jhc-2019-jul-v-3-n-2-analysis-ray-s

Analysis of the Diagnostic Value of Cytological Smear Method Versus Cell Block Method in Pleural Effusion Fluid of Suspected Lung Cancer Patients

 *Ray S,1 Zinnah SA,2 Awal N,3 Hassan I,4 Dewan RK,5 Jeba R,6 Hussain M7

 *Dr. Subrata Ray, Assistant Professor, Department of Pathology, Jashore Medical College. subrataray29@gmail.com

  1. Shahed Ali Zinnah, Associate Professor, Department of Pathology, Dhaka Medical College.
  2. Dr . Naila Awal , Assistant Professor, Department of Pathology, Greenlife Medical College.
  3. Imran Hassan, Assistant Professor(CC), Department of Pathology, (Col) Malek Medical College, Manikgonj.
  4. Rezaul Karim Dewan, Professor and Head, Department of Pathology, Dhaka Medical College
  5. Dr . Ruksana Jeba, Associate Professor, Department of Pathology, Dhaka Medical College.
  6. Dr Maleeha Hussain, Professor, OSD, DGHS.

 *For correspondence

 Abstract

Background: For any kind of lungs pathology accompanying pleural effusion pleural biopsy guided with thoracoscopy, of course, may provide important results. Since biopsy is an invasive procedure, the priority is to do,  a cytological examination of pleural fluid obtained with thoracentesis, which  is very crucial for the disease staging and treatment strategy. In this purpose, even though conventional cytosmear techniques have been used generally till this day, cell block technique is also being used recently.
Methods: In our study, 100 pleural effusion fluid sample were included. All the fluid specimen was subjected to cytosmear (CS) and cytoblock (CB).
Results: By using conventional CS method, 85 fluid specimens were diagnosed as negative for malignancy (85%), 5 cases were diagnosed as positive for malignancy (5%) and the remaining 10 cases were diagnosed as suspicious for malignancy (10%). On the contrary by using CB, complimented by combined PAS-Alcian blue stain where required, 90 cases were diagnosed as negative for malignancy (90%) and 10 cases were diagnosed as positive for malignancy. Thus, by the CB method, an additional 5 more cases were diagnosed as positive for malignancy, that is, 5% more diagnostic yield for malignancy.
 Conclusion: CB technique could be considered as a useful adjunct in evaluating malignant cells in MPE for a final cytodiagnosis, along with the routine CS method.

[Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):99-106]

 Key Wards: Cell block , Pleural effusion , Lung cancer 

Introduction

Development of pleural effusion is a common complication in patients with lung cancer either primary or secondary. There may be presence of neoplastic cells in the pleural effusion which is called malignant effusion (MPE) or it may be a para-malignant effusion, which is  pleural effusion with absence of cytological evidence of tumor cells in a known setting of malignancy. In UK, 40000 people per year are affected by MPE and it is estimated that up to 50% of the patients  with metastatic malignancy  develop a pleural effusion – either at the time of diagnosis or during the evaluation of cancer.1,2,3  The common etiologies of MPE are lung cancer, breast cancer, lymphoma, ovarian cancer and gastric cancer in order of decreasing percentage.

For any kind of lung lesion accompanying pleural effusion, pleural biopsy guided with thoracoscopy, of course, may provide important results, but has the risk of being an invasive procedure and it depends on the experience and efficiency of the surgeon. So, in a country with poor resource settings, the priority should be a cytologic examination of pleural fluid obtained by thoracentesis. It not only helps in diagnosis but also can help in disease staging and treatment strategy.4

In conventional cytological smear (CS), discrimination of the reactive mesothelial cells and malignant cells is the most important diagnostic problem. Distinguishing benign from malignant cellular changes may require meticulous screening, careful scrutiny of cellular features and an understanding of the range of reactive changes. Since the introduction of cell block (CB) technique by Bahernburg nearly a century ago,  it has been used routinely for processing fluid.5 CB has the advantage of recognition of the histological pattern of disease, possibility of study multiple sections by routine staining, special staining and immunological procedures.

In this study, we assessed the utility of CB and CS techniques and evaluated whether the CB, when it complements conventional CS, can increase the diagnostic yield.

Methods

This is an analytic study, carried out at the Department of Pathology, Dhaka Medical College, Dhaka from  July 2015 to  June 2017, over 100 pleural effusion fluid specimen of suspected lung cancer patients.

 10 ml of fresh pleural fluid sample was obtained by thoracentesis from each patient during clinical evaluation. Each sample was divided into two equal parts.

For conventional smear technique, 5 ml fluid specimen was centrifuged at 2500 rmp for 10 min. A minimum two smears were prepared from the sediment. They were immediately fixed in 95% alcohol and stained with the Papanicoloau and Haematoxylin- Eosin stain.

 

For cell block, we used AAF (95% ethyl alcohol + acetic acid + 10% formalin). After centrifuge at 2500 rmp for 10 min, cell sediments were mixed with thrice the volume of AAF fixative and again centrifuged at 2000 rmp for 10 min. The centrifuged tube was aside undisturbed for six hours. The cell button was scraped out and wrapped in filter paper and processed in automatic tissue processor for routine histopathological sections. The cell blocks were embedded in paraffin and sectioned at 4 micro meter thickness.

 The samples were studied in detail taking into account the available clinical and radiological data and various investigation reports. Each individual slide was objectively analyzed for cellularity, background blood, nuclear and cytoplasmic details and arrangement of the cells using the point scoring system described by Mair et al., (1989).6 All these criteria were put together and each cases was categorized as Benign, Suspicious for malignancy and Malignant effusion. Special stain of cell blocks, including combined PAS-Alcian blue stain was done whenever needed. In cases, where pleural biopsy of the same patients was available, used to confirm the diagnosis.

