The content of this article does not necessarily reflect the views or policies of the US Department of Health and Human Services or of the institutions affiliated with the authors. This research was supported by Paul G. Allen Family Foundation &#34;Enhanced Zero-Impact, Emergency Smart Pod&#34;. We deeply appreciate all the fruitful discussions and collaboration with the colleagues from Baylor College of Medicine, GSS Health, NASA&#39;s Johnson Space Center. We are sincerely thankful to Thermo Fisher Scientifics and its representatives for a loan of the RT-PCR machine, centrifuge, and automated pipettes to carry out a respiratory virus diagnostic test in the remote laboratory facility. The authors are thankful to Marta Storl-Desmond and Sidney Stephen Sorrell for their assistance in the manuscript preparation and videography.

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+real-time+RT-PCR+assay+for+the+detection+of+Ebola+virus+(Zaire)+during+an+Ebola+outbreak+in+Guinea+in+2014-2015.">Development and evaluation of a real-time RT-PCR assay for the detection of Ebola virus (Zaire) during an Ebola outbreak in Guinea in 2014-2015.</a> Journal of Virological Methods. 228, 26-30 (2016).</li><li>Cnops, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developement,+integration+of+a+quantitative+reverse-transcription+polymerase+chain+reaction+diagnostic+test+for+Ebola+virus+on+a+molecular+diagnostics+platform.">Developement, integration of a quantitative reverse-transcription polymerase chain reaction diagnostic test for Ebola virus on a molecular diagnostics platform.</a> Journal of Infectious Diseases. 214 (3), S192 (2016).</li><li>Keitel, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+research+response+to+the+2009+A(H1N1)pdm09+influenza+pandemic+(Revised).">Rapid research response to the 2009 A(H1N1)pdm09 influenza pandemic (Revised).</a> BMC Research Notes. 6, 177 (2013).</li><li>Parsons, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+diagnosis+of+tuberculosis+in+resource-poor+countries:+challenges+and+opportunities.">Laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities.</a> Clinical Microbiology Reviews. 24 (6), 314-350 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Expandable Bicon Shelter. Commercial Manual. Sea Box. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Safety.+Operating+the+Glovebox.">Lab Safety. Operating the Glovebox.</a> JoVE Science Education Database. , (2018).</li><li>World Health Organization (WHO). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Collecting, preserving and shipping specimens for the diagnosis of avian influenza A(H5N1) virus infection. , (2006).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li></ol>

Baylor College of Medicine holds a U.S. provisional patent application for Mobile Clinics (U.S. Patent Application No. 15/523,126, # 620078924). The authors declare that they have no competing financial interest.

