This work was supported by funds from the Midwestern University College of Health Sciences Research Facilitation Grant, awarded to EEH, and Midwestern University Office of Research and Sponsored Programs Intramural Grant, awarded to KJL. Additional support was provided by Affiliated Laboratories and Affiliated Dermatology. We thank Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel, and Ali Zaidi for their technical assistance.

All experimentation on human tissue samples was approved by Western IRB (WIRB Protocol #20142461). The single criterion for the collection of tissues for these procedures is a biopsy-proven diagnosis of SCC. No exclusion criteria were outlined in the original IRB protocol, but samples from patients with a known blood-borne communicable disease were not used. Informed consent was obtained prior to the collection of cutaneous tissue, blood, and saliva. Midwestern Institutional Review Board approved the validation work with...

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The authors have nothing to disclose.

To the author&#39;s knowledge, this protocol is the first of its kind that focuses on the clinical procurement of cutaneous tissue samples in both a cost-effective and fast approach. Patients undergoing Mohs microsurgery are typically scheduled during specific blocks of time, and collection is limited to these periods. Sample collection involves effort from the medical assistant involved in patient care, the Mohs histotechnologist who processes the tumor samples, and a designated laboratory staff member who handles sampl...

Cancer and biomarker research relies on a supply of quality human tissue samples, and limited supply has hindered research1,2. Many dermatologic studies are limited by the inadequate supply, variable quality, and costs associated with the use of human tissue. The cost of establishing a large, dedicated biobank has been estimated to be approximately two million dollars3, and these costs place the use of human tissue out of the reach of many researchers. Furthermore, the process of generating and storing research samples poses the risk of affecting clinical operations and delaying patient care if not carefully executed. A cost-effective, clinic-based biorepository has been established that focuses on skin cancer samples following recommended best practices and sample validation4,5,6.
This protocol has been developed in a dermatology clinic that performs a large volume of Mohs micrographic surgeries to remove squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma skin cancers. Volunteer donors can be recruited from this patient population. It is important to establish the biorepository at the site of collection to rapidly capture tissue and blood from consented patients without delaying treatment. Gathering samples from the same clinic minimizes variations in collection techniques and minimizes variations in the quality of samples, which can be problematic for downstream applications7,8.
The goal of the Mohs micrographic surgery technique is to ensure that all cancer tissue is removed while preserving as much healthy tissue as possible. The procedure involves the progressive removal of thin layers of tumor tissue. Each successive layer is histologically examined (after cryosectioning the tumor tissue and performing H&#38;E staining) by a dermatologist to determine if all cancer tissue has been removed. The excision and examination of subsequent layers of tissues is executed while the patient remains in the office. This technique is considered the best treatment option for SCC9. At this point, the wound is closed and, to improve healing and cosmetic appearance, adjacent normal tissue (ANT) is frequently excised. Thus, this surgical procedure to remove a tumor is ideally suited for collecting histologically characterized tissue for future studies.
The procurement procedure for obtaining tumor tissue, adjacent normal tissue, saliva, and blood samples has been designed to have minimal impact on normal staff duties (Figure 1). Medical assistants perform the blood draws while preparing the patient for the procedure. After completion of the Mohs procedure, the Mohs histotechnologist prepares additional histological slides of the specimen and transfers the tissue to the biorepository. Costs associated with establishing the biorepository include the purchase of cryopreservation freezers, the creation of modest clinical laboratory space, and the development of an inventory tracking program.
<img alt="Figure 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> 
Figure 1: Sequence of sample collection and responsible staff. Upon patient check-in and the attainment of patient consent, the medical assistant collects a buccal swab and performs a blood draw. The dermatologist and medical assistant then excise the tumor and close the wound, during which time SCC and ANT specimens are collected, respectively. A dedicated laboratory technician processes the blood and sections the SCC and ANT specimens for tissue culture, preservation, and entry into the biorepository. <a href="http://cloudflare.jove.com/files/ftp_upload/55583/55583fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The diversity of samples collected enables a variety of experimental approaches (Figure 2). Samples collected from the patient are buccal swabs (saliva can also be collected if needed), whole blood, and excised tissue. The buccal swabs and a sample from the whole blood are saved, without processing, for genotyping and tissue matching. Whole blood is separated into white blood cell (WBC) and plasma fractions for future analyses. After Mohs processing, the frozen tumor is placed directly into liquid nitrogen and transferred to a -80 &#176;C freezer. Fresh, viable tumor tissue and ANT samples are cultured using modifications of previous techniques10,11 and then cryopreserved. During collection, the number of each sample type is recorded on a spreadsheet prior to entry into the inventory tracking program to facilitate accurate processing (Table 1).
