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Laboratory Findings of Histopathologically Confirmed Tuberculous Lymphadenitis

Laboratory Findings of Histopathologically Confirmed Tuberculous Lymphadenitis

*Huda MM,1 Kamal M,2 Sultana AT,3 Yusuf MA,4 Taufiq M,5 Begum F6

Abstract

Laboratory findings are important for the tuberculous lymphadenitis patients for diagnosis and treatment. The purpose of the present study was to see the laboratory findings of tuberculous lymphadenitis patients. This cross-sectional study was done at the Department of Pathology, Banghabandhu Sheikh Mujib Medical University (BSMMU), Dhaka from January 2009 to March 2011 for a period of nearly two (2) years. All the patients irrespective of age and sex with the clinical features of tuberculous lymphadenitis and later confirmed by histopathological examination were selected for the study purposively. Relevant information was recorded in a prescribed data sheet and histomorphological findings were recorded accordingly. In cases where fresh specimen was available, caseous portion of lymph node was sent for culture. Fite Faraco staining was done on lymph node sections in all cases. A total of 50 cases tuberculous lymphadenitis patients were recruited for this study. Raised ESR was found in 42 (84.0%) cases. Mantoux (MT) test was positive in 37 (74.0%) patients. Out of 50 patients 34 (68.0%) cases had well-formed granuloma and 8 (16.0%) cases had both well-formed and ill-defined granuloma. Growth of Mycobacterium culture in Lowenstein-Jensen media was seen in 12 (60%) cases. In conclusion, majority of the tubercular lymphadenitis patients presented with raised ESR with positivity of Mantoux test (MT) and well defined granuloma.

[Journal of Histopathology and Cytopathology, 2017 Jul; 1 (2):116-119]

 Keywords: Tuberculous lymphadenitis; tuberculosis; lymph nodes; laboratory findings

  1. *Dr. Mohammad Mahmudul Huda, Assistant Professor, Department of Pathology, Dhaka National Medical College, Dhaka, Bangladesh. mmhuda.himel@gmail.com
  2. Mohammed Kamal, Professor, Department of Pathology, Banghabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh.
  3. Akhand Tanzih Sultana, Assistant Professor, Department of Respiratory Medicine, Bangladesh Institute of Child Health & Dhaka Shishu Hospital, Dhaka, Bangladesh.
  4. Md. Abdullah Yusuf, Assistant Professor, Department of Microbiology, National Institute of Neurosciences & Hospital, Dhaka, Bangladesh.
  5. Mohammad Taufiq, Associate Consultant (Pathology), Square Hospital, Dhaka, Bangladesh.
  6. Ferdousy Begum, Associate Professor, Department of Pathology, Banghabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh.

*For correspondence

Introduction

Lymph nodes are usually involved in the early stages of the pulmonary disease or as secondary tuberculosis by hematogenous spread.1 However, tuberculous lymphadenitis may arise without a detectable preceding pulmonary involvement.2 Tuberculous lymphadenitis affects mainly the cervical lymph node group and is an important cause of lymphadenopathy worldwide.3 The clinical as well as the demographic characteristics are varied. To confirm the diagnosis histopathological examination or FNAC is needed. These investigations help to arrive at an early diagnosis of tubercular lymphadenitis and institution of treatment before a final diagnosis is made by culture.4

Histopathological examination is suggestive of tuberculous lymphadenitis where Langhans’ giant cells, caseation necrosis, coalescing granuloma are present.5 The physicians treat these cases with anti-tubercular chemotherapy. In cases which are reported as suggestive of tuberculosis, the physician needs additional laboratory findings such as positive Mantoux test and other laboratory tests to start anti-tubercular chemotherapy.6 Therefore this present study was to see the laboratory findings of tuberculous lymphadenitis patients.