The remote laboratory facility described above is logistically-oriented, expandable, rapidly deployable, multifunctional, and based on human-centered design concepts that have been geared to protect laboratory personnel and workspace efficiency. The detailed protocol for quick laboratory set-up and safe respiratory virus isolation and diagnosis was developed and presented.
For optimal equipment functioning, the following conditions must be maintained in the laboratory units: ambient temperature of 21 &#177; 2 &#176;C, permissible temperature of 5 to 40 &#176;C, humidity of 14 &#177; 5% RH, permissible maximum relative humidity of 80% RH (noncondensing), and an altitude between 0 and 2,000 m above sea level.
Energy consumption is one of the most important parameters for management of an off-grid laboratory. For core laboratory equipment, the power efficiency can differ 15-40%; however, average energy consumption is estimated here to deliver an appropriate service. The highest power rate (1,500-2,000 W) relates to the air conditioner, the glovebox system, the PCR machine, and the autoclave sterilizer. Considering 8 hours of intensive work carrying out the protocol and 16 hours of the laboratory environment control, the daily energy consumption of laboratory units is approximately 36 kWh/day for BSL-2, about 43 kWh/day for BSL-3, and 73 kWh/day for the connected BSL-2/BSL-3+ facilities. For a single unit, we recommend providing a source of electrical power with capacity of running/continuous power &#8805;8 kW, surge/starting power &#8805;10 kW; for the connected facility, running/continuous power &#8805;12 kW, and surge/starting power &#8805;14 kW. Note, in the BSL-3 laboratory facility, a backup energy source is strongly recommended to avoid accidental power outage and guarantee steady work of the glove box and negative pressure system during a diagnostic test.
A gasoline powered electric generator is a cost-effective solution for emergency energy supply. Assume that fuel efficiency of a gasoline generator is approximately 1.5 gallons per hour at 100% load. Then, if the average daily energy consumption is 8 hours of 40% load and 16 hours of 10% load, the laboratory unit BSL-2 or BSL-3 requires 7-9 gallons of fuel per day, correspondingly, and the connected facility needs ~15 gal/day.
The remote laboratory units are designed to fit capabilities of off-grid solar panel systems. Solar panels do not require additional fuel and can be operated with high productivity in the tropical and subtropical regions of Africa, Asia and Latin America due to the high solar irradiation. Currently, one unit of a commercially available solar panel system allows a daily power usage of up to 44 kWh/day.
Regardless of the selected type of alternative electrical energy source, dirty electricity filters are strongly recommended and preinstalled in the laboratory facilities to improve power quality and to protect laboratory equipment. Keep the PCR system away from sources of strong and unshielded electromagnetic radiation because strong electromagnetic radiation may interfere with the proper operation of the device. It is also important do not use the PCR system near strong vibration sources, such as a centrifuge or pump because excessive vibration will affect instrument performance. The laboratory equipment may only be installed in an environment that has nonconductive pollutants, such as dust particles or wood chips. Ensure the room is away from any vents that could expel particulate material onto the instrument components.
The laboratory water usage depends on number of diagnostic tests running daily and number of laboratory technicians working in the facility. Nuclease free water is required for preparation of mixers during diagnostic procedure including extraction and PCR test and must be delivered in advance as other supplies and chemicals. At least 50 mL of nuclease free water is needed to run one diagnostic test; the required volume of nuclease free water depends on work load (i.e., on number of samples). Distilled water is needed to run the autoclave sterilizer. Autoclave water consumption in one cycle is 160-180 mL; the autoclave is recommended for daily use. Most of the plastics (tubes, pipette tips, etc.) are disposable, but some are re-usable and need to be washed (large containers, racks, etc.). Regular running water is used for washing hands between procedures and its minimal volume is estimated to be 15-20 L daily. The water needs to be pumped for pressure; sediment pre-filter system is recommended to protect the water appliances from the damaging effect of sediment and to improve the quality of running water.
For cold storage, at least one 5.1 cubic feet refrigerator (+4 &#176;C) and one 4.9 cubic feet (-20 &#176;C to -30 &#176;C) freezer are required in each laboratory unit to store samples/ RNA.
