Leishman stain in detection of hydatid hooklets in liver space occupying lesions.
Hydatid disease (HD) caused by Echinococcus granulosus is endemic in the cattle and sheep-raising regions of the underdeveloped and developed world.  It is endemic in India, as well as other parts of the world, including the Middle East, Africa, South America, New Zealand, Australia, Turkey, and Southern Europe. 
Human beings are usually infected as intermediate hosts when they ingest egg contaminated food or water. Infestation by HD in humans most commonly occurs in the liver (70%), but also in the lungs (20%), and 10% of cysts can occur almost anywhere in the body. 
Hydatid cysts in imaging studies can be confused with hepatic tumors, abscesses, cystadenomas, liver cysts, or other lesions. Three-quarters of the infected individuals develop one or more hepatic cysts; the majority occur in the right lobe.  Guided aspiration is a simple, safe and cost-effective screening test for deep-seated lesions with a significant role in the detection of clinically unsuspected malignancy making it an investigation of choice for early confirmation or exclusion of pathology. 
Fine needle aspiration cytology (FNAC) has been used as a diagnostic tool for human echinococcosis since the late 1980s. This technique is usually performed under ultrasound guidance and is useful in the pre-operative diagnosis of HD by demonstrating the diagnostic hooklets and scolices in the aspiration fluid, which are pathognomonic.  Conventionally Direct Air dried smears from the aspirated material are stained with May-Grunwald Giemsa (MGG) stain and alcohol fixed smears are stained with the Papanicolaou (Pap) and hematoxylin-eosin (HE) stain for cytodiagnosis. 
Leishman stain is a simple, quick and inexpensive staining technique easily available in both urban and rural laboratories which can be used as an efficacious tool to interpret cytosmears. We have been using Leishman stain to interpret cytosmears for more than 30 years. [5-8]
With this background, this study was aimed to assess the utility of Leishman stain for diagnosis of HD.
MATERIALS AND METHODS
A retrospective analysis of records of consecutive patients attending a pathology laboratory for ultrasound-guided FNAC was performed for 4 years from August 2012 to July 2016.
All the ultrasound-guided FNAC smears were stained with Pap stain, HE stain, and Leishman stain. Stained smears were examined under the microscope. Histopathology confirmation was done wherever required.
HE Stain: Air dried FNAC smears were fixed with ethanol followed by cleaning under running water. The slides were stained by 10 dips in freshly prepared hematoxyline. Extra stain was removed by running water following counter staining with Eosin. The slides were cleaned, dried and mounted before examination under the microscope.
Leishman stain: Air dried FNAC smears were kept on the staining tray, and Leishman stain was poured in a manner similar for blood smear staining. After 30 seconds, an equal amount of distilled water was added and kept for 5-7 minutes. The slides were cleaned with running water, dried and examined under the microscope.
We reviewed Pap, HE, and Leishman stained smears from hepatic space occupying lesions (SOLs) of patients which were diagnosed as hydatid cysts. The presence of hooklets on a necrotic and degenerated background was considered as pathognomonic for diagnosis of HD (Figure 1).
The study included 340 patients during a period of 4 years from August 2012 to July 2016. All the cases who presented with hepatic SOL were subjected to ultrasound-guided ultrasonography (USG)-FNAC. Out of 340 cases, 80 cases (24.11%) were diagnosed as hepatocellular carcinoma, hepatoblastoma was diagnosed in eight cases (2.35%), and metastatic carcinoma of the liver was diagnosed in 226 cases (66.47%). 24 cases of benign lesions were identified out of which six cases were diagnosed as HD (Table 1).
The patients of HD were in the age range of 29-60 years and all were male. All the patients were non-vegetarian and from rural background. In all the cases, the right lobe of liver was affected with well-defined hypoechoic lesions. The diameter of the lesions varied from 1.5 to 5.2 cms. Four cases were diagnosed as hepatoma and two cases as HD under USG. Aspirates from all the cases were necrotic in naked eye examination (Table 2).
Hemogram and biochemical parameters of all the cases were within normal limits. However, serum was positive for IgG anti-Echinococcus antibody in all six cases of HD.
In all the cases, the microscopic examination revealed the presence of hooklets scattered on a degenerated necrotic background. Few lymphocytes were, however, present too.
