How Do We Do It?: Another Optimal Methodology for Endobronchial Ultrasound Sample Handling

Abstract

We read with great interest the article by Nakajima and Yasufuku1Nakajima T Yasufuku K How I do it—optimal methodology for multidirectional analysis of endobronchial ultrasound-guided transbronchial needle aspiration samples.J Thorac Oncol. 2011; 6: 203-206Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar in the January issue of the Journal of Thoracic Oncology. These authors reported a methodology for analysis of samples obtained by endobronchial ultrasound (EBUS), not only initially developed for mediastinal staging of non-small cell lung carcinoma (NSCLC) but also widely used for the initial diagnosis of mediastinal metastatic lymph nodes. In this article, they report a multidirectional analysis to obtain precise diagnosis and molecular testing, particularly emphasizing the critical use of freshly stored material. We would like to report our own experience of a cell aspirate freezing method, allowing multiple cell analyses. This method, although it presents certain similarities with that described by Nakajima and Yasufuku, has a number of notable differences. Our sampling method has already been described.2Gounant V Ninane V Janson X et al.Release of metal particles from needles used for transbronchial needle aspiration.Chest. 2011; 139: 138-143Crossref PubMed Scopus (23) Google Scholar The entire procedure is performed by a cytopathologist for rapid on-site examination and specimen handling. One to three smears are performed for each aspirate, and in the case of abundant material, the extra aspirate is flushed into a tube containing Roswell Park Memorial Institute cell culture medium. After the last aspirate, the needle is rinsed with culture medium to obtain a cell suspension. Clots or tissue fragments, when observed are removed, fixed in 10% formalin, and paraffin embedded for cell blocks. Representativeness of cell suspensions is checked by a stained cytospin examination. Cell suspensions are centrifuged, and cell pellets are frozen at −80°C in 20% dimethylsulfoxide (Sigma, France) as usually performed for cell lines: one to four frozen aliquots are stored per specimen. From March 16, 2007 to December 9, 2010, 450 patients underwent conventional transbronchial needle aspiration (TBNA; N = 160) or EBUS-guided TBNA (N = 280). As systematic storage at −80°C was started in May 2007, 425 cell aspirates have now been frozen and stored (96% of all TBNA performed over the last 38 months). Two hundred sixteen of these stored samples have been thawed for complementary techniques. The other samples have been kept frozen for possible subsequent analysis, if necessary. When complementary techniques are required, the samples are thawed, and cytospin is performed to check the quality of the cells. It must be emphasized that this method of cell freezing is usually associated with very well-preserved cell morphology. Depending on the suspected diagnosis, frozen and fresh cells can be investigated by various methods. Flow cytometry is used for the diagnosis of lymphoma (lymphocyte immunotyping), small cell lung carcinoma (CD56 expression), or DNA ploidy. Tumor cell differentiation markers (TTF1, P63, CK7/CK20, estrogen receptors) are studied in NSCLC or in patients with a history of extrathoracic cancer, either on smears, cytospins from frozen cells or cell blocks. Molecular analysis for EGFR and Ras mutations is performed on thawed cell pellets in NSCLC, and fluorescence in situ hybridization analysis is performed on cytospins from frozen cells. In conclusion, we agree with Nakajima and Yasufuku that an optimal methodology is essential for the management of specimens obtained by EBUS-TBNA, particularly when rapid on-site examination is not possible. Fresh cell aspirates in cell culture medium stored at −80°C in dimethylsulfoxide, which is a very simple method, ensure optimal cell preservation for morphology, allowing a wide range of complementary techniques (flow cytometry, molecular genotyping, etc.).