Journal of Cytology

: 2013  |  Volume : 30  |  Issue : 2  |  Page : 121--124

Immunocytochemistry: It's role in diagnosis of undifferentiated neoplasms by fine needle aspiration cytology

Palash Kumar Mandal1, Santosh Kumar Mondal1, Shravasti Roy2, Anindya Adhikari1, Nandita Basu3, Swapan Kumar Sinha1,  
1 Department of Pathology, Medical College, Thakurpukur, Kolkata, India
2 Department of Pathology, Saroj Gupta Cancer Centre and Research Institute, Thakurpukur, Kolkata, India
3 Department of Pathology, School of Tropical Medicine, Paschim Midnapur, India

Correspondence Address:
Santosh Kumar Mondal
Teenkanya Complex, Flat 1B, Block B, 204 R N Guha Road, Dumdum, Kolkata - 700 028


Background: Fine needle aspiration cytology (FNAC) is a rapid, cheap and reliable method for diagnosing any accessible lesion. However, there remains a group of malignant undifferentiated neoplasms, which can only be categorized with the help of immunocytochemistry (ICC). The categorization is important due to their vast difference in treatment and prognosis. Aim: To evaluate the effectiveness of ICC in categorizing the undifferentiated neoplasms diagnosed on routine FNAC smears. Materials and Methods: Thirty six cases of undifferentiated neoplasms were selected from a group of total 78 cytology cases of undifferentiated tumors from different sites like head and neck, lymph node, soft tissue etc. These were then subjected to a panel of ICC markers based on the clinical and cytomorphological features. Results: Of these, 21 were simple, ten were computerized tomography guided and five were ultrasound guided FNACs respectively. All the 78 cases were confirmed by histopathological examination and immunohistochemistry. Of the 36 cytological cases, final diagnosis correlated in 30 cases histologically. The six cases were incorrect either due to inadequate material on the smears (three cases) or false positive staining (three cases). Conclusions: Our study found that ICC is a sensitive and specific method for early and definitive diagnosis of undifferentiated neoplasms. However, selection of antibodies must be judicious to make it cost effective.

How to cite this article:
Mandal PK, Mondal SK, Roy S, Adhikari A, Basu N, Sinha SK. Immunocytochemistry: It's role in diagnosis of undifferentiated neoplasms by fine needle aspiration cytology.J Cytol 2013;30:121-124

How to cite this URL:
Mandal PK, Mondal SK, Roy S, Adhikari A, Basu N, Sinha SK. Immunocytochemistry: It's role in diagnosis of undifferentiated neoplasms by fine needle aspiration cytology. J Cytol [serial online] 2013 [cited 2020 Apr 3 ];30:121-124
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Full Text


Fine needle aspiration cytology (FNAC) is a rapid and convenient method of diagnosing any accessible lesion. [1],[2] However, there are some tumors like malignant round cell tumors, which always pose a diagnostic problem to a pathologist. For quick diagnosis of such lesions, specialized techniques like immunocytochemistry (ICC) on FNAC smears can be used. [3] If FNAC and ICC are used properly, a quick and correct guideline can be provided to the clinicians for selecting the right protocol for treatment, avoiding immediate surgical intervention and thus save additional expense and time. [4]

Apart from FNAC smears, ICC can be done on smears prepared from centrifuged cell deposits obtained from body fluids and cells grown in culture. [5]

 Materials and Methods

The study was carried out in the Departments of Pathology in our institutes from June 2008 to June 2011.

Seventy eight cases of patients presenting with tumors of various sites were selected who attended the out-patient department in our institutes. Case details were recorded which included clinical presentation, radiological findings etc., FNAC was done in all cases and the diagnosis was given as either malignant round cell or undifferentiated neoplasm. All 78 cases were subjected to histopathological evaluation, special stains (Periodic acid Schiff, reticulin) and immunohistochemistry.

FNAC was performed by 10 mL syringe with a 22G or 24G needle following the standard technique and the smears were stained by Leishman-Giemsa and hematoxylin and eosin or Papanicolaou (Pap) stains.

