We would like to thank Joana Ribeiro for filming of the specimen.

The protocol followed the ethical guidelines of Southampton University Hospital NHS Foundation Trust. Informed consent was obtained for the use of the tissue for teaching and research purposes.
NOTE: The relevant steps are summarized in Table 2 and the relevant materials in Table of Materials.
1. Specimen orientation
NOTE: In order to aid the histopathologist in accurate specimen orientation, ensure that the operating surgeon places orientation sutures to mark the posterior dissection plane, the splenic vessel margins, and the pancreatic transection margin.
<ol>
	<li>Upon receipt, fix the specimen in formalin, at room temperature, for 24&#8722;48 h. 
	NOTE: Fixation may cause distortion of the specimen which makes accurate orientation difficult as the anterior peritoneal surface and retroperitoneal (posterior dissection) margin may have similar appearances. Furthermore, some centers take fresh tumor samples for research purposes. In this situation, care should be given to minimize distortion by inking, before fixation, one or more margins, if these are to be incised while the specimen is fresh.</li>
	<li>Identify posterior dissection plane by orientation suture (placed by the operating surgeon) or by the presence of the spleen, which is lateral, and the splenic vessels on the postero-superior border, the suture marking the posterior dissection margin, and staples marking the (medial) transection margin. Identify also other margins/surfaces. Finally identify the spleen. 
	NOTE: In this specimen the anterior surface, transection margin, posterior margin, superior border, inferior border, and additional gastric wedge can be identified.</li>
	<li>Ensure that the peripancreatic fat remains intact and attached to the specimen.</li>
</ol>
2. Measurements
<ol>
	<li>Measure all dimensions in millimeters.</li>
	<li>Measure the pancreas from medial to lateral (95 mm for this specimen), cranial to caudal (30 mm), and anterior to posterior (20 mm).</li>
	<li>Measure the spleen also from cranial to caudal (110 mm for this specimen), anterior to posterior (60 mm), and medial to lateral (20 mm). In addition, weigh the spleen if possible (see the note below). 
	NOTE: If the tumor is in close proximity to the splenic hilum, the spleen should not be separated from the pancreas and weighing of spleen should be done en bloc with the pancreas.</li>
	<li>Measure any potential additional organs, including the adrenal gland. 
	NOTE: In this protocol, the gastric wall was resected and measured. The wedge stomach measures 35 mm x 10 mm x 5 mm.</li>
</ol>
3. Inking
<ol>
	<li>Having measured the pancreas and spleen in three dimensions, ink the posterior dissection margin, transection margin, anterior peritonealized surface, and additional organ margins using different colors. If the splenic vessel margins appear grossly involved, ink these in order to identify the true margin with &#39;en face&#39; sections.</li>
	<li>Though, not a requirement for margin assessment, ink the superior and inferior border of the pancreas for improved specimen orientation and to microscopically group lymph nodes into the relevant anatomical stations in single transverse sections on hematoxylin and eosin stained slides.</li>
	<li>Furthermore, ink any additional margin (e.g., the gastric wall margin) with a different color. 
	NOTE: The specimen is inked in the following manner: the anteriorperitonealized surface is inked in yellow, the gastric wall margin is inked in blue, the transection margin is inked in red, the superior border of the pancreas is inked in green in order to identify station 11 lymph nodes, the inferior border is inked in orange to identify station 18 nodes, and the posterior dissection margin is inked in black.</li>
</ol>
4. Dissection
<ol>
	<li>Following measurement and inking of the specimen, use a blade to cut blocks from the transection and splenic vessel margins. Remove with the blade the staples in order to take an &#39;en face&#39; section of the transection margin. Cut closely to the staple line to preserve as much of the proximity to the true margin. 
	NOTE: The staple line of the transection margin must be removed as metal staples cannot be cut by the microtome blades.</li>
	<li>Cut with the blade a single &#39;en face&#39; section of 3&#8722;5 mm of the transection margin of the splenic artery and vein. Alternatively, cut bread slices of 3&#8722;5 mm of the transection margin perpendicular to the stapled margin. 
	NOTE: The splenic artery and vein are in close proximity to the transection margin and are usually marked with clips. This can be used to give accurate distances of tumor to the margin.</li>
	<li>Remove any additional margins using the blade. 
	NOTE: In the accompanying video, the staple line from the gastric wedge resection is removed.</li>
	<li>Using a long knife slice the specimen from medial to lateral (transection margin to spleen) in 3&#8722;5 mm intervals, and number the slices (for example 1&#8722;15, with 15 slices). Do not remove any of the posterior pancreatic fat.</li>
