English
SpanishSpecimen Processing Core for BSL-4 Research Projects
The Specimen Processing Core and its associated microbiology, molecular biology, chemistry and hematology laboratory equipment and capabilities are designed to support NEIDL Investigators engaged in the study of emerging infectious diseases including category A, B and C agents. The laboratory is designed to handle specimens obtained from animals housed in the Animal-BSL3 and Animal-BSL4 core facilities and cultures of BSL-3 and BSL-4 agents. Coordination with the Animal Cores (including Pathology/Necropsy) will be important so that specimens obtained can be appropriately processed immediately following collection and transportation to the laboratory in a safe and timely manner. This will include the use of digital scales to weigh tissue at the point of collection, the ability to use viral or other transport media such as transport media with chaotropic agents for subsequent nucleic acid extraction, the need to flash-freeze the specimen, to autoclave the specimen, or to treat it with gamma radiation prior to transportation out of the BSL-4 area.
Specimen and isolate tracking
In all instances barcode identification of specimens at the time of collection, accessioning, and when examined or manipulated will be used. In addition, digital video records of all work performed with samples or agents at BSL-4 will be maintained in order to enhance safety, prevent unauthorized activities, and to ensure that a chain-of-custody trail is adequately supported. The entire set of laboratory procedure manuals will be available in hard copy and on the information system.
Microbiology
The laboratory will be equipped for both classical, non-molecular diagnostic microbiology and for state-of-the-art molecular diagnostics that are designed to complement the research work and to serve, as needed, in the event of a national need for these methods. Classical microbiology requires the availability of a media preparation area, a tissue culture facility, the ability to perform both aerobic and anaerobic bacteriology, and those immunodiagnostic and other specialized tests that are needed (e.g., ELISA, fluorescent antibody testing, phage typing) to identify bacterial and viral agents and to characterize the host immune response to these agents. Identification of bacteria on the basis of whole-cell fatty acid profiles (i.e., the MIDI system) and on the basis of biochemical utilization of a broad range of substrates (i.e., BioLog) will be employed.
Molecular biology laboratory
This facility will use molecular methods to identify the presence of select agents in tissue, in cell culture, or in environmental specimens. In addition, the characterization of the agents will include the ability to perform nucleic acid sequencing. An automated nucleic acid extraction and purification system will be used to extract nucleic acid from experimentally infected animals and from cell cultures. For PCR-based assays, there will be a specimen preparation area (at BSL-4), and separate specimen preparation (at BSL-3) and amplification areas for molecular work. Whenever possible, the system will be used with uracil-n-glycosylase (UNG) in order to minimize the risk of contamination. In addition to standardize PCR-based assays, there will be real-time PCR instrumentation with a high throughput, such as the Applied Biosystems ABI Prism 7900 HT system, which uses 96-well or 384-well plastic plates. Sequencing of PCR amplification products corresponding to the 16S rRNA gene will assist in the identification of bacteria. Other uses of sequencing include studies of gene function in which site-directed mutagenesis is used to generate mutants, sequencing of genes that encode proteins that are vaccine candidates, sequencing genes that are potential virulence factors, and sequencing of actual agents used in a bioterrorism event. Plasmid DNA will be able to be isolated, both in order to support research studies and in order to fully characterize any bacterial etiologic agent of a bioterrorism incident. In designing the biological specimen processing and microbiology core facility we have incorporated the possibility that the laboratory will need to serve a “surge capacity” function in the event of a bioterrorism or emerging infectious disease emergency. As detailed in the Facility Plan and specimen flow diagram an entrance for delivery of specimens to this core has been incorporated. We have also included secure storage freezer and refrigerator space in the specimen processing anteroom where bar coding and video/photo documentation systems are located. The possibility that an engineered or chimeric agent has been used as an agent would have to be evaluated as rapidly as possible, and high throughput systems will be installed in order to meet these needs. Examples of situations in which this has to be considered as more than just a theoretical possibility are when there has been an expansion of the tissue or host range of a known pathogen (e.g., influenza), there is a new mode of spread of an agent, or an agent has been able to circumvent diagnosis, therapy, or vaccination (e.g., SARS). In these settings, there may have been engineering to identify new molecular targets, such as specific eukaryotic cell surface receptors or to alter specific targets in the pathogen.
Clinical Chemistry Laboratory
The Clinical Chemistry portion of the Specimen Processing Core will employ the Advia Integrated Modular System (IMS) for routine analysis of samples obtained from control and experimental groups of animals. This instrumentation is designed to utilize a single sample transport and processing system. The Advia IMS uses built-in robotics to transport samples individually from the loading area to the individual modular processing "engines". These analytical "engines" can perform either clinical chemistry plus homogeneous immunoassay testing, or heterogeneous immunoassay testing. Each module can accommodate up to 36 different reagent areas in individual reagent storage, as well as pipetting stations, incubation and measurement capabilities. Since each module functions independently, the ADVIA IMS is designed for customized configuration by selecting analytical modules as needed to meet the individual demands of NEIDL investigators. This system is also designed to offer on-site upgrade capability enabling addition, removal, or reconfiguration of modules, should laboratory requirements change over time. In the past, random access allowed individualized testing of samples across a broad menu of tests, on separate Clinical Chemistry or Immunoassay systems. With the Advia IMS capability, routine and special chemistries, therapeutic drugs, proteins, and homogeneous and heterogeneous immunoassays can all be mixed and matched on individual samples as required for individual experimental protocols.
Hematology
The hematology unit within the Specimen Processing Core will be responsible for the quantitation of white blood cell count, red blood cell count, hemoglobin, hemacrit, and platelet counts for all experimental animals enrolled in protocols at the NEIDL. When possible, targets utilized are derived from all-participant mean values as calculated by a robust statistical technique. All data will be reviewed prior to the establishment of acceptable limits and “peer group” specific targets will be used where appropriate. Analyte values (i.e., WBC count, RBC count, hemoglobin, hemacrit, and platlet counts) and acceptable limits will be established using criteria specified by CLIA 1988 regulations, allowing for rounding to appropriate significant digits. The hematology unit will also be responsible for determination of prothrombin time, partial prothrombin time, and quantitative fibrinogen levels. As described above, targets utilized are derived from all-participant mean values as calculated by a robust statistical technique, and acceptable limits are established using the criteria specified by CLIA 1988 regulations allowing for rounding to appropriate significant digits.
