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Ali Mukherjee, et al. Expanding the Utility of High-Sensitivity Dried Blood Spot Immunoassay Testing with Single Molecule Counting. J Appl Lab Med 2017;2:674-86.
Dr. Jeff Bishop is Senior Vice President of Diagnostics Operation at Singulex in Alameda, CA.
Hello, and welcome to this edition of “JALM Talk” from The Journal of Applied Laboratory Medicine, a publication of the American Association for Clinical Chemistry. I’m your host, Randye Kaye.
Clinical testing using whole blood dried on filter paper has been performed since the 1960s. The use of dried blood spots reduces the cost of sample acquisition and offers numerous benefits including simplified storage, transport, and reduction in the risk of infection for laboratory personnel.
Despite these clear advantages, there are significant pre-analytical challenges related to errors from improper collection: sample-to-sample variations in hematocrit, interference from blood cells, and the need for efficient analyte extraction. These challenges coupled with a small sample volume available for analysis have restricted the adoption of dried blood spots for analytes.
Overcoming the issues requires advancements in both dried blood spot quality control and analysis. Diagnostic applications of single molecule counting (SMC) technology make detection of low abundance biomarkers possible that were previously undetectable using dried blood spots.
Recently, a method was described that combines the sensitivity of single-molecule counting with the convenience of dried spot collection. The study evaluates three conventional protein analytes routinely measured in the clinical lab: Troponin I, Prostate Specific Antigen (PSA), and C-reaction protein. The authors detailed the assay method that incorporates spot-scanning to account for dried blood spot variability followed by analysis with single-molecule counting. “Expanding the Utility of High-Sensitivity Dried Blood Spot Immunoassay Testing with Single Molecule Counting” was published in the March 2018 issue of The Journal of Applied Laboratory Medicine.
The corresponding author is Jeff Bishop. Dr. Bishop is Senior Vice President of Diagnostics Operation at Singulex and has worked in the diagnostics industry for more than 18
years. Dr. Bishop has led the development of many drug
and biomarker assays on numerous platforms across a wide
range of disease states. He is our guest for today’s podcast.
Welcome, Dr. Bishop.
So, what is new or special about the assay method
described in this paper?
The assay method is really, I think, a win-win situation. We
took two things which are dried blood spots sample
collection and then single-molecule counting technology and
we combine them in a way that makes both of them better.
So dried blood spot sample collection is easy and
inexpensive but it has limited utility, because many of the
assays require either more or better controlled samples in
order to provide meaningful results. So single-molecule
counting technology is a high-sensitivity immunoassay
technology developed by my company, Singulex, that
overcomes the limitations of dried blood spot sample and
provide the clinically meaningful results for many protein
assays that couldn’t otherwise be measured with a small
So it’s a case of the whole is greater than the sum of its
parts. Can you explain in case somebody doesn’t know,
what exactly is single-molecule counting technology and
how does it work compared to other immunoassay
Sure, I’d be happy to. So single-molecule counting
technology has about a thousand times more sensitivity
than existing technologies, and it’s great because it reveals
the presence or absence of disease more clearly and
definitively than was possible before it came along. To
explain how it works, the technology uses a combination of
a brightly fluorescent dye and a confocal microscope along
with extremely sensitive single-photon avalanche photo
diode detector to gather signals from individual molecules.
The design of the optical system combined with our
proprietary analysis methods provides a much greater signal
compared to the background noise than other traditional
immunoassay methods. All that sounds complicated, so one
of the examples I use to try and illustrate this is, imagine
that you’re in a concert hall with a thousand people and
they all begin applauding at the end of a performance. If I
ask you to listen to the sounds created by one individual in
that group, you couldn’t do it because there’s too much
background noise created by the other 999 people.
Now, imagine you’re in that same concert hall and it’s
empty except for one person. Now if that person claps you can hear them even though the noise that they’re making is
the same as it was before. So if I bring that back to single-molecule
counting technology, what our optical system,
what our instrument does, is it allows as to focus both the
light source and the detector on a very small area so that
we can count individual molecules and because of that we
can see more of them than anybody else.
That’s a great analogy, thank you. That really helped me to
understand it. So, why would you combine this single-molecule
counting technology with dried blood spot
sampling? What’s the advantage of that?
Yeah. So as I said before, dried blood spot sampling has
some limitations, so I think the biggest of that is the overall
sample volume, so I think, probably, the most common
reason people might have been exposed or have seen dried
blood spot sampling before is in newborn infant screening.
If you have a child, they grab the child, turn him upside
down and prick their heel and get as much blood out of the
screaming baby as they can, and you don’t get a lot of
blood. And also, it’s not really easy to measure that
amount, it’s just there’s a lot of limitations associated with
that sample. And because single-molecule counting
technology provides ultra sensitivity, one of the applications
of that sensitivity is that a lot of people don’t think about is
you can then dilute a sample down if you need to have more
volume there and then still we have enough sensitivity to
measure analytes in that sample even if you didn’t have
much sample to begin with or you had to dilute it down.