	<li>With the transverse sections of the specimen laid out from medial to lateral, record the number of tumors, including their location (body, and/or tail), appearance(s), and maximum tumor size(s). 
	NOTE: Unlike tumors of the pancreatic head where the significance of named vessel involvement is reflected in TNM staging, with superior mesenteric artery and/or coeliac axis involvement corresponding to pT4 disease, the clinical significance of splenic artery invasion remains under investigation11. For the purpose of future investigation, it is recommended to record the splenic artery and vein involvement within the macroscopic description, and to confirm it microscopically. The splenic artery lies in the superior border of the pancreas, while the vein runs along the posterior border of the pancreas.</li>
</ol>
5. Tumor assessment
<ol>
	<li>Assess the tumor and its involvement of margins and/or (splenic) vessels. 
	NOTE: Here, the tumor is present in slices 9&#8722;14. The cut surface of the tumor is pale and homogeneous.</li>
	<li>Measure the macroscopic tumor size in three dimensions (cranial to caudal, medial to lateral, and anterior to posterior). 
	NOTE: Here, the tumor measures 30 mm cranial to caudal plane, 25 mm anterior to posterior, and 13 mm medial to lateral. The measurement is based on slice thickness. Most commonly maximum tumor size is in the medial to lateral plane. This measurement is assessed adding up the slice thickness of each slide containing tumor. Distances to margins can be difficult to assess macroscopically as ductal adenocarcinoma of the pancreas have a highly dispersed growth8,12. On this basis, while an estimation of the distances to the margins can be recorded in the macroscopic description, corroboration of these findings microscopically based on adequate tumor sampling is important.</li>
	<li>Slice the spleen with perpendicular section with 3&#8722;5 mm intervals starting from its outer convexity and proceed inwards towards the splenic hilum. In cases where the tumor is in close proximity to the spleen, keep the spleen intact with the specimen and take section in continuity between the tumor and spleen.</li>
</ol>
6. Tissue sampling
<ol>
	<li>As is done for pancreatoduodenectomy specimens, rationalize block selection to demonstrate tumor type, size, large vessel and other organ involvement, nodal staging, margin status, and tumor regression in cases where neo-adjuvant therapy has been given. Make close-up photographs of the specimen slices. 
	NOTE: The block selection includes the transection margin, (splenic) vascular resection margins, gastric wall margins, blocks of tumor demonstrating maximum size and distance to margins, background pancreas, representative section of spleen, and lymph nodes.</li>
	<li>Take tissue samples from the tumor en-bloc with the peripancreatic fat from the superior and inferior borders that harbors lymph nodes from stations 11 and 18. Dissect out individual lymph nodes from the peripancreatic fat in parts of the specimen that are well away from the tumor site. 
	NOTE: Especially take additional samples from what macroscopically seems to be the periphery of the tumor and the adjacent tissues that appear tumor-free. It is in the latter that microscopically invasive tumor is most often found to be present. The same rationale for thorough block taking applies to the assessment of the margins. In contrast to colorectal carcinomas that typically have a more well-circumscribed tumor front, pancreatic ductal adenocarcinomas have an infiltrative, discontinuous pattern of growth. The interface of tumor with normal pancreas is often obscured by the presence of chronic pancreatitis, placing further onus on margin sampling3. It is strongly recommended not to remove the posterior peripancreatic fat, as this will compromise assessment of the posterior margin.</li>
	<li>For tumors 20 mm or less, sample the entire tumor to allow for accurate pT staging. For larger tumors, sample sufficient tissue. When sampling tumor, include the peripancreatic fat, lymph nodes and the relationship with margins and adjacent structures. 
	NOTE: In the accompanying video, the relationship between tumor and gastric wall is highlighted. Regional lymph nodes for the distal pancreas are station 11 along the superior border of the pancreas, station 18 along the inferior border, and station 10 within the splenic hilar fat13. For tumors located in the pancreatic body the surgeon will additionally resect lymph node stations 8a and 9, which are submitted in separate pots.</li>
	<li>Take perpendicular sections of the spleen at 3&#8722;5 mm intervals, followed by sampling of splenic hilum (station 10) lymph nodes. 
	NOTE: There is no recommendation on whether to remove the spleen from the specimen or not. In case the tumor is located in the tip of the pancreas, do not remove the spleen and extent the sagittal slicing into the spleen.</li>
	<li>Take blocks from additional organs.</li>
</ol>