<img alt="Figure 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> 
Figure 2: Outline of clinic-based biorepository sample collection and processing. A buccal swab and blood sample are collected from the patient and stored for downstream genotyping and tissue matching. Whole blood is further processed for white blood cell (WBC) isolation and CTC analysis, as well as for plasma collection and liquid biopsy analysis. Tissue excised during the Mohs procedure is histologically processed for diagnostic purposes, after which the histologic slides can be used experimentally for further immunohistochemical analyses. Provided that the excised tissue sample is large enough, a portion of fresh tissue is removed and sectioned for protein and RNA isolation, and for the establishment of cultured cell lines. <a href="http://cloudflare.jove.com/files/ftp_upload/55583/55583fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collection Date:</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Patient 1</td>
			<td>Patient 2</td>
			<td>Patient 3</td>
			<td>Patient 4</td>
			<td>Patient 5</td>
		</tr>
		<tr>
			<td>Initials and Birth Date</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Color</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Type</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Location</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saliva</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whole Blood</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor Viable</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Normal Viable</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor Mohs Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Normal Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Checklist to record sample collections. Data tracked and recorded with each sample collected include patient initials, birth date, and eye color (for skin typing), as well as the location of specimen removal. The number of saliva samples, blood collection volumes, and the number of viable and preserved tissue specimens collected are also recorded as references for allocations to later uses. <a href="http://cloudflare.jove.com/files/ftp_upload/55583/Table1.xlsx">Please click here to download this file.</a>
To validate sample collection procedures, each sample type has been tested in downstream applications. Using modifications of previous techniques12, tumor and ANT have been successfully used in protein and RNA isolation and can potentially be used for DNA isolation. Viable explants established from the tissue sections have been evaluated by microscopy, while stored histological slides have been used for immunohistochemistry and immunofluorescence.
By following the protocol described here, it is possible to extend this model to other dermatology clinics, other tumor types (such as melanoma), and other surgical specialties and practices to provide human tissue samples for multifaceted research into human cancers. Slight modifications of this protocol are likely to be necessary for other practices but, in principle, this protocol is applicable to any surgical practice that routinely discards patient samples gathered in the course of patient treatment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza Walkersville KGM Keratinocyte Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9791321</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1950 Cryostat</td>
			<td>Leica Biosystems</td>
			<td>14047743909</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Rapid-Chrome H&#38;E Frozen Section Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>99-900-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>50-195-822</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>05-401-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>12-141-3</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CFH monoclonal antibody clone OX-24</td>
			<td>Abnova</td>
			<td>MAB12583</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p40 (p63 delta) antibody</td>
			<td>Abnova</td>
			<td>ABX-144A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

Tumor and ANT have been used successfully in protein isolation and tested by Western blotting. As shown in Figure 3, cutaneous squamous cell carcinoma markers (Muc114, FHL-115, and p4016,17) can be detected in patient samples collected and stored in our clinic-based biorepository.
<img alt="Figure 3" src="/...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

Cutaneous tumors are often discarded following Mohs micrographic surgery. A protocol is described here that enables clinical support staff to effectively process and store cutaneous tumor (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) samples for downstream laboratory applications without interfering with clinical operations.