As part of this, we also introduced all of the known and
documented quality control steps that others have used
before us in order to ensure the quality as much as possible
of these samples. So this is things such as punching out a
precise area of the paper, of the dried blood spot paper
using efficient extraction buffers and then visually reading
the optical density of the blood spot before we begin in
order to know how much blood we actually started with. So
we did all of the things that other people have done but
then by combining that with the sensitive assay technology
of single molecule counting, we just get better and more
meaningful results than a lot of people have gotten in the
So you chose to validate this method with specific assays
that you chose. Why did you choose to do that?
Yeah. So, the three analytes, the three biomarkers that we
measured in this specific paper were cardiac troponin, cTnI,
Prostate Specific Antigen, or PSA, and then C-reactive
protein, or CRP. So we had several reasons for choosing each one of these and maybe I’ll just explain each one really
First, these three biomarkers are all very well known in the
clinical chemistry field. I think anyone reading the journal
or listening to this podcast has probably heard of each of
these three markers, so that was first. Specifically cardiac
troponin I, it’s really the gold standard biomarker for
detecting cardiac injury in something like a heart attack or
also known as a myocardial infarction.
So this test is been an important one and if you look at any
issue of Clinical Chemistry or Journal of Applied Laboratory
Medicine in the last five years, there’s almost undoubtedly
an article about cardiac troponin in there. It’s a very
popular and very good marker. We have a cardiac troponin
assay in our laboratory here at Singulex so we know a lot
about it and it made it easier for us to gauge the
performance of our dried spot assay because we had
something already in hand to compare it to, so that’s
PSA, Prostate Specific Antigen, it’s an important marker in
the detection of prostate cancer and it’s not a perfect
marker in terms of specificity, meaning just because you
have an elevated PSA doesn’t mean you have prostate
cancer, but it’s still an important marker and its widely
used. But in prostate cancer patients that have had their
prostate removed either through surgery or to radiation
treatments, it has been shown that very small amounts of
PSA remaining after that procedure are predictive of cancer
recurrence. Therefore, it would be beneficial to periodically
monitor these patients, and the dried blood spot collection
method is a simple inexpensive way that could make this
periodic monitoring much easier.
And then, C-reactive protein, CRP, it’s a very widely used
marker of inflammation across a wide variety of disease
states and it’s present at relatively high concentrations
compared to troponin and PSA, so it seems like just a good
addition to the paper to show that we can measure analytes
at low concentration and high concentrations and that the
method is not limited to any one marker or one range.
Wow. So you’ve actually already started to answer my next
question I think, which is about some of the unique
applications for this assay technology. Are there any more
you’d like to add to what you’ve all ready told me?
Sure. This application of the technology is ideal in situations
where blood is either difficult to obtain in large quantities or
when it’s going to be needed frequently like in the case of
monitoring that I just mentioned or when the biomarker to be measured is present in very low concentrations, for
example, cardiac troponin.
One example is chronic disease management. If I wanted
to test your blood for a specific biomarker, let’s say, cardiac
troponin, and I needed to do so on, let’s say, a weekly or a
monthly basis, you’d quickly get tired of having someone
sticking a needle in your arm every time you needed blood
to do that. Also, depending upon the volume that was
needed and the frequency that you are going to do that, it
might actually pose a health risk to you in terms of anemia
or your general well-being.
Another example might be for use in resource-limited
setting such as under-resourced countries of any other
location where the patient and the laboratory are not in the
same place. The dried blood spot samples are stable at
room temperature. They can even be mailed through
regular postal couriers for the cost of a stamp, so it makes
transporting blood much easier.
One of my personal hobbies is running marathons, and so,
one of my favorite examples of an application is a paper
that we’re hoping to publish later on, but we did a study
where we actually went to a marathon and we collected
samples from runners both before and after they ran the
race. Many of these runners wouldn’t wanted to have had a
traditional blood draw prior to running the race but they
were happy to have a small finger stick sample taken. We
were then able to measure their cardiac troponin levels
before and after the race and compared those results to
some of the training and health history questions that we
asked them. So studies like that show the promise of this
technology and help us see things that would have been
difficult or impossible to do in the past.
To summarize, the new method makes sample collection
cheaper, easier and more accessible and then the assay
technology makes it possible to measure biomarkers that
were not previously measurable and this study validates the
method and then we also suggest the number of these
possibilities in the paper for how it might be used in the
Wow, that is so interesting and so much less invasive to the
person being tested, especially before and after a marathon.
I can’t even imagine that. Thank you so much for this very
interesting interview and have a great day.
Thanks, you too.
That was Dr. Jeff Bishop from Singulex talking about the
JALM publication, “Expanding the Utility of High-Sensitivity Dried Blood Spot Immunoassay Testing with Single Molecule
Counting” for this podcast. Thanks for tuning in for “JALM
Talk.” See you next time and don’t forget to submit
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