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Technical Proposal |
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Purpose: This project is designed to evaluate the Bactobridge as a diagnostic tool in determining the chemosensitivity of various tumors in order to select specific chemotherapeutic agents. The reference test for the instrument will involve detecting the presence of microorganisms within 1-24 hours from food sources, which will impact on industrial microbiological analyses, a secondary application of the instrument. Results to be achieved: We expect to develop a processing and analysis methodology utilizing the Bactobridge to rapidly determine the chemosensitivity of various tumor types in individual patients. It will be shown that rapid analysis utilizing a closed system and the principles of conductance/impedance measurement can be achieved prior to the onset of the possibility of tumor cell mutation over time.
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1. |
The initial study will be an eight (8) week study; in which, twenty (20) non-neoplastic (normal) tissue/cell suspensions and twenty (20) neoplastic tissue/cell suspensions will be run on the Bactobridge to determine electrical impedance characteristics. Currently no such data exists. |
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2. |
For each sample cell suspension, electrical impedance characteristics will be determined at different concentrations and under two conditions; namely, viable, and killed with mitomycin-C. |
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3. |
This laboratory experimentation will be performed at the Univ. of Maryland School of Medicine, Allied Health Building, Department of Medical & Research Technology (DMRT) Laboratory Research Facility which is located at 100 Penn St.-Rm. 306, Baltimore, Maryland. |
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4. |
Tissue Procurement/ Storage/ Processing |
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A. |
Normals (Utilization of home site) Peripheral blood samples will be obtained from faculty members of the DMRT. Approximately 5-8 ml. of blood will be collected in one Purple-Top tube (EDTA), and centrifuged. Followed by collection of the buffy coat for preparation of cell suspension. |
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B. |
Neoplastic Tissue (Utilization/Analysis of remote site) Collection site Mercy Medical Center, 301 St. Paul Place, Department of Pathology- 2nd Fl. Baltimore, Maryland. At the pathology gross bench, a Pathologist will assess specimen adequacy. If after all tissue requirements for routine pathologic evaluation have been fulfilled and excess tumor is present, 1-2 grams of tumor tissue will be sampled from an area identified by the Pathologist as representative of the tumor. As a control, sampling from the same site will be submitted for permanent section/frozen section to ensure proper representation of the tumor. Sampling will be done under sterile conditions with tumor immediately placed in culture/transport media for immediate transport or temporary storage, the latter not to exceed 16-20 hours. |
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C. |
Preparation of Cell Suspension (Developing methodology) Upon receipt of the specimen at the DMRT Laboratory Research Facility, it will be assigned a unique identifier with all clinical information logged into the database. Processing will proceed under sterile conditions in producing a single cell suspension [for Procedure, see Appendix B]. Cell viability will be established by the trypan-blue exclusion method. Cell suspensions with known cell counts [washed in non-nutritive Hank’s balanced salt solution without phenol red and sodium bicarbonate-to ensure serum-free preparations eliminating possible spurious impedance changes]. |
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5. |
Testing (see Appendix B for Bactobridge description - Users Guide) (Bactobridge parameters per company specifications) |
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A. |
Initial phase will be performed on twenty normal/non-neoplastic and twenty neoplastic samples. |
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B. |
Two types of preparations from each sample will be run on the Bactobridge to evaluate performance characteristics of the cells and each Bactobridge- (1) viable cell suspension; and, (2) killed cell suspension with mitomycin-C. |
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C. |
Following optimization of the procedure for sample type, exposure of the cell suspensions to pre-determined anti-cancer drugs utilizing different concentrations will be performed. Note: specific drug(s) to be determined by tumor type. For a complete list of anti-cancer drugs, please see - Samples will be run as six matched pairs (see Table #1). Electrical impedance changes between each matched pair will be measured (See Table #1) |
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Table #1 |
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Pair 1 |
Pair 2 |
Pair 3 |
Pair 4 |
Pair 5 |
Pair 6 |
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Control Cell (C Cell) Run 1 - Viable cells Run 2 Killed cells |
Run 1 Run2 |
Run 1 Run2 |
Run 1 Run2 |
Run 1 Run2 |
Run 1 Run2 |
Run 1 Run2 |
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Reaction Cell (R Cell) |
Viable Cells |
Conc. 1 |
Conc. 2 |
Conc. 3 |
Conc. 4 |
Conc. 5 |
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D. |
Expected Results Run 1 in which C cell contains a viable cell suspension, changes in electrical impedance will indicate tumor cell chemosensitivity. Run 2 in which C cell contains a killed cell suspension, a lack of change in electrical impedance will indicate tumor cell chemosensitivity. |
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6. |
Comparative/Reference Methodology [ PHASE I] Various tumor types will be analyzed by the above methodology and procedures optimized per specific tumor type. The length of this phase will vary depending upon tumor amount, resultant viable cell count. This phase will also determine the appropriate panel of chemotherapeutic agents to be tested. Tissue collection site to include University of Maryland Baltimore Tissue Bank (UMH-NBW). Following optimization phase of study, all tumor tissue submitted for Bactobridge testing will also be simultaneously submitted for testing for cell culture drug resistance testing using a reference laboratory. Prospective clinical data for each patient will be followed during chemotherapy 6-12 months after to monitor for persistence/recurrence. |
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7. |
Trial Testing Phase [PHASE II] Following optimization per specific tumor type and with appropriate chemotherapeutic panels established, prospective trials would be initiated. Collection sites to expand to multiple institutions and to major laboratories. |
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Tumor cell evaluation will show that isolating the glycolytic pathway of metabolizing tumors within the closed system of the reaction cell can provide a convenient indicator of alterations in the cellular milieu (following exposure to chemotherapeutic agents); and, that the Bactobridge utilizing bridge technologies and principles of conductance/impedance will display resultant characteristic patterns correlating with chemosensitivity, in real time. We expect that each tumor type will produce subtle but specific conductance curves. We also expect that some tumor types may not be viable candidates for evaluation by the Bactobridge.
DURATION OF PHASE I Tumor material for processing is expected to be limited. The scope of testing for each individual sample will be defined by the viable cell count following processing. "Optimization" requires the identification of the requirements of each specific tumor type for media, incubator parameters, and sufficient data to have statistical predictability power. These factors are dependent on tumor biology specific for each tumor type. A period of ten months is considered an adequate period to accumulate the necessary number of test specimens needed. |
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Technology to Improve Food Safety Beta Testing/Clinical Trial Center Products Valuation of Bactobridge Technology Investment Information |
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The Department of Medical and Research Technology, |