ISAC tries to forget San Diego and Harry Crissman?

Hi All,
 
I was thumbing through the new ISAC membership handbook, looking at the
color pages on the different ISAC meetings and there seems to be an ISAC
meeting missing.  ISAC XXI, 2002, San Diego, Harry Crissman, President.

 
Is this discrepancy just my book or everyone's?  I thought we had a
pretty good time at the banquet with the Mardels and doesn't anybody
remember riding the wave at the PFS booth?
 
Regards,
 
Kevin
 
C. Kevin Becker
Phoenix Flow Systems, Inc.
6790 Top Gun St. #1
San Diego, CA 92121
858 453-5095
fax 858 453-2117
www.phoenixflow.com
 
-----Original Message-----
From: Arthur Roberts [mailto:robertar@umdnj.edu] 
Sent: Friday, May 30, 2008 9:33 AM
To: cyto-inbox
Subject: SUMMARY: PI viability dye during sorting
 
Dear Flow-ers,
Thank you for all your useful replies. They ran quite the gamut, with
all sorts of interesting suggestions. I am posting a synopsis here. 

The original question:	I was wondering about the risks in using PI in a
stream-in-air sorter. Since PI is a DNA intercalater, and therefore,
could have nasty effects on the operator's DNA if it were inhaled as an
aerosol, is there a way to use it in sorting that will minimize
exposure?  I would wash the cells before sorting, but it seems that the
PI intensity would drop off quickly after washing, since the binding is
reversible.  

Thanks for any insight you might have.

- Art

Arthur Roberts
Robert Wood Johnson Medical School
Dept. of Molec. Genetics, Microbiol & Immunol
SRB rm 113
675 Hoes Lane
Piscataway, NJ 08854
phone: 732-235-4502

REPLIES:

Actually the PI binding is quite stable; we see little loss in  
fluorescence over a couple of hours.

I would bet the amount of PI aerosolized during pipetting far exceeds  
what you would get from a sorter.
______________________________
As an alternative you may want to use the food dye that is used to
discard dead and damaged sperm during sperm sorting for sex
predetermination.  A 0.002% solution of this dye, FD&C #40, (Johnson and
Welch. Theriogenology 1999;52:1323-1321; Garner and Seidel. Reproduction
2002;124:733-743)  is added to Hoechst 33342 stained sperm.  This dye
identifies the dead and damaged sperm so that they can be discarded in
the waste fluid at the time of sorting.  This system is being used
commercially and we have had no indication that it damages the sorted
living sperm (Garner and Seidel, Theriogenology, 2008;69:886-895)  
The red dye quenches the Hoechst 33342 and with the dual detectors ends
up as a smeared population in the lower quadrant of the bivariate plot
of UV emission detectors.
  
The FD & C #40 powder is procured from the Warner Jenkinson Co., 2526
Baldwin St., P.O. Box 14538, St.Louis, MO 63178-0538 .  
One dissolves 5 g of the FD&C #40 in 100 mL nanopure water to make a 5%
stock solution to use for staining samples.



______________________________________



We use PI all the time, it is at a rather low concentration and
so I  have never worried about it. we have a stock that is at 1mg/ml
which we dilute into a working  
solution 50 fold less concentrated, then add that to 200 to 400µl of  
cells. This works out to a final concentration of 0.5-1µg/ml if I did  
my math right.
_______________

Yes, it is called aerosol containment devices, no sort operator should
be caught without it. If your instrument doesn’t have one then you
should be seriously consider using a Personal Protective device. What do
you do when you sort other live material? I think I would be more
concerned about aerosol inhalation of other biohazardous materials from
live cells. Kevin Holmes did a very nice evaluation of aerosol
generation from an Aria cell sorter characterizing both size and
quantity of aerosols generated under a variety of conditions and showed
a good percentage are just the right size for inhalation into the lung.
He presented this at ISAC and will be presenting it at the upcoming
Chesapeake Cytometry meeting next Monday in Rockville, MD. Sorting
without some type of aerosol containment practice is at best risky.
_________________________

A correctly set up stream-in-air sorter should pose no risk at all. 
I ran a MoFlo for 3 1/2 years, when PI was the viability & cell cycle
dye of choice.	 
I agree aerosols are generated if using mixed populations such as blood,
especially at the maximum event rate for sorting.  However, the amount
of PI in your sample that is being liberated as an aerosol throughout
the sort is minute, it would be an interesting exercise to even try and
measure it. 
My exposure was simply limited by a perspex box that was fitted to the
front of the MoFlo during a sort.  Any basic air extraction (with Hepa
filters) would also be worthwhile.  My current sorter (an Aria - which
has a sealed sort chamber) came with a very neat extraction system that
is never used.
 
Invitrogen are selling some very nice dyes for viability/cell
cycle/live-dead now that are supposed to be considerably safer than PI. 
__________________________

Last advice that I had was that PI hazard is mainly from the powder
form. If the operator only works with PI solution then the hazard is
greatly reduced. I still would not handle the PI solution without
gloves, though, and I feel sympathy for the poor person who has to make
up the solution from powder! I did some tests a while ago as to the
washout effects on PI, and found no significant leaching in the cells I
was using.

_______________________________

PI would be the last of my worries in live samples.
________________________________