<ol>
	<li>Ilic, M., Ilic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+pancreatic+cancer.">Epidemiology of pancreatic cancer.</a> World Journal of Gastroenterology. 22 (44), 9694-9705 (2016).</li><li>Strobel, O., Neoptolemos, J., J&#228;ger, D., B&#252;chler, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+outcomes+of+pancreatic+cancer+surgery.">Optimizing the outcomes of pancreatic cancer surgery.</a> Nature Reviews Clinical Oncology. 16 (1), 11-26 (2019).</li><li>Verbeke, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resection+margins+in+pancreatic+cancer.">Resection margins in pancreatic cancer.</a> Der Pathologe. 34, 241-247 (2013).</li><li>van Roessel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Validation+of+the+Eighth+Edition+of+the+American+Joint+Committee+on+Cancer+(AJCC)+TNM+Staging+System+in+Patients+With+Resected+Pancreatic+Cancer.">International Validation of the Eighth Edition of the American Joint Committee on Cancer (AJCC) TNM Staging System in Patients With Resected Pancreatic Cancer.</a> JAMA Surgery. 153 (12), e183617 (2018).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+size+classification+of+the+8th+edition+of+TNM+staging+system+is+superior+to+that+of+the+7th+edition+in+predicting+the+survival+outcome+of+pancreatic+cancer+patients+after+radical+resection+and+adjuvant+chemotherapy.">Tumor size classification of the 8th edition of TNM staging system is superior to that of the 7th edition in predicting the survival outcome of pancreatic cancer patients after radical resection and adjuvant chemotherapy.</a> Scientific Reports. 8 (1), 1-9 (2018).</li><li>Verbeke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operative+Specimen+Handling+and+Evaluation+of+Resection+Margins.">Operative Specimen Handling and Evaluation of Resection Margins.</a> Pancreatic Cancer. , 67-87 (2017).</li><li>Barreto, S., Shukla, P., Shrikhande, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+of+the+Pancreatic+Body+and+Tail.">Tumors of the Pancreatic Body and Tail.</a> World Journal of Oncology. 1 (2), 52-65 (2010).</li><li>. Dataset for the histopathological reporting of carcinomas of the pancreas, ampulla of Vater and common bile duct from the Royal College of Pathologists Available from: <a target="_blank" href="https://www.rcpath.org/uploads/assets/34910231-c106-4629-a2de9e9ae6f87ac1/g091-pancreasdataset-mar17.pdf">https://www.rcpath.org/uploads/assets/34910231-c106-4629-a2de9e9ae6f87ac1/g091-pancreasdataset-mar17.pdf</a> (2017)</li><li>Strasberg, S. M., Drebin, J. A., Linehan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radical+antegrade+modular+pancreatosplenectomy.">Radical antegrade modular pancreatosplenectomy.</a> Surgery. 133 (5), 521-527 (2003).</li><li>Abu Hilal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+radical+'no-touch'+left+pancreatosplenectomy+for+pancreatic+ductal+adenocarcinoma:+technique+and+results.">Laparoscopic radical 'no-touch' left pancreatosplenectomy for pancreatic ductal adenocarcinoma: technique and results.</a> Surgical Endoscopy. 30 (9), 3830-3838 (2016).</li><li>Kanda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+the+splenic+artery+is+a+crucial+prognostic+factor+in+carcinoma+of+the+body+and+tail+of+the+pancreas.">Invasion of the splenic artery is a crucial prognostic factor in carcinoma of the body and tail of the pancreas.</a> Annals of Surgery. 251 (3), 483-487 (2010).</li><li>Verbeke, C. S., Knapp, J., Gladhaug, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+growth+is+more+dispersed+in+pancreatic+head+cancers+than+in+rectal+cancer:+Implications+for+resection+margin+assessment.">Tumour growth is more dispersed in pancreatic head cancers than in rectal cancer: Implications for resection margin assessment.</a> Histopathology. 59 (6), 1111-1121 (2011).</li><li>Tol, J. A. M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+a+standard+lymphadenectomy+in+surgery+for+pancreatic+ductal+adenocarcinoma:+A+consensus+statement+by+the+International+Study+Group+on+Pancreatic+Surgery+(ISGPS).">Definition of a standard lymphadenectomy in surgery for pancreatic ductal adenocarcinoma: A consensus statement by the International Study Group on Pancreatic Surgery (ISGPS).</a> Surgery. 156 (3), 591-600 (2014).</li><li>Demir, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R0+Versus+R1+Resection+Matters+after+Pancreaticoduodenectomy,+and+Less+after+Distal+or+Total+Pancreatectomy+for+Pancreatic+Cancer.">R0 Versus R1 Resection Matters after Pancreaticoduodenectomy, and Less after Distal or Total Pancreatectomy for Pancreatic Cancer.</a> Annals of Surgery. 268 (6), 1058-1068 (2018).</li><li>Verbeke, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resection+margins+and+R1+rates+in+pancreatic+cancer+-+Are+we+there+yet?.">Resection margins and R1 rates in pancreatic cancer - Are we there yet?.</a> Histopathology. 52 (7), 787-796 (2008).</li><li>Verbeke, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+the+R1+resection+in+pancreatic+cancer.">Redefining the R1 resection in pancreatic cancer.</a> British Journal of Surgery. 93 (10), 1232-1237 (2006).</li><li>Malleo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Number+of+examined+lymph+nodes+and+nodal+status+assessment+in+pancreaticoduodenectomy+for+pancreatic+adenocarcinoma.">Number of examined lymph nodes and nodal status assessment in pancreaticoduodenectomy for pancreatic adenocarcinoma.</a> Annals of Surgery. , (2018).</li><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stapler+vs+suture+closure+of+pancreatic+remnant+after+distal+pancreatectomy:+A+meta-analysis.">Stapler vs suture closure of pancreatic remnant after distal pancreatectomy: A meta-analysis.</a> American Journal of Surgery. 200 (4), 529-536 (2010).</li></ol>

