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>

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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

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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. ...

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7.3K Views.  Southampton University Hospital NHS Foundation Trust. 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.

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

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

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 ...

A Standardized Obstacle Course for Assessment of Visual Function in Ultra Low Vision and Artificial Vision

We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. Six sighted controls and 36 completely blind but otherwise healthy adult male (n=29) and female (n=13) subjects (age range 19-85 years), were enrolled in one of three studies involving testing of the BrainPort sensory substitution device. Subjects were asked to navigate the course prior to, and after, BrainPort training. They completed a total of 837 course runs in two different locations. Means and standard deviations were calculated across control types, courses, lights, and visits. We used a linear mixed effects model to compare different categories in the PPWS (percent preferred walking speed) and error percent data to show that the course iterations were properly designed. The course is relatively inexpensive, simple to administer, and has been shown to be a feasible way to test mobility function. Data analysis demonstrates that for the outcome of percent error as well as for percentage preferred walking speed, that each of the three courses is different, and that within each level, each of the three iterations are equal. This allows for randomization of the courses during administration.
Abbreviations: 
preferred walking speed (PWS) 
course speed (CS) 
percentage preferred walking speed (PPWS)

We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. The course is relatively inexpensive, simple to administer, and has been shown to be reliable and reproducible.

We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. Six sighted ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

Robotic Central Pancreatectomy with Roux-en-Y Pancreaticojejunostomy

Central pancreatectomy is a parenchyma-sparing alternative to distal pancreatectomy in patients with a benign or low-grade malignant tumor in the body of the pancreas. The aim of central pancreatectomy is to prevent postoperative life-long endocrine and exocrine insufficiency. The downside of central pancreatectomy is the high rate of postoperative pancreatic fistula, which is the main reason that many surgeons do not routinely use central pancreatectomy in eligible patients. Most studies report open or laparoscopic central pancreatectomy with a pancreatico-gastrostomy anastomosis in adults. This is the first description of a standardized approach to robotic central pancreatectomy with Roux-en-Y pancreaticojejunostomy reconstruction in an adolescent (16-year-old boy) with a pseudopapillary tumor in the body of the pancreas. The operation time was 248 min with 20 mL of blood loss. The postoperative course was uneventful except for the short-term medical treatment for a grade B pancreatic fistula. Robotic central pancreatectomy can be safely applied in selected patients in experienced centers.

Robotic central pancreatectomy may be used in selected patients in experienced centers. This protocol presents all steps and the feasibility of a robotic central pancreatectomy with Roux-en-Y pancreaticojejunostomy in a 16-year-old adolescent patient.

Central pancreatectomy is a parenchyma-sparing alternative to distal pancreatectomy in patients with a benign or low-grade malignant tumor in the body of ...