From: robert ashcroft (cytomat@netcore.com.au)
Date: Mon Sep 28 1998 - 08:49:05 EST
Dear Howard and List members,
As you can have too much of a good thing, this will be my final word on this
topic, unless something gratuitous turns up.
=======================================================================
RESET and REVIEW
=======================================================================
The Sydney water situation is of concern to many of us, and I raised the issue
because I did not believe that the FACSCalibur is the best instrument for the
task at hand.
Having examined the responses of Howard Shapiro and Mark Gauci, I still don't.
There is clearly a gap between my opinion and that of Duncan Veal, Mark Gauci
and the Australian Environmental Flow Group, AEFG, at Macquarie University, and
they are obviously committed to travelling all the way with a FACSCalibur, for
water or money.
I am not attacking the quality of the AEFG's work, but rather suggesting that
they
might be better able to do it with a better performed sorter. Indeed, they are
doing
great work in a difficult area, and like everyone else, I want them to succeed.
What I have particularly wanted to do is dissect the situation enough to enable
the key
measurement and diagnostic questions to be aired, and the choice of a fluidics
sorter against a droplet sorter to be fully reviewed.
The response of the List has been almost non-existent, which suggests that
no-one is coming to Sydney in 2000.
I am surprised.
The best result for all of us, and surely for Duncan and Mark, could well be
that BD would provide to
Macquarie's AEFG, a Vantage droplet sorter (plus operator) as well as the
existing Calibur, should there be superior performance features intrinsic to
the droplet sorters; so let's see if that might be the case.
I strongly assert that the use of modern droplet sorters in this application
should not be discarded by Sydney or the world at large in favour of a fluidics
sorter, after an inappropriate comparison against a long-superseded 1980's
EPICS droplet
sorter, the machine used by Duncan's colleague and pioneer of much of this
work, Dr Graham Vesey.
We do operate in a world of tight budgets I know, but
the long term costs of Sydney's outbreaks might be best contained by an
independent assessment. See the US$36 million reference below.
CONTEXT
The record of events and consequences...
In the past three consecutive months, Sydney's water has been declared unsafe,
on the basis of assay results using the AEFG method.
The cost to the NSW State government of these outbreaks already exceeds US$36
million (source= The Age Newspaper, 26th Sept, 1998) . A whole lot more cost is
going to come through in a plethora of class actions being led by a prominent
law firm, Slater and Gordon.
According to newspaper and TV reports, the AEFG method has detected and
reported the presence of high numbers of parasite oocysts, declared as
infective, and being at hazardous concentrations in some samples, from 100 to
10,000 organisms per 100 litres of water.
Inspite of these alarming reports, not one case of Giardia or Cryptosporidium
infection has been reported that
can be attributed to drinking Sydney's water.
In no way is this due to the people's rapid uptake of the warnings and adopting
safe drinking practice, as compliance would not have attained even 75% in the
first few days.
None of the authorities has been able to provide any clue as to the origin of
each of these outbreaks, nor why there were no associated illnesses.
Technically, the concentrated sample of 0.1 to 1.0 mL is obtained from a
starting volume of 100L.
The resultant sample contains lots of particles (plant matter and grit) which
non-specifically bind the (added) fluorescent antibodies and/or are themselves
intrinsically fluorescent, so that a predominance of false positives (what is
the figure, Mark, Duncan?) is
reported by the detection method. Sorting is imperative at this stage of the
program's development.
The Calibur uses the green/orange collection bands excited by 488nm laser
light, plus light scatter parameters as its discriminators.
It does not employ any live/dead discrimination during measurement. It does not
measure any intracellular property of the target parasites by flow cytometry; a
second (UV) laser would allow both these avenues to be explored.
QUESTIONS STILL TO BE ANSWERED
Was the AEFG method reporting genuine parasitic organisms?
No one has got sick, though many people drank contaminated water, so what is
wrong with the AEFG assay?
Does the AEFG method detect the infective state of the particles?
Does the AEFG method detect/report the live/dead status of the
organisms?
Was mass screening undertaken of a range of possible originating sources by the
AEFG method?
If so, what were the results?
If not, why weren't they?
Are there any plans to attempt this in the future?
If it wasn't sorting, what was the rate-limiting step in sample processing?
50 samples in one day at 15' a sample suggests a long day for the
operators.
How can the assay be bettered and the water kept safer?
ADVANTAGES OF A DROPLET SORTER
In measurement alone, the droplet sorter provides these advantages over the
Calibur,
more fluorescent properties per cell at each measurement (eg, via a
second UV
laser),
faster sample volume throughputs and higher measurement rates,
In sorting, the modern droplet sorter has further advantages over the Calibur
sorting directly onto slides
sorting at higher thoughput rates, using large diameter nozzle tips (0.2
&
0.4mm)
automated control of droplet break-off on some machines.
The combined effects of these properties are the following:
The assay's machine time for a 1 ml sample could be reduced to <5 minutes,
Additional discriminating properties could be measured simultaneously with
antibody markers, then used in sorting,
No specialised operator would be needed for some types of droplet sorting
machines (eg., SortMaster on MoFlo),
Large scale screening programs could be undertaken,
The capacity to develop diverse new cellular markers would become an option.
SUMMARY
As far as it goes, the assay is effective, but it needs additional work to
achieve the following,
simultaneous reporting of live/dead status of the parasites,
simultaneous reporting of infective status of the detected organisms,
suitability for large scale screening of water samples.
A droplet sorter would immediately allow mass sample screening according to the
third goal. It would immediately allow a
thorough investigation of possible ways of achieving goal number one. The
second goal would likely also benefit, given the enhanced measurement
capability of
modern droplet sorters.
The effect of using the present assays has been to put Sydney Water $36 million
behind the 8 ball.
With what is at stake, there is surely a case for reviewing the present
instrumentation.
===================================================================
Finally, I do rebutt Howard's claim that I was shouting at him, or being
zealous. My days of zealotry are long past, but that's another story.
In responding to Howard, I was vigourous and assertive, YES, but shouting, NO.
Howard did, however, put my email to mischievous use, but that's politics!
I used the CAPS simply to discriminate what Howard had written formerly from
what I WROTE IN REPLY.
It makes it simpler for the visually impaired, such as I am these days. I
apologise though, for any offence he may have taken.
===================================================================
Let's stay on track here.
Bob
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Dr Bob Ashcroft, Research Consultant
Page+61(3) 9625 3692; Fax(03) 9521 0841
r.ashcroft@unsw.edu.au
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