Laboratory decontamination includes several levels: cleaning &#62; antisepsis &#62; disinfection &#62; sterilization. Simple cleaning can be performed using soap and water while scrubbing with a gloved hand or brush. Antisepsis includes washing with liquid antimicrobial chemical in order to inhibit the growth and multiplication of germs. Alcohol solutions (70%) can be used as an antiseptic liquid. Disinfection is the application of a liquid chemical to eliminate nearly all pathogenic microorganisms (except bacterial spores) on work surfaces and equipment. Chemical exposure time, temperature, and concentration of disinfectant are important. Sodium hypochlorite solution (0.5%), or bleach, is an effective disinfectant on a large scale for surface purification and water purification. Ultraviolet germicidal irradiation is another method of disinfection. A germicidal lamp produces UV-C light and leads to the inactivation of bacteria and viruses. Sterilization employs a physical or chemical procedure to destroy all microbial life -- including highly resistant bacterial spores. Sterilization can be performed with an autoclave sterilizer.
All laboratory waste must be segregated at the point of generation. Place solid, non-sharp, infectious waste in leak-proof waste bags marked as biohazard. If generated waste is sharp, it must be placed in puncture-resistant containers. Collect potentially infectious liquid waste in properly labeled biohazard containers for liquids. Containers and bags should not be filled more than 2/3 the volume. The disposal of all bleach products must be sorted into their own designated waste bin. Laboratory waste must be handled gently to avoid generating aerosols and breakage of bags/containers. Collection bags/bins with biohazard waste must be sealed and external surfaces decontaminated after use with 0.5% sodium hypochlorite solution. Sterilize all laboratory waste in autoclave at 121 &#176;C for 30 minutes prior to incineration. Refer to functioning manual for the proper use of an autoclave. If possible, add a chemical or biological indicator to the autoclave to ensure proper sterilization. All autoclaved solid and liquid waste must be clearly labeled as sterilized with the setting, date, time, and operator. The labeled waste must then be placed in a secure, separate area prior to incineration.
As expected, workflow of diagnostic test depends on the disease and specimen. If it is recommended for virus identification to collect blood samples (e.g., Ebola19), sample aliquots can be stored at -20 &#176;C instead of -80 &#176;C (necessary for respiratory viruses). It is always better to take more than one specimen when sampling from a patient than to subdivide specimens later. If possible, for each type of specimen at least two specimens must be taken in separate specimen tubes. Specimens must be sub-divided if additional sampling is not possible.
If alternative specimens cannot be stored at appropriate temperatures (e.g., no freezers are available), swabs should be stored in pure (100%) ethanol or 99% methylated spirit (methanol additives only). In this case, the swab tip must be put into a vial with 1-2 mL of ethanol. Note that such specimens are suitable only for PCR. Also, note that a well-established assay is necessary for each particular virus diagnosis8,23, and unknown virus samples must be sent to assigned laboratories for further investigations19,20,21.
Mandatory and recommended requirements to the list of laboratory equipment for respiratory virus diagnostic PCR tests must be recognized. Table 3 underscores basic and minimally advanced (recommended) equipment and requirements for the RT-PCR diagnostic test. For BSL-3 practice, extra negative pressure protection (e.g., glove box) of personnel is crucial and necessary.
The connected laboratory modules are preferable to increase the number of personnel involved in laboratory testing and speed up the time required for a single test. Replacing the time-consuming manual RNA extraction is possible with automated qPCR (e.g., QiaCube). While this instrument is cumbersome (width 65 cm, length 62 cm, height 86 cm), it can fit the mobile laboratory workspace after rearrangement of furniture in BSL-2 or BSL-3 units.
Future work will be focused on the development of augmented reality (AR) and virtual reality (VR) trainings. The AR/VR glasses will be used to provide an interactive platform to teach requisite skills needed to become a well-trained laboratory worker. Helpful tips to perform some of the difficult, multistep procedures in laboratory diagnostic tests will be included in the software guide. This approach to personnel training should improve the quality of diagnostic test performance and management in remote laboratory facilities, especially remote and resource constrained areas.