 Methods

This cross-sectional study was done at the Department of Pathology, Banghabandhu Sheikh Mujib Medical University (BSMMU), Dhaka from January 2009 to March 2011 for a period of nearly two (2) years. All the patients irrespective of age and sex with the clinical features of tuberculous lymphadenitis and later on proved to be the same histologically were selected for the study purposively. The patients without having history of anti-tubercular drugs, malignancy and symptoms other than TB were excluded from this study. Relevant information was recorded in a prescribed data sheet and histomorphological findings were recorded accordingly. In cases where fresh specimen was available, caseous portion of lymph node was sent to ICDDRB for culture in conventional egg based Lowenstein- Jensen medium7. This part was done maintaining high level of sterility. Ziehl-Neelsen stain was done in smear prepared from 20 fresh cases. Fite Faraco staining was also done on lymph node sections in all cases. Computer based statistical analysis was carried out with appropriate techniques and systems. All data were recorded systematically in preformed data collection form (questionnaire) and quantitative data were expressed as mean and standard deviation and qualitative data were expressed as frequency distribution and percentage. Statistical analysis was performed by using window based computer software devised with Statistical Packages for Social Sciences (SPSS-22.0) (SPSS Inc, Chicago, IL, USA).

 Results

A total of 50 patients were recruited for this study. Raised ESR was found in 42 (84.0%) cases. Mantoux (MT) test was positive in 37 (74.0%) patients. Two (4.0%) patients had chest X-ray (CXR) suggestive of pulmonary TB; however, no case was positive for AFB on sputum (Table I).

 Table I: Laboratory and radiological findings

 

Investigations Frequency Percentage
Raised ESR 42 84.0
Mantoux test 37 74.0
X-ray chest 2 4.0
Sputum for AFB 0 0.0

 

In this study, out of 50 patients 34 (68.0%) cases had well-formed granuloma and 8 (16.0%) cases had both well-formed and ill-defined granuloma, and the remaining 8 (16.0%) cases had ill-defined granuloma (Table II).

Table II: Distribution of study sopulation according to types of granuloma

 

Types of granuloma Frequency Percentage
Well formed 34 68.0
  Coalescing 06 17.6
Discrete 06 17.6
Both coalescing & discrete 22 64.7
Ill defined 8 16
Both well formed and ill defined 8 16

Culture was done in 20 fresh cases in Lowenstein-Jensen media. Caseous portion of the lymph node was taken for culture. Growth of Mycobacterium was seen in 12 (60%) cases (Table III).

Table II: Culture of lymph node material in Lowenstein-Jensen media (n=20)

 

Culture Frequency Percentage
Growth present 12 60.0
Growth absent 8 40.0
Total 20 100.0

Discussion

Tuberculosis is a major cause of unpleasant health and mortality worldwide.1 The risk of infection however is a lot larger in midst of communities of inferior socioeconomic groups.2 Yearly 2.2 million individuals acquire TB in India of which approximately 0.87 million are infectious cases and responsible for about more than 330,000 per annum.3 TB is considered as the most usual opportunistic infection in belts where HIV infection is rampant. Someone in the world is newly infected with TB bacilli every second. Overall, one-third of the world’s population is currently infected with the TB bacillus.5 Among the various diagnostic tools of tubercular lymphadenitis, histological diagnosis is an important one.  As the morphological features are variable, this descriptive study was performed in 50 cases of tubercular lymphadenitis to find out histomorphological features and correlate with clinical profile and treatment outcome.

In this present study histomorphological examination shows a well formed granuloma in majority of the study population (68.0%). Among the well formed granuloma 64.7% had both coalescing and discrete granuloma, 17.6% had only coalescing granuloma and 17.6% had only discrete granuloma. Pahwa et al6 found well formed granuloma in 89.0% cases, which is higher than the percentage found in this present study. Extent of caseous necrosis was also observed in present study. Of 50 patients, 18% patient had focal area of caseous necrosis, 54.0% had moderate caseous necrosis and 28.0% had extensive caseous necrosis. This present study showed variation in percentage of caseous necrosis in individual cases.

Out of 50 cases caseous material of 20 fresh cases were cultured on Lowenstein-Jensen medium for Mycobacterium tuberculosis. In this study culture positivity was 60.0%. Similar to the present study result Iqbal et al7 found 62.0% culture positive cases in their study. However, Sathekge et al8 found 45.0% culture positive cases in their study. Fite Faraco stain was performed on lymph node sections in all 50 cases. No case was positive for AFB.  Jayalakshmi et al9 found 49.2% AFB positive cases in tissue section. They have performed Fite Faraco stain in several sections. Further analysis of the cases through several sections might have revealed positive cases. Five culture positive cases (41.7%) out of 12 needed treatment for nine months. Bacterial load in culture positive cases might play a role in requiring long duration of treatment.

