1. Sheath Contamination Survey: An Examination of Common (Though Not Necessarily Best) Laboratory Practices A Study of the Flow Cytometry Research Group (FCRG)
Dave Adams, University of Michigan; Alan Bergeron, Dartmouth College; Laura Blunk, Stowers Institute for Medical Research; Kathleen Brundage, West Virginia University; Karen Clise-Dwyer, MD Anderson Cancer Center; Matt Cochran, University of Rochester Medical Center; Monica DeLay, Cincinnati Children’s Hospital; Roxana Del Rio Guerra, University of Vermont; Maris Handley, Massachusetts General Hospital; Peter Lopez, New York University Langone Medical Center; E. Michael Meyer, University of Pittsburgh Cancer Institute; Alan Saluk, The Scripps Research Institute
EB: Frances Weis-Garcia

2. Previous FCRG Study looking at common sorter contaminants
Develop effective endotoxin cleaning procedure
Test for additional contaminants
Poll SRLs beyond the FCRG and evaluate common practices

3. The Callout Survey Monkey Platform
Advertised on PUCL mailing list and Google+ Cytometry Community
Bribed with chance to win Starbucks!
Participants asked about their testing procedures, operating procedures and maintenance procedures
Participants asked to submit samples for testing

4. How’d We Do? 61 Respondants 106 Sorters Described
3 from Non-SRLs
106 Sorters Described
Several Lessons About How Not to Ask a Question in a Survey
3 Happy Coffee Drinkers

5. Instrumentation Quick Facts
Of the 106 cell sorters described in the survey:
54 are housed within Biosafety Cabinets
55 are operated solely by laboratory staff while the remaining 51 allow trained users to operate the instrument
Average reported instrument usage is % capacity though that number falls to 51-75% as the number of sorters within the facility increases.
Sheath Sources break down as follows:
51% of respondents use a commercial 1x saline product
35% use a commercial concentrate saline product which is then diluted in-house
14% manufacture their saline in-house
68% of sorters over 5 years old and 74% rely on service contracts for maintenance

6. Testing Frequency Testing Frequency Responses Percentage Daily 2 4
Weekly 11 21
Monthly 7 13
Never 8 15
Only Upon Reported Contamination 25 47
When asked about sheath fluid testing, 62% either test only when they receive reports of possible contamination or do not test their sheath fluid at all.

7. Testing Location Collection Location Responses Percentage Sheath Tank24 53
Flow Cell 40 89
Sample Introduction Area 15 33
Bulk Sheath container 9 20
When Testing, the most common point of collection was the flow cell (89% of respondents collect from this location) followed by the sheath tank (53% of respondents).
Other samples collected by individual respondents include the sheath filter, the vacuum line and the DI water source.
By far the majority of respondents are looking for bacteria or fungus (see below). Only four of the 45 responses indicated an interest in endotoxin, and only five indicated an interest in mycoplasma. One respondent noted that they always look for particulates in the sheath and another noted they look for DNA.
Thirty-six of the forty-four respondents (82%) noted they complete their own testing while the remainder hand the samples off to another institutional group for testing.

8. Thirty-six of the forty-four respondents (82%) noted they complete their own testing while the remainder hand the samples off to another institutional group for testing.
Of those that tested, 21 of 47 responses indicate no positive results over the last twelve months. Those that did show positive results break down as follows. (Yikes for the poor fellow in the final column – They test for bacteria and fungus via culture). Overall little interest is paid to endotoxin.

10. User Operation

12. Bacteria Testing

13. Negative + ++ +++ Sheath Tank 2 6 4 5 Stream 12 3
17 respondents submitted samples for bacterial testing. Each individual submitted a sample from the flow cell as well as the sheath tank.
Negative + ++
+++
Sheath Tank 2 6 4 5
Stream 12 3

14. RNase Testing 47 samples were submitted for RNase testing.
47 samples were submitted for RNase testing. 
Two samples (1 from an Aria stream and another from an Aria sheath tank) were positive for RNase activity.​

15. Where to From Here?
Nearly half the survey respondents do not perform any proactive sheath testing and yet over half the received samples tested positive for bacterial/fungal contamination.
The prevalence of RNase in our testing pool was low, but it is worth noting that both positive tests were recorded in facilities that do no RNase testing.
Survey results at this point are limited and additional responses will present a better idea of current standard practices.

16. Follow the QR code to The current survey
Acknowledgements
We would like to thank all the individuals who answered our survey and provided samples for our study.
Follow the QR code to
The current survey
Follow the QR code to
The current survey