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Principles of Exposure Assessment

we're going to discuss exposure biomarkers

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in environmental epidemiology.
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So the term biomarker can be used
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to describe a marker of exposure
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or the marker of effect
among other things,
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but we're really gonna be focusing on
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how to utilize these biomarkers
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to assess exposure in humans
to environmental chemicals.
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So the way the lecture
is structured first,
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we're gonna talk about the
different types of biomarkers,
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how we go about selecting them,
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then how to deploy these measurements,
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and then once we've measured
these concentrations
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or levels in individuals,
how then to analyze the data.
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And then finally some of the pitfalls
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that we need to think about.
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So considerations that
we need to think about
0:53
when we select these
biomarkers, first of course
0:56
science should drive
our choice of biomarker.
0:59
We need to think about what
the suspected mechanism is
1:03
and what the target system or organ is.
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So for example, if we're interested
1:09
in how methylmercury affects the brain,
1:11
the ideal biomarker might be levels
1:16
of methylmercury actually in the brain.
1:20
Now, that's really not
feasible unless we're dealing
1:23
with deceased individuals.
1:28
So, what's the next best thing?
1:30
Well, often we rely on circulating
concentrations in blood.
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We might rely on hair for example
1:38
as a biomarker of that exposure.
1:40
But we should be thinking about how well
1:42
that exposure represents the
true level of methylmercury
1:45
in that suspected target system or organ.
1:50
Secondarily, hopefully
to that is feasibility.
1:55
So I mentioned that we
can't assess methylmercury
1:59
in the brain.
2:03
So what's the next and
most feasible option?
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Collecting it in hair,
collecting it in blood.
2:09
Again, maybe we would want to
collect a biomarker in blood,
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but perhaps we're dealing with infants.
2:17
We don't wanna draw blood
2:19
so we might use another
biomarker such as hair.
2:20
So in relation to that
invasiveness for example,
2:24
that I mentioned like venipuncture
2:28
in infants or children is cost.
2:30
So some of these persistent
organic pollutants like PCBs,
2:33
perfluorinated compounds,
DDT, DDE are quite expensive.
2:39
So if you send a single sample,
2:43
plasma sample or serum sample
2:47
to a lab, it might cost about $750
2:49
to ascertain all of those
various concentrations
2:53
so it can get expensive
2:57
and that's something that
we also need to think about.
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And again related to invasiveness,
3:03
for some of these concentrations
like furans and dioxins,
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if the concentrations are very low,
3:12
it might take a lot of blood
3:14
in order for us to determine
the actual concentration
3:16
so seven or eight, 10ml tubes.
3:20
So a 10ml tube, typically
if you got your blood drawn,
3:24
this is the small tube that is used
3:26
to collect whole blood, imagine
seven or eight of those.
3:30
I've already mentioned
blood several times.
3:37
So blood itself is not a biomarker,
3:40
but it is a matrix in
which the biomarker exists.
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So really what we're
gonna be talking about
3:48
are these different matrices
3:50
that you can assess these biomarkers in.
3:52
So blood, we think about
whole blood, plasma, serum.
3:56
So the difference
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is that let's say you have a test tube
 4:03
and you might draw an
entire tube of whole blood
4:08
which contains clotting factors,
red blood cells, et cetera.
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If we separate off the red blood cells,
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what we're left with
4:31
is rather plasma.
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Serum is plasma minus
the clotting factors.
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Why does this matter in terms
4:53
of assessing different
environmental exposures?
4:54
For some environmental
exposures like lead,
4:57
we're gonna use whole blood.
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For something like persistent chemicals
5:05
such as perfluorinated compounds and PCBs,
5:08
we're gonna use either plasma or serum.
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It doesn't really matter,
5:15
these clotting factors
that get removed as you go
5:17
from plasma to serum don't
really affect the determination
5:21
of these concentrations very much.
5:24
So, usually either of
these matrices are fine.
5:26
So again, whole blood is used
for things like blood lead
5:31
and other metals.
5:36
It's easily obtained.
5:37
Nobody likes getting a skin prick,
5:40
but it's generally easy compared
5:41
to other more invasive measures.
5:44
In some cases, we can
do a fingerstick option.
5:47
So for determining blood
lead, often in the field,
5:52
you can do a little finger
prick to draw that small amount
5:55
of whole blood.
5:59
Often an issue there is
surface contamination.
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If there's a lot of handling
6:04
of lead materials for example,
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people who are taking apart batteries,
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there may be quite a bit
of service contamination
6:15
of lead on their hands.
6:17
So finger stick would
not be the ideal option.
6:19
And as I mentioned
before, plasma or serum,
6:24
these persistent often
lipid soluble compounds
 6:27
so things like PCBs that are bound
6:31
to lipids are more
amenable to determination
6:35
in plasma or serum.
6:40
The nice thing about plasma
and serum is it can be stored
6:42
for decades without much loss
in terms of these chemicals.
6:45
If you take something like
whole blood and you store that
6:52
for a while, there's lots of problems.
6:55
The red blood cells burst
and it can't be stored.
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So typically if you're gonna
measure something like lead,
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you need to do it right away
7:06
whereas if you're
measuring other compounds,
7:07
you can collect blood
7:09
and then subsequently analyze it later.
7:11
Urine again is another matrix
for which we can determine
7:17
these different exposure biomarkers.
7:22
Urine is typically used for
readily metabolized compounds.
7:25
Compounds like Bisphenol A (BPA)
7:29
which I'm sure you've all heard of.
7:32
This is in water bottles
and lots of other consumer
7:34
and personal care products, perfumes.
7:38
Things like phthalates
also again are plasticizers
7:41
in personal care products and perfumes.
7:46
And organophosphate pesticides
that are used on crops.
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These compounds typically
have short half lives.
7:55
They're not typically lipid soluble.
7:58
They're ingested, they get glucuronidated
8:01
in the liver and then they are excreted.
8:05
So for example, Bisphenol
A has a half life
8:08
of about six hours in urine
8:11
so it's ingested and it's
it's readily excreted.
8:15
The benefit of using urine as a matrix
8:19
for determining exposure
8:22
is that it's easy to obtain in adults.
8:25
I say in adults,
8:29
in infants obviously who
aren't toilet trained,
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it's a lot more complicated.
8:35
There's different ways to assess it
8:38
and you'll see these mentioned
 8:40
in the epidemiologic literature,
8:41
the exposure assessment literature.
8:44
First morning void.
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This is what it sounds like,
8:48
it's the first urination of the day.
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And that's thought to maybe
better represent exposure
8:53
in the last 24 hours
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because you haven't
been ingesting anything
8:59
for the last eight hours or so.
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A spot urine is just a
collection at any time.
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So if for example,
9:09
you go to the doctor and they
ask for a urine specimen,
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what you're giving them
is a spot urine specimen
9:14
so just one point in time.
9:17
There are in more complicated
exposure assessment studies,
9:21
this idea of 24 hour urine
9:26
and this is collection
9:29
of every urination throughout the day.
9:30
So for example, EPA does some studies
9:33
where they measure 24 hour
urine in pregnant women
9:36
and they do so for several weeks
9:40
in order to determine what the half life
9:43
is of a lot of compounds.
9:46
Obviously, if you have
an epidemiologic study
9:48
and you're asking people
9:51
to collect urine each time
they go to the bathroom,
9:52
it gets a little complicated.
9:58
The problem with urine, because
it involves self-collection
10:00
is this non-trivial
contamination potential
10:04
which we'll talk about a little
bit later in the lecture.
10:07
But again, because we're
typically measuring compounds
10:10
like Bisphenol A and phthalates,
10:14
these are compounds that are in house dust
10:17
and in many other products around.
10:20
And so, when an individual
is donating the specimen
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and they open up the jar
and they open up the cap,
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there's the possibility
to introduce quite a bit
10:30
of contamination that way.
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I'm gonna spend a little bit
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of time talking about less common matrices
10:40
in which we assess biomarkers.
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One example is meconium.
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Those of you who don't know,
10:49
meconium is the first bowel movement
10:50
that the infant has after
birth or right around birth.
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Obviously there are
some collection problems
10:57
because first, sometimes
this occurs very early.