 Conclusion

In conclusion majority of the histologically confirmed tubercular lymphadenitis patients presents with raised ESR. Furthermore, maximum shows positivity of Mantoux test (MT). Such laboratory findings with lymphadenitis should lead to histological confirmation of tubercular lymphadenitis.

 References

  1. Swaminathan S, Rekha B. Pediatric tuberculosis: global overview and challenges. Clin Infect Dis 2010;50(Supplement 3):S184-94
  2. Kumar V, Abbas AK, Fausto N, Aster JC. In: Robbins and Cotran Pathologic Basis of Disease. Elsevier Health Sciences; 2014 Aug 27
  3. Seth V, Kabra SK, Jain Y, Semwal OP, Mukhopadhyaya S, Jensen RL. Tubercular lymphadenitis: clinical manifestations. Indian J Pediatr 1995;62(5):565-70
  4. Chao SS, Loh KS, Tan KK, Chong SM. Tuberculous and nontuberculous cervical lymphadenitis: a clinical review. Otolaryngology–Head and Neck Surg 2002;126(2):176-9
  5. Raviglione MC, Narain JP, Kochi A. HIV-associated tuberculosis in developing countries: clinical features, diagnosis, and treatment. Bull WHO 1992;70(4):515
  6. Pahwa R, Hedau S, Jain S, Jain N, Arora VM, Kumar N, Das BC. Assessment of possible tuberculous lymphadenopathy by PCR compared to non-molecular methods. J Med Microbiol 2005;54(9):873-8
  7. Iqbal MA, Subhan AN, Aslam AS. Frequency of tuberculosis in cervical lymphadenopathy. J Surg Pakistan 2010;15(2):107-09
  8. Sathekge M, Maes A, D’Asseler Y, Vorster M, Gongxeka H, Van de Wiele C. Tuberculous lymphadenitis: FDG PET and CT findings in responsive and nonresponsive disease. Europ J Nuclear Med Molecul Imag 2012;39(7):1184-90
  9. Fontanilla JM, Barnes A, Von Reyn CF. Current diagnosis and management of peripheral tuberculous lymphadenitis. Clin Infect Dis 2011;53(6):555-62

 

 

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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

 

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Comparing PAP Smear Cytology with High Risk Human Papilloma Virus DNA Test in Patients of Cervical Lesions

Comparing PAP Smear Cytology with High Risk Human Papilloma Virus DNA Test in Patients of Cervical Lesions

*Ansari NP,1  Rahman AN,2  Saleh AM,3 Shahida SM4

 Abstract

Cervical cancer is the second most frequent type of cancer worldwide. More than eighty eight percent deaths from cervical cancer occur in developing countries. In developed countries, the cases and deaths have declined markedly due to their extensive screening programs. The present study was undertaken to assess precancerous and cancerous cervical lesions by cytology as well as Human Papilloma virus (HPV) DNA identification and their comparison with histopathology in Visual Inspection of Cervix with Acetic Acid (VIA) positive cases. This observational study was carried out at the Department of Pathology in collaboration with the Department of Gynaecology and Obstetrics of Mymensingh Medical College Hospital and Department of Microbiology and Hygiene of Bangladesh Agriculture University for HPV DNA detection during the period of July 2012 to June 2013. Study was carried out among 160 VIA positive patients and selected by non-random judgment sampling from the colposcopy clinic. Out of 160 cases, only 40(25.00%) were found HPV DNA positive, while the rest 120(75.00%) cases were negative. Among positive cases 77.50% were cancerous cases and 22.50% were precancerous cases. It was further revealed that in cancerous cases, 86.11% were HPV DNA positive. PCR showed low sensitivity, probably due to sampling error and inclusion of all cases (chronic cervicitis, precancerous and cancerous lesion). The statistical value of accuracy, sensitivity and specificity of Pap smear cytology, HPV DNA test and histopathology yielded some important directives. The sensitivity values of Pap smear cytology and HPV DNA were found 87.50% and 88.89% respectively.  Thus Pap smear test showed almost equal sensitivity to DNA test. The accuracy of the Pap smears and HPV DNA in this study was 88.13% and 96.88% respectively. The accuracy of Pap smears is lower than HPV DNA tests. The present study showed the significant relationship between cytological with HPV DNA test and histopathological diagnosis. But cytology and HPV DNA testing are not suitable as a single test. In conclusion, it can be stated that combination cytology (Pap smear), histopathology and new technologies such as HPV DNA typing would ultimately be more useful.

