Fine Needle Aspiration Cytology With Ziehl Neelsen Staining Biology Essay

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Objective: To describe the pattern of presentation of tuberculous lymphadenitis, to correlate the sensitivity of Fine Needle Aspiration Cytology (FNAC) and Ziehl Neelsen (ZN) stain in the diagnosis of Tuberculosis (TB) lymphadenitis and to assess the diagnostic yield of FNAC in cervical lymphadenopathy.

Study Design: Prospective cross sectional study.

Methods: Total one hundred and sixty four patients of clinically suspected TB lymphadenitis, referred to Dow Diagnostic Reference & Research Laboratory (DDRRL) for diagnostic services for lymphadenopathy from Jan 2008 to Oct 2009 were included. Cytological evaluation for identification of Acid Fast Bacilli (AFB) was done on separate slides with Hematoxylin & Eosin (H & E) & ZN (hot method) stain.

Results: Mean age was 24.49; Female were 69%; Male were 31%. Out of 164, tubercular were 117(71.3%) & 17(10.4%) were of reactive nature. The remaining 9(5.5%) cases were of malignant lymphadenopathy, consisting 1(0.6%) case of primary malignancy i.e. lymphoma and 8(4.9%) of metastasis to lymph nodes. Overall 11 cases (6.7%) were found positive for AFB on ZN stain.

Conclusion: Cytomorphologic diagnostic features of FNAC on H&E stain have significant diagnostic yield. FNAC is an optimally selected, efficient, easy to perform and economical test for initial diagnostic workup in patients with TB Lymphadenitis in Pakistan. Supplementation of ZN stain does not add to the overall Positive Predictive Value of FNAC.

Keywords: Tuberculosis, lymphadenopathy, fine needle aspiration cytology.

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Department of Pathology, Institute of Basic Medical Sciences, Dow International Medical College, Dow University of Health Sciences, Karachi, Pakistan.

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Correspondence: Dr Sulaiman Ahmed. E-mail: docsalman93@yahoo.com

INTRODUCTION.

Globally, TB accounts for approximately nine million new cases of disease and three million deaths every year. Tuberculosis is a disease of poverty, with 90% of the cases occurring in developing countries 1. The WHO has declared tuberculosis a global emergency, mainly in developing countries where tuberculosis is still responsible for 25% of all adult deaths 2. Pakistan is a developing country with high burden of tuberculosis and limited resources to combat with disease of such magnitude. Extra-pulmonary tuberculosis is a major health problem and the most common presentation is cervical lymphadenitis, relatively more common among the Asian populations 3, 4. Empirical use of anti-tuberculosis therapy for mere lymph node enlargements without a pathological diagnosis should not be undertaken because it may lead to undue delay in the diagnosis of malignancy 5. Diagnosis of extrapulmonary tuberculosis is often made on conventional histopathology. Histopathological diagnosis is made when there is caseous necrosis in a granulomatous lymphadenitis. ZN staining on the histopathology section is an adjunct. However, the chances of identifying acid-fast bacilli in a tissue section are much lower. The effect of formalin and xylene on the stainability of mycobacteria by ZN method results in extremely low sensitivity of detection of mycobacteria in histopathology sections 6. FNAC provides an easy, quick, highly reliable and cost-effective tool for diagnosing the disease. It is a simple and rapid diagnostic technique with early availability of results, simplicity, minimal trauma and complications 7. It has been reported that aspiration biopsy diagnosis of tuberculous lymphadenitis has a sensitivity of more than 70% when the aspirate contains purulent material 8. This is of particular importance in view of the high prevalence of tuberculosis in our country, atypical presentation of tuberculosis and because AFB are mostly seen in purulent aspirate smears, which do not show granulomas, necrosis or epithelioid cells and which in absence of ZN staining can be dismissed as acute suppurative lymphadenitis 4.

The aims of this prospective study were to describe the distributional patterns of tuberculous lymphadenitis and to assess the correlation between FNAC and the ZN staining technique in diagnosing TB lymphadenitis and to evaluate the usefulness of FNAC as a diagnostic tool in patients of lymphadenopathy in a referred laboratory setting.

SUBJECTS AND METHODS.

Study Population and Setting: One hundred and sixty four patients from January 2008 to September 2009 were inducted in this study. Patients included in the study were those who were clinically suspected of TB lymphadenitis. The variables included in the study were age, sex, site of lesion. Relevant history and examination of nodes were recorded. Nodes were aspirated after all aseptic measures with sterile disposable 24 G needle attached with 10 cc disposable syringes. Two smears were prepared with part of aspirated material, one stained with H & E & other unstained. In 94 cases out of 164 were stained with ZN. The slides were examined under oil immersion objective. The remaining part of the aspirate was used to prepare paraffin embedded cell block & after processing sections were stained using H & E. The results of FNAC were further correlated with paraffin embedded sections of tissue blocks. All data grouped and analysed by use of SPSS version 16.0. Descriptive statistical tests were used to analyse the differences within the data.

Procedure: Prepared smears were examined by one microbiologist & one cytopathologist concomitantly for presence of granuloma, necrosis, langerhans giant cells, plasma cells, lymphocytes, macrophages, neutrophils and presence or absence of AFB in ZN stained slides under oil immersion objective.

Granulomatous adenitis was diagnosed when sheets of epithelioid cells were visualized with lymphocytes and plasma cells with or without multinucleated giant cells; and caseous necrosis was defined as eosinophilic granular material intermingled with inflammatory cells and necrotic cell debris 8. The tuberculous abscess was described as degenerate caseous necrosis and/or liquefied necrotic material with marked degenerating and viable inflammatory cell infiltration without epithelioid granulomas 9.

