Dr Tim Bracey.
Abbreviations:
PMCB = poor man’s cell block
EUS = endoscopic ultrasound
UBUS = endobronchial ultrasound
FNA = fine needle aspiration
ROSE = rapid on site evaluation
Endoscopic and Endobronchial Ultrasound (EUS and EBUS) methods of sampling lesions lesions in the retroperitoneum and mediastinum otherwise difficult to reach without more invasive procedures such as mediastinoscopy, thoracoscopy, laparoscopy. The needle used is of a wider gauge than conventional percutaneous FNA, and uses a repetitive chiselling technique unlike the static bite of a core biopsy. Multiple fragments of lesional material are liberated with the technique for microscopic examination which is traditionally prepared as a series of cytology slides plus or minus a cell block for histology.
The endoscopist/bronchoscopist may choose to increase or limit the number of passes performed, depending on the clinical indication. Sampling is often required for diagnostic, prognostic, or predictive testing and require extensive immunohistochemical, microbiological or molecular testing. “Rapid On Site Evaluation” (ROSE) of cytology slides enables dynamic examination of aspirated material during the procedure to determine whether the lesion has been successfully sampled and guiding further testing. ROSE is considered by many to be the gold standard, and is now practised by many well staffed specialist centres in the UK and abroad.
When I first started supporting EUS in an ad hoc fashion 8 years ago, I was working with a radiologist who was new to performing the technique and was very grateful for the ROSE support I was providing. We were both on a learning curve, and for me being able to look at diffquik slides on site meant I could prompt additional sampling of a lesion and say when I had enough material to make a diagnosis. My colleague was able to improve his aim and get a feel for the number of passes he needed to perform to get a diagnostic sample with immediate feedback. The sample was delivered to me through the scope in a long column of partly clotted liquid and using preparing conventional smears I found the chunks of thick stained lesional material difficult to visualise on cytology slides. In addition, non-lesional mucosa from the needle transit through the gastric or oesophageal wall “contaminated” the potential lesional material and it was easy to mistake gastric mucosa on a cytology sample for a mucinous epithelial lesion, for example when a cystic pancreatic lesion was being targeted.
It was clear to me from the outset that precious samples from anatomical “tiger country” required emphasis on an excellent cell block, since many of the lesions aspirated required immuno-histochemical analysis on multiple serial sections. I decided to use the poor man’s cell block technique where the entire sample is aspirated into the lid of an inverted universal tube and fixed with formalin vapour (see figure). This technique was developed for percutaneous FNAs to effectively “converts” a cytology specimen into a solid histology block and uses simple equipment found in all labs and clinics. I had used the technique myself when performing FNAs which I knew were primarily for immunohistochemical or molecular analysis where a smeared slide was mainly only necessary to let me know I had hit the target or needed an immediate repeat. Knowing that EUS and EBUS samples were more voluminous than conventional FNAs in most cases were likely to need histology as well as cytology analysis, it seemed to me an obvious choice. The technique also allowed me to take a representative aliquot of the entire mixed sample for ROSE, and simply fix the remaining sample for histological examination without the need for dilution and centrifuging. Using this technique I could avoid putting too much material on slides and keep as much of the sample as possible for the cell block.
With our limited departmental capacity for ROSE at the time in a short staffed department, and with my growing suspicion that the endoscopist I was supporting was beginning to get a good sample regardless of whether I attended or not, we decided to test whether or not we could dispense with cytology slides altogether and maximise the yield from our PMCBs.
In our pilot study we looked at paired cytology and histology samples (the latter prepared as a PMCB) which were booked in and reported separately. Although the cytology and histology samples both showed good adequacy rates, the cytology samples were most frequently in the “probably benign” or “probably malignant” categories rather than a definitive diagnosis. In contrast the PMCB preparations gave us a diagnosis in about 80% of cases and allowed a more confident diagnosis over a range of benign and malignant pathology. The PMCB cancer cases often showed stromal invasion and even perineural invasion, and most provided enough serial sections for immuno-histochemistry and molecular studies.
