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Courage to Leap Group

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Chris Carney
Chris Carney

Post-Show Protocol Ebook.pdf


Ethical clearance for the study was obtained from the Health Research Ethics Committee(HREC) of Stellenbosch University (South Africa) (reference: N19/03/043, project ID: 9521). The experimental objectives, risks, and details were explained to volunteers and informed consent were obtained prior to blood collection. Strict compliance to ethical guidelines and principles Declaration of Helsinki, South African Guidelines for Good Clinical Practice, and Medical Research Council Ethical Guidelines for Research were kept for the duration of the study and for all research protocols.




Post-Show Protocol Ebook.pdf



ELISA analysis was performed on PPP from 12 controls, 11 individuals with acute COVID-19 and 11 individuals with Long COVID/PASC. The Human SAA ELISA Kit (SAA1) (E-EL-H2183, Elabscience Biotechnology Inc.) was used in this analysis following manufacturer prescribed protocol. SAA1 is one of the two acute phase SAA proteins [39].


All PPP samples were exposed to a first step of trypsin digestion of the plasma proteins. The samples were diluted in ten times in 10 mM ammonium bicarbonate and protein concentration determined. The samples were standardised to 1 mg/mL total protein. A total of 1 µg trypsin (New England Biosystems) was added to the plasma for 1:50 enzyme to substrate ratio. No reduction or alkylation was performed. After this first trypsin digestion protocol, both COVID-19 and Long COVID/PASC samples formed a visible pellet deposit at the bottom of the tubes after centrifugation for 30 min at 13,000g. Healthy PPP samples and the T2DM PPP samples did not form any visible deposit at the bottom of the tube.


After the first trypsin digestion protocol, supernatants were removed and the remaining 10 µL was exposed to ThT (as previously described) and the rest of the supernatants were analysed using mass spectrometry (seen methods below).The 10 µL of healthy PPP and T2DM PPP contained no pellet deposit, while the 10 µL of the COVID-19 and Long COVID/PASC sample did contain a visible pellet deposit. These samples were also visualized using the Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).


After the first trypsin digestion protocol, the supernatants were subjected to C18 and solid phase extraction (SPE) and proteomics were performed. After the pellet deposit was solubilized in the second trypsin digestion protocol, the now soluble pellet deposits were also studied using proteomics.


Plasma samples of healthy individuals, T2DM, COVID-19 and Long COVID/PASC patients were then exposed to the first trypsin protocol. From each sample the supernatant was removed and ThT was added to 10 µL of the remaining sample and viewed with fluorescence microscopy are shown in Figs. 4 and 5. In this remaining 10 µL, COVID-19 and Long COVID/PASC PPP samples, a visible pellet was present.


bioRxiv is for the distribution of preprints, which are complete but unpublished manuscripts. Research articles reporting new, confirmatory, or contradictory findings may be posted. Individual components of research articles such as figures, tables, and data sets are not appropriate for posting to bioRxiv. In the Scientific Communication and Education subject category, research articles on professional standards may be posted. bioRxiv does not permit the posting of news, product advertisements, announcements, teaching/instructive materials, meeting reports, white papers, policy statements, human or veterinary case reports, theses, dissertations, student projects, recipes and simple protocols.


Research articles summarizing new experimental or computational methods are appropriate and may include step-by-step protocols. Step-by-step protocols alone are not sufficient and must be placed in the context of a complete research article that includes elements such as introduction, results, and discussion.


The State of Maine uses statewide EMS protocols for the care of patients in the pre-hospital environment. Protocols are developed by the Maine EMS Medical Direction & Practices Board (MDPB) and are reviewed/updated on a 2-year cycle.


Maine EMS protocols are available via a free app for smartphones. It can be found in the App Store (iPhones/iPads) or Google Play (Android) as the "Maine EMS Protocol App" from Maine Emergency Medical Services. Statewide protocols are no longer printed by Maine EMS but can be viewed, saved, and printed via the PDF link (below).


The MDPB has spent countless hours researching, drafting, and revising these EMS protocols. In addition to monthly public meetings and volunteering many work hours, the MDPB held six statewide public protocol discussion forums.