The authors have nothing to disclose.

Adequate histopathological assessment of a resected specimen is essential for the stratification of disease prognosis and guidance of further treatment strategies. Standardized protocols for the assessment of specimens resulting from distal pancreatectomy for PDAC are lacking. This potentially creates a considerable variability among the reported histopathological findings14. Differences in definitions and practice between centers limit the comparability of studies15. Furth...

Pancreatic ductal adenocarcinoma (PDAC) is associated with a very poor prognosis1. Surgery, in combination with (neo)adjuvant therapy remains the only curative treatment2. Following surgery, adequate histopathological assessment of the resected specimen is essential for prognostic stratification and in addition it may guide further treatment strategies3. Furthermore, the recent 8th edition of American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) staging system for pancreatic tumors has incorporated significant changes to tumor (pT) stage, which is predominantly based on tumor size4,5. While maximum tumor size is assessed macroscopically, adequate specimen sampling is required in order to corroborate these findings, particularly as chronic pancreatitis can mimic tumor appearance with the naked eye.
As the majority of pancreatic ductal adenocarcinomas (up to 80%) are encountered in the head of the pancreas, most of the literature is based on the assessment of pancreatoduodenectomy specimens6,7. In the United Kingdom, the Royal College of Pathologists (RCPath) have published datasets that provide evidence-based guidelines on the specimen handling, dissection and reporting of pancreatic cancer, with focus placed on the more common pancreatoduodenectomy specimens8. Nonetheless, international consensus regarding specimen grossing is still lacking and practice is still highly divergent between centers6. The equivalent process of standardizing pathology assessment of specimen originating from a distal (i.e., left) pancreatectomy is now of growing clinical interest.
The Distal Pancreatectomy, Minimally Invasive or Open, for malignancy (DIPLOMA, ISRCTN44897265) trial is an international multicenter, randomized controlled trial comparing open versus minimally invasive surgical approach for the management of PDAC of the pancreatic body and tail. The DIPLOMA pathology protocol has been developed as a means of standardizing pathology assessment and reporting for this trial. The protocol describes the assessment of distal pancreatectomy specimens, including specimen orientation, inking, lymph node sampling, assessment of splenic vessel involvement (and other organs if present), and block selection.
The described method was developed during four meetings of the DIPLOMA study group (April 2015 Manchester, December 2016 Amsterdam, May 2017 Mainz, and April 2018 Amsterdam) with highly experienced 20&#8722;40 surgeons and pancreatic pathologists from 10 countries across Europe. Discussions included the relevance of the various margins, the transection plane and especially the dissection plane between the posterior part of the body and tail.
Patient characteristics
A 79-year-old woman presented with an incidental finding of a 34 mm tumor in the body of the pancreas, which was suspicious for malignancy. The CT scan showed no radiological evidence of tumor involvement of major vascular structures or the presence of (distant) metastases. Only adjacent small sized lymphadenopathy was noted. The patient was discussed in the multidisciplinary team meeting where it was decided that she was eligible for surgery. An open radical distal pancreatectomy, splenectomy and wedge resection of the stomach was performed within the DIPLOMA trial.
Macroscopic assessment of distal pancreatectomy specimens and nomenclature of margins