Results

In our study, 100 pleural effusion fluid sample were included. The age-rang from 26 to 82 years. Most of the patients are between the age group 51-60 years (30%). The male female ratio was 1.8:1. All the fluid specimen was subjected to CS and CB. By using connectional CS method, 85 fluid specimens were diagnosed as negative for malignancy (85%), 5 causes were diagnosed as positive for malignancy (5%) and the remaining 10 causes were diagnosed as suspicious for malignancy (10%).On the contrary by using CB, complimented by combined PAS-Alcian blue stain where required , 90 cases were diagnosed as negative for malignancy (90%) and 10 cases were diagnosed as positive for malignancy. Thus by the CB method, an additional 5 more cases were diagnosed as positive for malignancy, that is, 5% more diagnostic yield for malignancy .

Among the 10 malignant cases, diagnosed by CB, 9 cases were subcategorized as adeno carcinoma (90%) and 1 cases was diagnosed as lymphoma.

The malignant effusion was more common in male (70%) than female (30%). The male female ratio was 7:3.

When the cytological smear and cell block techniques were studied for their quality using the point scoring system of Mair et al, (1989).6  it was noticed that 5% of the CS and 6% of the CB was inadequate and unsuitable for diagnosis. 50% of the CB and 32% of the CS showed highest cellularity. 41% of the CB and only 11% of the CS yielded highest morphological details. Wilcoxon Signed Rank test was done to measure the level of significant, which was highly significant in CB compared to CS regarding cellularity and morphological details.


Table V: Distribution of patients according to point scoring system adopted by Mair et al,(1989)6 in CS

 

 

Discussion

Lung cancer, whether primary or secondary, is invariably accompanied by pleural effusion. The cell population in sediment of pleural fluid represent much larger surface area than obtained by needle biopsy.  Malignant cells first involve the visceral pleura and tend to be focal in the parietal pleura.7 This explains why, pleural fluid cytology is a more sensitive diagnostic test than closed percutaneous pleural biopsy.8,9

Thoracentesis followed by cytological examination is the first investigation performed in a pleural effusion fluid of a suspected lung cancer patients and has been accepted as a routine laboratory procedure. The differentiation of a malignant effusion from a para-malignant effusion is extremely important, not only in the diagnosis of a malignant lesion, but also in staging, prediction of prognosis and to reform a proper treatment protocol.10

In our study, 100 pleural effusion fluid specimen of radiologically and clinically suspected lung cancer patients were examined by using conventional CS and CB techniques. In CS, of 85 patients, the fluids were diagnosed as negative for malignancy (85%).

All the 85 negative cytosmear cases were confirmed by CB. In conventional CS method 10 cases were diagnosed as suspicious for malignancy, of which 5 cases turned to be negative and another 5 cases   proved to be positive for malignancy in CB. In conventional CS method, reactive mesothelial cells, an abundance of inflammatory cell and paucity of representative cells contributed to the considerable difficulties in making conclusive diagnosis of malignancy.

The false positive diagnosis in CS was secondary to the marked atypia of the mesothelial cell which may be due to the microbiological, chemical, physical, immunological or metabolic insult to the serous membrane or due to the subtle cytomorphological features of some malignant neoplasm, particularly well differentiated adenocarcinoma.11 The problem was compounded by artifact   caused by poor fixation, preparation and staining technique. In our settings, fixation and transportation are contributory factors. Generally the reactive mesothelial cells appears rounded and have single centered or eccentric nucleus. Some of the mesothelial cells form cell ball, clusters and takes a signet-ring cells appearance, thus closely mimicking malignancy. The malignant cells have irregular nuclear membrane, nuclear molding and prominent nucleoli with absence of windows. However, these differentiating features are sometimes difficult to identify.

By using CB method, 5 more malignant cases were diagnosed, which were diagnosed as suspicious for malignancy by conventional CS, thus increasing the diagnostic yield by 5% (5/10). Further special stain (PAS-Alcian blue) supported the diagnosis of malignancy.

Another 5 cases were diagnosed as negative for malignancy by CB, which were diagnosed as suspicious for malignancy by conventional CS. The benign cells did not take PAS-Alcian blue stain.

In a parallel study on effusion fluid, Shivakumaraswamy et al, (2012)5 found   15% more diagnostic yield for malignancy on CB.   Bhanvadia et al., (2014)12 in another study observed    10% more diagnostic yield in CB. Thapar et al, (2009)4  also   showed 13% more diagnostic yield by CB.

In our study, after the final diagnosis as benign or malignant effusion, a critical evaluation was made for diagnostic yield taking a consideration on cellularity, morphology, architecture and background blood. The conventional CS and CB technique were studied for their quality by using point scoring system of Mair et al, (1989).6 In CB, 41% ensured highest morphological details where as in CS, highest morphological detail was acquired in only 11% cases. The comparative result of our point scoring system was analyzed by Wilcoxson Signed Ranks test which was highly significant in respect of cellularity and cellular morphology.

Reactive mesothelial cells have in the past been responsible for simulating malignancy in CS, largely due to the formation of rosettes, pseudo acini or acini, with or without the presence of prominent nucleoli. The CB effectively puts both the features in their proper prospective. That is, the nucleoli does not appear as prominent in CS and the pseudoaciner or acinar strictures can be better appreciated when present in the CB. Similar findings were noticed in the Dekker and Bapp (1978)(13) study. More important is, this CB is a valuable tool in the evaluation of well differentiated adenocarcinoma, where the presence of true acini is seen in the CB with mucin. Positive stain  for mucin in CB  indicated  malignancy.

In our study, we noticed a significant number of macrophages admixed with malignant cells having large pleomorphic nuclei simulating malignant cells. Multiple large PAS positive granules were identified by special stain that were diagnosed as suspicious for malignancy by CS. We also noticed pericellular lacunae in many of the cases of adenocarcinoma specially of mucin secreting type, characterized by cell clusters and Bull’s eye (Target) inclusion like finding was seen in one case of metastatic carcinoma.