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is that excised tissues that are normally discarded and destroyed are instead collected and preserved for research, using procedures, which minimally impact routine clinical operations.The variety of samples collected enables correlations between protein and RNA biomarkers in the tissue and plasma samples with patient outcome data. In principle, this protocol, once slightly modified is applicable to any surgical practice, that routinely discards patient samples, gathered in the course of patient treatment. Demonstrating the procedure will be Stefani Fawks, a Mohs histotechnician, and Dr.Mitchell Manway, a research fellow in our practice.To begin, use the tissue received from the Mohs surgeon and with a scalpel, excise a 10 to 50 milligram sample from the center of the apical side of the tissue. With tweezers, transfer the viable tissue to a labeled 1.5 milliliter tube, prefilled with culture medium. Make sure the tissue is entirely immersed in the medium.Place the sample in a refrigerator and store it at four degrees Celsius, for no longer than 24 hours before further processing. Harvest at least a 10 cubic millimeter tissue fragment from the apical side of the excised tissue, when the tumor sample is large enough, and such removal will not interfere with routine Mohs processing. Next, place the remaining unprocessed tumor sample on a cryostat tissue holder.Embed the tissue in an optimal cutting temperature compound, and freeze it within the holder to prepare histological slides. Using a cryostat, cut the tissue into seven micron thick sections, and layer up to three sections over a slide. After sectioning, fix the tissue by immersing the slides in acetone for one to two seconds.Then, allow the slides to air dry. Keeping the remaining sample in a cryostat chamber, pry the tissue from the cryo embedding medium, and transfer it to a labeled cryogenic tube. Immediately transfer the labeled vial into liquid nitrogen.Transfer of the tissue to a cryo vial after Mohs processing must be performed in the negative 20 degrees Celsius cryostat chamber. If performed properly and the tissue is kept frozen, this step will enable the successful extraction of RNA from post-Mohs tissue samples. Next, obtain adjacent normal tissue from the Mohs surgeon.Excise a sample from the apical side of the adjacent normal tissue. Then, using tweezers, transfer the excised sample to a labeled 1.5 milliliter tube filled with culture medium. Immerse the tissue in the same culture medium.Place it into an appropriate sample bag, and store at four degrees Celsius, until subsequent processing. Collect approximately 10 milliliters of venous blood into EDTA-coated collection tubes. Combine the blood samples in a sterile 15 milliliter centrifuge tube.Then, transfer approximately one milliliter of the blood sample into a 1.5 milliliter cryogenic tube, avoiding bubbles. Store this whole blood sample at minus 80 degrees Celsius, until subsequent genotyping and tissue matching. Next, centrifuge the remaining blood sample at 1000 times G for 30 minutes to obtain blood plasma.After spinning, collect and divide the plasma until one milliliter aliquots in 1.5 milliliter cryogenic tubes. And store the samples at minus 80 degrees Celsius for use during future liquid biopsy analyses. Enter the data from the checklist of patient samples, along with patient information and diagnosis, subtracted from the patient chart to the biorepository database.Finally, record the localization of the de-identified samples in the biorepository database, and move the samples to the research lab. Immediately after, transfer the tissue samples into 1.5 milliliter tubes, prefilled with appropriate extraction solutions, making sure the tissue is completely immersed. Store the samples at minus 80 degrees Celsius until further processing.Presented here are the images of representative Western blots of known squamous cell carcinoma markers, mucin one, FHL-one, and p40, obtained for adjacent normal tissue and a tumor sample. The quality of the RNA isolated from Mohs samples was first assessed using gel electrophoresis, followed by RNA integrity analysis. RNA integrity number values achieved for normal and tumor samples indicate that high quality and intact RNA was obtained.Cytometry profiles of the bands were plotted as fluorescence units versus nucleotide number for representative RNA samples from tumor and normal patient tissue. A squamous cell carcinoma tissue explant was imaged using phase contrast microscopy and shown to be a mixed keratinocyte and fibroblast culture, that might be subcultured or used for future cell line development. Finally, in the squamous cell carcinoma tissue sections, positive staining for the epithelial to mesenchymal transition marker was shown.Once mastered, this protocol will extend the standard work flow of the clinic staff to less than 30 minutes, if it is performed properly. The majority of the steps are performed by the existing clinical staff after patient care is completed. While attempting this protocol, it is important to maintain accurate record keeping, linking the samples with the patient's history and diagnosis.As this information is often needed for downstream applications. This technique will help pave the way for researchers in the field of dermatology for the development of liquid biopsies and biomarkers in squamous cell carcinomas and melanoma. After watching this video, you should have a good understanding of how to implement a clinic-based biorepository for cutaneous cancer samples, generated as part of daily practice.

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6.4K Görüntüleme.  Midwestern University. The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is...