The relevant margins that should be assessed in a distal pancreatectomy specimen include the transection margin, splenic artery and vein margins, posterior dissection margin, and additional margins in the case of multivisceral resections as shown in Table 1.
The transection margin is the surface where the pancreatic body was separated from the neck. Mainly in laparoscopic, but also in increasing numbers of open, surgical specimens, this margin is a linear staple line. The splenic artery and vein margins are in close proximity to the stapled transection margin and are marked with vascular clips or small staples. The posterior margin is the dissection plane between the posterior part of the body and tail of the pancreas and the frontal plane of the renal fascia, within the retroperitoneum. Between the anterior and posterior renal fascia is the perirenal space, within which lie the kidney and adrenal gland in a loose fibrofatty connective tissue compartment. The posterior dissection margin varies depending on the exact surgical procedure performed. This may include the anterior renal fascia, with or without the adrenal gland and posterior renal fascia9,10. While the anterior, peritonealized surface is not considered a surgical margin, tumor breaching of this surface is associated with an increased risk of local recurrence3.

<table><tbody><tr><td>Formalin</td><td>Genta</td><td>BFNC50</td><td></td></tr><tr><td>Gloves</td><td>Healthline</td><td>FTG182, FTG183, FTG184 (depending on size)</td><td></td></tr><tr><td>Blade Handles</td><td>Swann Morton</td><td></td><td></td></tr><tr><td>Blades</td><td>Swann Morton</td><td>FSF440</td><td></td></tr><tr><td>Scales</td><td>Ohaus</td><td></td><td></td></tr><tr><td>Long Knives</td><td>Cellpath</td><td>KMY811</td><td></td></tr><tr><td>Ruler</td><td>Solmedia</td><td>RUL003</td><td></td></tr><tr><td>Scissors</td><td>Weiss</td><td>FGP8939</td><td></td></tr></tbody></table>

diploma approach for standardized pathology assessment of distal pancreatectomy specimens

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. Accurate pathology assessment of the pancreatic specimen is essential for providing prognostic information and it may guide further treatment strategies. The recent 8th edition of the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) staging system for pancreatic tumors has incorporated significant changes to tumor (pT) stage, which is predominantly based on tumor size. This change emphasizes the importance of careful block selection. Owing to the greater prevalence of tumors in the head of the pancreas, efforts are made to standardize the assessment of pancreatoduodenectomy specimens. However, consensus regarding the macroscopic assessment of distal (i.e., left) pancreatectomy specimens is lacking. The DIPLOMA approach includes the standardized measurement of pancreas and other resected organs, inking of relevant surgical margins and anatomical surfaces without removing covering layers of fat, measurement of tumor size (for T-stage), together with assessment of splenic vessel involvement (and other organs if present). All relevant margins are assessed, and relevant blocks are selected to confirm these parameters microscopically. The current protocol describes a standardized approach to the macroscopic assessment of distal pancreatectomy specimens. This approach was developed during several meetings with pathologists and surgeons during the preparation phase for an international multicenter trial (DIPLOMA, ISRCTN44897265), which focuses on radicality of distal pancreatectomy for pancreatic ductal adenocarcinoma. This standardized approach can be instrumental in the design of studies and will uniform reporting on the outcomes of distal pancreatectomy. The described technique is used in the DIPLOMA trial for pancreatic ductal adenocarcinoma but may also be useful for other indications.

