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Discover new possibilities in lymph node analysis for colorectal cancer – unleash the potential of quantitative assessment

Question & Answer

You will find answers to your questions as provided by the experts after their presentations.
They provide more details of the methods and more insight that provided in the webinar!

1. How do you organise logistic and resources for fresh lymph nodes (LNs) harvesting after surgery?

Dr Märkl: “At our institution, the surgeon works at the same hospital. The way from the surgery to the pathology laboratory is very short and pathologists receive the specimen immediately after resection. Therefore, it is very easy to organise the logistics.”

Dr Aldecoa: “At the Hospital Clínic of Barcelona, pathologists are in close contact with the chief nurse surgery and the anaesthesiologist who contact the pathologists by mobile phone when a specimen is ready. Usually, a pathologist or a technician will go to the operating room to get the fresh specimen. In our experience, the way of communicating varies according to the staff involved: surgeons, pathologists, anaesthetists, nurses and technicians. In terms of resources, the pathologists teach technicians how to harvest the LNs in fresh tissue. It is a feasible approach, at least in our case, and it is a great aid to manage the workload in the laboratory.”



2. Can we use methylene blue when a specimen arrives at the lab and analyse the LNs with OSNA?

Dr Märkl: “Using methylene blue requires several hours until the ink reaches its optimal effect. I can only see the LNs coloured in blue clearly after a couple of hours, which does not help for the dissection of the LNs in the fresh state, neither for the use of OSNA.”



3. What is the difference between the tattooed LNs and the SLN (sentinel lymph node) described in the literature?

Dr Aldecoa: “The difference is a clinical perspective. For example, to find the SLN using Indian ink, tattooing is made in a specific setting just prior to surgery (the day before surgery or even on the same day). This can ensure finding a tiny number of LNs that must be the first echeloned nodes of the patient, i.e. the sentinel lymph nodes. On the other hand, endoscopic tattooing is used during the endoscopy to mark the base of a resected polyp with high risk factors or unresectable lesions, to spot them in subsequent endoscopies or surgery. In this setting, the time between endoscopic tattooing and surgery might differ from some weeks to even a couple of months. This period of time lets one find more LNs other than the usual SLNs. Another difference is that endoscopic tattooing is performed at some distance from the lesion, not in its stalk or base, so that may also influence in the detection of the SLN.”



4. Is there any correlation between OSNA positivity and high-risk factors?

Dr Aldecoa: “In our study (1) including in situ lesions to pT1 and pT2 tumours, OSNA positive cases showed larger tumours (p = 0.02) and higher tumour grade (p<0.01). In an additional study (2) with stage I and II colon carcinomas, i.e. including pT3 and pT4 tumours, not only the two factors, tumour size and high grade, but also the presence of signet ring-cell areas, as well as high grade areas, were also predictors of greater OSNA positivity. In addition, multivariate logistic regression showed independent correlation of molecular positivity with gender, tumour grade and number of fresh LN.In low grade tumours, the total tumour load (TTL) assessed by CK19 increased along with the tumour size and the pT stage. Henceforth, the correlation between the OSNA positive result and the presence of different high-risk factors has been shown by our studies and by other authors indicating that the TTL may be used as an additional factor for a better selection of stage I-II patients at risk of recurrence.”



5. Have you had any kind of issue regarding contamination of the specimen? If yes, how did you handle that?

Dr Märkl: “At our Institute, pathologists have never experienced problems with contamination. Pathologists try to work as cleanly as possible and clean the specimen first. They do no open the specimen; this is very important. Contact with the mucosa layer can cause a diverticulitis and pathologists should be very careful about it. Finally, always using a clean table, single material and fresh plates may avoid any contamination of the specimen.”

Dr Aldecoa: “At the Hospital Clínic of Barcelona, the fresh cases were initially difficult to handle as fresh LN procurement was not the standard technique in our pathology department. Nowadays, we are using a clear and standardised protocol, which recommends the use of a clean space, sterilised gloves and scalpels. Besides, we ensure we do not open the specimen - leaving the peritoneum and the mucosa intact to avoid risk of contamination. To date, pathologists from the Hospital Clínic have currently assessed more than 300 cases of colorectal specimens without any incidences, which demonstrates that our protocol is robust in terms of contamination.”



6. Can we perform the immunoscore on the tattooed tumour?

Dr Aldecoa: “In our clinical setting, the tumour itself is not tattooed since the endoscopist usually tattoos the specimen a few centimetres next to the tumour. In case the Indian ink leads to an inflammatory reaction, this is usually mild. Hence, the immune reaction in the tumour should remain unaltered. Therefore, the immunoscore can be performed on a tattooed specimen.”



7. Can we correlate the number of LNs one can find based on patient characteristics: gender, age, BMI, location of the tumour, size of the specimen?

Dr Märkl: “There are many features from the patient or from the tumour itself which are in relation to the number of LNs found. This is valid if no special techniques such as methylene blue or fat clearance are used. If such techniques are applied, the patient’s features should be neglected.
Furthermore, the location of tumour is important: the right colon cancers always have more lymph nodes and the left colon cancers less. Neoadjuvant chemotherapy or radiation therapy play a big role. In this case, it is very difficult to find enough LNs. The type of tumour (MSI or MSS) is also important.

Finally, many parameters can play a big role on the number of harvested LNs: for example, a study by Morikawa et al. with a cohort of 900 node-negative patients showed that factors such as specimen length, tumour size, ascending colon location, and T3 stage, as well as PIK3CA and KRAS mutations were significantly associated with higher LN yield.(3)”




  1. Aldecoa I, Montironi C, Planell N, Pellise M, Fernandez-Esparrach G, Gines A, et al. Endoscopic tattooing of early colon carcinoma enhances detection of lymph nodes most prone to harbor tumor burden. Surg Endosc Other Interv Tech. 2016;
  2. Aldecoa I, Atares B, Tarragona J, Bernet L, Sardon JD, Pereda T, et al. Molecularly determined total tumour load in lymph nodes of stage I-II colon cancer patients correlates with high-risk factors. A multicentre prospective study. Virchows Arch. 2016 Jul;
  3. Morikawa T, Tanaka N, Kuchiba A, Nosho K, Yamauchi M, Hornick JL, et al. Predictors of lymph node count in colorectal cancer resections: data from US nationwide prospective cohort studies. Arch Surg [Internet]. NIH Public Access; 2012 Aug [cited 2017 Dec 24];147(8):715–23. Available from:

Dr Iban Aldecoa, Pathologist, Hospital Clínic de Barcelona, Spain

PD Dr Bruno Märkl, Chief Pathologist, Klinikum Augsburg, Germany