Given that our cell block adequacy rates remained high with PCMB preparations, we decided to dispense with cytology samples altogether for EUS, and later EBUS. Our new respiratory physician was offered ROSE support but he was skeptical of its benefit, having considerable prior experience from other high volume centres placing his samples directly into formalin. The only arguable benefit for continuing with cytology samples was if we were able to offer ROSE, but it seemed unlikely that the considerable expense and time commitment would be worth the possible slight increase in percentage adequacy. In our experience the only lesions which commonly gave us an inadequate PCMB sample were those difficult cystic (usually pancreatic) lesions which even more difficult to diagnose with cytology.
Fixation and sample quality from the PMCBs is superb, with most specimens being cellular enough to grade and perform prognostic IHC and molecular tests. One unexpected benefit was that non-lesional sampling (the inevitable accompanying mucosa picked up en route, for example gastric mucinous epithelium) was spatially separated from the lesion sampled on the PCMB rather than being mixed up with it (and potentially confused with a mucinous neoplasm) on cytology preparations. Perhaps controversially following the pilot study, we decided to dispense with ROSE and process all the EUS and later EBUS specimens as histology specimens without cytology. While as a group several of us reported cytology, cytologists did not always report the specialities relevant to the lesions aspirated at EUS/EBUS. Using the PMCB gave us the freedom to distribute the resultant specimens to pathologists who did not report cytology. For example, pancreatic lesions and could be reported by the GI/HPB team, lung cancers by the thoracic team, and even difficult mesenchymal lesions and lymphomas could be reported by respective specialist “non-cytologists”.
Since our pilot study there have been many studies showing benefits of ROSE and an improvement in adequacy rates but recent meta-analyses have failed to show a statistically significant benefit of ROSE despite obvious publication bias towards studies showing benefits and more papers from better staffed, well funded departments in specialist centres. Although statistics do not tell the whole story and there are benefits from ROSE relating to training and quality control, it is clear from our experience that using our alternative PMCB sample preparation technique is safe and reliable and enables our users to get the answers they need to manage their patients effectively. It has been pointed out that those showing improved adequacy with ROSE do not have a particularly high non-ROSE adequacy rates. In order to benefit from ROSE, a department such as ours would have to show a considerable benefit, given that we have maintained diagnostic yield in excess of 80% for both EUS and EBUS, to justify the considerable time, expense and expertise to adequately fund and staff these services.
Cytology will always been an important skill in the armoury of a pathologist and I don’t want people to get the idea that I am dead against it as a sub-speciality of cellular pathology. There are many situations where FNA with air dried slides and or pap stains are all that is needed to make a diagnosis. In staging head and neck cancers for example, a simple FNA is enough to stage oral cavity SCC in combination with clinical examination and imaging. Papillary thyroid carcinoma can be confirmed with FNA cytology in an appropriate context. Most benign salivary gland tumours can be accurately diagnosed with FNA and screening serous fluids can be done cheaply and rapidly without resorting to cell block preparations in the majority of cases. The number of situations where cytology alone is enough without cell block preparations is however dwindling and with pathologists becoming increasingly sub-specialised, cytologists who also have diagnostic expertise in thoracic, pancreaticobiliary and GI pathology are an increasingly rare species. ROSE can be a valuable service in support of EUS and EBUS, particularly during the aspirating physician’s early learning curve, but our experience highlights that ROSE is not necessary when an alternative reliable sample preparation method such as the PMCB is available. Success and happiness is not only measured by wealth so if you work with limited resources but still want to support EUS/EBUS, the poor man’s technique may bring you rewards without ROSE.
| Advantage of PMCB versus | Limitations of ROSE cytology |
| Cheap, with no special equipment or attending pathology staff needed | Expensive and time consuming with cytology staff and expertise required on site |
| The entire specimen is available for further testingLesional and non-lesional (mucosal) sampling are spatially separated in the block and slides | A considerable proportion of the specimen remains on slidesLesional and non-lesional material can be mixed and gastric epithelium potentially confused with mucinous tumour. |
| Stromal, vascular and nerve invasion can be demonstrated | Cytology cannot distinguish in situ and invasive neoplasia except by inference |
| GISTs commonly sampled and often enough material to grade and prognosticate | Lesional material on cytology slides not sufficient for mitotic counts or IHC |
| Rare neoplasms, lymphoma and sarcoma can be diagnosed and subtyped | Rare neoplasms may be suspected using cytology but cannot be confirmed |
| Specimens can be allocated by speciality and expertise to non-cytologists | Cases can only be reported by those with cytology experience and expertise |