[Journal of Histopathology and Cytopathology, 2017 Jul; 1 (2):102-109]

 Key words: Pap smear cytology, Human Papilloma virus, Cervical lesions

  1. *Dr. Nazma Parvin Ansari, Assistant Professor of Pathology, Community Based Medical College, Mymensingh. palashansaripath@gmail.com
  2. AJE Nahar Rahman, Honorary Professor, Department of Pathology, BIRDEM, Dhaka.
  3. AFM Saleh, Professor of Pathology, Mymensingh Medical College, Mymensingh.
  4. S M Shahida, Assistant Professor of Gynae & Obs, Dhaka Medical College, Dhaka

 *For correspondence

Introduction

Cervical cancer is the second most frequent type of cancer and the leading cause of mortality among women worldwide.1 According to WHO/ICO Report2 the   incidence rates of cervical cancer in Bangladesh are highest compared to other countries of South Asia and the annual mortality is 11.6 per 1,00,000 women. The report further stated that 50.19 million women are at risk of developing cancer. Each year 17,686 women are diagnosed with cervical cancer and 10,364 die from the disease. It is observed that the cases and deaths have declined markedly in developed countries from approximately 1.4 to 1.7 per 1,00,000 women and this reduction was mainly as a result of extensive screening programs.3,4

 There is worldwide agreement that screening test for cervical cancer is a necessity because it separates healthy persons from those with a high probability of having the disease. The lower incidence in developed countries is due to the access of women to screening test, which allows precancerous lesions to be detected and treated before they develop into full blown cancer.5 Popular screening tests are Papanicolaou (PAP) smear, visual inspection of cervix with acetic acid (VIA) and HPV DNA test. The uptake of screening test in many developing countries is still poor.6,7,8,9  VIA has been introduced by the Government of Bangladesh to screen mass population because it is easy procedure and cost effective. All the government medical college hospitals, district hospitals, maternal and child welfare centers and some of the urban primary health care centers have facilities for VIA test free of cost by the support of UNFPA Bangladesh. Bangabandhu Sheikh Mujib Medical University (BSMMU) is collaborating with Government of Bangladesh in expansion of these facilities.10

At the present time a good deal of attention is being paid to screening for early confirmatory detection of cancer. The present study was undertaken to assess precancerous and cancerous cervical lesion by cytology in VIA positive cases and the relationship between the high risk Human Papilloma viruses.

 Methods

This was a descriptive, cross-sectional type of observational study. The study was carried out in the Department of Pathology, Mymensingh Medical College. Patients of different age groups were selected from colposcopy clinic of Mymensingh Medical College Hospital. Duration of study was one year ranging from July 2012 to June 2013. A total of 160  patients were selected in the mentioned study period with follwing criteria which were (1) VIA positive married married females aged 30-60 years and (ii) who were married for at least 10 years but below 30 years of age. The study excluded patients who were pregnant, below twenty years of age and patient at menstruating period.  PAP smears were collected with Ayer’s spetula and cytobrash by gynaecologists in the colposcopy clinic in the department of Mymensingh Medical College Hospital. Colposcopy guided punch biopsy were taken by same gynecologists from the suspected lesions of the VIA positive cases. Histopathological examination of the biopsy samples were done in the department of Pathology, Mymensingh Medical College. HPV DNA detection by Polymerase Chain Reaction was done from paraffin embedded tissue blocks. HPV type 16, 18, 31 and 52 primers were used.

Results

The study was undertaken to assess the precancerous and cancerous cervical lesion by cytology in VIA positive cases and compare their results with histopathology and high risk Human Papilloma Virus (hr-HPV) DNA test. Table I shows that 36 (22.50%) patients were in 20 to 29 years age group, 72 (45.00%) patients belonged to the age group 30 to 39 years, 37 (23.10%) cases in 40 to 49 years age group and 15 (9.40%)  cases in 50-59 years group.

Table I: Age distribution of the subjects

Age Groups in Year Frequency %
20-29 36 22.5
30-39 72 45
40-49 37 23
50-59 15 9.4
Total 160 100

*Mean () = 38.00 Years; Standard Deviation (SD) = 8.15 Years.