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Second of all, if you're collecting this
11:05
for the purposes of ascertaining
exposure concentrations,
11:08
there's again the potential
11:15
for quite a bit of contamination.
11:17
Why?
11:19
If it's collected in a diaper,
11:20
there are already
contaminants in that diaper.
11:22
The question though is
why is this interesting?
11:25
It's interesting because it captures
11:28
what that infant was exposed
to throughout gestation.
 11:31
Think about all of the products that
11:36
that infant has metabolized
during that process
11:38
and been exposed to in utero,
11:41
that meconium is a representation
of all that exposure.
11:44
So sometimes this is
used in studies as a way
11:50
of assessing exposure during pregnancy.
11:53
Another less common biomarker
11:58
that's used are dried blood spots.
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So in New York State, from each infant,
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a dry blood spot is collected.
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A few drops are spotted onto a card.
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It gets sent to Albany
for genetic testing.
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And those cards are then stored
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for I believe 20 or 25 years in Albany.
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One could go back with proper
approval from human subjects
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and measure concentrations
of some pollutants
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on those cards.
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So for example, there is an individual
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at Wadsworth lab in Albany, Dr. Kannan
12:41
that I collaborate with,
 12:45
who has pioneered using
these dried blood spots
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to measure some persistent
organic pollutants
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and also some non persistent
organic pollutants.
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Not typically well measured,
12:57
the non persistent pollutants
in these dry blood spots,
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but certainly a good measurement
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for things like perfluorinated
compounds and PCBs.
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Something like this might
be particularly useful
13:10
when you have a large population
with maybe a rare outcome.
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So let's say we're looking at brain cancer
13:20
and we're doing a case control study.
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And so we identify cases of individuals
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who develop this brain cancer
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or some other cancer
in their early twenties
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or maybe late teens,
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but we're interested in the effect
13:39
of an early life exposure.
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So, there's not a way to go
back and easily ascertain what
13:43
that person was exposed to
say, 15 or 20 years prior.
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Dry blood spots are useful for this
13:55
because you have an
existing record potentially
13:57
of exposure during that early life period
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even what that person was
exposed to during gestation.
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So sometimes we can think
about if you had a rare disease
14:09
and you had a large population
14:13
from which those cases accrued,
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using these storage dried blood
spots might be a useful way
14:19
of going about assessing exposure.
14:24
Another less common matrix
here is amniotic fluid
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and this is very similar to meconium.
14:34
So again, this is a representation
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of what that exposure was like
14:40
in the fetal compartment,
in utero during gestation.
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Breast milk is more commonly used
14:49
than these other biomarkers mostly
14:51
because it's readily available.
14:54
Most women try at least to breastfeed
14:58
even if they don't continue for very long,
 15:01
they do at least try and so
early in life it's possible
15:04
to collect breast milk.
15:08
So colostrum refers to
milk very early in life,
15:11
transitional milk is
then a little bit later
15:16
and then mature milk after a
couple of weeks after birth.
15:20
Breast milk is useful because
it's a very lipophilic.
15:25
There's a lot of fat,
cholesterol in breast milk.
15:28
And so compounds that are bound
15:31
to these lipids like PCBs,
other organochlorines,
15:35
breast milk is a useful way
15:41
for measuring those concentrations.
15:43
And also, if breast milk is
really the only dietary source
15:45
for an infant, it's also
gonna be just about one
15:51
of the only sources of exposure
for many of these compounds.
15:55
Bone is much less used,
16:01
but it is used quite
frequently in studies of aging,
16:04
particularly for metals like lead.
16:07
There's been some studies
16:11
in the normative aging study out of Boston
16:12
that have measured lead levels in bone
16:16
and that can be done through x-ray.
16:20
Typically by x-raying
bone in the long part
16:22
of the leg and in the patella in the knee.
16:28
That's a useful way to measure lead
16:34
because again, it's not invasive
16:36
and the nice thing about that
16:39
is that concentrations of metals like lead
16:41
in bone represent a cumulative measurement
16:45
of lead throughout the life period.
16:50
So, if you're thinking about the exposure
16:52
as being a cumulative process,
16:56
bone might be a good way of
going about measuring them.
16:59
I mentioned before that for
organochlorines like PCBs,
17:06
they're lipid bound.
17:14
As a result,
17:16
using something like adipose
tissue may be a great way
17:18
to assess body burden.
17:22
So this is a title from an actual paper,
17:24
"A Prospective Study of Organochlorines
17:26
"in Adipose Tissue
17:29
"and risk of Non-Hodgkin Lymphoma."
17:30
So concentrations of PCBs
were measured directly
17:33
in adipose tissue.
17:37
This is not commonly used much anymore.
17:40
It was thought
17:43
that maybe adipose tissue
better represents body burden,
17:44
but as you can imagine,
it is somewhat invasive.
17:49
I do have a collaborator
again in Albany, New York
17:53
who has used adipose tissue
to do some bio monitoring
17:57
and they have a noninvasive
way of collecting that.
18:03
They actually get adipose
tissue from plastic surgeons
18:07
who have been doing liposuction
18:11
and who would otherwise
discard that medical waste.
18:13
And they've used it
18:16
to ascertain different
chemical concentrations.
18:17
Nail clippings, these are useful
for a lot of trace elements
18:23
and metals that sequester in
areas of the body like hair,
18:27
teeth and nails.
18:34
This is mostly used again
for trace element analysis.
18:36
And those of you who are clinically aware,
18:40
you'll know that
sometimes changes in color
18:44
of the nails may represent
different types of poisonings.
18:48
The teeth, we'll talk about in a minute.
18:52
And I also mentioned that
hair is a useful biomarker.
18:57
Hair is useful for a couple of reasons.
19:01
It's first of all, noninvasive.
19:03
Second of all, there's
not much contamination
19:05
if you treat it and handle it properly.
19:08
And thirdly, hair grows at
a pretty predictable rate.
19:11
So if you think about
an individual's scalp
19:15
and you think about the hair
growing out of their scalp,
19:18
and this is the longest most distal date,
19:23
and this would be obviously
the most recent date,
19:30
you can count up the amount
of time that has elapsed.
 19:34
So let's say that each of
these is one centimeter
19:44
and you know that hair
grows one centimeter,
19:49
let's say every two weeks.
19:55
You can say something
about exposure here that,
20:00
that was say 20 weeks ago,
18 weeks ago, 16, et cetera.
20:04
So if you think about measuring
that during pregnancy,
20:12
it may be that if this is hair collected
20:16
from a woman right after pregnancy,
20:20
and let's say this is three months,
20:23
and this is six months,
20:27
capturing that hair in this period
20:29
is gonna give you the
concentrations of mercury
20:32
that she was exposed to during pregnancy.
20:35
So hair is also a very useful biomarker.
20:38
I previously mentioned teeth,
20:43
and they're a pretty
interesting biomarker.
20:45
Teeth grow a lot like
concentric rings on a tree.
20:50
So on a tree,
20:55
this is when the tree
starts beginning growth,
20:57
and then each year or
approximately a new ring is added.
21:03
And teeth grow approximately the same way.
21:11
So if we think about a tooth as a matrix
21:15
in which to measure exposure
to environmental contaminant,
21:19
we can imagine that varying the depth
21:25
at which we assess the concentration
21:27
will give us different
information about when
21:31
that particular exposure occurred.
21:35
So a less deep measurement
would tell us something
21:37
about more recent exposure
and a deeper assessment
21:42
in that tooth would tell us
more about later exposure
21:47
or exposure that occurred in the past.
21:52
That's why I'm calling this
retrospective exposure.
21:55
So it can give us
information about the past.
21:58
These are data from work by Manish Arora
22:05
who is the primary investigator on this
22:10
at Mount Sinai, New York
City and his collaborators.
22:14
This was published in "Nature" in 2013.
22:17
What this is looking at
are barium/calcium ratios.
22:20
The reason that they look at this ratio
22:26
of barium to calcium
22:29
is that the placenta restricts barium.
22:31
So if we think about,
22:35
say these two different
ratios for example,
22:38
and we have a ratio that is in utero
22:43
and then after birth so potnatally,
22:49
the difference here is that
there's no longer a placenta
22:57
so the placenta is gone after birth.