Rapid diagnostics is a critical instrument in timely viral infection control, especially if early symptomatology is indistinguishable to a variety of infection diseases. The recent Ebola outbreak (2014-2015) in West Africa1,2, Zika virus epidemics (2015-2016) in Asia and Latin America3,4, the emergence of the Middle East Respiratory Syndrome (MERS) coronavirus infections5,6, and the unusually deadly flu (influenza) epidemics (2017-2018) in the U.S.7,8 uncovered the need for rapidly-deployable, laboratory facilities that address a multitude of issues from transportation, access, facilities, equipment, and communication.
Off-grid capability (autonomous power and water supply, etc.) is crucial in rural, resource-constrained global settings9,10,11. Our experience in clinical operations and global programs at Baylor College of Medicine was used to design and build a container-based mobile laboratory with capabilities for easy deployment, set-up and multifunctional usage (BSL-2 and BSL-3). Images of this versatile, logistically-enhanced laboratory facility is shown in Figure 1.
This rapidly-deployable, laboratory facility has an expandable design similar to the previously described container clinic (The &#39;Emergency Smart Pod&#39;)12,13,14, developed by Baylor College of Medicine and sponsored by USAID. A single packed unit (in Transport Mode) has the dimensions of 9 feet 9 inches x 8 feet x 8 feet (Figure 1A, B), and expands to an area of 170 square feet (15.75 m2) (Figure 1C, D). The unit can be deployed by two to four people in less than ten minutes.
The remote laboratory is built for a BSL-2 lab facility (Figure 2A) with a separate, modular, attachable, BSL-3 unit (Figure 2B) designed for work with infectious agents that may cause serious or potentially lethal disease through inhalation15. The connectivity of the two laboratory modules enables optimization of experimentation workflows, sharing of resources, and cost savings (Figure 2C-E).
The modules are air-tight and water-tight to create a comfortable, energy efficient mobile shelter. Heating, ventilation, and air conditioning (HVAC) system is used for centralized and temperature-controlled units. In general, the design of the laboratory units minimizes power consumption by usage of their own alternate power sources such as solar panels and/or an independent electrical generator. Each unit includes a sink and eyewash station, electrical power and water connectors (Figure 3A-C). The ICT platform delivers an optional, tablet-based (Android phone/Tablet or iPhone/iPad) documentation app for supply tracking and laboratory result documentation (Figure 3D) developed in partnership with Baylor&#39;s Information Technology (IT) research group that is well-experienced in working in remote environments with limited connectivity. The system can function using cellular or wireless signals, and allows documentation without connectivity, with immediate backup or transmission to a secure-cloud based server when connectivity is re-established.
The laboratory has several key infection-control features including: (a)&#160;negative pressure air flow, (b) a glove box or biosafety cabinet, (c) a health risk management system: a germicidal ultraviolet (UV-C) lighting system using 4 hierarchies of defense proven to eliminate 99.7% of pathogens that cause healthcare-related infections. The facility is easily disinfected using hydrogen peroxide or sodium hypochlorite (bleach) systems for efficient and effective decontamination.16
The assurance of quality laboratory results depends on a commitment to assess all aspects of the entirety diagnostic testing process. Here, we present a checklist for the BSL-2 and BSL-3 laboratory workflow, and a protocol for rapid respiratory virus diagnostic test. The proposed diagnosis of viral diseases relies on the detection of viral RNA or DNA in specimen (nasal wash, blood, stool, and urine, etc.) through real-time reverse-transcription polymerase chain reaction (RT-PCR). The ability to rapidly estimate viral loads in a specimen makes PCR an efficient tool for viral disease screening17,18. The implementation of novel, molecular diagnostic assays allows expansion of diagnostic capabilities for viruses such as Ebola19,20,21, influenza8,22, and tuberculosis (TB)23.
The goal of this work is to validate a novel modular and rapidly-deployable laboratory facility and provide a training guide for laboratory personnel working in remote, low-resource environments during an epidemic, natural disaster or other emergency relief situation. Here, we present a protocol for respiratory influenza diagnosis in this innovative, portable laboratory.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclave Sterilizer &#39;BioClave&#39;</td>
			<td>Benchmark Scientific, Edison, NJ, USA</td>
			<td>B4000-16</td>
			<td>16 liter, Benchtop, Dims: 22x17.5x15.7 in, Fully automatic, Extremely Compact</td>
		</tr>
		<tr>
			<td>Barcode Scanner</td>
			<td>Zebra Technologies ZIH Corp., Lincolnshire, IL, USA</td>
			<td>Symbol LS2208</td>
			<td>Handheld, lightweight</td>
		</tr>
		<tr>
			<td>Breaker Box Panelboard Enclosure</td>
			<td>Square D (Schneider Electric), France&#160;</td>
			<td>MH62WP&#160;</td>
			<td>NEMA 3R/5/12, Dims: 20 W x 62 H x 6-1/2 in. D, Electrical distribution board</td>
		</tr>
		<tr>
			<td>Centrifuge - Microcentrifuge 17,000 x g</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>75002440</td>