There had not been yet any standard method for the preparation of CB from effusion fluid. Our study was done by using 10% alcohol formalin fixative. In our paraffin embedded CB section our attention was drawn by the presence of large clusters of cells either malignant or reactive mesothelial cells in a separate peripheral layer leaving a  thick amorphous proteinaceous material in the center. These helped in reducing background artifact and ensured more nuclear details for the distinction between reactive mesothelial cells and malignant cells. Few inflammatory cells were noticed in a less peripheral separate layers admixed with other cells in malignant cases. The other advantage of CB was concentration of cellular materials in one small area that can be evaluated at a glance with all cells lying in the same focal plane of microscope. It bridges the gap between cytology and histology

Conclusion

The cell block made by using 10% formalin-alcohol as a fixative, is a simple, inexpensive method, and does not require any special training or instrument. Multiple sections could be obtained required, for special stain. Therefore, CB technique could be considered as a useful adjunct in evaluating malignant cells in MPE for a final cytodiagnosis, along with the routine CS method.

References

  1. Kastelik JA. Management of malignant pleural effusion. Lung, 2013;191(2):165-75.
  2. Bennett R & Maskell N. Management of malignant pleural effusions. Curr Opin Pulm Med, 2005;11(4): 296-300.
  3. Maskell N, Gleeson F & Davies R. Standard pleural biopsy versus CT-guided cutting-needle biopsy for diagnosis of malignant disease in pleural effusions: a randomised controlled trial. The Lancet, 2003;361(9366):1326-30.
  4. Thapar M et al. Critical analysis of cell block versus smear examination in effusions. Journal of Cytology, 2009;26(2):60–64.
  5. Shivakumarswamy U, Arakeri SU, Karigowdar MH & Yelikar BR. Diagnostic utility of the cell block method versus the conventional smear study in pleural fluid cytology. Journal of Cytology, 2012;29(1):11–15.
  6. Mair S, Dunbar F, Becker PJ & Plessis WD. Fine needle cytology–is aspiration suction necessary? A study of 100 masses in various sites. Acta Cytologica, 1989;33(6): 809-13.
  7. Rodrîguez-Panadero F, Naranjo FB & Mejîas JL, 1989. Pleural metastatic tumours and effusions. Frequency and pathogenic mechanisms in a post-mortem series. European Respiratory Journal, 1989;2(4):366-9.
  8. Johnston WW. The malignant pleural effusion. A review of cytopathologic diagnoses of 584 specimens from 472 consecutive patients. Cancer, 1985;56(4):905-9.
  9. Prakash UB. & Reiman HM. Comparison of needle biopsy with cytologic analysis for the evaluation of pleural effusion: analysis of 414 cases. Mayo Clinic Proceedings, 1985;60(3):158-64.
  10. Rivera MP, Mehta AC & Wahidi MM. Establishing the diagnosis of lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest, 2013;143(5):e142S-e165S.
  11. Price BA, Ehya H & Lee JH, 1992. Significance of pericellular lacunae in cell blocks of effusions. Acta Cytologica, 1992;36(3):333-337.
  12. Bhanvadia VM, Santwani PM & Vachhani JH, 2014. Analysis of Diagnostic Value of Cytological Smear Method Versus Cell Block Method in Body Fluid Cytology: Study of 150 Cases. Ethiopian Journal of Health Science, 2014;24(2):125–131.
  13. Dekker A & Bupp P A, 1978. Cytology of serous effusions. An investigation into the usefulness of cell blocks versus smears. American Journal of Clinical Pathology, 1978;70(6):855-60.

Fine Needle Aspiration Cytology in the Diagnosis of Focal Liver Lesions

Fine Needle Aspiration Cytology in the Diagnosis of Focal Liver Lesions

*Saem AM,1 Saha NK,2 Begum F,3 Hye AA,4 Islam N,5 Anam T6

 Abstract

Fine needle aspiration cytology (FNAC) assisted by cell block examination might be more accurate method for the definitive diagnosis of focal liver lesions (FLL). This study was designed to find out the role of FNAC in the diagnosis of FLLs in comparison to cell block preparations. This cross sectional observational study was carried out in the department of Pathology in collaboration with the department of Radiology & Imaging at Sylhet MAG Osmani Medical College. Study period was from 1 July, 2015 to 30 June, 2016. Clinically & radiologically diagnosed patients of focal liver lesions were study populations. The age of the study patients ranged from 15 to 80 years with a mean of 53.58 years. On FNAC, 10% cases were unsatisfactory, 8% cases were cystic lesion, 4% cases were benign tumor and 78% cases were malignant tumor. Among 39 malignant cases, 30.77% cases were hepatocellular carcinoma (HCC) and 69.23% cases were metastatic adenocarcinoma. Unsatisfactory samples were 18.18%, 6.82% were benign tumors and 75% were malignant tumors. Among the malignant lesions, 18.18% were HCC and 81.82% were metastatic adenocarcinoma. The sensitivity, specificity, positive predictive value (PPV), negative predictive value(NPV) and accuracy of FNAC in the evaluation of FLLs were 100%, 66.67%, 97.06%, 100% and 97.22%, respectively. The sensitivity, specificity, PPV, NPV and accuracy of FNAC in the detection of HCC were 66.67%, 85.18%, 50%, 92% and 81.82% respectively. FNAC of focal liver lesions has high sensitivity and accuracy in the detection of malignancy but it has low sensitivity in the detection of HCC. Cell block preparations were found superior to cytomorphology as immunostaining can be done on cell block preparations.

[Journal of Histopathology and Cytopathology, 2017 Jul; 1 (2):110-115]

 Key words: Focal liver lesions, FNAC, Cell block, Immunohistochemistry, HCC, and Metastatic carcinoma.

  1. *Dr. Abu Mohammad Saem, Lecturer, Department of Pathology, Comilla Medical College, Comilla. saemshampa@yahoo.com
  2. Naba Kumar Saha, Professor & Head, Department of Pathology, MAG Osmani Medical College, Sylhet.
  3. Ferdousy Begum, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.
  4. Azizul Qadar Md. Abdul Hye, Associate Professor Department of Pathology, MAG Osmani Medical College, Sylhet.
  5. Nazmul Islam, Assistant Professor, Department of Pathology, Army Medical College, Comilla.
  6. Tasmina Anam, Scientific Officer, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.

