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Cell block, Cytopathology, Diagnostic utility, Immunohistochemistry, 20.1001.1.17355303.2021.16.4.7.5, Main Subjects Breast Pathology, IntroductionCarcinoma of liver has a prevalence of 2-8% worldwide (1,). Metastatic lesions are more common than primary tumors arising from the liver. Most of the liver masses prototype can be suspected by the clinician with history, signs and symptoms, exami-nation and correlation with radiological aids like USG, CT or MRI. However, confirmation needs a definitive pathological report, previously considered to be a histopathological report following a biopsy (1,, 2,). Can that be replaced by FNAC with cell block with or without ancillary studies especially in resource limited areas? This is applicable more in cases of liver lesions because of the following reasons, a) Firstly, as com-pared to the other common site for secondaries, that is, lungs, a liver biopsy is technically more difficult and also has more chances of complications including massive bleeding and biliary peritonitis which can lead to death. b) Secondly, primary hepatocellular cancer, specially the poorly differentiated forms is difficult to differentiate from poorly differentiated metastatic carcinomas without the help of markers (immuno-histochemical studies) even in biopsy samples as the morphology appears similar (2,). Of the primary cancers of liver, Hepatocellular Carcinoma (HCC) is the most common. HCC develops mostly after chronic hepatitis (Hep B, Hep C infection). FNAC from liver mass can be obtained either blindly or by the aid of imaging technique. FNAC is quick, easy and helps the oncologist to plan out the management of patients. To differentiate between benign and malignant as well as primary and metastatic liver lesion is important because treatment approach varies in these cases, as does prognosis.Diagnostic sensitivity of FNAC of liver varies from 67-100% and specificity 93-100% (3,). So FNAC has gained increased acceptance as surgical procedures are invasive and requires general anesthesia and hospitalization. The yield of FNA sampling in some cases is scanty and may not provide sufficient information for an accurate diagnosis as the histological architecture is lost. Thus, the major drawbacks include the risk of false negative and indeterminate results (4,, 5,). This leads to diagnostic dilemma especially in differentiating primary and metastatic hepatic tumors and also primary and regenerating liver nodules. FNAC is cost effective, rapid, minimally invasive and yields better architectural pattern and morphological feature with cell block (6,). Here, we make an attempt to overcome the deficiencies of FNAC using cell block technology as an adjunct and compare that with a core needle biopsy. This technique refers to processing of sed-iments or grossly visible tissue fragments from cytolo-gyical specimens into paraffin blocks which can be further processed, cut and stained by the same methods used for routine histopathology. Cell block preparation has helped in studying the architecture and also performs immunohistochemistry and special staining, if required (4,, 7,, 8,). If properly done, it is very helpful especially using a small-bore tube and essentially converts cytology to histopathology, thus can be called Fine needle aspiration histopathology. Although FNAC with cell block may be costlier than a biopsy, it is logistically easier on the patients and has a much better compliance as sometimes biopsy has a negative psychological impact. Though final diagnosis in most difficult cases still remains through trucut biopsy, which is a minimally invasive procedure under anesthesia, requiring a TruCut needle of 18 gauze size or an automatic biopsy gun and biopsy material is obtained after an ultra-short incision.The Aim of this study was to evaluate the scope and accuracy of cell block following FNAC with or without immunohistochemistry along with ancillary studies for diagnosing various liver lesions (especially SOL, space occupying lesions). Also, we aimed at evaluating the role of cell block for differentiating primary hepatic malignancy from metastatic lesions of the liver along with the use of cell block as an adjunct to FNA in sub typing the various metastatic carcinomas and identifying the source or the origin of the malignancy.Material and MethodsThis is a retrospective descriptive study carried out at both KPC medical college, Kolkata and Medical College, Patna over a period of 3 years. As it was a retrospective study no ethical issue or patient consent was needed. A detailed previous history of any other preexisting liver disease and record of serological viral marker, where available, were collected from the surgery department. FNAC was carried out either blindly or with USG/CT guidance in the radiology department. Direct air-dried smear were stained with MGG. Some smears were immediately fixed in 95% alcohol and stained with Pap. The remaining material in the syringe was allowed to clot to form cell block, where aspiration was adequate for cell block formation (Figure 1,).Fig. 1.Illustration to show cell block formation methodology in our laboratoryResults were analyzed by two independent senior pathologists and a final conclusion of the diagnosis was derived after discussions with a third senior faculty. All the procedures were performed following the standard operating procedures with routine and con-sistent checks to identify and address various types of errors and omissions, ensuring data integrity, correct-ness and completeness of all the available records. The quality control checks included accurate patient identification, proper fixation time, adequate processing measures, appropriate embedding techniques, precision in microtome sectioning, unacceptable artifacts and regular inspection of controls used in IHC and special stains to determine the correctness in our method.Statistical analysis was done using Chi-square to compare various parameters. The P-value was calcula-ted using the sampling distribution of the test statistics under the null