Name: establishment of a clinic-based biorepository
Uploaded: 2017-05-29T14:00:00
Description: Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository Video at JoVE.com

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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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="/...

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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.

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Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice Video (Video) | JoVE

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts Video (Video) | JoVE

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy Video (Video) | JoVE

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Establishment and Characterization of UTI and CAUTI in a Mouse Model Video (Video) | JoVE

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis Video (Video) | JoVE

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits Video (Video) | JoVE

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct Video (Video) | JoVE

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Establishment and Evaluation of a Porcine Vein Graft Disease Model Video (Video) | JoVE

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging Video (Video) | JoVE

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...