The hooklets were better visualized in Leishman stained smears than Pap and HE stained smears.
The hooklets on the Leishman stained smears were visible under lower magnification, whereas in the Pap and HE stained smears it was difficult to locate the hooklets under lower magnification. However, hooklets were clearly identified under higher magnification in all the smears stained with Leishman, Pap, and HE stains.
In this study, the age of the patients ranged from 29 to 60 years, and in none of the cases, there was any clinical suspicion of HD. Our observations are on par with two previous studies in which the age of patients ranged from 3rd to 6 decades, and none of the cases was diagnosed clinically as HD. [9,10]
All the six cases encountered in this study had lesions in the right lobe of the liver. All the SOLs presented as uniform solid lesions on USG, and four of the six lesions were in fact diagnosed as hepatoma. In agreement to our observation, other workers stated that imaging studies of hydatid cyst confused with hepatic tumors and majority were in the right lobe. 
Ultrasound guided FNAC has been found to be safe, inexpensive and useful in the diagnosis of hydatid cysts in our study. Jha et al.  have also documented that guided aspiration is a simple, safe, and cost-effective screening test for deep-seated lesions with a significant role in the detection of clinically suspected malignancy making it an investigation of choice for early confirmation or exclusion of pathology.
In our study, we have stained the dry air dried cytosmears with Leishman and HE stain and wet-fixed cytosmears with Pap stains. Earlier direct air dried smears from the aspirated material were stained with MGG, and alcohol-fixed smears were stained with the Pap and HE stain. 
With Pap and HE stained smears, the hydatid hooklets were not clearly visible under lower magnification, the typical glistening hooklets were, however, visible under high-power objective. In contrast, in Leishman stained smears, the hooklets could be clearly identified under lower power objective and still better with higher magnification. Thus, Leishman stain was found to be very useful to identify the hooklets, and was superior in comparison to the Pap and HE stain for this purpose.
Leishman staining which is an easy, inexpensive, quick, and ubiquitous technique is ideally suited to diagnose the hooklets of HD. This study shows that it is superior to the conventionally employed HE and Pap stains for diagnosis of hydatid hooklets.
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Niranjan Rout (1), Manorama Swain (2), Sashibhusan Dash (1), Omprakash Agarwal (3), Chudamani Meher (3), Shivaram Prasad Singh (4)
(1) Department of Pathology, A.H. Regional Cancer Centre, Cuttack, Odisha, India, (2) Department of Biochemistry, S.C.B. Medical College, Cuttack, Odisha, India, (3) Beam Diagnostics, Cuttack, Odisha, India, (4) Department of Gastroenterology, S.C.B. Medical College, Cuttack, Odisha, India
Correspondence to: Niranjan Rout, E-mail: firstname.lastname@example.org
Received: May 16, 2017; Accepted: June 01, 2017
Caption: Figure 1: (a) Photomicrograph is showing hooklet of hydatid. (Leishman, x400); (b) photomicrograph showing hooklet of hydatid (hematoxylin-eosin, x400)
Table 1: Cytodiagnosis profile of liver SOL (n=340) Types of lesions Number of cases (%) Neoplastic lesions Hepatocellular carcinoma 82 (24.11) Hepatoblastoma 08 (2.35) Metastatic carcinoma 226 (66.47) Total 316 (92.94) Nonneoplastic lesions Liver abscess 15 (4.41) Hydatid cyst 06 (1.76) Hemangioma 03 (0.88) Total 24 (7.06) Table 2: Clinical presentation of six cases of HD Age (years) Symptoms Diameter of USG diagnosis SOL (in cm) 39 Nil 5.0 Hepatoma 60 Nil 2.5 HD 52 Nil 5.2 Hepatoma 29 Abdominal pain 1.5 HD 39 Nil 5.0 Hepatoma 49 Abdominal pain 3.8 Hepatoma SOL: Space occupying lesion, HD: Hydatid disease, USG: Ultrasonography
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|Title Annotation:||Research Article|
|Author:||Rout, Niranjan; Swain, Manorama; Dash, Sashibhusan; Agarwal, Omprakash; Meher, Chudamani; Singh, Shi|
|Publication:||International Journal of Medical Science and Public Health|
|Date:||Aug 1, 2017|
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