23:00
So what we would expect if we look
23:03
at the barium to calcium ratio
23:05
is if barium is being
restricted by the placenta,
23:14
we would expect a smaller fraction
23:19
in the in utero period and after birth
23:27
because barium is no
longer being restricted,
23:30
we would expect a larger fraction.
23:36
So if we take that information
and apply it to these figures
23:41
on the left,
23:44
what we see here are measurements
in two different parts
23:46
of the tooth, one in the
dentin and one in the enamel
23:52
and that's not particularly important.
23:57
On the Y axis, we have the
barium to calcium ratio.
24:00
And on the Y axis here we have time,
24:05
and this is in days.
24:11
What the vertical lines represent
in each figure is birth,
24:16
and so this would be the postnatal period,
24:22
and this would be the prenatal period.
24:26
So again, what I mentioned
before is if this ratio
24:33
is smaller during the in utero period
24:41
which we would expect because
of the restriction of barium,
24:44
we would have a smaller fraction,
24:48
which is exactly the case
here in both of these,
24:50
the fraction is smaller.
24:56
Than after birth,
24:58
because barium is no
longer being restricted
25:01
by the placenta, we see larger
ratios of barium to calcium.
25:06
This was simply a demonstration
 25:14
that by measuring the
barium/calcium ratio in a tooth,
25:17
we can document exactly
when birth occurred relative
25:23
to the development of the tooth.
25:27
So they took this knowledge
and they applied it
25:30
to environmental exposures.
25:33
And basically the data
look something like this.
25:37
So there's a lot of information
contained in this figure.
25:46
We have data that we can use to ascertain
25:53
when the peak exposure
occurred and how high it was.
25:59
We can think about a
cumulative exposure metric.
26:04
We can think about average.
26:09
And we can think about these
for the postnatal period
26:13
as well as the prenatal period.
26:19
So for example,
26:24
if we wanna think about
the peak concentration,
26:26
this is obviously the peak level,
26:31
we can talk about when it occurred.
26:35
We can also measure cumulative exposure.
26:39
For example, by taking
area under this curve.
26:44
We could think about cumulative
exposure perhaps only
26:49
in the prenatal period.
26:56
We could think about
maybe average exposure
26:59
which would be something like this.
27:04
And again, we can apply this idea
27:09
of peak and cumulative
average specifically
27:13
to the postnatal and prenatal period.
27:17
So as I showed you here,
27:20
this is cumulative just
for the prenatal period.
27:23
And we could think
about perhaps an average
27:26
for just the postnatal period.
27:29
So again, this one tooth
27:32
is providing lots of
different exposure data
27:35
for this individual.
27:40
Peak exposure, cumulative,
average, time specific.
27:42
So let's say that the outcome
of interest you thought
27:46
was a neurodevelopmental outcome,
27:51
and let's say that based on animal studies
 27:54
and other mechanistic studies,
27:57
you believe that the critical period
28:00
for brain development occurs
at 15 days before birth
28:03
so somewhere in here.
28:09
So using this metric we can say,
28:11
"I know exactly what the
concentration was on day 15."
28:15
And so that might be
the relevant measurement
28:20
that you're gonna use in your study.
28:22
So this is pretty incredible
if you think about
28:25
how we're going to assess
exposure in an individual.
28:28
This is extremely costly.
28:32
It takes a lot of analytic
expertise to do this.
28:34
But again, really interesting information.
28:39
And the example here is for lead.
28:42
Again, they have it adjusted
by the calcium concentration
28:45
and that's for analytic reasons,
28:49
but you can just think about this
28:50
as a relative measurement
of the amount of lead
28:52
that this particular infant is exposed to.
 28:55
So really, really some nice work here
28:58
by Manish Arora at Mount Sinai.
29:01
Here's an example for
methylmercury exposure
29:07
and fish consumption.
29:10
And there's a lot of information here.
29:12
This is from a paper published in 1983,
29:15
and this is a single individual.
29:20
There's a couple messages
here in this figure
29:23
that I wanna discuss.
29:27
So what happened here is
this individual ate fish
29:29
for some amount of time, about 100 days.
29:34
Then measured methylmercury
in three different matrices,
29:41
blood, hair stubble and head hair.
29:46
So here are the blood
measurements in blue.
29:51
We have the hair stubble
here, these small dots.
30:04
And then we have the head hair.
30:13
And I talked about this example
30:15
of measuring hair during
pregnancy and the ability
30:18
to ascertain discrete
time windows of exposure.
 30:23
So on the X axis, we
have time, we have days.
30:30
On this Y, we have mercury
concentration in blood.
30:35
And on the other Y axis,
30:40
we have the measurement
of mercury in hair.
30:41
And they're just different concentrations.
30:47
One is parts per billion and
the other is parts per million.
30:51
So there's several takeaway points.
30:57
First of all, we can say that fish intake
30:59
is positively associated
with methylmercury exposure.
31:02
Why?
31:06
As fish intake goes up over time,
31:07
so does methylmercury concentration.
31:11
We see that here in blood,
31:15
we see that in the stubble
31:19
and we see that in the head hair.
31:21
It's the first takeaway.
31:24
The second piece is
that once fishing intake
31:26
is discontinued, mercury
concentrations decrease.
31:30
So again, that supports causality
31:34
in terms of the methylmercury
exposure is caused
31:38
by the increased fish consumption.
31:42
Another point to take away from this
31:45
is that even though these
are different biomarkers
31:47
of exposure, we're using
hair from the head,
31:53
stubble from the face and blood,
31:56
they're all correlated.
31:59
There's a little bit of a
lag here with head hair.
32:02
It seems to peak a little
bit later than the others
32:07
so it's kind of slid this way,
32:10
but it is this kind of 20 day mean.
32:12
But in general, these
are highly correlated.
32:15
Concentrations say at
day 40 look very similar.
32:18
Concentrations at about day 100 or 120
32:24
and concentrations at day 200.
32:28
The level may not all be the
same but relatively speaking,
32:31
they track and would thus be correlated.
32:36
So we can think about
these different matrices,
32:39
but at the end of the day, as
an investigator, you may say,
32:44
"Which of these am I going to use?"
32:47
Well, certainly getting head
hair is probably the easiest.
32:49
So maybe you're gonna
use head hair, right?
32:56
Stubble, that's gonna be problematic
32:59
so you probably don't wanna use that.
33:02
Maybe you wanna use blood.
33:04
So even though we're talking
about these different methods
33:07
to assess exposure,
33:12
these different matrices
at the end of the day,
33:13
they may be highly correlated
33:16
and that's exemplified on the next slide.
33:19
So this isn't methylmercury,
33:24
this is looking at PCBs
and they're measured
33:26
in several matrices.
33:32
These are mother-infant pairs.
33:33
And we have maternal milk,
placenta, cord tissue,
33:36
and cord serum so these
four different matrices.
33:40
And we also have measured
maternal serum so five in all.
 33:46
So these four are on the Y axis.
33:54
And on the X axis, we have maternal serum.
33:59
So maternal serum is being
compared with concentrations
34:01
in these other different matrices.
34:06
So what do we know about PCBs?
34:11
Well, PCBs are lipophilic.
34:13
These are lipid based concentrations.
34:15
Because they're lipophilic,
34:18
they're persistent and they
tend to stick around for a while
34:20
and they're readily available
in these different matrices.
34:25
So overall, what do
you see in this figure?
34:33
Well, overall you see this positive trend.
34:37
In other words,
34:41
maternal serum concentrations
are well represented
34:42
by these concentrations in
maternal milk and fetal tissues
34:46
so as one goes up, so does the other.
34:51
so again, as an investigator,
as an epidemiologist,
34:54
you may look at this and say,
34:58
"Well, do I really need
34:59
"to collect five different
exposure matrices?
35:01
"Do I need to collect serum
from mom, and milk from mom,
35:04
"and the placenta at
birth, and cord tissue,
35:08
"and cord serum and analyze these?"
35:11
This is going to get very expensive.
35:14
Rather than do that, you
may consider this and say,
35:17
"Well, which of these
35:20
"do I think best represents the underlying
35:22
"and most relevant
process that I'm after?"
35:25
So again, we can measure it in
all these different matrices,
35:30
but we should in some cases just pick one.