 * For correspondence

 Introduction

A focal liver lesion (FLL) is a solid or cystic mass or area of tissue that is identified by radiological or imaging techniques as an abnormal part of the liver. It may be either a benign lesion such as focal nodular hyperplasia, hepatocellular adenoma and hepatic cyst or a malignant lesion such as hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma and metastatic carcinoma.1

Pathological examination is an important aspect in the evaluation of an FLL. FNAC is the preferred method for diagnosis of focal liver lesions and needle core biopsy (NCB) for evaluating diffuse liver diseases where architectural details are important.2 In recent years FNAC has emerged as an effective tool for diagnosis of a hepatic mass.

 

Cell blocks prepared from residual materials of fine needle aspirations can be useful adjuncts to smears for establishing a more definitive cytopathological diagnosis.3 Use of cell blocks improves diagnostic accuracy as it facilitates study of architecture details of multiple sections, use of special stains and immunohistochemistry.4

The distinction of moderately to poorly differentiated hepatocellular carcinoma from metastatic carcinoma may be a major problem for cytologists and this distinction is clinically important. Immunohistochemistry is required in this situation to differentiate hepatocellular carcinoma from metastatic carcinoma.5

With this background the study was designed to find out the role of FNAC in the diagnosis of focal liver lesions and to correlate its efficacy with cell block preparations using H&E and immunohistochemistry.

Methods

This cross sectional observational study was carried out in the department of Pathology in collaboration with the department of Radiology & Imaging at Sylhet MAG Osmani Medical College from 1 July, 2015 to 30 June, 2016. Clinically and radiologically diagnosed patients of focal liver lesions attending the department of Radiology & Imaging from different departments during the study period were the target population and those who fulfilled the inclusion and exclusion criteria were considered as study population. Patients of all ages and both sexes were included. Patients with bleeding diathesis, suspected liver abscess, hydatid cyst and hemangioma were excluded from the study. 22 gauge needle was placed in the lesion under ultrasound guidance and the material was aspirated with a 10 ml disposable syringe. After placing aspirates on the slides, thin smears were prepared by gentle friction of two slides. Then smears were fixed in 95% ethyl alcohol for at least 30 minutes and stained with Papanicolaou stain. After preparation of smears, the residual material was secured for clot preparation. It was then transferred into 10% formalin and processed as a cell block.6 Then, the cell blocks were cut at 5 micrometer thickness and were stained with Harri’s Haematoxylin and Eosin stain. From the paraffin block 3 micrometer sections were cut and stained for immunohistochemistry with Glypican-3 antibody. The immunohistochemistry was performed in the Immunohistochemistry Laboratory of Bangabandhu Sheikh Mujib Medical University (BSMMU) following their staining protocol. All the data were organized by using scientific calculator and Statistical Package for Social Science (SPSS) version 23.

 Results

The age of the study patients ranged from 15 to 80 years with a mean of 53.58 years (SD +15.32). Out of 50 cases, 33 (66%) were male and 17 (34%) were female with male to female ratio of 1.94:1. Among these patients, the highest number of patients 13(26%) were in the age group 51-60 years (Table I).

Table I: Age and sex distribution of study cases (n=50)

 

Age Groups (years) Male
No (%)
Female

No (%)

Total

No (%)

11-20 2(4) 1(2) 3(6)
21-30 1(2) 0(0) 1(2)
31-40 2(4) 4(8) 6(12)
41-50 9(18) 3(6) 12(24)
51-60 7(14) 6(12) 13(26)
61-70 10(20) 2(4) 12(24)
71-80 2(4) 1(2) 3(6)
Total 33(66) 17(34) 50(100)

 

Out of 50 focal liver lesions, 5 cases were unsatisfactory, 4 cases were cystic lesion, 2 cases were benign tumor and 39 cases were malignant tumor in cytology. Among the malignant cases, 12 were hepatocellular carcinoma (HCC) and 27 were metastatic adenocarcinoma (Figure 1).

 Figure 1. Pie diagram showing distribution of study cases according to FNA cytomorphology

Finally, 8 unsatisfactory, 3 benign and 33 malignant cases were diagnosed in cell block preparations. Among 33 malignant cases 6 were diagnosed as hepatocellular carcinoma (HCC) and 27 were diagnosed as metastatic adenocarcinoma (Figure-2).

 Figure 2. Pie diagram showing distribution of 44 cases according to combined cell block preparations.

 36 cases were conclusive on both cytomorphology and cell block preparations. On evaluation of cytomorphological diagnosis of 36 cases, 33 were true positive diagnosis, 2 were true negative diagnosis, 1 was false positive diagnosis and there was no false negative diagnosis (Table II). Sensitivity, specificity, PPV, NPV and accuracy of FNAC in the diagnosis of malignant focal liver lesions were100%, 66.67%, 97.06 %, 100 % & 97.22 %, respectively.

Table II: Statistical evaluation of cytomorphological diagnosis of 36 conclusive cases.

 

Combined cell block preparations (H&E and IHC) Cytomorphological diagnosis
Disease positive (Malignant) Disease negative(Benign)
Positive(Malignant)   33 TP               33 FP                  1
Negative(Benign)       3 FN                0 TN                  2
Total                         36                    33                        3

 

TP= True positive, TN= True negative, FP= False positive, FN= False negative

33 cases were diagnosed as malignant by both FNAC and cell block preparations. On evaluation of cytomorphological diagnosis, 4 were true positive, 23 were true negative, 4 were false positive and 2 were false negative in the detection of HCC (Table III). Sensitivity, specificity, PPV, NPV and accuracyof FNAC in the detection of HCC were 66.67%, 85.18%, 50%, 92% and 81.82%, respectively.

Table III: Statistical evaluation of cytomorphological diagnosis in the detection of HCC.