ICC was done with smears fixed in cold acetone. [6] ICC was done by two step peroxidase labelled polymer method. [7] Monoclonal antibodies to pancytokeratin, CK7, CK20, leucocyte common antigen, desmin, vimentin, CD3, CD20, CD30, neurone specific enolase, chromogranin A, CD99, terminal deoxynucleotidyl transferase, TTF 1, BCl 2, CD117 and others along with Novolink polymer detection kit by Novocastra (UK) were selectively used depending on the case. [8],[9],[10],[11],[12],[13]

The ICC protocol was as follows:

First the slide was washed in TRIS buffered saline (TBS, 0.005M, pH 7.6), 10 min each for 3 changes. Then the slide was treated with 3% hydrogen peroxide in methanol for 10 min (peroxidase blocking), then again washed in TBS (as before), followed by protein blocking for 10 min. Then it was incubated with primary antibody (pre diluted) at room temperature in moist chamber for 1 h. It was then washed in TBS (as before). Then post primary blocking was done for 30 min. The slides were washed in TBS (as before). Then it was treated with Polymer for 30 min, which was followed by washing in TBS (as before). It was then treated with prepared DAB -chromogen for 5-10 min (till appearance of brown color). Then after washing in distilled water, counterstaining was done with hematoxylin for 1 min. Then it was washed under running tap water followed by graded dehydration in alcohol, cleared in xylene and mounted with DPX.


Of the 78 cases, 51 (65.3%) were plain, 17 (21.8%) were computerized tomography (CT) guided and 10 (12.8%) were ultrasonography (USG) guided FNACs respectively. Of these we could perform ICC in only 36 (46.1%) cases. Of the 36 cases of study group, plain FNAC was done in 21 (58.3%) cases, CT guided FNAC in 10 (27.8%) cases and USG guided FNAC in 5 (13.9%) cases [Table 1].{Table 1}

Patients' age varied from 2.5 years to 76 years. Male: Female ratio was 1.2:1. Our diagnosis by ICC on FNA smears was confirmed by histopathological evaluation as well ase immunohistochemitry when required [Figure 1] and [Figure 2]. The diagnosis correlated in 30 (88.9%) cases but didn't match in 4 (11.1%) cases, because they were either inadequate or necrotic smears or showed equivocal staining with more than one antibody.{Figure 1}{Figure 2}

Lesions according to most common sites- was head and neck 11 (30.5%), then soft tissue 10 (28%), lung 08 (22%), breast 02 (5.5%), mediastinum 02 (5.5%), ovary 02 (5.5%) and urinary bladder 01 (03%) respectively [Table 2].{Table 2}

Sensitivity of this study was 93%, specificity 83% and accuracy 92% respectively.


Definite diagnostic criteria in different lesions using FNAC was first applied by Zajicek [14] and immunostaining method using enzymes as labels was first used by Nakane and Pierce in 1966. [15] One of the most popular recent techniques Streptavidin-Biotin technique was first used by Guesdon et al. in 1979. [16] Undifferentiated neoplasms very often pose diagnostic problem to pathologists specially when only FNAC is used as a diagnostic tool. FNAC in conjunction with ICC can solve most of these problems.

Abendroth and Dabbs, [5] used immunoperoxidase staining with selected antibodies in 75 cases of smears made from various neoplastic or non-neoplastic tissues received as surgical specimens. ICC showed expected positive staining in these cases. Dabbs and Wang [9] also used more than one antibody on a single pap stained slide to prove the use of ICC on limited smears. Brahmi et al. [3] used ICC on FNAC smears to confirm the diagnosis of 51 cases of malignant small round cell tumors. However, ICC may have some inherent problems and may give false positive or false negative results. Causes of false positive staining may be due to insufficient peroxidase blocking, necrotic cells, dried preparation, cross reactivity of antibodies. False negative results may be due to low antibody concentration, denaturation of aspirated material etc., Last but not the least, in both the categories there is misinterpretation of neoplastic and reactive cells by the cytopathologist. [6] These problems may be resolved to some extent by use of cell block preparation. [14],[17] However, even ICC on cell block may not have 100% diagnostic accuracy. Moreover, processing time for cell block preparation (like small biopsy) and subsequent ICC may be more time consuming compared to ICC on smears. The material on cell block also may be limited. [6]


ICC is a sensitive and rapid method for diagnosing undifferentiated tumors of various sites. It is less time consuming (report is ready within 24 h) and a cheap outdoor procedure. As the number of specimen is somewhat limited, conventional light microscopy should not be ignored for judicious selection of antibodies.

ICC can thus be established as a routine technique in the diagnosis of undifferentiated tumors.


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