35:34
So now that we've decided on
your biomarker for your study,
35:40
now what do we do?
35:44
Well, we have to decide how
often we're gonna collect this?
35:48
It may not be relevant just
to collect the biomarker
35:52
at a single time point.
35:55
That's dependent on the
scientific question,
35:57
feasibility and the
reliability of the measurement.
 36:00
So for example,
36:03
let's say that you're
interested Bisphenol A exposure.
36:05
This is a non-persistent compound.
36:09
It's in plasticizers and things like that.
36:13
It's readily excreted.
36:16
The half life in urine is about six hours.
36:18
And you're interested in
kind of the total exposure
36:21
to BPA during pregnancy.
36:25
You don't have a particular
time period in mind,
36:28
but you're interested
in exposure throughout
36:31
that entire pregnancy,
36:33
say an assessment of average
or cumulative exposure.
36:34
Because of that,
36:40
your question is about all of pregnancy.
36:41
So measuring it at one
time during pregnancy
36:43
is probably not going to
well represent average
36:47
or cumulative exposure.
36:51
So then you have to think about,
36:52
"How often am I gonna measure it?
36:54
"Am I gonna measure it once per trimester
36:56
"so three measurements?"
36:59
Ideally, maybe you'd measure it every day,
37:01
several times a day.
37:04
Well, that isn't really feasible.
37:05
The third issue that is related
37:08
to some of these other
issues is the reliability
37:11
of the measurement and that's dependent
37:15
on how long this compound sticks around.
37:18
This is an example of BPA.
37:23
So we talked a little
bit about BPA previously.
37:26
And in this study, there
were thousands of women,
37:30
but we selected 80.
37:33
And for each woman,
37:37
three urine specimens were
collected during pregnancy
37:38
about wondering each trimester.
37:42
And we had 240 specimens total.
37:45
So a little more background on BPA.
37:53
It's a weak estrogenic chemical.
37:55
It's largely a dietary exposure
37:58
so we're exposed through
containers that we eat food from
 38:01
and from the food itself which
has already been contaminated
38:07
with BPA through its handling.
38:12
As I mentioned before,
it has a short half life,
38:16
six or less hours in urine and
low ICCs have been reported
38:20
in the peer-reviewed literature.
38:25
So an ICC is an intraclass
correlation coefficient
38:28
and we'll talk a bit about
that on the next slide.
38:33
And in the peer reviewed literature,
38:36
this ICC has ranged from 0.09 to 0.43.
38:38
So this is the correlation for
three or more measurements.
38:43
And in pregnant women,
38:48
three studies have reported
ICCs of 0.10 to 0.32
38:51
so not a strong correlation
between these measures
38:56
of BPA in urine.
39:01
By contrast, if you look at the ICC
39:03
for a persistent compound
like DDT, DDE, or PCBs,
39:05
and if you measured that at
three time points in pregnancy,
39:12
you would have an ICC of at least 0.9.
 39:14
So what is the ICC?
39:20
It's the intraclass
correlation coefficient.
39:22
You can think about it as
a correlation coefficient
39:25
when you have more than two measurements.
39:28
And again, just like a
correlation coefficient,
39:31
it ranges from a zero to one.
39:34
There aren't technically negative ICCs
39:42
so any of the ICC that you see
39:45
because of the way it's calculated
are going to be positive.
39:48
So in that sense, it's slightly different
39:52
from a typical correlation coefficient,
39:54
but the range of measurements
are still the same.
39:57
So why am I introducing
this concept of the ICC?
40:02
There are some simple methods
that take into account the ICC
40:07
when thinking about sample size needed.
40:13
So let's just walk back
from this for a minute.
40:18
Let's say that you have
three measures of BPA
40:20
in urine during pregnancy,
one in each trimester.
 40:26
So you develop a sample
size calculation and in it,
40:32
you determined that you have
four different outcomes.
40:40
And for each of these outcomes,
let's say, hypothetically,
40:44
you need 50 participants for one outcome,
40:48
100 for another, 200 and 500.
40:53
So basically you'd need a
study with a sample size of 500
40:57
in order to accomplish all these aims
41:03
and to analyze all these outcomes.
41:07
The assumption there with
that power calculation
41:11
is that you have perfect
measurement of your exposure.
41:14
But the ICC is a measurement
of exposure reproducibility
41:21
and that ICC is related to the sample size
41:26
that you need to adequately
power your study.
41:33
And it's not important how
this is exactly calculated,
41:38
but really what you're doing
is inflating the sample size
41:41
by the sample size over the ICC.
41:46
So let's say you assume perfect
measurement of the exposure,
41:51
the ICC is one.
41:57
So it's perfectly correlated
41:58
across those three measurements.
42:01
Well, what if it's less correlated?
42:04
What if the ICC is 0.50?
42:06
Well, using this formula
of, N over the ICC,
42:10
we would end up with 50 over 0.50,
42:18
and so we would require 100 individuals.
42:24
So that's forcing us to
inflate by a factor of two.
42:29
The issue then becomes
42:36
when you have these
substantially lower ICCs.
42:37
And this is the territory
we're in for exposures like BPA
42:42
that are measured in urine.
42:49
The ICC is fairly low.
42:51
As I mentioned on the previous slides,
42:52
the ICCs are somewhere
between 0.10 and 0.3
42:55
for measurements taken during pregnancy.
43:02
So initially we're thinking
about a sample size of say 500.
43:05
But, if the ICC has really 0.10,
43:12
you have to think about
inflating our sample size
 43:16
by a factor of 10.
43:18
So this really affects
the statistical power
43:20
and it really makes us think
about how many people we need
43:25
to adequately power our study
43:29
when there is such a degree
of measurement error,
43:31
when there is so much
misclassification of exposure.
43:35
So I'm showing you this
because I want you to recognize
43:39
that yes, these measurements
are not very reproducible.
43:43
What does that mean for
epidemiologic study design?
43:50
Well, what that means is we're
gonna need more individuals.
43:53
We've touched on this a little bit already
44:00
when we talked about
measuring say lead in teeth
44:02
that we're able to ascertain
these measures of cumulative,
44:08
and peak and average exposure.
44:12
What do these really mean?
44:15
So we can measure cumulative exposure
44:17
through something like
area under the curve
44:21
and I showed you that example.
44:23
But what does this really measure?
44:27
Typically, it's a cumulative
or irreversible effect.
44:29
Think about cigarette smoking.
44:34
So, in contrasting these two
44:36
or these three rather.
44:42
If you smoke say two
packs a day for 20 years,
44:45
three packs a day for 10 years
44:56
and maybe one pack a day for 20 years,
45:03
if it's the cumulative effect
45:13
of the cigarette smoking we're after,
45:15
it would be essentially
if we had 50 years.
45:19
So 10, 20, 30, 40, 50,
45:27
and one, two and three packs a day.
45:36
We can think about for 20
years, smoking two packs a day,
45:40
for 10 years, smoking three packs a day
45:47
and then for 20 years, one pack a day.
45:51
So this cumulative process
45:56
is really the area
under this entire curve.
45:59
So maybe that's really what matters
46:08
for our outcome of interest
 46:10
and let's say it's lung cancer.
46:11
Maybe it's that total cumulative effect.
46:13
On the other hand, it may
just be the peak exposure.
46:17
And its peak is an assessment
of it's the greatest exposure
46:21
as the causal agent.
46:25
So the two pack a day smoking
isn't enough to kind of cross
46:27
that exposure threshold to start
46:31
that underlying physiological process
46:35
which moves us toward lung cancer.
46:38
Maybe what we really need
to do is hit this threshold
46:41
of three packs a day.
46:44
We can also think about the average.
46:48
And maybe the average is somewhere here.
46:51
So we can think about that as slow
47:01
or partially reversible effects.
47:04
It's related to cumulative exposure
47:06
in the case of persistent exposures.
47:08
Cumulative and average
are essentially the same.
47:12
But instead of cumulative exposure
47:16
which encompasses more
of the time element,
 47:21
the average exposure
47:25
is really only encompassing
kind of the average dose.
47:26
What does that look like?
47:34
Well, these are actual data.
47:35
The Y axis didn't come out very well,
47:38
but these are mean blood
lead concentrations
47:40
and micrograms per deciliter.