 

Combined cell block preparations (H&E and IHC) Cytomorphological diagnosis
Disease positive (HCC) Disease negative

(Non HCC)

Positive (HCC)                   6 TP               4 FP                   4
Negative (Non HCC)        27 FN               2 TN                  23
Total                                 33                     6                        27

 

TP= True positive, TN= True negative, FP= False positive, FN= False negative

Discussion

In the present study, USG guided FNAC was compared with cell block preparations (H&E and immunohistochemistry) in differentiation of focal liver lesions. FNA smears were available in all the 50 cases, but cell blocks were available in 44 cases.

Age of the study patients ranged from 15 to 80 years with a mean of 53.58 years. Nazir et al. (2010) and Kuo et al. (2004) showed 55 and 58.1 years as mean age in their studies which are close to the mean age of present study.7,8 Highest number of patients (26%) was in the age group of 51-60 years in our study. Nazir et al. (2010) reported that maximum number of cases was seen between 55-65 years of age which is nearly similar to present study.7 Out of 50 cases, 33 (66%) were male and 17 (34%) were female with male to female ratio of 1.94:1. Similar findings were reported by Swamy et al. (2011).9 Nazir et al. (2010) showed a male to female ratio of 1.7:1 which is also close to present study.7

Out of 50 cases, 5 (10%) cases were unsatisfactory, 4 (8%) cases were cystic lesion, 2 (4%) cases were benign tumor and 39 (78%) cases were malignant tumor on cytomorphology. Further categorization of benign tumors was not done as in Khurana et al. (2009).6 Among 39 malignant cases, 12 (30.77%) cases were HCC and 27 (69.23%) cases were metastatic carcinoma. All the cases of metastatic carcinoma were adenocarcinomas. Nearly similar findings were found on cytomorphology in the study of Mohmmed et al. (2012), Nazir et al. (2010), Khurana et al. (2009) and Ceyhan et al. (2006).6,7,10,11 Ozkara et al. (2012) found 9.9% of cases as unsatisfactory on cytomorphology which is similar to the unsatisfactory smear (10%) of the present study.12

In final diagnosis of 44 cases by combined cell block preparations (H&E and immunohistochemistry), 8 (18.18%) were unsatisfactory, 3 (6.82%) were benign tumors and 33 (75%) were malignant tumors. Nazir et al. (2010) reported 85% cases as malignant which is nearly close to the malignant cases found in the present study.7 But Mohmmed et al. (2012) showed 39% cases as malignant which is lower and Khurana et al. (2009) showed 93.75% cases as malignant which is higher than that of present study.6,10 Among the malignant lesions, 6 (18.18%) were HCC and 27 (81.82%) were metastatic adenocarcinoma in our study. Khurana et al. (2009) found 17.78% cases as HCC and 82.22% cases as metastatic tumor which are concordant with the present study.6

The sensitivity, specificity, and accuracy of USG guided FNAC in the evaluation of focal liver lesions were 100%, 66.67% and 97.22%, respectively. Sensitivity of the present study (100%) is similar or close to the sensitivity of studies done by Khurana et al. (2009), Nazir et al. (2010), Swamy et al. (2011) and Mohmmed et al. (2012).6,7,9,10 Specificity of the present study (66.67%) has concordance with the specificity found by Mohmmed et al. (2012).10 The specificity shown by Khurana et al. (2009), Nazir et al. (2010) and Swamy et al. (2011) has discordance with that of current study.6,7,9The present study showed an accuracy of 97.22% which is similar to that of Nazir et al. (2010) and Swamy et al. (2011).7,9

The sensitivity, specificity, and accuracy of FNAC in the detection of HCC were 66.67%, 85.18% and 81.82% respectively in our study.  Sensitivity of FNAC in the detection of HCC described by Ozkara et al. (2013) was 68.2% which is similar to the sensitivity of present study.12 Khurana et al. (2009) and Nazir et al. (2010) showed the sensitivity in the detection of HCC as 72.3% and 96% respectively which are higher than the sensitivity of present study.6,7 Specificity and accuracy showed by Nazir et al. (2010) were 100% and 97.5% respectively which are also higher than those of the present study.7

 Conclusion

FNAC of focal liver lesions has high sensitivity and accuracy in the detection of malignancy but it has low sensitivity in the detection of HCC. No significant complication was observed during aspiration. FNAC is a relatively safe, quick, cost effective and patient compliant procedure which has high accuracy in the differentiation between benign and malignant focal liver lesions. Simultaneous cell block preparations can improve the efficacy of FNAC in the subtyping of malignancy.

