whereby slides containing microbiopsies were examined on those very smears, without squashing and removal as for processing in cell/paraffin blocks. The advantage of this technique was lesser time in reporting while preserving the cytology smears as such. Further, we could analyze and interpret the smears with tissue fragments, on both cytological and histological perspectives in the same slide.
Lungs were the most common primary site accounting for 14 cases (17.3%) while lymph nodes were the most common secondary site with 19 cases (23.5%). Mravunac et al., also found similar results in their study.
Microbiopsies were of great help in establishing the diagnosis and tumor typing which was not possible by cytology alone. Cases where microbiopsies facilitated in exact diagnosis included soft tissue neoplasms (12 cases) diagnosed as MPNST, fibrosarcoma, spindle cell sarcoma and malignant melanoma. In a study, Kilpatrick et al., have presented the extent to which cytology can be utilized in effective diagnosis of soft tissue neoplasms. Fourteen cases of lungs were typed into small cell, large cell, squamous cell and adenocarcinoma, and eight cases of breast were classified as ductal, medullary, and lobular carcinoma. Five cases of thyroid swellings were diagnosed as follicular neoplasm (4 cases) and metastatic squamous cell carcinoma (1 case) while three cases of parotid mass were reported as mucoepidermoid carcinoma (2 cases) and a single case of adenoid cystic carcinoma. Liver accounted for two cases of hepatocellular carcinoma and one case of metastatic adenocarcinoma from gallbladder. There were one case each from ovary (papillary serous cystadenocarcinoma) and rectum (primary adenocarcinoma). Microbiopsies also helped in the diagnosis of two cases of adenocarcinoma prostate and three cases of primary adenocarcinoma gallbladder. In conjunction with history and clinical examination findings, assessment of microbiopsies led to identification of primaries in prostate and thyroid in metastatic bone tumors. Lymph node metastases were mainly in the cervical region from squamous cell carcinoma of lung and larynx, and adenocarcinoma of upper gastrointestinal tract and follicular neoplasm of thyroid.
The cyto-histopathologic concordance was raised from 81.2% in the absence of microbiopsies to 93.2% in their presence. Thus, the evaluation of FNA smears with microbiopsies provided a definite edge over those without them. However, the interpretation from tissue fragments was rendered difficult in the presence of degenerative changes, obscuring inflammatory cells, and crushing artifacts.
| Conclusion|| |
Apart from cytological features, microbiopsies provided additional information on the tissue architecture, thus aiding in diagnosis, tumor typing, and also in predicting possible primary sites in metastatic tumors. Malignant lesions have been diagnosed with the ''time honored'' histopathology that is recognized as the gold standard for their evaluation. However, in the current era, where ''needle is preceding the scalpel'' and the biopsy material is getting limited, it would be prudent to careful evaluate smears with tissue fragments to enhance the diagnostic accuracy of FNAC. Microbiopsy technique can easily be put into practice in evaluating routine cytology smears, even in under resourced laboratories and in places where the scope of liberal use of immunocytochemistry is limited due to financial constraints.
| References|| |
|1.||Orell SR, Sterrett GF, Whitaker D. Fine Needle Aspiration Cytology, 4 th ed. Philadelphia: Churchill Livingstone; 2005. |
|2.||Verbeek DH, Smedts F, Wijnen-Dubbers CW, Mravunac M. Histologic processing of thick tissue specimens from cytology slides- A novel new technique. Acta Cytol 1996;40:1198-204. |
|3.||Hertzog PJ, Pilbrow SJ, Pedersen J, Polglase AL, Lawson M, Linnane AW. Aberrant expression of intestinal mucin antigens associated with colorectal carcinoma defined by a panel of monoclonal antibodies. Br J Cancer 1991;64:799-808. |
|4.||Mravunac M, Verbeek D, Van Heusden C, Reuterink A, Hop G, Smedts F. Applications of the microbiopsy technique in non-cervical cytology: where cytology and histology meet. Histopathology 1998;33:174-82. |
|5.||Koss LG. Koss' Diagnostic cytology and its histopathologic basis. 5 th ed. Philadelphia: Lippincott Williams and Wilkins; 2006. p. 1591-2. |
|6.||Thapar M, Mishra RK, Sharma A, Goyal V. Critical analysis of cell block versus smear examination in effusions. J Cytol 2009;26:60-4. |
|7.||Takenaka A, Kaji I, Kasugai H, Sasaki Y, Ishiguro S, Wada A, et al. Usefulness of diagnostic criteria for aspiration cytology of hepatocellular carcinoma. Acta Cytol 1999;43:610-6. |
|8.||Buscarini L, Fornari F, Bolondi L, Colombo P, Livraghi T, Magnolfi F, et al. Ultrasound guided fine-needle aspiration biopsy of focal liver lesions: techniques, diagnostic accuracy and complications. A retrospective study of 2091 biopsies. J Hepatol 1990;11:344-8. |
|9.||Nosanchuk JS, Posso M. Salvaging the too thick fine-needle aspirate smear. Diag Cytopathol 1993;9:232-33. |
|10.||Kilpatrick SE, Cappellari JO, Bos JD, Gold SH, Ward WG. Is fine needle aspiration biopsy a practical alternative to open biopsy for the primary diagnosis of sarcoma? Am J Clin Pathol 2001;15:59-68. |
Department of Pathology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh - 202 002, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]