			<td>Holds 24 x1.5 or 2 ml tubes, Dims: 8.9x9.6x13.8 in</td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>NuAire, Inc., Plymouth, MN, USA</td>
			<td>NU-602-400</td>
			<td>4 Ft. Class II Type A2 Cage Changing Biological Safety Cabinet, 12&#34; Access Opening, HEPEX Pressure Duct&#160;</td>
		</tr>
		<tr>
			<td>Class III Biological Safety Cabinet (Glove box)</td>
			<td>Germfree Laboratories, Ormond Beach, FL, USA</td>
			<td>Model #PGB-36, Serial #C-2937</td>
			<td>Glove box, Portable, 36&#34;, Class III BSC. Dims: 36x20x23.75 in, Includes 2 interior outlets</td>
		</tr>
		<tr>
			<td>Cryo Coolers</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>414004-286</td>
			<td>0.5 or 1.5 ml tube benchtop coolers</td>
		</tr>
		<tr>
			<td>Freezer (30&#176;C freezer)</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>Model ULT430A</td>
			<td>To occupy 4.9 Cubic feet</td>
		</tr>
		<tr>
			<td>Laminar Flow Cabinet</td>
			<td>NuAire, Inc., Plymouth, MN, USA</td>
			<td>NU-126-300</td>
			<td>3 Ft. Vertical Laminar Airflow Cabinet, 8&#34; Access Opening, HEPA filter supply, 99.99%</td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>75004061</td>
			<td>Dims: 4.1x5.0x6.0 in</td>
		</tr>
		<tr>
			<td>Pipettes automated</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>05-403-151</td>
			<td>Pipet 4-pack (2.5,10, 100 and 1,000&#956;L volume)</td>
		</tr>
		<tr>
			<td>Pipettes automated &#39;Finnpipette&#39;</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>4700880</td>
			<td>Pipet 4-pack (2, 20, 200 and 1,000&#956;L volume), Advanced Volume Gearing(AVG), Ultra durable</td>
		</tr>
		<tr>
			<td>Power Generator</td>
			<td>Cummins Power Generation, Minneapolis, MN, USA</td>
			<td>C60 D6</td>
			<td>60 kW, 60 Hz, 1 Phase, 120/240V, Diesel</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>BioMedical Solutions, Inc., Stafford, TX, USA</td>
			<td>BSI-HC-UCFS-0504W</td>
			<td>Standard Undercounter Refrigerators &#38; Freezers</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>05LRAETSA&#160;</td>
			<td>To occupy&#160; 5.1 Cubic feet</td>
		</tr>
		<tr>
			<td>RT-PCR machine &#39;Step-one plus&#39;</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>4376598</td>
			<td>Holds 96 samples, Dims: 9.7x16.8x20.2 in&#160;</td>
		</tr>
		<tr>
			<td>Vortex Mix</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>88880017TS</td>
			<td>Dims: 6.1x8.3x3.3 in</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AgPath-ID One-Step RT-PCR Reagents</td>
			<td>Applied Biosystems, Foster City, CA, USA</td>
			<td>4387391</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol Koptec Pure 200 Proof</td>
			<td>Decon Labs, Inc., King of Prussia, PA, USA</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>Ambion, Inc., Carlsbad, CA, USA</td>
			<td>AM9906</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>52906</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Platinum One-Step qRT- PCR Kit</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>11732-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL cryogenic tubes</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>03-337-7X&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>10025-726</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L Filter Tips</td>
			<td>Neptune, VWR, Radnor, PA, USA</td>
			<td>Neptune, BT10XLS3</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L Filter Tips</td>
			<td>Multimax, BioExpress, VWR, Radnor, PA, USA</td>
			<td>MultiMax, P-3243-30X</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Filter Tips</td>
			<td>ART, Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>ART, 2770</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Filter Tips</td>
			<td>Phenix Research Products, Candler, NC, USA</td>
			<td>TS-059BR</td>
			<td></td>
		</tr>
		<tr>
			<td>AB custom probes</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>N/A</td>
			<td>Custom probes</td>
		</tr>
		<tr>
			<td>Combitips</td>
			<td>Eppendorf, Hauppauge, NY, USA</td>
			<td>89232-972</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrated DNA Technology (IDT) custom probes and primer</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Custom probes</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>490003-978 CS</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast Reaction Tubes (8 tubes/strip)</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>4358293</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical 8-Cap Strip</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>4323032</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific, Carlsbad, CA, USA</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biohazard waste bags</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>14220-046</td>
			<td>20.3 x 30.5 cm Biohazard bags</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Denville Scientific, Holliston, MA, USA</td>
			<td>G4162-250</td>
			<td>Small, meduim or large Nitrile or latex gloves</td>
		</tr>
		<tr>
			<td>Lab coat</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Customizable</td>
		</tr>
		<tr>
			<td>Masks</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>414004-663</td>
			<td>Advanced protection mask</td>
		</tr>
		<tr>
			<td>Protective shoes</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Customizable</td>
		</tr>
	</tbody>
</table>