 References

  1. Marrero JA, Ahn J, Reddy KR. ACG clinical guideline: The Diagnosis and Management of Focal Liver Lesions. Am J Gastroenterol, 2014; 109(9): 1328-47.
  2. Conrad R, Prabhu SC, Cobb C, Raza A. Cytopathologic diagnosis of liver mass lesions. J Gastrointest Oncol, 2013; 4(1): 53-61.
  3. Nathan NA, Narayan E, Smith MM, Horn MJ. Cell block cytology: Improved Preparation and its Efficacy in Diagnostic Cytology. Am J Clin Pathol, 2000; 114: 599-606.
  4. Ali SR, Jayabackthan L, Rahim S, Sharel MB, Prasad K, Hegdekatte N. Role of fine needle aspiration cytology in the diagnosis of hepatic lesions. Muller J Med Sci Res, 2015; 6(2): 125-128.
  5. Ahuja A, Gupta N, Srinivasan R, Kalra N, Chawla Y, Rajwanshi A. Differentiation of Hepatocellular Carcinoma from Metastatic Carcinoma of the liver – Clinical and Cytological features. J Cytol, 2007; 24(3): 125-129.
  6. Khurana U, Handa U, Mohan H, Sachdev A. Evaluation of Aspiration Cytology of the Liver Space Occupying Lesions by Simultaneous Examination of Smears and Cell Blocks. Diagn Cytopathol, 2009; 37(8): 557-563.
  7. Nazir RT, Sharif MA, Iqbal M, Amin MS.Diagnostic Accuracy of Fine Needle Aspiration Cytology in Hepatic Tumours. J Coll Physicians Surg Pak, 2010; 20(6): 373-376.
  8. Kuo FY, Chen WJ, Lu SN, Wang JH, Eng HL. Fine Needle Aspiration Cytodiagnosis of Liver Tumors. Acta Cytologica, 2004; 48(2): 142-148.
  9. Swamy MCM, Arathi CA, Kodandaswamy CR. Value of ultrasonography-guided fine needle aspiration cytology in the investigative sequence of hepatic lesions with an emphasis on hepatocellular carcinoma. J Cytol, 2011; 28(4): 178-184.
  10. Mohmmed AA, Elsiddig S, Abdullhamid M, Gasim GI, Adam I. Ultrasound- guided fine needle aspiration cytology and cell block in the diagnosis of focal liver lesions at Khartoum Hospital, Sudan. Sudan JMS, 2012; 7(3): 183-187.
  11. Ceyhan K, Kupana SA, Bektas M et al. The diagnostic value of on-site cytopathological evaluation and cell block preparation in fine-needle aspiration cytology of liver masses. Cytopathol, 2006; 17: 267–274.
  12. Ӧzkara SK, Tuneli IӦ. Fine Needle Aspiration Cytopathology of Liver Masses: 101 cases with Cyto-/Histopathological Analysis. Acta Cytologica, 2013; 57:332-336

 

Evaluation of Space Occupying Lesion of Liver by Fine Needle Aspiration Cytology and Cell Block Examination

Evaluation of Space Occupying Lesion of Liver by Fine Needle Aspiration Cytology and Cell Block Examination

*Sultana SS,1Dewan RK,2 Ferdousi F,3 Sarker R,4 Jinnah SA,5 Jeba R,6 Haque N,7 Hussain M8

 To differentiate between benign from  malignant  tumor and hepatocellular carcinoma from metastatic carcinoma in hepatic space occupying lesion on the basis of cytology and cell block examination this study was done. This was a descriptive cross sectional study comprising of 48 cases, carried out at the department of pathology, Dhaka Medical College during the period of July 2013 to June 2015. Results of all patients were collected and tabulated. Statistical analysis was performed  on tabulated data. Out of 48 cases, the cytological diagnosis  revealed the highest number of cases of hepatocellular carcinoma 22(45.8%), followed by metastatic carcinoma 13(27.1%), abscess 6(12.5%), hepatocellular dysplasia 3(6.3%) and negative for malignant cell 4(8.3%). Of the total 42 cases of space occupying lesion evaluated by cytology, the diagnoses were similar in cell block, the another six cases contains necrotic debris and cytologically proved as abscess. This measure of agreement is statistically significant with substantial agreement between cell block and cytology status in evaluation of space occupying lesion in liver. Fine needle aspiration cytology in case of space occupying lesion of liver can be relied upon to differentiate between benign and malignant lesion and also primary from secondary lesion. Simultaneous preparation of cell block give no hazard to the patient but provide maximum benefit.

[Journal of Histopathology and Cytopathology, 2018 Jan; 2 (1):11-18]

 Key words: Liver, Lesions, cytology, Cell block

  1. *Dr. Sk Salowa Sultana, Assistant Professor, Department of Pathology, Ad-Din Women’s Medical College, Dhaka. salowasultana257@gmail.com
  2. Professor Dr. Rezaul Karim Dewan, Professor & Head, Department of Pathology, Dhaka Medical College, Dhaka
  3. Farjana Ferdousi Lecturer, Department of Cytopathology, National Institute of Cancer Research & Hospital.
  4. Rabindranath Sarker, Associate Professor, Department of Radiology and Imaging, Dhaka Medical College. Dhaka
  5. Shahed Ali Jinnah, Associate Professor, Department of Pathology, Dhaka Medical College, Dhaka
  6. Ruksana Jeba, Associate Professor, Department of Pathology, Dhaka Medical College, Dhaka
  7. Najmul Haque, Former Associate Professor, Department of Pathology, Dhaka Medical College, Dhaka
  8. Professor Dr. Maleeha Hussain, Former Professor & Head, Department of Pathology, Dhaka Medical College, Dhaka.

 

*For correspondence

 Introduction

Liver diseases are common health problem throughout the world. Liver diseases are broadly categorized as diffuse and focal lesion. The differential diagnosis of focal lesions are primary liver tumors (benign and malignant), metastatic deposits, congenital and acquired cysts and abscess.1 Appropriate clinical management depends on accurate diagnosis but evaluation of the lesion is a common clinical problem.2 Imaging techniques and serological markers are useful in narrowing the differential diagnosis. Fine needle aspiration cytology (FNAC) mainly indicated in the diagnosis of malignant focal lesions both primary and secondary. FNAC also performed to rule out neoplasm from inflammatory lesion when radiologically inconclusive.3-6

Hepatocellular carcinoma (HCC) is responsible for a large proportion of cancer death worldwide. Also there are demographic variation in the incidence of HCC.7 GLOBOCAN global analysis published moderately high incidence (11-20 per 100,000) in Southeast Asia and also shows 82% of liver cancer cases occurring in developing countries. HCC is preceded by cirrhosis of the liver in most cases. The majority of them are due to viral hepatitis. Indeed, worldwide 50-80% of HCC is due to HBV and (10-25)% of cases are due to HCV infection respectively.8 Dual infection with HBV and  HCV is not uncommon in southeast Asia.9 Other causes include alcoholic liver disease, nonalcoholic steatohepatitis, intake of aflatoxin contaminated foods, diabetes and obesity.10

Liver is the most common site of distant metastasis as it filters most of the blood from the body.11 Metastasis commonly arise from tumor of colon, pancreas, breast and lung. Accurate diagnosis of the metastatic lesions is essential in determining the stage of tumor and also for therapeutic and prognostic purposes.  The  treatment vary  from  palliative care to partial hepatectomy, specially in those which are potentially chemosensitive or hormonally manipulable. Correlation of clinical, laboratory and radiologic findings is necessary. Radiologically multiple nodules of various sizes distributed randomly suggest metastases5.