Table II shows the results of Pap smears diagnosis. On cytological examination, 63 (39.40%) cases were diagnosed as Negative for Intraepithelial Lesion or Malignancy (NILM), followed by 5 (3.10%)  cases diagnosed as lesions with  Atypical Squamous Cells of Undetermined Significance (ASCUS) and  other 5 (3.10%) cases were found Atypical Squamous Cell  that cannot exclude HSIL (ASC- H), 40 (25.00%)  patients with Low Grade Squamous Intraepithelial Lesions (LSIL); 17 (10.60%) patients with high grade squamous intraepithelial lesion (HSIL)and 30 (18.75%)  patients  were diagnosed as Squamous Cell carcinoma (SCC).

Table II: Cytological (Pap smear) diagnosis of cervical lesions

Serial no. Pap smear test

result

 Frequency %
1 NILM 63 39.40
2 ASCUS 05 3.10
3 ASC-H 05 3.10
4 LSIL 40 25.00
5 HSIL 17 10.60
6 Squamous Cell Carcinoma 30 18.75
Total 160        100.00

NILM = Negative for Intraepithelial Lesion or Malignancy, ASCUS = Atypical Squamous cells with Undetermined Significance, LSIL = Lowgrade Squamous Intraepithelial Lesion, HSIL = High grade Squamous Intraepithelial Lesion, ASC-H = Atypical Squamous Cell  that cannot exclude HSIL

The histopathological result of 160 cases is presented in Table III. It was revealed that 56 (35.0%) cases had chronic cervicitis, 51 (31.9%) cases  mild dysplasia (CIN-I), 14 (8.8%)  cases diagnosed as  moderate dysplasia (CIN II), 3 (1.9%) patients were severe dysplasia / CIN III and the remaining 36 (22.5%)  cases show invasive squamous cell carcinoma (SCC).

 Table III: Histological diagnosis of patients with cervical lesions

SL. no. Histological

Diagnosis

  Frequency %

 
1 Chronic Cervicitis 56 35.00
2 CIN – I 51 31.90
3 CIN II 14 8.80
4 CIN III  3 1.90
5 Squamous cell carcinoma 36 22.50
Total 160 100

 

CIN = Cervical Intraepithelial Neoplasia

Table IV shows that on histological examination of 160 cases, a total of 56 (35.00%) cases were found with chronic cervicitis, among them 50 (79.37%) cases were correctly diagnosed previously cytologically as negative for intraepithelial lesion. Out of 51 (31.9%) diagnosed CIN I cases, 34 (85.0%) cases were diagnosed  cytologically as LSIL. Out of 14  (8.8%) cases diagnosed as CIN II, 10 (58.82%) cases were diagnosed cytologically HSIL. Out of 3 (1.90%) cases of histologically diagnosed  CIN III, 2 (11.76%) cases were diagnosed cytologically as HSIL. The 30 cases were diagnosed cytologically as carcinoma and those cases were histologically confirmed as squamous cell carcinoma. There is highly significant relationship in respect to cytological (PAP smear) and histological diagnoses of cervical lesions in the study group (P<0.005).

Table IV: Comparison of Cytological (PAP smear) and histological diagnoses of cervical lesions

 

Frequency in PAP  smear diagnosis Frequency in histological diagnosis
Pap smear

Test

 Frequency Chronic Cervicitis CIN I CIN II CIN III Squamous cell carcinoma
NILM 63 50(79.37) 13(20.63) 00 00 00
ASCUS 05 00 3(60.00) 2(40.00) 00 00
ASCH 05 00 00 2 (40.00) 1(20.00) 2 (40.00)
LSIL 40 6 (15.00) 34 (85.00) 00 00 00
HSIL 17 00 1 (5.89) 10 (58.82) 2 (11.76) 4 (23.52)
Squamous cell carcinoma 30 00 00 00 00 30 (100.00)
Total 160 56 51 14 03 36

NILM = Negative for Intraepithelial Lesion or Malignancy, ASCUS = Atypical Squamous cells with Undetermined Significance, LSIL = Lowgrade Squamous Intraepithelial Lesion, HSIL = High grade Squamous Intraepithelial Lesion, ASC-H = Atypical Squamous Cell  that cannot exclude HSIL

*Figures within parentheses indicate percentage

 The HPV DNA test of cervical lesions was conducted on all 160 cases. The result is shown table V. Out of 160 cases, only 40 (25%) cases were positive and the rest 120 (75%) cases were negative for HPV DNA.