remote laboratory management: respiratory virus diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

The goal of this study is to demonstrate that the proposed BSL-2 and BSL-3 mobile laboratory facilities provide an adequate environment allowing respiratory virus diagnostic tests with representative results identical to tests performed in high-quality stationary laboratories. The laboratory facilities are designed to comply with the test requirements given in Occupational Health and Safety (OHS) recommendations. As soon as the remote laboratory facility is deployed (Figure 4) and all equipment and supplies are installed (Figure 5), laboratory tests can be run.
In accordance with laboratory standard operating procedures, PPE (lab coats, protective shoes, gloves, advanced mask, protective eyewear, etc.) appropriate for BSL-2 practice is required. For BSL-3 practice, the PCR laboratory module of negative pressure is equipped with a certified glove box. The laboratory units are upgraded by external pass-through windows to protect personnel at the step of sample receiving. The registration process can be simplified with previously developed tablet-based application (Figure 3D). Other acceptable applications that run on a laptop can be used as well.
This particular respiratory virus diagnostic test can be performed in the connected laboratory modules to separate steps of the diagnostic procedure on purpose to avoid contamination or potential interference between biochemical reagents, which may affect the testing results. To maximize the quality of diagnosis, the rapid diagnostic test practice utilizes (i) both the basic laboratory BSL-2 and the traverse connected PCR room (Section 3) or (ii) the GB and PCR rooms connected by pass-through window (Section 4). The diagram of the proposed laboratory workflow is presented on Figure 6 and emphasizes personal protection. The diagram recognizes the importance of each indicated step for personnel protection, especially if laboratory staff in remote areas is minimally trained.
The rapid diagnostic test of influenza is accomplished via the RT-PCR technique. The procedure contains four main steps. Note that individual workspaces are assigned for each stage of the protocol.
The first step is to obtain a sample and sub-divide it into several aliquots. The aliquots can then be marked with barcodes to improve effectiveness of data control and stored in the freezer for further investigations. The second step is to inactivate a sample in lysis buffer by centrifuging and heating. The first and second steps must be carried out in biosafety cabinets. Utilize individual pipette sets and equipment. A PCR test is proposed to be performed in the PCR room, if available. The third step is the documentation of results. Step four is the maintenance after the equipment usage, and reminder of personnel protection at the end of experiment.
If the specimen is expected to be classified as BSL-3+ (e.g.,Ebola, Zika, MERS, TB) the glove box facility must be used. In the remote laboratory, the GB room has its own pass-through window to receive specimens and a laptop or tablet for sample registration. The sample aliquot and virus inactivation must all be performed in the glove box chamber. UV-C lighting is recommended to avoid contamination during procedure. After inactivation of a sample, further steps for protocol are similar to the basic laboratory BSL-2 and BSL-3 test and follows Checklist Part III (Table 1, Figure 6). 
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59188/59188fig1.jpg" /> 
Figure 1.&#160;Laboratory facility prototype. (A, B) Transport mode; (C) Deployed mode: outside; (D) Deployed mode: interior. <a href="https://www.jove.com/files/ftp_upload/59188/59188fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59188/59188fig2.jpg" /> 
Figure 2.&#160;Schematics. (A) The basic laboratory BSL-2; (B) The BSL-3 module includes the glove box and PCR laboratories, which have a common pass-through window for protected specimen transfer; (C) Connected laboratory facilities (A) and (B) with shared utilities. (D,E) Photographs of the connected units from opposite sides. <a href="https://www.jove.com/files/ftp_upload/59188/59188fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59188/59188fig3.jpg" /> 
Figure 3. (A) Interior of the BSL-3 facility has (1) a pass-through window, a sink and (2) an eyewash station at the inlet; (B) Electrical power connectors, (C) Water connectors; (D) Tablet-based software for supply tracking and laboratory result documentations. <a href="https://www.jove.com/files/ftp_upload/59188/59188fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59188/59188fig4v2.jpg" /> 