The present study was done to evaluate the space occupying lesion of liver by fine needle aspiration cytology accompanying with cell block examination. There are some pitfalls in cytology associated with aspiration of necrotic material and presence of  regenerative atypia in hepatocytes. Some of these pitfall can be minimized by using cell block.  In cell block, architectural pattern,  thickening of cell plate and traversing blood vessels with their lining endothelial cells can be seen. Simultaneous preparation of cellblock  from the residual material after smear preparation can help to evaluate the difficult cases.12

 Methods

This is a descriptive cross sectional study which was carried out at the department of pathology, Dhaka Medical College, during the period of July, 2013 to June, 2015. The study was done on fine needle aspiration material of liver SOL that were received from Dhaka Medical College and Hospital, and Bangabandhu Sheikh Mujib Medical University (BSMMU).

Patients with radiologically diagnosed SOL in liver and suspected as a case of hepatic neoplasm were included in this study. Patient with bleeding disorders, prolonged prothrombin time,  Patient with liver abscess, cyst, haemangioma and already diagnosed cases were excluded from this study.

Relevant clinical informations were recorded. Patients having suspected hepatic neoplasm with good coagulation profile underwent ultrasound guided FNAC. According to standard protocol FNAC was done by an expert pathologist or radiologist and USG  guidance was provided by an expert radiologist.

Several cytologic smears were prepared and fixed immediately in 95% alcohol. The smears were left in alcohol at least for 30 minutes at room temperature before staining. The residual material remaining after completion of cytological smears  were fixed in 10% formalin and later processed to prepare paraffin embedded blocks .

 Smears prepared were stained by papanicolaou stain. Cell blocks were prepared by fixed sediment and bacterial agar method and stained by Hematoxyllin and eosin. Cytopathological examination of the stained slides of hepatic SOL were carried out under light microscope on the same day or next day. Satisfactory smears contained adequate number of representative cells from the target sites. Stained cell block sections were examined to compare  the cytological diagnosis.

Results

Table I shows age of the study patients, half of the patients belonged to age 51-70 years. The mean age was found 53.0±15.0 years with range from 18 to 90 years.

It was observed that three fourth (36, 75.0%) patients were male and 12(25.0%) patients were female. Male female ratio was 3:1.

Table II shows cytological diagnosis of the study patients. It was observed that almost half of the patients (45.8%) were found HCC followed by 13(27.1%) were metastasic Ca, 6(12.5%) were abscess and 3(6.3%) were hepatocellular dysplasia .

Table III shows presenting complaints of the study patients. Total 29 patients present with abdominal pain only. Nine patients presented with abdominal lump and  pain.

Table IV shows 22 patients were cytologicaliy  diagnosed as HCC. Among them 11(50.0%) were HBsAg positive and  2(9.1%)  were Anti HCV positive. No case was dual positive. In case of, 42 patients the tumor size was >3 cm, among them 2(66.7%) patients cytologically diagnosed as hepatocellular dysplasia, 22(100.0%)  HCC, 13(100.0%) metastatic carcinoma and 5(83.3%)  abscess. Total 28 patients presented with multiple SOL. Among them 10 (45.5%)  were HCC and 11(84.6%)  were metastatic carcinoma.

The association between cell block and cytology status in evaluation of space occupying lesion of liver is given in table VI. Of the total 42 cases of space occupying lesion evaluated by cytology, the diagnosis of 3(75.0%) negative for malignant cell, 2(66.7%) hepatocellular dysplasia, 20(90.9%) HCC and 8(61.5%) metastatic tumour were also similar by cell block. The results of the two methods (cell block and cytology status) analysis found Kappa value = 0.680 with p<0.05. This measure of agreement is statistically significant with substantial agreement between cell block and cytology status in evaluation of space occupying lesion in liver. One of the three cytologically diagnosed hepatocellular dysplasia one was finally proved as HCC after cell block examination.

 Table I: Distribution of the study patients by age (n=48)

 

Age (years) Number of patients Percentage
≤30 4 8.3
31-50 17 35.4
51-70 24 50.0
>70 3 6.3
Mean±SD 53.0 ±15.0
Range (min-max) 18 -90

 

Table II: Distribution of the study patients by cytology (n=48)

 

Cytology Number of patients %
Negative 4 8.3
HD 3 6.3
HCC 22 45.8
Abscess 6 12.5
MetastasicCa 13 27.1
        Adenocarcinoma 9 18.8
        Sq. CC 1 2.1
        RCC 1 2.1
        GIST 1 2.1
        Small cell Ca 1 2.1

 

Table I11: Distribution of the study patient with different  cytological diagnosis according to  clinical feature (n=48)

 

Clinical feature Cytological diagnosis
Negative

(n=4)

HD

(n=3)

HCC

(n=22)

Metastatic Ca

(n=13)

Abscess

(n=6)

 

n % N % n % n % n %
Abd lump only 0 0.0 0 0.0 0 0.0 3 23.1 0 0.0
Abd pain only 3 75.0 1 33.3 15 68.2 5 38.5 5 83.3
Lump + pain 0 0.0 1 33.3 7 31.8 1 7.7 0 0.0
Pain + ascitis 0 0.0 0 0.0 0 0.0 1 7.7 0 0.0

 

Table IV: Distribution of cytologically diagnosed cases  with viral marker (n=48)

 

Viral marker Cytologcal diagnosis
Negative

(n=4)

HD

(n=3)

HCC

(n=22)

MetastasicCa

(n=13)

Abscess

(n=6)

n % n % N % N % n %
HBsAg
Positive 1 25.0 0 0.0 11 50.0 0 0.0 0 0.0
Negative 3 75.0 3 100.0 11 50.0 13 100.0 6 100.0
Anti HCV
Positive 0 0.0 0 0.0 2 9.1 0 0.0 0 0.0
Negative 4 100.0 3 100.0 20 90.9 13 100.0 6 100.0