47:43
So I'm gonna say mean blood lead levels
47:44
in micrograms per deciliter.
47:49
And this is an individual at
different ages of assessment
47:54
in months, six months, 12, et cetera,
47:57
all the way up to 72 months or six years.
48:01
And what you can see
is for this individual,
48:10
their concentrations start
48:13
around three micrograms per deciliter.
48:15
They peak between 24 and 36 months
48:17
and subsequently declined.
48:22
So again, like the two flood example,
48:26
we can ascertain a lot
of information here.
48:29
So we can calculate the peak concentration
48:31
which is about nine
micrograms per deciliter.
48:34
We can calculate an AUC
or cumulative effect
48:39
and that would be this
total area under the curve.
48:44
And we can also calculate
a mean concentration
48:50
which is gonna be related to the AUC.
48:54
But instead of it being
a total measurement,
49:03
the mean is going to be something like,
49:10
say five micrograms per deciliter
49:13
which isn't necessarily gonna take
49:17
into account the time element.
49:21
And you can see this on the next slide.
49:23
Again, we have the same individual
49:29
and we would calculate the AUC
49:34
by taking the total area under the curve.
49:38
So now that we've estimated
these different exposure metrics
49:43
of cumulative and peak
49:48
and average exposure levels,
49:51
we can incorporate that
into a data analysis.
49:55
So in this case, you can see we have peak,
50:00
we have concurrent
 50:04
which is an assessment
occurring at the same time
50:06
as the outcome assessment
50:10
and we have infancy average.
50:13
So this is from a table from
a paper published in 2003
50:15
and it's looking at blood lead levels
50:21
and how they relate to child IQ.
50:27
And these are data from a cohort study
50:34
in Rochester, New York.
50:36
Same data from the slides I
showed you just previously
50:37
where we constructed the
blood lead concentration.
50:43
And so if we just take a
look at these estimates here
50:47
in the far right column,
this is the overall estimate.
50:51
IQ was assessed at two time
points, at three and five,
50:54
but we'll just look at the
overall for simplicity.
50:57
We can look at different estimates,
51:00
one for lifetime average, one for peak,
51:02
one for this concurrent measurement
51:08
and one for infancy average.
51:11
Well, what you see is that
there's not much difference
51:15
between the lifetime average
and the infancy average.
51:17
Also, not too much difference
between lifetime average
51:21
and infancy average and
the concurrent measurement.
51:25
But really peak, not as
strong in association.
51:29
So for example,
51:33
for every 10 microgram
per deciliter increase,
51:34
you have a decline in IQ of 4.4 points
51:37
for the peak exposure.
51:41
And for these other metrics of exposure,
51:44
you have about an eight point
decline in child IQ overall
51:48
for each 10 micrograms per deciliter.
51:54
So this is useful information
for a variety of reasons.
51:57
First of all,
52:02
it's informative from a
public health standpoint.
52:03
So if we think about how
do we regulate blood lead?
52:07
Well, this says it's not so
much the peak concentration
52:11
that matters, it's the average
exposure during a period.
52:14
It's also not just the exposure
that occurs during infancy.
52:19
It seems to be exposure
that occurs later as well.
52:24
So maybe we don't regulate
52:29
the child's highest
blood lead concentration,
52:31
but what we regulate
52:34
are average blood lead
concentrations or something.
52:35
It's a little hard to do,
52:41
but this does provide useful
public health information.
52:43
It also provides useful
information to kind of go back
52:46
and think about what the mechanism may be.
52:51
Also what the critical period
52:53
for brain development might be.
52:55
You know, you might assume
52:57
that concentrations higher during infancy
52:59
are really what matter,
53:01
but this would suggest
53:02
that it doesn't really matter any more
53:03
or any less than average exposure
53:06
throughout the whole time
period we're considering.
53:09
So we've talked about how many times
53:16
to measure this exposure.
53:19
We've talked about after
you've measured it,
53:21
how to construct these
different variables.
53:25
A related topic is this idea
of pharmacokinetic modeling
53:28
or physiologically based
pharmacokinetic models.
53:35
What these are, is using data
53:40
so measured exposure data.
53:44
Other information that's
known about the kinetics
53:48
of the exposure.
53:52
Also information about in this case,
53:54
we're thinking about PCBs,
PCB exposures that occur
53:58
in a child in utero and postnatally.
54:08
We also can incorporate child variables
54:11
like gestation weight, birth weight
54:14
in these exposure variables.
54:15
So the actual measured
concentrations, when it was sampled
54:19
and breastfeeding, length
of breastfeeding, et cetera.
 54:24
What this allows us to do is
through mathematical models are
54:30
to derive additional data about exposure.
54:35
So let me show you what I mean.
54:40
In this figure here,
54:43
we have measured levels
identified by the open circle
54:45
which I'm gonna make red.
54:50
So in this example,
54:54
we have exposure measured
at six months in the child.
54:56
We have it measured again at 16 months.
55:03
This is the second dot here.
55:08
And then again at say about 45 months.
55:11
So this is really all we have
55:19
are these measured concentrations.
55:21
By utilizing the information here
55:27
and knowledge about the
toxic kinetics of PCBs,
55:31
we can actually simulate
kind of a time course
55:36
of what exposure would look like.
55:40
So for this individual,
55:43
they start at some level, say in utero
55:45
and that's based on a maternal
specimen or a cord specimen.
 55:49
So I should say we actually
55:53
have four concentrations measured here.
55:54
So we can using this model,
estimate these profiles,
56:01
these exposure profiles or
curves for an individual.
56:06
So based on the measured concentrations,
56:13
we know that it peaks and
we know that it declines.
56:15
Why?
56:18
Because we've got a measurement up here
56:19
and measurements down here.
56:20
We also might know that
for this individual,
56:23
that breastfeeding occurred
for let's say eight months.
56:26
Because of that,
56:33
concentrations are peaking
during that eight month period.
56:36
Subsequent to that, exposure
may only be occurring
56:40
through normal dietary exposure,
56:44
not through breastfeeding
56:46
because breastfeeding is lipophilic
56:48
and the exposure dose
for persistent compounds
56:50
like PCBs matter a lot during that period.
 56:54
So if I go back and clean
this up a little bit.
57:02
So I know that this individual
breastfed for eight months.
57:14
What I can do is derive
information about peak exposure.
57:21
So this measurement was done
at six months and we know that,
57:27
but it looks like exposure
peaked again if I clean this up.
57:34
It looks like exposure
peaked just before that.
57:40
Why might it have peaked just before that?
57:46
Well, maybe exclusive breastfeeding peaked
57:48
so let me just say at five months.
57:56
And so the peak exposure
occurred at five months.
58:03
We measured concentrations
in the infinite six months,
58:06
but because we have this model
58:10
and we have information
about breastfeeding,
58:12
we know that the peak
occurred slightly before
58:15
that measurement and then tapered down.
58:18
Now, going back, the only
measurements that we had
58:22
on this individual are
these maternal six month,
 58:26
16 month and 45 months.
58:32
But using this model,
58:35
what we're able to do
58:37
is ascertain information
about peak exposure.
58:39
And if you recall,
58:42
we can also say something
about an area under the curve
58:44
for this individual.
58:49
Now you might imagine a case
58:52
where we have two different individuals.
58:56
And let's say that these
are fictitious Y axis.
59:01
We have time on the X axis in both cases.
59:09
And this is person one
59:13
and person number two.
59:18
Maybe individual two was never breastfed
59:23
and they had maternal concentrations
59:29
that were starting at one
and maybe stayed at one.
59:31
And during that time period
didn't really change much.
59:40
So their AUC, let's just say
that this time was one year.
59:45
Their AUC was say 1.0
over that time period.
59:52
On the other hand, you
may have an individual
1:00:01
who for (clears throat)
six months was breastfed.
1:00:03
So they had 1.5 for six months.
1:00:15
And then for the last six
months had minimal exposure,
1:00:20
but their average AUC
is still gonna be 1.0.
1:00:27
What this pharmacokinetic
model does is allow us
1:00:34
to say something about
these exposure profiles.
1:00:37
So even though the AUC is the same,
1:00:41
the peak concentration is different.