Red 8: Under 13.a., the dose was hidden by the pediatric teddy bear logo. It read "VF/VT: amiodarone 300 mg IV/IO; may consider additional mg IV/IO one time". It has been corrected to read "VF/VT: amiodarone 300 mg IV/IO; may consider additional 150 mg IV/IO one time". This has been updated to the full protocol pdf and protocol app. If you have printed the entire protocol pdf, you can print the single replacement Red 8 corrected page (PDF) (12/7/21).


Red 12: Under Amiodarone, the dose read "Can repeat in 3-5 minutes up to a total dose of". This has been corrected to read "Can repeat in 3-5 minutes up to a total dose of 15 mg/kg". This has been updated to the full protocol pdf and protocol app. If you have printed the entire protocol pdf, you can print the single replacement Red 12 corrected page (PDF) (11/23/21).


Yellow 3: Under PEARLS, "MSDS" was changed to "SDS". This has been updated to the full protocol pdf and protocol app. If you have printed the entire protocol pdf, you can print the single replacement Yellow 3 corrected page (PDF) (11/23/21).


Please note that for existing smartphone apps, users should manually update the apps on or after December 1, 2021. Maine EMS does not print protocol books. To download the free 2021 protocol app, visit the Apple store (iPhones/iPads) or Google Play store (Android devices) and search for the "Maine EMS Protocol App". Please make sure you update regularly for any updates/changes/improvements in the app.


Did participants in each treatment group adhere to the protocols for assigned interventions? For example, if Group 1 was assigned to 10 mg/day of Drug A, did most of them take 10 mg/day of Drug A? Another example is a study evaluating the difference between a 30-pound weight loss and a 10-pound weight loss on specific clinical outcomes (e.g., heart attacks), but the 30-pound weight loss group did not achieve its intended weight loss target (e.g., the group only lost 14 pounds on average). A third example is whether a large percentage of participants assigned to one group "crossed over" and got the intervention provided to the other group. A final example is when one group that was assigned to receive a particular drug at a particular dose had a large percentage of participants who did not end up taking the drug or the dose as designed in the protocol.


Changes that occur in the study outcomes being assessed should be attributable to the interventions being compared in the study. If study participants receive interventions that are not part of the study protocol and could affect the outcomes being assessed, and they receive these interventions differentially, then there is cause for concern because these interventions could bias results. The following scenario is another example of how bias can occur. In a study comparing two different dietary interventions on serum cholesterol, one group had a significantly higher percentage of participants taking statin drugs than the other group. In this situation, it would be impossible to know if a difference in outcome was due to the dietary intervention or the drugs.


For example, retrospective self-report of dietary salt intake is not as valid and reliable as prospectively using a standardized dietary log plus testing participants' urine for sodium content. Another example is measurement of BP, where there may be quite a difference between usual care, where clinicians measure BP however it is done in their practice setting (which can vary considerably), and use of trained BP assessors using standardized equipment (e.g., the same BP device which has been tested and calibrated) and a standardized protocol (e.g., patient is seated for 5 minutes with feet flat on the floor, BP is taken twice in each arm, and all four measurements are averaged). In each of these cases, the former would get a "no" and the latter a "yes."


As you assess this criterion, think about whether it is likely that the person(s) doing the outcome assessment would know (or be able to figure out) the exposure status of the study participants. If the answer is no, then blinding is adequate. An example of adequate blinding of the outcome assessors is to create a separate committee, whose members were not involved in the care of the patient and had no information about the study participants' exposure status. The committee would then be provided with copies of participants' medical records, which had been stripped of any potential exposure information or personally identifiable information. The committee would then review the records for prespecified outcomes according to the study protocol. If blinding was not possible, which is sometimes the case, mark "NA" and explain the potential for bias.


For example, a retrospective self-report of dietary salt intake is not as valid and reliable as prospectively using a standardized dietary log plus testing participants' urine for sodium content because participants' retrospective recall of dietary salt intake may be inaccurate and result in misclassification of exposure status. Similarly, BP results from practices that use an established protocol for measuring BP would be considered more valid and reliable than results from practices that did not use standard protocols. A protocol may include using trained BP assessors, standardized equipment (e.g., the same BP device which has been tested and calibrated), and a standardized procedure (e.g., patient is seated for 5 minutes with feet flat on the floor, BP is taken twice in each arm, and all four measurements are averaged). 041b061a72


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