 

Table V: Association between cytology status with USG finding (n=48)

 

USG finding Cytologycal diagnosis P value
Negative

(n=4)

HD

(n=3)

HCC

(n=22)

Metastatic Ca

(n=13)

Abscess

(n=6)

 

 

n % N % n % N % n %
Size
        ≤3 cm 4 100.0 1 33.3 0 0.0 0 0.0 1 16.7 0.001s
        >3 cm 0 0.0 2 66.7 22 100.0 13 100.0 5 83.3
SOL
        Single 1 25.0 1 33.3 12 54.5 2 15.4 4 66.7 0.122ns
        Multiple 3 75.0 2 66.7 10 45.5 11 84.6 2 33.3
Diagnosis
        Primary 0 0.0 0 0.0 13 59.1 0 0.0 0 0.0
        Secondary 3 75.0 2 66.7 6 27.3 12 92.3 0 0.0 0.001s
        Not diagnosed 1 25.0 1 33.3 3 13.6 1 7.7 6 100.0

 

Table VI: Association between cytological diagnosis with cell block (n=42)

 

Cell block Cytological diagnosis
Negative

(n=4)

Hepatocellular dysplasia

(n=3)

HCC

(n=22)

Metastatic Tumour

(n=13)

Inconclusive
n % N % n % n %
Negative for malignah 3 75.0 0 0.0 0 0.0 0 0.0 0
Hepatocellular dysplasia 0 0.0 2 66.7 0 0.0 0 0.0 0
HCC 0 0.0 1 33.3 20 90.9 0 0.0 0
Metastatic tumour 0 0.0 0 0.0 0 0.0 8 61.5 0
Inconclusive 1 25.0 0 0.0 2 9.1 5 38.5 0

 

 

 

 

 

Figure 1. Sex distribution of the study patients

 

 

 

 

 

 

Figure 2. Photomicrograph showing hepatocellular carcinoma (Cytology, Pap stain x40)

 

 

 

 

 

 

Figure 3. Photomicrograph showing hepatocellular carcinoma (Cell Block, H&E stain x20)

 

 

 

 

 

 

 

Figure 4. Photomicrograph showing hepatocellular carcinoma (Cell Block, H&E stain x10)

 

 

 

 

 

Figure 5. Photomicrograph showing adenocarcinoma (Cell Block, H&E stain x40)

 

 

 

 

 

 

Figure 6. Photomicrograph showing adenocarcinoma (Cytology, H&E stain x20)

 Discussion

Liver diseases particularly neoplasia  form focal lesion and are often asymptomatic. Even relevant biochemical tests may not show significant changes.13 Diagnosis and management of space occupying lesions in liver is a great challenge. The present study was carried out to evaluate the space occupying lesion of liver by USG guided FNAC.

The mean age of this study cases with hepatic SOL was forth to fifth decades. In Bangladesh Rahman et al (2014)14 showed similar observations. Similar findings were also observed by Nasit et al (2013)3 in India and Nazir et al (2010)4 in Pakistan. In this study, the incidence was more in male than female. According to World Factbook 2014, the finding is similar. In Asia socioeconomic condition and lifestyle favour exposure to hepatitis more in male than female. Risk factors for HBV and HCV infection such as transfusion related spread, sharing  of needle and syringes which is common for drug abuser, unprotected sex etc are more prevalent in male.18

Common clinical features were abdominal pain, lump and other constitutional symptom. Most of the patients with HCC presented with only abdominal pain. In metastatic group abdominal pain was frequently accompanied by lump in abdomen. Most of the patients with abscess presented with the complain of abdominal pain.  Similar observations regarding the clinical presentations were observed in the study done by Nasit et al (2013)3 and Hossain et al (2010).15

Viral marker was significantly positive in patients with hepatic malignancy. Half of the patients with HCC were HBsAg positive and 9% were anti-HCV positive in this study. Rahman et al (2014)13 and Rahman et al (2010)15 also observed similar findings in Bangladesh.

In present study, most of the cytological diagnosis cases of metastatic carcinoma  had multiple SOL in USG. Similar findings regarding distribution of cases according to cytological diagnosis was observed by Mohammed et al (2013)18 and Najir et al (2010). Hepatocellular carcinoma were more than metastatic neoplasm. Also metastatic cases include mostly adenocarcinoma. Similar findings regarding distribution of cases according to cytological diagnosis was observed by Mohammed et al (2013)15 and Najir et al (2010).4 To evaluate the accuracy of USG guided FNAC in hepatic SOL, the cytological diagnosis was compared with cell block.  Dysplastic nodules are precursor lesions of HCC and need careful evaluation. In such difficult cases cell block can help in examining architectural pattern as well as ancillary studies. Sometimes cirrhosis, progressing to HCC may have SOL. Cytology of cirrhosis may reveal pleomorphism, multinucleation, stippled cytoplasm and mimic HCC. Cell block in these cases show  hepatocytes and bile duct epithelial cells in monolayered sheets.  Other pitfall of FNAC related to  the diagnosis of well differentiated HCC and poorly differentiated HCC to be distinguished. Architectural pattern, thickening of cell plate, lining and traversing endothelial cell can determine and differentiate in such cases.18 Thus, cell block in addition to smears improve the diagnostic performance and decrease the non diagnostic result.

Conclusion

Treatment modalities are rapidly developing worldwide. Long term survival requires detection of small tumors. The patients with chronic liver diseases and other known primary need regular and proper evaluation. FNAC is a safe, minimum invasive procedure and multiple sample can be obtained with the small diameter needle. FNA cytology in case of SOL of liver can be  relied upon to differentiate between benign and malignant lesion and also from primary from secondary. However the indeterminate or  inconclusive report is a pitfall , which needs to be minimized. The result can be improved considering with availablity  of cell block examination. To get maximum benefit combined approach of FNAC and cell block   can be applied.

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