From: Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@Unilever.com)
Date: Tue Sep 29 1998 - 13:50:34 EST
Dear colleagues
Can't resist a comment. Keeping the long story short I have
to agree with Howard that speed is not everything and ease
of use is definitely with the non-adjustable instruments. As
the event rate is low, so are your number of sort decisions.
High purity is also not required as verification is still
done microscopically.
I do not know the current state of measurement, but also was
a bit disappointed by the lack of multiparametric
measurements that would indeed improve the differentiation
of the oocysts. A better discrimination would also
eventually make the sorting step obsolete anyhow and
alternatively there might be better ways of detection
anyhow. You do not need any UV to do so and the measurement
of viability (my favourite subject) is to complex to discuss
here. However in combination with viability determination
droplet sorters could be of use if you are prepared to run a
bioassay. All you have to do is to sort set numbers of
potentially viable oocysts into capsules which you can than
feed into an appropriate animal model (or to give to humans
if you have someone specific in mind).
If anyone has an open cheque book about this issue I am
happy to get in touch.
All the best
Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com
______________________________ Reply Separator _________________________________
Subject: Sorting Sydney's water: List input requested!!
Author: cytomat@netcore.com.au at INTERNET
Date: 23/09/1998 19:58
Dear Mark & Duncan,
Your spirited defense of the FACSCalibur and your dismissal of modern droplet
sorters are much as I would have expected, and I will reply to that in due
course, once we have had some input from other List members.
In the meantime, just two things,
#1
Contrary to your request to direct all correspondence to you, I would request
that the List members air all their replies on this List, as "the discussion we
need to have" will benefit much more from the wider experience and input of the
List members than from what might be perceived (then used to dismiss the points
raised, perhaps) as our two sets of vested interests. My vested interests arise
from 20 years in high speed droplet sorting, along with my known Cytomation
connection; and your Australian Environmental Flow Group's vested interest,
arising from the active involvement of BD (manufacturer of the FACSCalibur) as
a funding source in your program of development, where they have also provided
the cytometer for the methodological development process itself.
In this context, you might provide details of what type and vintage (not
vantage) droplet sorter you used in developing detection methods prior to
switching to the FACSCalibur, and what were the "problems inherent in its use
for such work as this project requires".
I think the stakes involved are bigger than either of these sectional
interests.
Moreover, the List will be a fine moderating influence on the validity of what
any of us may put forward.
Further, a healthy exchange here is certainly preferable to its becoming
politicised as is now happening in Sydney, where various Members of Parliament
are calling for scalps from the present NSW Government. There are clearly many
questions still to be raised and addressed.
I would like your thoughts on this.
#2
Your reply to the List referred to the culminating material from a series of
additional email exchanges and sets of replies between me and Howard Shapiro,
which he, then I (replying to Howard) copied to Duncan.
As Howard humourously pointed out, there is indeed a Ferrari employed on the
project; keep up the good work, Belinda!
I include the relevant trigger email now, between the pair of lines,
thus========
Let's keep it rolling.
Cheers,
Bob
========================================================
Friday, 18 Sept 1998; EDITS FROM ME ARE INDICATED AS (asdfgh jkl; etc)
Dear Howard,
Even if you concentrate (the 100L sample), and I concede you must, there are
superior features in modern sorters that are necessary for this task.
You need to measure the whole volume (of the concentrated sample), if the
incidence of targets is low.
Compare acquisition rates of 5,000/s to 50,000/s
You need to sort every cell that looks like a positive
Compare sorting rates of possible positives at 400/s vs 40,000/s
You need direct sorting of the cells for immediate inspection and assay
Compare recovery of sorted cells onto a slide vs into a large collection volume
which itself has to be concentrated and so will have large adherence losses.
You need live/dead discrimination simultaneous with Mab positivity
Compare no UV-Ho33342 capability to having a UV source on the machine
You need to find the origin of the contamination, so... a rapid multi-sample
processing system
Compare T model Calibur to Ferrari Vantage or MoFlo
I still think the Calibur is the wrong machine.
Cheers
Bob
========================================================
-----Original Message-----
From: robert ashcroft [SMTP:cytomat@netcore.com.au]
Sent: Friday, September 18, 1998 10:10 PM
To: cyto-inbox
Subject: RE: Sorting Sydney's water
Dear Howard,
Even if you concentrate, and I concede you must, there are superior features in
modern sorters that are necessary for this task.
You need to measure the whole volume, if the incidence of targets is low.
Compare acquisition rates of 5,000/s to 50,000/s
You need to sort every cell that looks like a positive
Compare sorting rates of possible positives at 400/s vs 40,000/s
You need direct sorting of the cells for immediate inspection and assay
Compare recovery of sorted cells onto a slide vs into a large collection volume
which itself has to be concentrated and so will have large adherence losses.
You need live/dead discrimination simultaneous with Mab positivity
Compare no UV-Ho33342 capability to having a UV source on the machine
You need to find the origin of the contamination, so... a rapid multi-sample
processing system
Compare T model Calibur to Ferrari Vantage or MoFlo
I still think the Calibur is the wrong machine.
Cheers
Bob
-----Original Message-----
From: Howard Shapiro [SMTP:hms@shapirolab.com]
Sent: Wednesday, September 16, 1998 8:17 AM
To: cyto-inbox
Subject: Re: Sorting Sydney's water
Bob-
I think that for water analysis, it's probably better to do extreme
concentration and analyze a very small volume of fluid; there are few enough
parasites, even in the concentrated sample, so that high speed sorting
doesn't offer a substantial advantage. Also, they are trying to optimize
the reagent technology (better monoclonals, etc.) so they don't need to sort
at al. B-D seems to have taken an active interest in this, or at least in
Duncan Veal's program at Macquarie, which is where the methodology was
developed. He'd be the best person with whom to continue this discussion.
-Howard
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