1:00:45
This information can be incorporated again
1:00:48
into our analysis.
1:00:51
And rather than say just
using AUC or something else,
1:00:53
we can say something more
specific about peak exposure
1:00:57
or exposure at a specific time point,
1:01:01
say at one month, or two months,
or three months, et cetera.
1:01:04
So that goes to the next slide.
1:01:10
So these are actual
data from a study I did.
1:01:17
And the exposure of interest
in this study was PCB exposure.
 1:01:21
And we were looking at anti BCG levels.
1:01:26
So anti BCG levels are essentially levels
1:01:31
of antibody specific to the BCG vaccine.
1:01:35
And we measured this our
outcome at six months.
1:01:43
And we measured PCBs at birth
1:01:50
and at six months.
1:01:57
What we did is we used
a pharmacokinetic model.
1:02:02
And from those exposures, we were able
1:02:07
to derive several different
metrics of exposure.
1:02:11
So we were able to do
month specific estimates
1:02:15
and we were also able to calculate an AUC
1:02:22
for that six month time period
1:02:25
and also peak exposure
for that time period.
1:02:28
So let's ignore this side
of the figure for now.
1:02:34
But you can see here,
1:02:39
these are the different
month specific exposures,
1:02:40
one, two, three, four, five and six.
1:02:43
And we also have an AUC
measure and a peak measure.
1:02:47
Each of these estimates here
represents the percent change
1:02:53
in the outcome for a change
1:02:58
in exposure across the
intercore tile range.
1:03:01
And don't worry too much about this,
1:03:08
but these are two
different outcomes specific
1:03:10
to the anti BCG.
1:03:12
But essentially what you see
1:03:15
is the closer the exposure assessment
1:03:17
is to the outcome
assessment which is again,
1:03:20
the outcome is assessed at six months,
1:03:23
the stronger the association.
1:03:26
So as we go further back in time,
1:03:28
we see that the
association is less strong.
1:03:32
So this tells us
something about the nature
1:03:35
of this association
between PCBs and anti BCG.
1:03:38
It suggests that concurrent exposure
1:03:43
is most strongly related to the outcome.
1:03:47
It also suggests that
exposure around six months
1:03:50
might matter most.
1:03:55
When we also looked at AUC,
1:03:58
AUC was not as strongly associated
1:04:00
as the six month measurement
1:04:03
which again, provides information
1:04:05
that it's not just the
dose, the cumulative dose,
1:04:07
it seems to be timing specific.
1:04:11
And same thing with peak.
1:04:14
We did observe associations with peak,
1:04:15
but again, not as strong as we did
1:04:18
with the six month time point.
1:04:20
So this provides by analyzing
data using these models,
1:04:22
we're able to drive these
additional metrics of exposure
1:04:27
and capitalize on these
when we analyze our data.
1:04:32
So this would suggest that
PCBs matter in terms of timing
1:04:35
and not just dose.
1:04:42
So it's not just the dose,
1:04:44
it's not the AUC or the peak.
1:04:46
It seems to mean that
timing really matters.
1:04:48
Let's look at another issue
in environmental epidemiology
1:04:54
and for measuring biomarkers of exposure.
 1:05:00
Let's assume that you
have a case control study
1:05:06
of breast cancer in serum
pesticide concentrations.
1:05:08
So pesticides are your
exposure of interest
1:05:12
and your outcome here is breast cancer.
1:05:20
So what we really want to know,
1:05:25
we have 500 cases of breast
cancer in 500 controls.
1:05:28
So my question to you
is what is the measure
1:05:34
of association you want to know?
1:05:36
Well, what we really want to know
1:05:38
to make inferences about causality
1:05:41
is whether the exposure concentrations
1:05:45
to these pesticides are higher
1:05:48
among breast cancer cases
relative to non case controls.
1:05:51
So, is the concentration higher
1:05:59
in these 500 cases
versus the 500 controls?
1:06:03
That really is at the end of the day,
1:06:14
what we want to know.
1:06:16
Now, typically how we
would conduct this study
1:06:18
is we would determine concentrations
 1:06:21
in each of the cases,
in each of the controls.
1:06:23
And we would arrange people
into a two by two table.
1:06:27
So what does that look like?
1:06:34
Here's our two groups,
1:06:35
the case group, the control group,
1:06:40
and we can divide people
1:06:43
into high and low
pesticide concentrations.
1:06:45
So we've analyzed the
500 cases, 500 controls.
1:06:51
So we've analyzed 1000.
1:07:01
And I told you earlier that
this might cost around $750
1:07:03
so this is $750,000 to do.
1:07:08
This is quite expensive.
1:07:13
At the end of the day, you could say,
1:07:16
"Well, why don't I just take
blood from all of the cases,
1:07:18
"pool them into a single
aliquot and analyze one aliquot?
1:07:25
"I could do the same for controls,
1:07:34
"aliquot out a sample
from each of the controls
1:07:36
"and pool them and
analyze that single pool."
1:07:43
So this should say one pool
1:07:47
and this really should say one pool.
1:07:52
So an aliquot from each individual.
1:07:53
Then what have I done?
1:07:57
Well, I've analyzed two specimens.
1:07:58
It's gonna cost me $1,500, right?
1:08:02
You could think about doing that,
1:08:08
only analyzing two specimens.
1:08:10
There are lots of issues with that
1:08:13
and that's to the extreme.
1:08:17
But what if we look at this
a little bit differently
1:08:20
and let's say that we have pools of 10.
1:08:24
Pools of 10 specimens.
1:08:33
That means four cases.
1:08:37
We would have 50 sets.
1:08:43
And for controls, we would have 50 sets.
1:08:46
Remember there's 10 in each.
1:08:49
So, we pool 10 people together,
1:08:53
another 10, another 10 and
we do that for 50 sets.
1:08:57
We then distribute each set
into a higher-low group.
1:09:01
So maybe set one goes
here, set two, set three,
1:09:06
set four, set five, six, seven, et cetera.
1:09:10
Then we get to the end of those 50 sets.
1:09:14
And let's say it turns out
that we've evenly divided them
1:09:17
into high and low groups based
1:09:22
on their average concentration.
1:09:24
Same thing with the controls.
1:09:27
We have 50 sets and we
analyze set number one
1:09:29
with the 10 specimens.
1:09:33
And let's say we put it in this group,
1:09:35
set number two, and three,
four, five, et cetera.
1:09:37
So, rather than analyzing
individual concentrations,
1:09:43
we're now analyzing pools of 10 specimens
1:09:47
and then distributing them
1:09:51
into the two by two table dependent
1:09:53
on where that mean concentration lies.
1:09:56
So again, instead of taking for example,
1:10:00
we have five specimens,
1:10:05
rather than analyzing each
of those five individuals,
1:10:07
we take this whole group,
1:10:11
we mix them together simplistically
1:10:13
and we analyze a single specimen
 1:10:16
that might be a pool of five.
1:10:19
What this does is this reduces the cost.
1:10:22
So now we're analyzing a total
of 100 sets rather than 1000
1:10:24
so we've decreased our
costs substantially.
1:10:31
Now, there are some issues with this.
1:10:34
You can't look at effect modification
1:10:36
and there are some methods
1:10:39
that you need to apply
in the data analysis.
1:10:40
But in this case,
1:10:43
you can certainly adopt
the idea of pooling
1:10:45
and it is done on occasion,
1:10:49
something that you should be aware of.
1:10:51
So you've decided on how to collect them.
1:10:55
You've decided on how
often to collect them.
1:10:59
You've decided on the matrices
1:11:01
in which you're going to measure them in.
1:11:06
Now you have to actually
analyze the specimens
1:11:09
or probably send them off
to an analytic chemist
1:11:12
who can do it for you.
1:11:17
So what are the issues there?
1:11:19
This is a picture from
the cleanup of serum.
1:11:23
So serum cleanup for several individuals.
1:11:30
We have this person here, here, et cetera.
1:11:38
So you can see the color of the serum.
1:11:49
So we have one, two,
three, four, five, six,
1:11:54
seven, eight, nine, 10.
1:11:58
We have one that is a standard
1:12:04
which contains known amounts
1:12:06
of the different PCBs we're gonna analyze.