hypothesis and our sample data as in a two-sided test. In our analysis, an alpha of 0.05 was used as the cut off for significance. When the P-value was less than 0.05, we rejected the null hypothesis that there is no difference between the means thus, we concluded that a significant difference exists. So, in our study, P-value below 0.05 was taken as significant and over 0.05 as not significant. Fischer&,rsquo s exact test was also done to compare various parameters in the patients.ResultsOut of 416 cases who underwent guided FNAC from liver, 15 cases were considered inconclusive for reporting due to very scanty cellularity or blood only aspirate. Among the adequate aspirations which were 401 in number, the aspirate was enough to make cell block in 349 cases. Others were reported on FNAC as benign or malignant and were not included in our study.Age range varied from 42 to 84 years, with a mean age of 65.5 years. Hepatocellular carcinoma was in the range of 48-84 years with a mean of 67.2 years while metastatic age range was 42-81 years with a mean of 58.4 years. Highest amount of inadequate and inconclusive smears was when the lesion size was &,lt 1 cm. Male to Female ratio was 6,4. In 251 out of 349 cases, immunohistochemical study could be done on cell block preparation. Among the rest 98 cases, 54 did not require IHC due to clear morphology on H&,amp E staining for a final diagnosis. Of these cases, 23 were non-compliant for IHC study, mostly due to economic reasons and decided to go for direct incision biopsy as it is the gold standard. Of the patients, 18 opted for further investigation and treatment in an oncology center, while 3 were lost in follow up after H&,amp E reporting on cell block. So, the total biopsy results were obtained for 323 cases which remained our study sample (Figure 2,). Fig. 2.Distribution of the cases in study population with selection of sample populationOn cell block, with or without immunohistoche-mistry, 43 cases (13.31%) were positive for hepato-cellular carcinoma, 254 cases (78.63%) were positive for metastatic lesions, 7 cases (2.1%) were suspicious of malignancy and 19 cases (5.8%) were designated as benign lesions (Figure 3,).Individual comparison of cell block results with that of biopsy, which is the final diagnostic tool, showed a few discrepancies in interpretation of individual lesions. In biopsy, 52 cases (16.09%) were primary hepato-cellular carcinoma, 253 cases (78.32%) were metastatic lesions while 15 cases (4.64%) were actually benign and 3 cases (0.9%) were regenerative nodules (Figure 4,). A detailed correlation of individual lesions is given in the Table 1,. Fig. 3.Distribution of the cases in cell block preparationFig. 4.Comparison of the cell block results with the biopsy resultsCell blockBiopsyHCC(43)Control-known HCC caseMarker-Hep Par 1, pCEA, &,alpha feto proteinPoorly differentiated1HCC1others42HCC42METASTATIC(254)a) AdenocarcinomaControl-AppendixMarker-CK7, CK20,pCEAPoorlydifferentiated13Adenocarcinoma Gall Bladder-34Adenocarcinoma others-Colon-6Stomach-1Ovary-1Pancreas-2HCC-0741022HCC2Well -moddifferentiated169Adenocarcinoma othersColon-58Stomach-24Pancreas-10Ovary-0Unknown Primary-29456231212Adenocarcinoma Gall Bladder-7575b) undifferentiatedControl- known poorly differentiated carcinomaMarker-CK7,CK20,pCEA,&,alpha feto protein,SMA,HepPar148Undifferentiated-4240HCC-23Adenocarcinoma others-02SCC-22Sarcoma-31c)SCCControl-Seborrheic keratosisMarker-CK7,CK2016SCC-1616d)Round cellControl-Ewings sarcomaMarker-Synaptophysin, Chromogranin6Round cell-66e) sarcomaControl-FibroidMarker-SMA2Sarcoma-12Suspicious of malignancyControl-AllIHC-All7HCC4Regenerative nodule3Benign (19)Inflammatory5Round cell/Neuroendocrine tumor1Hematological malignancy1abscess3Necrosis4Adenocarcinoma others1Adenocarcinoma GB1Abscess2benign10cirrhosis6abscess4Table 1.Correlation of the cases in cell block with that of biopsy with immunohistochemical markers, control used and source of origin of metastasisThere was occasional variance between both the results of cell block and biopsy in almost all lesions, however the disparity was obvious in undifferentiated carcinoma with eight false positive cases. Hepatocellular carcinoma was diagnosed when polygonal cells with eosinophilic cytoplasm, large vesicular nucleus with prominent nucleoli were seen in the smears. When smears showed malignant cells arranged in loose clusters or sheets of pleomorphic cells with moderate to abundant cytoplasm, they were diagnosed as metastatic adenocarcinoma. Adenocarcinoma metastasis from GIT, ovary, and pancreas with metastatic adenocarcinoma from gall bladder was differentiated with IHC and other ancillary studies like radiological imaging, history along with clinical examination of the patient. Similarly, for undifferentiated metastatic carcinoma, the site of origin of primary focus was determined by considering all the above parameters. Round cell tumor had tight clusters of monomorphic cells with nuclear molding and scanty cytoplasm. Sarcoma metastasis showed oval to spindle cells with indistinct cytoplasm. Regenerative nodules had hyperplastic hepatocytes with no distinctive cyto-architectural features and were mistaken as suspicious for malignancy on cytology. Hematological diagnosis was also missed in cell block technique. Due to aspiration from necrotic area, a few cases of metastatic adenocarcinoma were missed. Immunohistochemistry was utilized to arrive at the final diagnosis, as and when essential. Morphology was observed from the smears obtained with MGG, PAP and H&,amp E routinely from the cell block preparation. Special stain was PAS (to look for mucin) and reticulin (to look for trabecular strand) was also performed on cell block preparation. Table 2, and 3, were utilized to differentiate between hepatocellular carcinoma, poorly differentiated metastatic carcinoma, moderately to well differentiated metastatic carcinoma and benign lesions of the liver.MorphologyHCCPoorly differentiatedMetastatic carcinomaModerately differentiated to well differentiated metastatic carcinomaBenign lesion 1) Cytological pattern |