Cell-block in EUS Guided FNA-Best method in a Resource Constraint Setting?

Background: Endoscopic ultrasound guided fine needle aspiration (EUS-FNA) has emerged as an effective tool for diagnosing suspected gastro-entero-pancreatic lesions. On site Diff stained smears though have improved the diagnostic accuracy, search have always been made for an effective method of cell block preparation in these cases where diagnosis can further be supplemented by immunohistochemistry (IHC) use.


Introduction
Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) cytology is an emerging imaging modality used in the histological diagnosis of gastero-enterico-pancreatic lesions [1,2]. The diagnostic efficacy of EUS-FNA depends on the expertise of gastroenterologist as well as on cytopathologists expertise. On site Diff stained smears have been widely used in EUS FNA's for increasing the diagnostic yield. The diagnostic accuracy can further be enhanced by taking material for cell block and confirming findings with immunohistochemical stains. Several methods of cell block preparations have been reported in literature; however there has always been a search for easy cell block preparation procedure. We conducted this study to standardize the methodology of cell block preparation in our laboratory and to prospectively analyze the utility of these cell blocks in diagnosing EUS cytology cases.

Study design
The study was an observational study designed to assess an ideal methodology of preparing cell blocks in a resource constraint setting. There after assessing their diagnostic accuracy in EUS FNA cases. As it was an ex vivo study with no risk involved as far as the methodology is concerned to human subjects, so ethical committee approval was not sought for.

Methods
Five samples of fluids with adequate cellularity were processed by four different methodologies for cell block preparation and a numerical score was calculated for all. The scores were analyzed for assessing and standardizing the methodology of CB preparation. The standardized cell blocks were then utilized in 22 cases of EUS guided FNA's to determine the diagnostic accuracy using immune histochemical markers. No funding was received for same.

Study methodology
Random body fluids with adequate cellularity on routine cytology and adequate amount were used for making cells blocks after 24 hours of reporting of these fluids. Each of these fluids was divided into 4 tubes, which were labeled as 1. Thrombin method 2. Formalin method 3. Cell Block fluid 1 method 4. Cell block fluid 2 method ( Figure 1).
Cell blocks were prepared from these 4 techniques. Selected histological parameters were evaluated in these cell blocks. All parameters were graded on a scale of 1+ to 3+ with 3+ indicating the best quality. The best quality cell blocks were than standardized and utilized in prospective EUS guided FNA cases. The diagnostic accuracy of these cell blocks for EUS FNA cases were evaluated.
Procedure-4 tubes were taken and labeled as
Fluid samples (ascitic/pleural fluid) with adequate cellularity as reported on routine microscopy and adequate volume (around 10 ml) were utilized for this study. About 2 ml of fluid was taken in each tube and all tubes were centrifuged at 1500 rpm for 10 min. Supernatant was discarded. Thereafter the samples were processed as follows:

1.
Thrombin method: To the sediment in tube 1, equal volume of normal patient plasma was added and than add double amount of thromborol S. Then the tube was dipped in water bath at 37 degree for few seconds. Thereafter a jelly like tissue was formed. To this, 10% formalin was added now for 2-3 hours for this jelly like material to harden in consistency.

2.
Formalin method: In tube 2, 10% formalin was added to the sediment and kept for 4-6 hours.

4.
Cell block fluid 2 method (70: 30): To sediment in tube 4, 70: 30 (7 part of alcohol and 3 part of 10% formalin freshly prepared) was added and kept for 6 hours. Thereafter the supernatant fluid was discarded and samples were scooped out from the tubes with the help of a thin stick and were processed in tissue processor as a routine tissue. After processing and slide preparation 7 histological parameters were analyzed for each slide ( Figure 2 and Table 1).
These parameters were: Cutting of block, tissue loss during cutting, cellularity of sections, cell dispersal, morphology of block, knife marks and section folds.
Each parameter was graded on a scale on 1+ and 3+, 3+ indicating easiest to cut, least tissue loss, rich cellularity, uniform dispersal of cells, best morphology, least knife marks, minimum tissue folds and 1+ indicating poorest score for all features. The overall score for all 4 methods of preparing cell blocks was calculated on total of 5 samples.
After standardizing the technique for CB preparation this technique was prospectively utilized on 22 cases of EUS guided FNA together with on site Diff staining for adequacy ( Figure 3).
Final diagnosis with IHC could be offered in 20 of these cases. EUS Guided FNA material were obtained from pancreatic mass, gall bladder mass, ampullary lesion, bile duct mass, liver lesion, lymph nodes (Periportal/hepatoduodenal/subcarinal/mediastinal), duodenal, gastric lesions. The material was taken for smears (Giemsa/Pap stains) and 2-3 passes were separately taken in a container for cell block preparation with normal saline.
Of the 22 cases of EUS FNA analyzed pancreatic malignancies are the most commonwith 8/22 cases of EUS FNA (Table 3).
Remaining cases were of neuroendocrine tumor, lymphoma, gall bladder carcinoma, gastrointestinal stromal tumor, gastric carcinoma and cholangiocarcinoma. Diagnosis could be supplemented with preparation of CB and with immunohistochemical stains in 20 of these cases (Figure 4).   Histological correlation was available in 7 cases with a 100% concordance.

Discussion
There have long been search for an effective method of making cell block in cytology specimens for improving efficacy and diagnostic accuracy of cytology material obtained. Different techniques for preparation of cell block have been studied by several authors. In the past decade techniques with automated CBP methods have also propped up. Nigro et al. studied CB from 12 non-gynaecologic specimens by 4 different methods-inverted filtration method, thrombin method, albumin method and simple sedimentation method. They found that thrombin cell block methods were easily prepared and produced the best cell blocks with regards to cellularity, cell distribution and background [1]. Nathan     on CB preparatory techniques and results obtained to members of the American Society of Cytopathology and other pathologists. Total 90 participants participated in survey, they found that histogel had the lowest satisfaction among cytopathologists and plasma thrombin cell block was found better than histogel. However, they concluded that there is no consistent method to prepare CB [3].
Jain et al. in 2014 reviewed different cell block preparatory techniques-Tissue coagulum clot method, plasma thrombin clot method, histogel/albumin/colloidin method and automated cell block method-Cellient CB method. They reviewed the advantages and limitations of various CB preparatory techniques [4].
Kulkarni et al. studied the feasibility and utility of thromboplastinplasma (TP) method for cell block preparation and to compare their efficacy with conventional smears. They found that absolute concordance was seen in 66 cases (94%) between the smears and cell blocks. They concluded that TP method is simple, cost effective, and reproducible method and a useful adjunct to routine cytology [5,6].
Studies on newer automated methods of cell block production include-Cellient TM Automated Cell Block method. These automated methods achieve higher cellularity and better cellular presentation in addition it is faster and more reliable due to lack of operator dependency [4,7,8].
However the newer techniques utilize methanol-based fixation, which interfere with immunohistochemical analysis especially for ER, PR, MIB 1 and Her 2 neu [9][10][11]. However this issue may be overcome by formalin pre-fixation prior to Cellient [7]. Thirty minutes pre-fixation seems to be preferable to longer fixation to ensure good morphological quality [7].

Conclusion
Cell-block preparation by Thrombin technique has been found to be ideal method of preparing cell block over other methods and offers high diagnostic yield and is a useful adjunct in EUS FNA cases in a resource constraint setting.