1. Johnson Space Center 1 Salivary Markers Skills Human Research Facility The Effect of Long-Term Exposure to Microgravity on Salivary Markers of Innate Immunity Skills Training Instructors: Brian Crucian281-483-7061brian.crucian-1@nasa.govbrian.crucian-1@nasa.gov Ali Schade281-483-9531alissa.z.schade@nasa.govalissa.z.schade@nasa.gov

2. Johnson Space Center 2 Salivary Markers Skills Human Research Facility Session Objectives The crewmember will be able to: Identify the science objectives of the Salivary Markers experiment. Demonstrate the unique techniques for collection of blood with the different types of blood tubes. Describe anomalies of past phlebotomy sessions using the ACD tube and strategies implemented to mitigate the anomalies for future sessions. Describe the need for obtaining full blood tubes. Identify and describe the Blood Collection Hardware and Lg Blood Sample Kit. Properly configure Refrigerated Centrifuge for processing of blood sample tubes. Identify which blood sample tubes will be stored in MELFI. Describe potential safety hazards associated with the experiment.

3. Johnson Space Center 3 Salivary Markers Skills Human Research Facility The Salivary Markers study will collect blood, saliva, urine and a health assessment before, during and after a 6-month mission to the ISS. These samples will be used to determine if spaceflight induced immune system dysregulation increases infection susceptibility or poses a significant health risk to crewmembers onboard the International Space Station. The saliva, urine and health assessment training has already been completed. The focus of this session will be to emphasize the differences in blood collection technique using the Salivary Markers experiment unique hardware. Introduction

4. Johnson Space Center 4 Salivary Markers Skills Human Research Facility Introduction FD-10FD-90* Soyuz Landing Handover Soyuz Undock R-1 7 Day Saliva Collection 24 Hour Urine Collection Health Assessment Blood Collection Operations: Activities occur on or around FD10, FD90, and R-1 FD-10 will be a Frozen Blood Collection FD-90 and R-1 will be an Ambient Blood Collection Total Collections: -3 blood collections (two are ambient return) *The FD-90 window will be flexible to permit ambient blood to be returned on a Soyuz.

5. Johnson Space Center 5 Salivary Markers Skills Human Research Facility Study Specific Information The Salivary Markers experiment will utilize two different types of blood collections. Ambient Frozen Ambient blood collections require different techniques than those taught in “generic phlebotomy.”

6. Johnson Space Center 6 Salivary Markers Skills Human Research Facility Importance of proper ambient blood collections Lessons Learned from Previous ISS Experiments

7. Johnson Space Center 7 Salivary Markers Skills Human Research Facility Post-Phlebotomy, Study Specific Skills Training Generic phlebotomy teaches the universal skill of “needle into vein” There are always specific caveats to each phlebotomy session: -Hardware difference -Tube order -Tube type (glass/plastic, anticoagulant volume, fill level, fill time) -Post-collection handling (mixing, temperature, time to ground) -Short sample tolerance -Auto vs. operator (pros, cons of each) -Study specific variations for techniques, optimization Previous Experiments: majority of on-orbit collections successful some subjects lost for preventable reasons training augment to assure acceptable samples

8. Johnson Space Center 8 Salivary Markers Skills Human Research Facility Study Specific Information Ambient blood collections will occur as close to hatch closure/landing as possible, so that the live cells may be returned for immune function assays. Ambient samples are never frozen. Samples are stowed in Lg Blood Sample Kit and then put in Insulated Blood Pouch for return to Earth.

9. Johnson Space Center 9 Salivary Markers Skills Human Research Facility Two missions: No lost science Ambient Blood Flight Samples

10. Johnson Space Center 10 Salivary Markers Skills Human Research Facility Two missions: Lost science Ambient Blood Flight Samples

11. Johnson Space Center 11 Salivary Markers Skills Human Research Facility How much blood is needed? Depends on:-WBC count -Hematocrit -Subject assay compatibility Given Salivary Markers cell requirements, it is imperative that all blood tubes be as full as possible. To assess crewmember immune status, we: Stain and count cells, culture cells, examine activation markers, assess cytokine production, extract RNA, extract viral DNA, measure virus-specific immune cells, challenge cells with viral proteins.