Microscopic assessment showed a 28 x 25 x 30 mm, moderately differentiated, pancreatic ductal adenocarcinoma as shown in Table 3. There was perineural and lymphovascular invasion without splenic artery or vein involvement. In total, 17 lymph nodes were found, of which 3 were involved (1 superior border, 2 inferior border). Distant lymph nodes (station 8 and hepatic artery) showed no evidence of metastatic malignancy. All resection margins were clear of tumor: transection ...

Watch this Scientific Journal Video about DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens at JoVE.com

DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens

The current study highlights a standardized approach to the macroscopic assessment of distal pancreatectomy specimens for pancreatic ductal adenocarcinoma, with special emphasis on the measurement of pancreatic dimensions and those of other organs, inking of margins, measurement of tumor size and proximity to margins, lymph node sampling and block selection.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant cancers. A minority (20%) of PDACs are found in the pancreatic body and tail. ...

diploma-approach-for-standardized-pathology-assessment-distal

Research

JoVE Journal

Medicine

7.4K Views.  Southampton University Hospital NHS Foundation Trust. Gestandaardiseerde pathologiebeoordeling van distale pancreatectomiemonsters, een verklarende video zoals uitgevoerd in de DIPLOMA-studie. Pancreas ductal adenocarcinoom wordt geassocieerd met een slechte prognose. Een minderheid van deze tumoren zijn te vinden in de alvleesklier lichaam en staart.Nauwkeurige pathologische beoordeling van alvleeskliermonsters is essentieel voor het verstrekken van prognostische informatie die de behandelingsstrategieën kan sturen. Het volgende protoco...

Video: DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Cancer Research

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection (DP-CAR) for Pancreatic Cancer: Surgical Planning and Technique

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), relying on collateral flow to the liver through the gastroduodenal artery (GDA). In the current manuscript, the technical conduct of robotic DP-CAR is outlined. The greater curve of the stomach is mobilized with care to avoid sacrificing the gastroepiploic vessels. The stomach and liver are retracted cephalad to facilitate dissection of the porta hepatis. The hepatic artery (HA) is dissected and encircled with a vessel loop. The gastroduodenal artery (GDA) is carefully preserved. The common HA is clamped and triphasic flow in the proper HA via the GDA is confirmed using intra-operative ultrasound. A retropancreatic tunnel is made over the superior mesenteric vein (SMV). The pancreas is divided with an endovascular stapler at the neck. The inferior mesenteric vein (IMV) and splenic vein are ligated. The HA is stapled proximal to the GDA. The entire specimen is retracted laterally with further dissection cephalad to expose the superior mesenteric artery (SMA). The SMA is then traced back to the aorta. The dissection continues cephalad along the aorta with the bipolar energy device used to divide the crural fibers and celiac nerve plexus. The specimen is mobilized from the patient's right to left until the origin of the celiac axis is identified and oriented towards the left. The trunk is circumferentially dissected and stapled. Additional dissection with hook cautery and the bipolar energy device fully mobilizes the pancreatic tail and spleen. The specimen is removed from the left lower quadrant extraction site and one drain is left in the resection bed. A final intra-operative ultrasound of the proper HA confirms pulsatile, triphasic flow in the artery and liver parenchyma. The stomach is inspected for evidence of ischemia. Robotic DP-CAR is safe, feasible and when used in conjunction with multi-modality therapy, offers potential for long-term survival in selected patients.

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection DP-CAR for Pancreatic Cancer: Surgical Planning and Technique

We present our operative approach to robot assisted distal pancreatectomy, splenectomy, and celiac axis resection (DP-CAR), demonstrating that the procedure is safe and feasible with proper planning, patient selection, and surgeon experience.

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), ...