Table V: Diagnosis of cervical Lesions by HPV DNA test (PCR)

HPV DNA test Frequency %
Positive 40 25
Negative 120 75
Total 160 100

Table VI gives a correspondence result of Histological Diagnosis with Pap smear and HPV DNA test. It was remarkable that out of 36 squamous cell carcinoma cases 31 patients were detected by HPV DNA test. The result further indicates that the cytological diagnosis nearer to the histological findings in almost all cases under this study and in case of high grade lesion and carcinoma it was almost close to positive reaction to HPV DNA test.

Table VI. Correspondence of histological diagnosis with PAP smear and HPV DNA test

 

Histological

diagnosis

 Frequency Frequency of Pap Smear diagnosis N0. of patients

positive to HPV DNA test
NILM ASCUS ASCH LSIL HSIL Squamous cell carcinoma Positive Negative
Chronic

cervicitis

 56 50 – – 06 – – 00  56
CIN – I 51 33 03 00 34 01 00 02  49
CIN – II 14 – 02 02 10 – 05  09
CIN – III 03 – 00 01 00 02 – 02  01
Squamous cell carcinoma

 

 36 – – 02 – 04 30 31  05
Total 160 63 05 05 40 17 30 40 120

 

NILM = Negative for Intraepithelial Lesion or Malignancy, ASCUS = Atypical Squamous cells with Undetermined Significance, LSIL = Lowgrade Squamous Intraepithelial Lesion, HSIL = High grade Squamous Intraepithelial Lesion, ASC-H = Atypical Squamous Cell  that cannot exclude HSIL

The result illustrates that the sensitivity value of this test is almost equal to both Pap test and HPV DNA test, those were found 87.50% and 88.89% respectively. In case of specificity value the Pap test yielded 89.29% and HPV DNA tests yielded higher value, 100%.

Table   VII: Comparison of statistical analysis of PAP smear and HPV DNA test

 

Statistical value Pap smear

Cytology in %

 HPV DNA test in %
Sensitivity 87.50   88.89
Specificity 89.29 100.00
Accuracy 88.13   96.88
Predictive value of a

positive test

 93.81 100.00

 
Predictive value of a

negative test

 79.37  95.83

 HPV DNA revealed that, HPV – 16 were present in the highest percent (75%), HPV-18 were in 15% and the lowest number of occurrences were HPV – 31 type in 5% and HPV – 52 type in 5 %.

 Discussion

The present study focused on traditional Pap smear method and HPV DNA type identification for cervical screening and their comparison with colposcopic biopsy. In this study, 160 VIA positive women were selected. Pap smear cytology, histopathological examination and HPV DNA test were done in every cases. On cytological (Pap Smears) examination it was revealed  that 63 (39.4%) cases were diagnosed as Negative for Intraepithelial Lesion or malignancy (NILM), 5 (3.10%) cases were diagnosed as Atypical Squamous Cell of Undetermined Significance (ASCUS) and 5 (3.10%)  cases were Atypical Squamous Cell  cannot exclude HSIL (ASC- H), 40 (25%)  patients with Low Grade Squamous Intraepithelial Lesions (LSIL); 17 (10.6%) patients with high grade squamous intraepithelial lesion (HSIL) and 30 (18.75%) cases were diagnosed as Squamous Cell carcinoma (SCC). A study on patients attending the OPD of BSMMU showed much lower incidence in Pap smear. The percentages of SCC, LSIL and NILM were 0.2%, 4.20% and 91.70% respectively. The lower incidence may be due to incorporation of normal cases in BSMMU study.11

The result of histopathological examinations of 160 cases revealed 56 (35%)  cases of chronic cervicitis, 51 (31.9%) cases of mild dysplasia (CIN-I), 14 (8.8%)  cases of moderate dysplasia (CIN II), 3 (1.9%) patients of severe dysplasia (CIN III) and the remaining 36(22.5%)  cases as invasive squamous cell carcinoma (SCC). Among cytologically diagnosed 30 (18.75%) squamous cell carcinoma cases all were confirmed histologically as squamous cell carcinoma. Highly significant relationship was found with cytological (Pap smear) and histological diagnoses of cervical lesions in this study group (p<0.005).