12. Johnson Space Center 12 Salivary Markers Skills Human Research Facility Performance by Tube Type Summary statistics from all in-flight samples: Acceptable EDTA: 86% Acceptable ACD: 59%

13. Johnson Space Center 13 Salivary Markers Skills Human Research Facility Flying in space does not alter tube vacuum characteristics

14. Johnson Space Center 14 Salivary Markers Skills Human Research Facility Study Specific Issue: Microgravity Filling of Vacutainers Tube TypeCap ColorMaterialCharacteristicFill direction EDTAPurplePlasticHydrophobicBottom to top HeparinGreenPlasticHydrophobicBottom to top ACDYellowGlassHydrophilicAmorphous (blood adheres to glass)

15. Johnson Space Center 15 Salivary Markers Skills Human Research Facility Video #1:Vacutainer filling in zero gravity (parabolic flight)Video #2:Vacutainer filling onboard ISS

16. Johnson Space Center 16 Salivary Markers Skills Human Research Facility -Please collect two yellow 8.5ml ACD tubes first and leave connected to vein for 45 seconds -Collect the green 6ml Heparin tube second and leave connected to vein for 20 seconds. -Collect the purple 6ml EDTA tube last and leave connected to vein for 20 seconds. Solution:

17. Johnson Space Center 17 Salivary Markers Skills Human Research Facility Operator-based vs. Self Phlebotomy You may be trained to perform auto phlebotomy for other studies. This training is usually based on need, and not preference (crew time, availability of US crewmembers, etc.). Nominal phlebotomy is a two handed procedure. This is based on -Ability to secure rolling veins -Angle of entry -Ease of utility -Availability to control adverse events -General safety. Success rates are generally much higher for operator based, than self phlebotomy. Consuming extra blood tubes over multiple attempts is a significant study risk, based on upmass limitations. Ultimate choice of method remains crew preference. For Salivary Markers, operator based phlebotomy is strongly advised.

18. Johnson Space Center 18 Salivary Markers Skills Human Research Facility FD 90, R-1 Ambient Collection Ambient Blood Collection: Lg Blood Sample Kit 2 Blood 8.5ml ACD 1 Blood 6ml Heparin 1 Blood 6ml EDTA Lg Blood Sample Kit Blood 8.5ml ACD Blood 6ml Heparin Blood 6ml EDTA

19. Johnson Space Center 19 Salivary Markers Skills Human Research Facility FD 90, R-1 Ambient Collection Draw blood (done by operator or yourself). 4 tubes must be filled. Place samples in Lg Blood Sample Kit Label tubes with Crew ID and GMT day and time to the minute. 90 AS

20. Johnson Space Center 20 Salivary Markers Skills Human Research Facility FD 90, R-1 Ambient Collection Ambient Blood Draw (in pictures) Place Lg Blood Sample Kit into Insulated Blood Pouch Stow Insulated Blood Pouch for return on Soyuz Lg Blood Sample Kit

21. Johnson Space Center 21 Salivary Markers Skills Human Research Facility FD 10 Frozen Collection Frozen Blood Collection: (2) 7ml Heparin tubes (2) 7ml EDTA tubes Blood tube Stowage: 3 x 5 Mesh Bags Blood 7ml Heparin Blood 7ml EDTA MELFI 3 x 5 Mesh Bag Place blood sample tubes in 3 x 5 Mesh Bag prior to MELFI stowage

22. Johnson Space Center 22 Salivary Markers Skills Human Research Facility FD 10 Frozen Collection Label and temp stow blood samples Properly balance tubes in 6K rotor and spin in HRF centrifuge for 30 minutes. Place samples in 3 x 5 Mesh Bag then insert into MELFI. Label tubes with Crew ID and GMT day and time to the minute. Draw blood, done by an operator or yourself, (4 tubes should be filled) 90 AS

23. Johnson Space Center 23 Salivary Markers Skills Human Research Facility Critical Points to Remember to Ensure Successful Science Return For Blood Collection Your medical trainer will be on console for private consultation Follow order of draw outlined in procedures Tubes must be labeled and barcodes called down, placed in crew notes or scanned via the BCR/RFID scanner Frozen Only: Blood must be collected, then centrifuged and placed in MELFI within 60 minutes of collection Ambient Only: Blood must not be frozen, and will be placed in Lg Blood Sample Kit which in stowed in Insulated Blood Pouch for return to Earth.

24. Johnson Space Center 24 Salivary Markers Skills Human Research Facility Blood Collection Hardware HRF Rack 2

25. Johnson Space Center 25 Salivary Markers Skills Human Research Facility Blood Collection Hardware The HRF Supply Kits (Purple and Green) will provide several of the consumables needed for the blood draw. HRF Supply Kits: Multi-user supply kits designed to support several HRF experiments Salivary Markers is just one of the users of these supply kits Contains supplies for blood sample collection Ambient blood tubes are in a different location HRF Supply Kits: Purple and Green

26. Johnson Space Center 26 Salivary Markers Skills Human Research Facility Blood Collection Hardware The HRF Supply Kit Purple (top) and Supply Kit Green (bottom): HRF Supply Kit Purple: Blood tubes (vacutainers) Disposable gloves Blood collection belts Sharps containers Liner bags (biohazardous trash) HRF Supply Kit Green: Butterfly needles Disinfectant wipes (Iodine & Biocide) Gauze Tape Sharpie Band-Aids Tourniquet Centrifuge counterweight

27. Johnson Space Center 27 Salivary Markers Skills Human Research Facility Blood Collection Hardware Blood Collection Butterfly Setup: Unassembled Assembled To assemble: screw clear tube holder onto needle. Butterfly Needle Tube Holder (not disposable) Blood Tube

