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Identify two or more independent biomarkers?

Supplemental Testing

Animal studies Antibiotic susceptibility Electron microscopy Nucleic acid sequencing Mass spectroscopy

Identify two or more independent biomarkers?

FIGURE 11.1 Flow of specimens in analytical laboratory (from ref. 9, with permission).

Supplemental Testing

Animal studies Antibiotic susceptibility Electron microscopy Nucleic acid sequencing Mass spectroscopy

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FIGURE 11.1 Flow of specimens in analytical laboratory (from ref. 9, with permission).

preparation of test plans for incoming samples or sample sets. Such test plans help ensure compliance with the laboratory's operational strategy, taking into consideration the samples' unique properties, available quantities, analytical requirements, and confounding circumstances, while protecting forensic evidence. Without a test plan, excessive use of sample material may result in inadequate quantities for subsequent archiving, retesting, testing for other threat agents, or confirmation assays. The test plan should thus reflect calculations

Variable Sample Types Soil Swab Clinical

Miscellaneous matrices

Extraction into PBS+0.3% Tween-20

More Flexible SOPs

Molecular

Molecular

Animal injection

More Rigid SOPs

Immunological Electron

Culture Microscopy

Standardized Analytical Assays FIGURE 11.2 Analytical strategy using flexible and rigid SOPs.

to determine quantities needed to best meet test requirements and to determine whether the operational strategy should be modified due to limited quantities.

Development and implementation of a test plan encompass three principles: 1) State what actions will be performed, 2) perform the stated actions, and 3) document the actions as performed along with any deviations from the test plan. In laboratories where analyses of certain sample types have become routine, test plans may merely follow a series of well-established or validated SOPs. However, attempts to implement an operational strategy with such stringent SOPs can be a daunting task for the vast myriad of biological threat agents, types of matrices, assay inhibitors, stabilization requirements, variations in concentration, etc. Therefore, flexibility may be required in the initial phases of a test plan so as to allow modification of the operational strategy and SOPs to accommodate variations or deviations (Figure 11.2). For example, during the investigations after the Bacillus anthracis spore-contaminated mail incident in the fall of 2001, items were received for analysis including clothing, computer keyboards, various forms of garbage, etc., for which there were no validated SOPs. In order to accommodate such variations in sample matrix type, a strategy was adopted by a number of laboratories in which samples were processed so as to convert all sample forms into a common sample matrix (i.e., suspension or extraction in phosphate-buffered saline + 0.3% Tween 20). This was done by using more flexible SOPs; after samples were extracted into a common buffer, more rigid SOPs were then used in the analyses (Figure 11.2). The flexible SOPs used in the initial stages of analysis are usually permitted as long as their details are well documented in the sample analysis records, are well controlled where possible (i.e., internal extraction positive controls, laboratory quality system in place, etc.), and in some cases, are approved by the submitting agency. It is important that documentation of laboratory actions, including technician's initials, date, and descriptions of deviations from SOPs be performed at the time they occur, or as soon as possible, rather than hours or days later.4,5

The development of test plans to accommodate requirements for rapid initiation of processing can be accomplished by using work sheets which are designed for rapid entry of pertinent information, such as the nature of the sample, quantity, biological threat agent(s) to be assayed, how the sample is to be extracted, etc. In many cases, portions of the test plan can be formulated before the samples arrive at the laboratory, as samples often arrive with advance notice, prior knowledge of their history, and an understanding of the testing required.

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