Robotic Pancreatoduodenectomy for Pancreatic Head Cancer: a Case Report of a Standardized Technique

Robotic pancreatoduodenectomy (RPD) for pancreatic cancer is a challenging procedure. Aberrant vasculature may increase the technical difficulty. Several studies have described the safety of RPD in case of a replaced or aberrant right hepatic artery, but detailed video descriptions of the approach are lacking. This case report describes a step-&#173;by-&#173;step technical video in case of a replaced right hepatic artery. A 58-year-old woman presented with an incidental finding of a 1.7 cm pancreatic head mass. RPD was performed using the da Vinci Xi system and involves a robotic-assisted pancreatico- and hepatico-jejunostomy and open gastro-jejunostomy at the specimen extraction site. The operation time was 410 min with 220 mL of blood loss. The patient had an uncomplicated postoperative course and was discharged after 5 days. Pathology revealed a pancreatic head cancer. RPD is a feasible and safe procedure in case of a replaced hepatic artery when performed in selected patients in high-volume centers by experienced surgeons.

Robotic pancreatoduodenctomy (RPD) has been highly standardized in recent years and may be used in selected patients with pancreatic head cancer, including those with a replaced right hepatic artery. This case report describes a standardized and reproducible technique for RPD, which includes the approach of the Dutch LAELAPS-3 training program to an aberrant vasculature.

Robotic pancreatoduodenectomy (RPD) for pancreatic cancer is a challenging procedure. Aberrant vasculature may increase the technical difficulty. Several ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Bioengineering

Technical Detail for Robot Assisted Pancreaticoduodenectomy

Since its first report in 2003, robotic pancreaticoduodenectomy (RPD) has gained popularity among pancreatic surgeons. Inherent advantages of the robotic platform, including three-dimensional vision, wristed instruments, and improved ergonomics, allow the surgeon to recapitulate the principles of open pancreatoduodenectomy allowing safe oncologic dissection, hemostasis, and meticulous reconstruction. Over the course of the past decade, significant strides have been achieved in outlining the safety, feasibility, and learning curve of the robotic Whipple. When performed by high volume pancreatic surgeons experienced in RPD, recent comparative effectiveness studies show potential advantages compared to the open technique, including reductions in hospital stay and morbidity. National data also show reductions in conversion rates compared to its laparoscopic counterpart. Although long-term oncologic data are still needed, short-term oncologic surrogates of margin resection and lymph node harvest suggest no compromise in oncologic outcomes. As pancreatic surgeons increasingly integrate robotics into their practice, proficiency-based training and credentialing will be necessary for the safe application and dissemination of RPD. Here, we provide the detailed steps of a robotic pancreaticoduodenectomy performed at the University of Pittsburgh Medical Center.&#160;

The following manuscript details a stepwise approach to the robot-assisted pancreaticoduodenectomy performed at the University of Pittsburgh Medical Center.

Since its first report in 2003, robotic pancreaticoduodenectomy (RPD) has gained popularity among pancreatic surgeons. Inherent advantages of the robotic ...

Inducing Pancreatic Injury in Mice Through Intraperitoneal Drug Injection

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 &#181;g/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis (SAP).

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological ...

Immunology and Infection

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic resection and reconstruction techniques, it is difficult to perform LPD for patients with locally advanced pancreatic head cancer after neoadjuvant therapy. Our team initiates LPD using the in-situ No-Touch isolation technique. The innovation and optimization of this modified No-Touch isolation technique emphasize exploring the distal section of superior mesenteric vein&#160;(SMV) and the left side of the superior mesenteric artery (SMA) prior to evaluating the resectability by subcolonic mesenteric approach, which is an ideal exploring approach. After that, we use the median-anterior, and left-posterior of SMA approaches to cut off the blood flow of the pancreatic head to make the tumor isolated intact, then move and dissect the tumor. It is a process fitting the surgical principle of tumor-free. This article aims to demonstrate the feasibility and safety of performing LPD using the in-situ No-Touch isolation technique, which might elevate the R0 resection rate. It is an oncological ideal operation process.

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

No-Touch isolation procedures might prevent the dissemination of cancer cells from the primary tumor. However, these techniques are not widely accepted in laparoscopic pancreatoduodenectomy (LPD) by now. We herein present in-situ No-Touch isolation LPD with partial resection and reconstruction of the superior mesenteric vein (SMV) for pancreatic cancer after neoadjuvant therapy.

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic ...

DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens

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