to the nasopharynx and held the tube at that location. A 0.6 mm619 mm6305 mm flexible sterile suction catheter was placed through the nostril to the posterior nasopharynx. One to 2 ml of sterile saline was instilled through the catheter 
and the aspirate was immediately collected with gentle suction while simultaneously removing the catheter. The NPA was collected into a sterile container of 1.5 ml of viral transport medium . Specimens were Ki-8751 price stored at BMC for no longer than 72 hours at 4uC before deidentification, transfer and storage at the Klapperich Laboratory. Specimens received at the Klapperich Laboratory were immediately centrifuged at 5000 rpm for 5 min and stored as 0.5 ml aliquots at 280uC. They were not spun again before testing. A subset of the samples Disposable Molecular Diagnostic for Influenza A was tested using rapid immunoassays as part of the standard of care, by request of the treating physician. When a rapid test was performed, the remainder of the sample was sent to the Klapperich Laboratory for further testing. Both NPA and NPS samples were taken at BIDMC, though the vast majority of the specimens were swab samples. In all cases, specimens were taken from patients in the course of routine clinical care, for testing ordered by the patient’s clinician. NPS specimens were taken using two Copan flocked swabs. The first swab was inserted flat and pushed forward with gentle downward pressure on the lower nasal floor to the posterior wall of the nasopharynx, where it was rotated for a few seconds to collect cellular material. The swab was withdrawn and placed into sterile 1X PBS. The collection procedure was repeated using the second flocked swab in the other nostril; the second swab was placed into M4RT media for viral culture. The two swabs were then submitted on ice to the BIDMC microbiology laboratory. After routine direct fluorescent antigen and culture testing, tested/resulted specimens were stored at 280uC. In a minority of patients, NPA specimens were collected instead of NPS specimens and submitted on ice to the BIDMC clinical microbiology laboratory for testing, after which they were frozen at 280uC. The frozen NPS and NPA specimens were later deidentified and sent to the Klapperich Laboratory where they continued to be stored at 280uC. Before testing, all specimens were routinely quick thawed from 280uC in a 37uC water bath. Then the specimens were spun at 13,000 rpm for 10 min at 4uC to remove human cell debris and mucous, and the supernatant was used for all downstream testing. was then cross-linked by UV irradiation for 5 min as above through both sides of the chip, achieved by flipping the chip over half way through irradiation. Before use, chips were cleaned with 500 mL of 100% methanol to rinse away excess porogen and to create the open PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189542 pore structure of the SPE. The Microfluidic Assay The first chamber of the chip is an SPE column for sample preparation; it is integrated directly with the RT and PCR steps. Sample preparation followed the same strategy as previously reported. Briefly, before introducing the sample, the channel was conditioned with 300 mL of buffer containing 1.5 M guanidine thiocyanate , 50% 2Propanol, and 1X RNASecure. Next, 100 ml of the NPA or NPS specimen was mixed with 300 ml lysis buffer ). This mixture was run though the channel and collected from the first outlet port. During this step, nucleic acids released from the lysed influenza particles in the specimen bind to the SPE column
