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Year : 2016  |  Volume : 59  |  Issue : 4  |  Page : 504-506
Impact of methylene blue staining in the retrieval of lymph nodes in resected colorectal cancer specimens

1 Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
2 Departments of Pathology and Surgical Oncology, Hyderabad, Telangana, India

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Date of Web Publication10-Oct-2016


Adequate lymph node harvest in resected colorectal cancer (CRC) specimens is important in staging and choosing appropriate therapeutic options. The yield of lymph nodes and metastatic nodes by methylene blue dye injection in 30 randomly selected resected CRC specimens was compared with an equal number of CRC specimens (control) with matched grade and stage.The mean number of lymph nodes retrieved in the study group was 22 ± 9 compared to 17 ± 8 in the control group which was statistically significant (P = 0.04).Methylene blue technique is an effective aid in increasing the yield of lymph nodes in the resected CRC specimens.

Keywords: Colorectal cancer, lymph node retrieval, methylene blue dye, Union for International Cancer Control

How to cite this article:
Vasala A, Nair HG, Rao ST, Tagore K R, Murthy SS, Fonseca D. Impact of methylene blue staining in the retrieval of lymph nodes in resected colorectal cancer specimens. Indian J Pathol Microbiol 2016;59:504-6

How to cite this URL:
Vasala A, Nair HG, Rao ST, Tagore K R, Murthy SS, Fonseca D. Impact of methylene blue staining in the retrieval of lymph nodes in resected colorectal cancer specimens. Indian J Pathol Microbiol [serial online] 2016 [cited 2022 Jul 4];59:504-6. Available from: https://www.ijpmonline.org/text.asp?2016/59/4/504/191804

   Introduction Top

Colorectal cancer (CRC) is a global malady with an increasing incidence in the Indian population. [1],[2] Accurate diagnosis, staging, and treatment options hold the key to good prognosis and enhanced survival benefits. One of the prime factors is histological lymph node staging which is vital in determining therapeutic strategies in CRC. The Union for International Cancer Control (UICC) recommends the examination of minimum twelve lymph nodes for appropriate staging. [3]

There are several techniques employed to improve the yield of lymph nodes which include fat clearing techniques, lymphatic system dye, specialized fixatives such as glacial acetic acid, ethanol, distilled water, and formaldehyde. [4] This study was designed to evaluate the effectiveness of methylene blue dye in augmenting lymph node recovery and metastatic nodes in CRC resected specimens.

   Materials and Methods Top

Resected specimens of histologically proven carcinoma involving the colon or rectum, operated at our hospital during 1 year period were included in the study. Emergency nonelective resections and specimens from patients who received preoperative radiotherapy or having concomitant inflammatory bowel disease were excluded from this study. Thirty specimens of resected CRC specimens were randomly selected for the implementation of methylene blue technique, which were received in fresh state immediately postoperatively or within 3 h of fixation in 10% formalin, following which the main artery was identified and cannulated. Subsequently, 10-30 ml of 0.5% methylene blue was gently injected [Figure 1]. The specimen was then allowed to fix in 10% formalin overnight. Four specimens were excluded due to failed technique. An equal number of CRC specimens which were matched for grade and stage were processed routinely, and lymph node dissection was done by a conventional method. The dissection of lymph nodes in both groups was performed by the same pathologist. Processing and reporting were performed as per standard protocol. Analysis of both groups with particular attention to a number of lymph nodes dissected and involved by tumor was performed.
Figure 1: (a) Left: Resected colorectal specimen with the main artery identified, cannulated and injected with 10-30 ml of methylene blue dye (grey arrow). Inset depicting the highlighted surface vessels visible through the intact mesorectal fascia (black arrow). (b) Right: Highlighted lymph nodes postfixation as blue circumscribed areas (arrows). Inset depicting dissected lymph nodes stained by methylene blue

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Statistical analysis

The mean and standard deviation were used to calculate the number of lymph nodes in each group. For correlation between means, two-tailed t-test or Mann-Whitney U-test depending on normality results was used. P ≤ 0.05 was considered as statistically significant. SPSS 13 and Graph Pad Prism 6 software, SPSS Inc. (IBM corp), GraphPad software, Inc was used for statistical analysis.

   Results Top

There were 26 CRC specimens in each group. There were ten each of low grade and seven each of high-grade tumors in both groups. The pathological stage was pT2 in 8, pT3 in 18 in the study group whereas there were 7 in pT2, 18 in pT3, and one in pT4 in the control group.

The mean number of lymph nodes retrieved in the study group was 22 ± 9 whereas it was 17 ± 8 in the control group and the difference was statistically significant (P = 0.04). The mean number of lymph nodes involved by metastases was three in the study group and 12 in the control group, which was not statistically significant (P = 1.0).

The number of specimens with less than UICC recommendation of 12 nodes was two in the study group and six in the control group, and this difference was not statistically significant (P = 0.24). There were no significant differences between the two groups with respect to gender, tumor location, and size.