Figure 4.&#160;Deployment of the laboratory facility. Instruction for panels unfolding on one side of the unit as illustrated (A-D). <a href="https://www.jove.com/files/ftp_upload/59188/59188fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59188/59188fig5.jpg" /> 
Figure 5.&#160;Schematics of the connectable laboratory: (A) BSL-2 module 1; (B) Glove Box and PCR module 2. <a href="https://www.jove.com/files/ftp_upload/59188/59188fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59188/59188fig6.jpg" /> 
Figure 6. Flow chart for a respiratory virus diagnostic RT-PCR test in the remote laboratory facility. <a href="https://www.jove.com/files/ftp_upload/59188/59188fig6large.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>Remote Laboratory BSL-2</td>
			<td>Remote Laboratory BSL-3</td>
		</tr>
		<tr>
			<td>Part I</td>
			<td>Part I</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; i.&#160; Lab tech to enter through door labeled entrance and put on lab coat, which is hanging on rack next to entrance door. Open shoes are prohibited, advanced mask and protective eyewear are encouraged.</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; i.&#160;&#160;&#160; Lab tech to look into Glove Box window from outside of unit to insure negative pressure is activated. (Pink Ball should be visible in the unit to show the negative pressure is working).&#160;</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; ii.&#160; Lab tech to wash hands in sink, put on disposable gloves and begin with intake of samples.</td>
			<td>&#160;&#160;&#160;&#160;&#160; ii.&#160;&#160;&#160; If negative pressure is working, lab tech to enter through only door and put on lab coat, which is hanging on rack next to entrance door.&#160; Open shoes are prohibited, advanced mask and protective eyewear are desirable.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; iii.&#160; Samples that were dipped in hypochlorite bath prior to being dropped at pass-through window are sitting in pass through for lab tech.</td>
			<td>&#160;&#160;&#160;&#160; iii.&#160;&#160;&#160; Lab tech to wash hands in sink, put on disposable gloves, PPE and begin with intake of samples.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; iv.&#160; Received in sample reception.</td>
			<td>&#160;&#160;&#160;&#160; iv.&#160;&#160;&#160; Samples that were previously dipped in hypochlorite bath prior to being dropped at pass through window are sitting in pass through for lab tech.</td>
		</tr>
		<tr>
			<td>Part II</td>
			<td>&#160;&#160;&#160;&#160;&#160; v.&#160;&#160;&#160;&#160; Received in sample reception.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; v.&#160; Depending on diagnostic procedure, specimens moved to biosafety cabinet and inactivated.</td>
			<td>Part II</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; vi.&#160; Specimens prepped for microscopy, centrifuge, or ROTs.</td>
			<td>&#160;&#160;&#160;&#160; vi.&#160;&#160;&#160; Specimens inactivated in Glove Box.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; vii.&#160; Appropriate diagnostic tests run.&#160;</td>
			<td>&#160;&#160; vii.&#160;&#160;&#160; Specimens executed for nucleic acid isolation.</td>
		</tr>
		<tr>
			<td>&#160;&#160; viii.&#160; Store specimens in 4&#176;C refrigerator or appropriate freezer.</td>
			<td>&#160; viii.&#160;&#160;&#160; After extraction, specimens moved to pass-through window.</td>
		</tr>
		<tr>
			<td>Part III</td>
			<td>&#160;&#160;&#160;&#160; ix.&#160;&#160;&#160; Lab tech enters through entrance in PCR side of unit (positive pressure).</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; ix.&#160; Use sink for staining &#38; washing of items.</td>
			<td>&#160;&#160;&#160;&#160;&#160; x.&#160;&#160;&#160;&#160; Lab tech to put on lab coat from rack next to entrance, wash hands in sink, put on gloves.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; x.&#160; Use laptop &#38; counterpace to perform analyses and documentation.&#160;</td>
			<td>&#160;&#160;&#160;&#160; xi.&#160;&#160;&#160; Receive samples from Glove Box room in pass-through window.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; xi.&#160; Sterilize equipment by running autoclave.</td>
			<td>&#160;&#160; xii.&#160;&#160;&#160; If necessary samples prepped in Laminar flow cabinet.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; xii.&#160; Dispose of any biohazardous waste in biohazard waste container.</td>
			<td>&#160; xiii.&#160;&#160;&#160; Appropriate diagnostics tests run.</td>
		</tr>
		<tr>
			<td>&#160;&#160; xiii.&#160; Wash hands in sink.</td>
			<td>&#160; xiv.&#160;&#160;&#160; Store specimens in 4&#176;C refrigerator or appropriate freezer.</td>