1:12:07
And we have a blank concentration.
1:12:10
So you've collected, let's say
serum from this individual.
1:12:15
You have it in the test tube.
1:12:21
Let's say you collect whole blood,
1:12:25
you spin it down and you
remove clotting factors.
1:12:27
And then you're left
was a serum you thought,
1:12:31
and you weigh it and you add
it to this extraction process.
1:12:36
So all those steps occur.
1:12:43
You then have to " do clean up."
1:12:44
So in here,
1:12:48
there's essentially kind of
like a cotton material that acts
1:12:50
as a filter and to clean
up anything in this matrix.
1:12:54
The next step here is to
dilute these concentrations.
1:13:02
So again, after the cleanup process,
1:13:07
you're left with considerably less.
1:13:09
And they further get diluted
under a nitrogen stream
1:13:16
and you're left with very small amounts.
1:13:20
These are each separate
steps with different columns.
1:13:23
After that, this is a GC-ECD machine.
1:13:29
This is gas chromatography with
electron capture detection.
1:13:37
This is an older technology
1:13:42
for measuring substances like PCBs
1:13:44
in different biological matrices.
1:13:49
So this is an oven, it heats up.
1:13:53
The sample is injected from the top.
1:13:55
And based on when that sample comes out
1:13:57
of the gas chromatograph,
1:14:00
it provides information
about the molecular weight
1:14:01
and the relative concentration.
1:14:04
So after the cleanup,
 1:14:08
you have to inject these into the machine
1:14:10
and you get a read out on the machine.
1:14:13
This for example is the
peak for para para DDE
1:14:16
which is probably the most
prevalent conjure related to DDT.
1:14:22
And then looking at one of these curves,
1:14:31
you have to then figure out
what the concentration is
1:14:34
by determining information on the peak
1:14:37
and the length of the peak.
1:14:42
So the point is in those previous slides
1:14:47
is that it takes quite a bit of time.
1:14:51
So to do 10 samples, it
might take a person a day
1:14:54
to extract them, to clean
up, to concentrate them,
1:14:58
to put them through the GC-ECD,
1:15:02
and then to integrate the curves.
1:15:06
So when you think about
measuring these specimens
1:15:09
and say on 1000 people
at three time points,
1:15:13
you think about 3000 specimens.
1:15:17
Well, that's a person
working every day for a year
1:15:19
so it takes quite a bit
of time to do this work.
1:15:23
And thus, when I say it's $750 a specimen,
1:15:26
you can easily see how
that cost quickly adds up.
1:15:30
So let's discuss some
of the potential issues
1:15:36
that occur when analyzing biomarker data.
1:15:39
The first and probably
the most common issue
1:15:44
is that biomarker
concentrations are subject
1:15:48
to a limit of detection.
1:15:52
When you're measuring compounds
1:15:55
that occur at the parts per million
1:15:57
or parts per billion level,
1:15:59
often some concentrations won't
be detected within a sample
1:16:02
of say serum, or plasma, or breast milk
1:16:09
because they're just not
a prevalent compound.
1:16:13
So one example is TCDD,
commonly referred to as dioxin.
1:16:16
And in this case,
1:16:23
you can see that these
are data from Ann Haynes
1:16:24
in multiple survey years.
1:16:28
So 1999 and 2000, et cetera.
 1:16:30
And if you look at these
percentiles, so the median here,
1:16:33
the 50th percentile,
1:16:37
you can see that the median
concentration for all years
1:16:39
for the entire group of individuals
1:16:45
is below the limit of detection.
1:16:46
So what do you do with concentrations
1:16:51
that are subject to a limit of detection?
1:16:55
Well, there are a few
ways to deal with it.
1:16:59
The first is what we
call simple substitution.
1:17:02
And this is useful when
some of the concentration
1:17:06
are below a limit of
detection, say less than 10%.
1:17:10
So there are methods such as
using one-half the LOD value,
1:17:14
or one-half divided by the square root,
1:17:20
or just substituting the
actual limit of detection.
1:17:25
These are fine
1:17:30
when the amount of essentially
missing data is trivial.
1:17:32
There are other methods when
the amount of missing data
1:17:38
is a bit more substantial,
more than 10% to about 50%.
1:17:42
Multiple imputation is one example
1:17:48
and there's a whole body
of literature on this
1:17:50
in the epidemiology
field and in epi methods.
1:17:53
And essentially this involves
simulating multiple data sets
1:17:59
and then impugning values based
1:18:03
on those simulated data sets.
1:18:06
It performs well,
1:18:08
multiple imputation does when
the range of missing this
1:18:10
is in this 10 to 50%.
1:18:14
And in fact, it's really the ideal method
1:18:18
when you have less than 50% of values
1:18:21
that are censored or are missing.
1:18:25
There are some other techniques out there
1:18:27
that can be used like Cox regression
1:18:30
where you're really treating exposure
1:18:33
as a censored measurement where
it's really left censored.
1:18:37
I was involved with a group
that published a paper
1:18:45
where they treated exposure as outcomes.
1:18:48
So they flipped X and Y
in the regression equation
1:18:52
and treated the exposure as
a Y value which was censored.
1:18:56
This gets complicated very quickly.
1:19:02
It limits the analysis that you can do.
1:19:04
And obviously if you
anticipate having lots
1:19:07
of values below the LOD,
1:19:11
you need to rethink
your method of analysis
1:19:12
or the matrix you're using,
1:19:16
or even if it's relevant
to measure that compound.
1:19:19
So a second problem or
really debate in this field
1:19:26
is how to adjust for the solvent.
1:19:30
So chemical concentrations
in urine for example,
1:19:33
are a function of urine output.
1:19:37
So in other words, if
your urine is very dilute,
1:19:40
say you just drank a lot of water,
1:19:44
the amount of chemical
concentration in there
1:19:48
is gonna be a function of
how dilute that urine is.
1:19:52
And so there are ways to
adjust for urine output.
 1:19:56
One is called creatinine concentration
1:20:00
and creatinine is a protein
that you can measure in urine.
1:20:03
And so it's a way of
adjusting for the dilution
1:20:08
of the urine.
1:20:13
And a second method that
you'll see is specific gravity.
1:20:14
So if you encounter publications
1:20:17
that are using non-persistent
chemicals measured
1:20:21
in urine such as BPA and phthalates,
1:20:24
you'll often see some correction made
1:20:30
for the concentration based
1:20:34
on either creatinine concentration
1:20:36
or specific gravity.
1:20:38
Same goes for lipid-soluble
compounds like PCBs, DDT.
1:20:42
And the most typical method is to adjust
1:20:54
for lipid concentration
1:20:57
so measuring total cholesterol
1:20:59
and some other specific types of lipids.
1:21:03
How you do that adjustment is debatable.
1:21:06
So one method is to add
the lipid concentration
1:21:11
as a separate term,
 1:21:16
as a covariate in a statistical model
1:21:18
and adjust for it that way.
1:21:20
Another method is just to
standardize concentration.
1:21:22
So if you just measure PCBs in blood,
1:21:26
you'll see something
like 5 nanograms per ml.
1:21:31
But if it's standardized per lipid,
1:21:37
you might see it as 5
nanograms per gram lipid.
1:21:41
So in that case,
1:21:47
the concentration is actually
standardized by the amount
1:21:48
of lipid in the blood.
1:21:53
The best way of adjusting
for this really is debatable.
1:21:55
And there are some methods,
papers dealing with this
1:21:59
by a group at NIHS,
also Enrique Schisterman
1:22:02
who's also at NIH.
1:22:08
This is a pretty typical
sensitivity analysis in papers
1:22:12
that adjust for creatinine
concentrations, specific gravity
1:22:18
or lipid adjustment is to do it one way
1:22:23
in the primary analysis
and in a secondary analysis
 1:22:26
or in a sensitivity analysis to say,
1:22:29
"We're doing it another way
1:22:32
"and do our results
really differ depending
1:22:34
"on the method of adjustment."
1:22:39
So you have your results from your essays
1:22:44
and you need to interpret those results
1:22:47
and you need to do so carefully.
1:22:51
So I'm gonna walk you through an example
1:22:55
of how you can go wrong.