28. Johnson Space Center 28 Salivary Markers Skills Human Research Facility Blood Collection Hardware Blood Collection Belt Disposal of non-sharps biohazardous trash (required) Temp stowage of supplies and samples (optional)

29. Johnson Space Center 29 Salivary Markers Skills Human Research Facility Blood Collection Hardware Liner Bags and the Glove Bag Assembly: Liner bags (biohazard bags) are used for disposal of any non-needle trash during blood collection, e.g.: Used iodine and Biocide wipes Band-aids Liner Bag holder fits on right side of Blood Collection Belt for convenient trash disposal Spare liner bags are found in Supply Kit Purple Disposable Gloves Used gauze Needle packaging

30. Johnson Space Center 30 Salivary Markers Skills Human Research Facility Blood Collection Hardware Sharps Container For disposal of used Butterfly Needles Remember to remove Tube Holder

31. Johnson Space Center 31 Salivary Markers Skills Human Research Facility Refrigerated Centrifuge Provides refrigeration from +4  C to ambient Currently, not cooling Provides selectable speed from 500 rpm to 5000 rpm Accommodates sample sizes from 0.5mL to 50mL with use of adapters

32. Johnson Space Center 32 Salivary Markers Skills Human Research Facility Refrigerated Centrifuge Stowage Drawer 6K Rotor Spanner Release tool 6K 50ml Adaptors Door Seal 18K Rotor Launch Spacer 18K Rotor adaptors (various) Compressor Launch restraint bolts 6K Rotor Adaptors (various)

33. Johnson Space Center 33 Salivary Markers Skills Human Research Facility Refrigerated Centrifuge Front Panel Stowage Drawer Manual Release point Door Unlock Led Chamber Door handle Chamber Door Launch Spacer Assembly Control Panel Power switch Circuit breaker switch Compressor Life Clock

34. Johnson Space Center 34 Salivary Markers Skills Human Research Facility Parameters: T/min – minutes of run time T/SEC – Seconds of run time RPM – Speed of run - Speed of acceleration - Speed of deceleration T/C – Temperature in Celsius To set parameters: use  key on control panel to cycle to parameter to be set. use and keys to set desired data. press START button once within 8 seconds to lock data into program. Setting Refrigerated Centrifuge Parameters

35. Johnson Space Center 35 Salivary Markers Skills Human Research Facility Blood borne pathogens – Observe universal precautions Needle precautions –Do not unsheathe needles until immediately prior to use. – Immediately ‘safe’ needle and discard in sharps container after use. Safety

36. Johnson Space Center 36 Salivary Markers Skills Human Research Facility ACDAcid citrate dextrose BCRBarcode Reader BDCBaseline Data Collection DCTData Collection Tool EDTAEthylenediaminetetraacetic acid FDFlight day GMTGreenwich Mean Time Hrhour HRFHuman Research Facility LLaunch MELFIMinus Eighty Laboratory Freezer for ISS METMission Elapsed Time OprOperator OSTPVObnoard Short Term Plan Viewer OvOverview PAOPublic Affairs Office PDLPayload Development Lab PIPrincipal Investigator P/LPayload RReturn Acronyms and Abbreviations

37. Johnson Space Center 37 Salivary Markers Skills Human Research Facility Auxillary Review Material

38. Johnson Space Center 38 Salivary Markers Skills Human Research Facility Veins and Arteries Arteries: Deeper, flow away from the heart Veins: Closer to surface, flow towards the heart

39. Johnson Space Center 39 Salivary Markers Skills Human Research Facility How a tourniquet functions Tourniquet constricts surface vein flow up the arm Blood pressure causes surface veins to expand Deep arteries unaffected, supply capped veins with full blood pressure Apply tourniquet tightly!

40. Johnson Space Center 40 Salivary Markers Skills Human Research Facility 1.Apply tourniquet, select vein Choose straight veins Avoid bends Avoid valves Proper site 2. Prep with biocide wipe After disinfection, don gloves and proceed with blood draw

41. Johnson Space Center 41 Salivary Markers Skills Human Research Facility 1.Secure needle/tube holder assembly Gauze can be temp stowed on subject’s arm with tape to allow for easier retrieval following removal of needle from arm 2. Prep with biocide wipe Secure vein with free hand to prevent ‘rolling’ Note: impossible during ‘auto- phlebotomy’

42. Johnson Space Center 42 Salivary Markers Skills Human Research Facility Fill blood tubes Insert tube in to tube adaptor. Fill yellow first, green second and purple last (tube order is provided in procedures) Blood will be drawn into tube due to vacuum Remove tube after filled and flow stops (~45 sec). Repeat until all required tubes are filled Important to gently mix all tubes Place gauze/remove needle

43. Johnson Space Center 43 Salivary Markers Skills Human Research Facility “Safe” the needles Safely dispose of sharps, stow dry trash where indicated