		</tr>
		<tr>
			<td>&#160; xiv.&#160; Hang lab coat back up on rack.</td>
			<td>Part III</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; xv.&#160; Exit through&#160; same door.</td>
			<td>&#160;&#160; xv.&#160;&#160;&#160;&#160; Use sink for staining &#38; washing of items.</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160; xvi.&#160;&#160;&#160; Use laptop &#38; counterpace to perform analyses and documentation.&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160;xvii.&#160;&#160;&#160; Transfer vials into pass-through window to PCR room and sterilize equipment by running autoclave.</td>
		</tr>
		<tr>
			<td></td>
			<td>xviii.&#160;&#160;&#160; Dispose of any biohazardous waste in biohazard waste container.</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160; xix.&#160;&#160;&#160; Wash hands in sink.</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160;&#160; xx.&#160;&#160;&#160;&#160; Exit through same entrance door.</td>
		</tr>
	</tbody>
</table>
Table 1. Checklist for the PCR diagnostics workflow.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Maintenance and calibrations</td>
		</tr>
		<tr>
			<td rowspan="2">Real-time PCR systems&#160;</td>
			<td>Monthly</td>
			<td>Perform background calibrations every month</td>
		</tr>
		<tr>
			<td>18 months</td>
			<td>Perform background, spatial and dye calibrations every 18th months</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>1 year</td>
			<td>Calibrate for revolutions per minute and temperature through external or internal calibration services</td>
		</tr>
		<tr>
			<td rowspan="3">Glove Box</td>
			<td>Daily</td>
			<td>Visually inspect elements, particularly for damage to the exposed surfaces of the HEPA filters, gloves, o-rings and hoses. Make sure duct clamps are tight and in place. Perform leak pressure test. Test the pressure alarm.</td>
		</tr>
		<tr>
			<td>6 months</td>
			<td>Change the HEPA filter</td>
		</tr>
		<tr>
			<td>1 year&#160;</td>
			<td>Calibrate the system</td>
		</tr>
		<tr>
			<td rowspan="3">Autoclave</td>
			<td>Weekly</td>
			<td>Clean the water tank and racks using a mild non-abrasive detergent</td>
		</tr>
		<tr>
			<td>3 months</td>
			<td>Calibrate timer and gauges</td>
		</tr>
		<tr>
			<td>1 year or every 50 cycles</td>
			<td>Inspect, clean thoroughly, test and calibrate</td>
		</tr>
		<tr>
			<td rowspan="2">Refrigerator and Freeezer</td>
			<td>6 months</td>
			<td>Check&#160; fan motor, evaporator coils, vacuuming condensing coils and condensor filters and replace batteries as needed</td>
		</tr>
		<tr>
			<td>1 year</td>
			<td>Calibrate freezer through internal or external calibration services</td>
		</tr>
	</tbody>
</table>
Table 2. Real-time PCR equipment maintenance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mandatory</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>Lab coat, protective shoes, gloves</td>
			<td>Lab coat, protective shoes, gloves, masks, eyewear</td>
		</tr>
		<tr>
			<td>Refrigerator 4 &#176;C, freezer -20 &#176;C</td>
			<td>Refrigerator 4 &#176;C, freezer -20 &#176;C, freezer -80 &#176;C</td>
		</tr>
		<tr>
			<td>One set of automated&#160; pipettes&#160;</td>
			<td>Three sets of automated pipettes</td>
		</tr>
		<tr>
			<td>Centrifuge, shaker, thermocycler</td>
			<td>Robotic system</td>
		</tr>
		<tr>
			<td>RT-PCR machine, ice bath</td>
			<td>RT-PCR with temperature control, ice-free cooler</td>
		</tr>
		<tr>
			<td>Biohazard waste bags</td>
			<td>Autoclave to dispose biohazard waste</td>
		</tr>
	</tbody>
</table>
Table 3. Minimum requirements for the RT-PCR respiratory virus diagnostic test BSL-2.

Watch this Scientific Journal Video about Remote Laboratory Management: Respiratory Virus Diagnostics at JoVE.com

Remote Laboratory Management: Respiratory Virus Diagnostics

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

remote-laboratory-management-respiratory-virus-diagnostics

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32.8K Views.  Baylor College of Medicine. A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

Video: Remote Laboratory Management: Respiratory Virus Diagnostics

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.

Breathing-controlled Electrical Stimulation BreEStim for Management of Neuropathic Pain and Spasticity

The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity.

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life.
The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI.
We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.

Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of ...

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...