   Discussion Top

Retrieval of a maximum number of lymph nodes from resected CRC specimens is crucial in patient care and management. Of the several techniques available, methylene blue staining technique was chosen in the present study because of its simplicity and cost-effectiveness. In this study, with the use of methylene blue, there was an increase in the yield of lymph nodes when compared to the control group which was statistically significant. Similar findings were reported earlier. [5],[6],[7],[8],[9],[10],[11],[12]

Jepsen et al. reported that with the increase in the retrieval of lymph nodes, the number of positive lymph nodes also increased raising the number of upstaged cases. [9] There was no significant difference in the number of metastatic nodes in both groups in this study as was reported earlier. [12]

Many factors affect the yield of lymph nodes such as age, location, obesity, immune response, microsatellite instability, surgical technique, and efficient dissection methods. [4],[13],[14] In this study, two cases in the study group and six cases in the control group did not meet the UICC recommended criteria; the older age group with a likely diminished immune response could be one of the contributing factors for a lower harvest. The methylene blue method has certain disadvantages, and technical difficulties can be encountered. Four specimens were excluded from the study due to ineffective cannulation, fragile blood vessels, and ineffective staining. Similar difficulties were reported earlier. [5] It is recommended that cannulation and injection of dye is preferably done by the surgeon himself in the fresh state to avoid the technical failures. [6],[15]

   Conclusion Top

Methylene blue staining can be regarded as an effective aid in improving lymph node yield in resected CRC specimens and can assist in meeting the UICC recommended guidelines. However, there was no significant influence in the detection of metastatic lymph nodes.


We are very grateful and sincerely thank Prof. Dr. C Sundaram for critical review and editing of the manuscript, Dr. Lavanya for statistical analysis, and lab technicians Sudhakar R, Sambasiva R, Ravinder K for their assistance.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. Globocan 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11. Lyon, France: International Agency for Research on Cancer; 2013. Available from: http://www.globocan.iarc.fr. [Last accessed on 2015 Nov 02].  Back to cited text no. 1
Mohandas KM. Colorectal cancer in India: Controversies, enigmas and primary prevention. Indian J Gastroenterol 2011;30:3-6.  Back to cited text no. 2
Märkl B, Roble J, Arnholdt HM, Schaller T, Krammer I, Cacchi C, et al. The clinical significance of lymph node size in colon cancer. Mod Pathol 2012;25:1413-22.  Back to cited text no. 3
Deodhar KK, Budukh A, Ramadwar M, Bal MM, Shrikhande SV. Are we achieving the benchmark of retrieving 12 lymph nodes in colorectal carcinoma specimens? Experience from a tertiary referral center in India and review of literature. Indian J Pathol Microbiol 2012;55:38-42.  Back to cited text no. 4
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Törnroos A, Shabo I, Druvefors B, Arbman G, Olsson H. Postoperative intra-arterial methylene blue injection of colorectal cancer specimens increases the number of lymph nodes recovered. Histopathology 2011;58:408-13.  Back to cited text no. 5
Märkl B, Kerwel TG, Wagner T, Anthuber M, Arnholdt HM. Methylene blue injection into the rectal artery as a simple method to improve lymph node harvest in rectal cancer. Mod Pathol 2007;20:797-801.  Back to cited text no. 6
Klepšyte E, Samalavicius NE. Injection of methylene blue solution into the inferior mesenteric artery of resected rectal specimens for rectal cancer as a method for increasing the lymph node harvest. Tech Coloproctol 2012;16:207-11.  Back to cited text no. 7
Kerwel TG, Spatz J, Anthuber M, Wünsch K, Arnholdt H, Märkl B. Injecting methylene blue into the inferior mesenteric artery assures an adequate lymph node harvest and eliminates pathologist variability in nodal staging for rectal cancer. Dis Colon Rectum 2009;52:935-41.  Back to cited text no. 8
Jepsen RK, Ingeholm P, Lund EL. Upstaging of early colorectal cancers following improved lymph node yield after methylene blue injection. Histopathology 2012;61:788-94.  Back to cited text no. 9
Frasson M, Faus C, Garcia-Granero A, Puga R, Flor-Lorente B, Cervantes A, et al. Pathological evaluation of mesocolic resection quality and ex vivo methylene blue injection: What is the impact on lymph node harvest after colon resection for cancer? Dis Colon Rectum 2012;55:197-204.  Back to cited text no. 10
Borowski DW, Banky B, Banerjee AK, Agarwal AK, Tabaqchali MA, Garg DK, et al. Intra-arterial methylene blue injection into ex vivo colorectal cancer specimens improves lymph node staging accuracy: A randomized controlled trial. Colorectal Dis 2014;16:681-9.  Back to cited text no. 11
Märkl B, Schaller T, Krammer I, Cacchi C, Arnholdt HM, Schenkirsch G, et al. Methylene blue-assisted lymph node dissection technique is not associated with an increased detection of lymph node metastases in colorectal cancer. Mod Pathol 2013;26:1246-54.  Back to cited text no. 12
Görög D, Nagy P, Péter A, Perner F. Influence of obesity on lymph node recovery from rectal resection specimens. Pathol Oncol Res 2003;9:180-3.  Back to cited text no. 13
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 2012;147:715-23.  Back to cited text no. 14
Albatanony AA, Alseesi AA, Ammar MS, Shaaban M. Improving lymph node harvest in colorectal cancer by intra-arterial injection of methylene blue: A randomized trial. Egypt J Surg 2015;34:99-102.  Back to cited text no. 15
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Correspondence Address:
Anjaneyulu Vasala
8-2-682/4/3, Plot No. 10, Anand Banjara Colony Road No. 12, Banjara Hills, Hyderabad - 500 034, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.191804

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