A study was performed in BSMMU on 70 histologically diagnosed cases of CIN. Out of 70 cases, 34 (48.6%) cases were CIN-I and 36 (50.4%) cases were CIN-II/III.12  Another study  from Brazil reported the percentage of CIN-I, CIN-II and squamous cell carcinoma to be 12.0%, 4.3% and 1.0% respectively13. The higher percentage of the present study reflects the inclusion of VIA positive cases only. In Bangladesh a cross sectional study was found and the above occurrences as 21.4%, 10.0% and 29.0% respectively.14

Therefore, out of 160 cases 91(56.88%) cases were correctly diagnosed by cytology, 13 (8.13%) cases were false negative, 6 (3.75%) cases were false positive and 50 (31.25) cases were negative for precancerous and cancerous lesions. There is a significant correlation between Pap smear and Histological findings. Others studies similarly found a significant concordance between Pap smear and histological tests for the diagnosis of precancerous cervical lesions.15,14,16

The result of the HPV DNA detection conducted on all 160 cases. Out of 160 cases, only 40 (25.00 %) were found positive and the rest 120 (75.00%) cases were negative. Present study shows that 86.11% positive in cancerous lesions. The technology can find the HPV DNA in almost 100.0% of the invasive cervical cancer cases, 75.00-100.00% of precancerous lesions and 50.00% of borderline lesions’ samples.17,18

.Comparison between HPV DNA test and Pap smear diagnosis represents none of the NILM and ASCUS positive cases exhibited positive reaction to PCR. Among 5(3.10%) ASCH cases 02(40.0%) were PCR positive. Out of 40(25.0%) LSIL cases only 2(5.00%) cases were detected positive. The detection of HSIL cases were found to 11(64.7%) cases out of 17(10.6%) cases. The Squamous cell carcinoma patient’s show out of 30(18.75%) cases 25 (83.33%) cases were positively detected by PCR. The present study indicated that Pap smear cytology is still better than only HPV DNA test for the diagnosis of chronic cervicitis, LSIL, HSIL.

However, a study, comparing the histological diagnosis and it was revealed that none of the chronic cervicitis cases exhibit positive for PCR test.19 Present study also revealed that none of the chronic cervicitis cases show positivity to PCR detection.

 The present study has shown that the occurrence of different types of HPV virus be responsible for the causation of cervical cancer. These are detected in samples of patients of cervical lesions. HPV DNA revealed that, HPV – 16 were present in the highest percent (75.00%), HPV-18 were 15.00% and the lowest number of occurrences were HPV – 31 type 5.00 % and HPV – 52 type 5.00 %.

The accuracy of the Pap smears and HPV DNA in this study show to 88.13% and 96.88% respectively. This demonstrates that the accuracy of Pap smears is lower in respect to HPV DNA tests. The accuracy thus determines that the probability of correct diagnosis could be detected by the later tests singly or combined.  Specificity  in case of Pap smear test is 89.29 and in case case of HPV DNA 100.0%. This means that specificity is higher in DNA test. The sensitivity values of Pap smear cytology and HPV DNA were found 87.5% and 88.89%   respectively. The sensitivity value focuses on the ability of a test to correctly diagnose the disease. In this case the cytological diagnostic test is almost equal to DNA test.

 Conclusion

It can be concluded that HPV DNA test is not suitable as a single test for detecting precancerous and cancerous lesion than routine Pap smear test. All the cytologically diagnosed NILM (negative for intraepithelial lesion or malignancy) cases and all histologically diagnosed chronic cervicitis cases were HPV DNA test negative. So, mass screening programme does not need HPV DNA test. This will reduce the cost of the screening programme. Combination of cytology (Pap smear) and new technologies such as HPV DNA test would ultimately be more useful. Based on findings, the study recommended that, PAP test should be routinely used in every patient attending the colposcopy clinic, biopsy should be taken in all VIA positive patient and combined pap test and histopathological examination is enough to detect cervical precancerous and cancerous lesion. In our country HPV DNA test is not mandatory as a routine test.

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