RESULTS.

Aspirates of 164 patients were enrolled in study with suspicion of extra pulmonary tuberculosis. Mean age was 24.49; most of the patients were females (69%) while males were 31% with female to male ratio approximately 3:1. The majority of patients were children and young adults 79.2% less than 30 years of age. (Table I). The most common involved lymph nodes in TB lymphadenitis were cervical 137 (83.5%) followed by submandibular 16 (9.8%) and axillary 9 (5.5%). Table II.

Tuberculous cases were found to be 117(71.3%) and 17(10.4%) were of reactive nature. The remaining 9(5.5%) cases were of malignant lymphadenopathy, consisting 1(0.6%) case of primary malignancy i.e. lymphoma and 8(4.9%) metastasis to lymph nodes. (Table III). Out of 164 cases, ZN stain positivity for AFB was found in 11 cases (6.7%) out of 94 cases.

Table I. Age frequency of FNAC samples

Age (yrs)

Frequency

Percentage (%)

1 to 10

4

2.4

11 to 20

75

45.7

21 to 30

51

31.1

31 to 40

20

12.2

41 to 50

5

3

51 to 60

7

4.3

>60

2

1.2

Table II. Lymph node involvement

Lymph node site

No. Of cases

Percentage (%)

Cervical

137

83.5

Submandibular

16

9.8

Axilla

9

5.5

Others

2

1.2

Total

164

100

Table III. Cytological and microbiological correlation of lymphadenitis cases

Cytological diagnosis

No. Of cases (%)

AFB smear positive cases (%)

AFB smear negative cases (%)

CGI suggestive of TB

107 (65)

5 (9.4)

48 (90.5)

Acute and CI

11 (6.7)

3 (33.3)

6 (66.6)

CN

09 (5.4)

3 (33.3)

6 (66.6)

RC

17 (10)

0 (0)

6 (100)

CI

03 (1.8)

0 (0)

2 (100)

MC

08 (4.8)

0 (0)

7 (100)

CAI

02 (1.2)

0 (0)

2 (100)

AI compatible with abscess

03 (1.8)

0 (0)

3 (100)

Lipoma

02 (1.2)

0 (0)

2 (100)

ASI

01 (0.6)

0 (0)

1 (100)

Lymphoma

01 (0.6)

0 (0)

0 (00)

Total

164

11

83

CGI, Chronic granulomatous inflammation; CI, chronic inflammation; CN, caseous necrosis; RC, reactive changes; MC, metastatic ca; CAI, chronic active inflammation; AI, acute inflammation; ASI, acute suppurative inflammation.

DISCUSSION.

In Pakistan, TB burden is ever increasing with added problems like poor socio-personal conditions, poverty, improper health care delivery system and poor education level. This collectively leads to an escalation of TB burden in Pakistan. Rise in incidence of HIV/AIDS is complicating the picture further. Pakistan is ranked as sixth among the 22 countries of the EMRO region with the highest burden of TB 10, 11. In 2001, the Government of Pakistan declared TB as a National Emergency. The diagnosis of extrapulmonary TB still remains to be more of a clinical decision. Not many clinically sensitive tests are available in Pakistan to assist the treating physician. For accurate diagnosis of Mycobacterium tuberculosis, isolation and culture of organism is gold standard but as Mycobacterium tuberculosis is slow growing organism so culture on conventional Lowenstein-Jensen (LJ) medium takes 6 to 8 weeks. Middlebrook medium isolates growth of organism comparatively more rapidly. Mean duration to yield positive culture is about 3 weeks. But the disease like TB, this is long enough to wait for results as it is necessary to start treatment at the earliest. Therefore, comparatively quicker methods need to be established to diagnose tuberculous lymphadenitis 12. FNAC is a well-established diagnostic method in the assessment of lymph node lesions. In areas where mycobacterial infections are prevalent, a diagnosis of tuberculosis can be made with confidence when its cytomorphological criteria are met. FNAC is inexpensive, safe and rapid method of diagnosing not only TB lymphadenitis but also reveals other pathologies. It also avoids the possible physical and psychological complications of an open surgical biopsy 13, 14.

In this study we examined 164 cases for diagnosis of TB lymphadenitis on FNAC. The majority of the patients were less than 30 years of age, which correlates the findings of other studies 5, 15-17. Females were more in number (69%) than males (31%), which were conversely reported by other researchers 5, 18.

Most common site involved was cervical region (83.5%), similar findings were reported previously 5, 8, 19. CGI was the commonest finding (65%), followed by reactive changes in 10% of cases. Over all ZN positivity was identified in only 11out of 94 cases (11.7%) probably due to the low concentration of mycobacteria in the aspirate. ZN positivity was only 9.4% in cases of CGI & it was more (33.3%) in CN. The concentration of organisms in the sputum in pulmonary tuberculosis has a direct relationship to the sensitivity of the ZN stain and a concentration of ≥10 4 organisms/mL would guarantee a positive smear 20. This is applicable to the aspirates from tuberculous lymphadenitis as suggested earlier 12. Some studies have reported ZN positivity of 37.4% to 59.4% 5, 21, 22.

Thus, FNAC may be an important diagnostic tool for TB lymphadenitis in developing countries with limited diagnostic and therapeutic resources. AFB-negative necrosis and/or abscess should be subjected to culture in areas where it can be carried out 5.

CONCLUSION.

FNAC is an excellent method, even with negative ZN stains for AFB, the cytomorphological features on FNAC are sufficient for diagnosis of TB lymphadenitis. It is an excellent first line method, for investigating the nature of the lesions, as it is economical and convenient alternative to open biopsy.

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