1:22:58
And the authors in this
study didn't go wrong,
1:23:01
but I think if they
hadn't thought about this
1:23:05
or knew as much as they did
about the pharmacokinetics
1:23:08
of perfluorinated compounds,
they might've gone wrong.
1:23:14
So their question of interest
were perfluorinated compounds
1:23:18
so these are the PFAs.
1:23:22
So things like PFOS and PFOA,
1:23:26
and these are chemicals that
are used as stain repellents.
1:23:32
They're used in products that carry food.
1:23:36
They were used in a lot of
consumer products like scotchgard
 1:23:40
and things like that.
1:23:45
They're very prevalent.
1:23:46
And there's decent experimental
evidence suggesting
1:23:48
that they're developmental toxicants.
1:23:52
So their study was looking
1:23:56
at these perfluorinated
compounds and fecundability.
1:23:58
And fecundability is the
probability of conception
1:24:03
in a given menstrual cycle.
1:24:07
So it's a reproductive outcome.
1:24:09
It's focused on the female
partner in this case.
1:24:11
And the outcome really
is time to pregnancy.
1:24:16
This reflects fecundability.
1:24:20
So time to pregnancy in this
case is time from initiation
1:24:23
of unprotected intercourse to conception.
1:24:27
And it's typically
self-reported as in the case
1:24:31
of this study.
1:24:35
So the construct here is this
fecundability or probability
1:24:36
of conception and how they
operationalize constructors
1:24:40
through this assessment
of time to pregnancy
1:24:44
which is self-reported.
1:24:47
So they're including 932 subjects.
1:24:51
They have a case group which
includes subfecund women
1:24:56
or it is a group of subfecund women.
1:25:01
And these are women
1:25:04
whose time to pregnancy
was longer than 12 months.
1:25:05
And then a control group
1:25:10
where time to pregnancy was
less than or equal to 12 months
1:25:11
and the women had no reported
fertility treatments.
1:25:15
The exposure of interest here
1:25:19
were maternal perfluorinated compounds
1:25:23
and they're measured at
17 weeks of gestation.
1:25:26
So they were measured after
the mother got pregnant.
1:25:29
So logistic regression analyses were used.
1:25:36
Results were stratified by parody.
1:25:39
It's a bit cutoff in the figure here,
1:25:42
but these come from a
paper by Christy Whitworth
1:25:45
and they were published in epidemiology.
1:25:48
So what did the authors find?
1:25:56
Let's just focus here on
the adjusted estimates.
1:25:58
And so these are odds ratios
and confidence limits here.
1:26:02
We have those for PFOs and those for PFOA.
1:26:07
So it looks like for PFOs
1:26:18
that as the concentrations increase,
1:26:22
that the odds of subfecundability increase
1:26:26
and that's also the same for PFOA.
1:26:34
And you can see here,
1:26:38
the tests for trend indicate
that there's dose response
1:26:39
and you can see that here.
1:26:44
So in the previous table, you
saw the results collapsed,
1:26:50
not stratified by parity.
1:26:56
These results are shown in graphical form,
1:26:59
and they are stratified by parity.
1:27:02
So these are the results for PFOs.
1:27:04
And the blue is for nulliparous women.
1:27:10
So these are women who have
not had a previous pregnancy.
1:27:15
And the green is for parous women.
1:27:20
So they've had a previous pregnancy.
1:27:23
So I'll say previous pregnancy positive,
 1:27:28
and I'll say previous pregnancy negative.
1:27:31
And what's modeled are
the odds of subfecundity.
1:27:38
And we have four exposure
categories, core tiles.
1:27:51
This is the reference category,
second, third and fourth.
1:27:56
And generally what we
see are elevated odds
1:28:02
for the parous women.
1:28:07
And so did this protective association
1:28:11
for nulliparous women.
1:28:19
So in other words,
1:28:23
parous women are at an
increased odds for subfecundity
1:28:25
whereas nulliparous women,
1:28:31
women who have never had a
previous pregnancy don't seem
1:28:33
to have that risk or it is protective.
1:28:38
So it almost looks generally
these confidence limits
1:28:41
are fairly wide.
1:28:44
They're fairly wide for
most of the estimates,
1:28:45
but it generally looks like no association
1:28:49
for the nulliparous women.
1:28:52
But for parous women,
 1:28:53
women who have had a previous pregnancy,
1:28:54
it looks like perfluorinated
compound PFOSs
1:28:59
in this case is associated
with an increased odds
1:29:02
of having this delayed time to pregnancy.
1:29:07
If we look at the results for PFOA now,
1:29:13
we see a similar trend.
1:29:18
Essentially no association
for the nulliparous women,
1:29:20
but a positive association
for the parous women.
1:29:29
So again, it looks like only
1:29:35
for the parous women
1:29:37
are these perfluorinated
compounds associated
1:29:40
with an increased odds of subfecundity.
1:29:43
So one explanation is that it's causal
1:29:51
that these perfluorinated
compounds are causing women
1:29:56
to have a delayed or a
longer time to pregnancy.
1:30:01
So these compounds somehow
interfere with reproduction.
1:30:06
So that is the causal explanation.
1:30:12
Another way of looking at this
1:30:17
is to think about the pharmacokinetics
 1:30:18
of these compounds.
1:30:21
So this is from a
publication by Olson in 2009
1:30:22
and it's looking at a hypothetical woman
1:30:30
who's trying to get
pregnant in the late 1990s.
1:30:32
So she has some body burden
1:30:42
of these perfluorinated chemicals.
1:30:45
So as we go up on the Y axis,
the body burden increases.
1:30:48
So this woman is trying to get pregnant.
1:30:58
Then she's kind of chugging along in time.
1:31:01
She isn't pregnant yet.
1:31:05
So there aren't changes, dramatic changes
1:31:07
in these body burden of
perfluorinated compounds.
1:31:10
Well, she gets pregnant.
1:31:14
And then during pregnancy,
the body burden decreases.
1:31:17
Why does it decrease?
1:31:21
Well, her body burden decreases
1:31:23
because she's off putting
these perfluorinated compounds
1:31:25
onto the fetus.
1:31:29
After she gives birth,
1:31:32
her body burden continues
to decline during lactation
1:31:36
because she's breastfeeding the infant
1:31:41
and breast milk is rich in
perfluorinated compounds.
1:31:43
And so again, she continues
1:31:47
to off put these compounds
onto this time the neonate.
1:31:49
So after she's finished with pregnancy,
1:31:54
she's finished with breastfeeding,
1:31:56
what happens is she then
begins to go back up
1:31:59
to her normal baseline which is up here.
1:32:03
This process of re-equilabration.
1:32:08
So she'll continue to ascend
to her typical body burden.
1:32:12
Why?
1:32:18
Because she'll resume her normal
diet and typical exposures.
1:32:19
So this nicely describes what happens
1:32:24
to the body burden during
pregnancy and lactation.
1:32:27
So now this is where things
get a little bit complicated.
1:32:32
And so again,
1:32:36
we have the woman who pre
pregnancy has a steady state,
1:32:37
drops during pregnancy,
drops during lactation.
1:32:41
Then when she's finished,
she begins to re-equilibrate.
1:32:46
Now let's say that we're
dealing with a parous woman,
1:32:50
so that woman will have
had a pregnancy here.
1:32:57
So this is pregnancy number one
1:33:02
and this would be pregnancy number two,
1:33:06
or this could be pregnancy
three and four et cetera.
1:33:11
So she, during this re-equilibration
process begins trying
1:33:17
to get pregnant again.
1:33:24
Now let's say there are
two different women here
1:33:27
and one woman has a short
time to pregnancy here,
1:33:31
and woman two has a
long time to pregnancy.
1:33:38
Now, recall that in the previous slide,
1:33:48
what we saw was
1:33:52
that this adverse
association was only present
1:33:53
in parous women.
1:33:59
So women like those in
this diagram, right?
1:34:00
Because parous women
 1:34:04
are going through this
re-equilibration process.
1:34:05
Nulliparous women are
always at a steady state.
1:34:09
So what I'm about to
show you does not apply
1:34:14
to the nulliparous women.
1:34:19
So let's say these two
women both begin trying
1:34:25
to get pregnant.
1:34:31
And for whatever reason,
1:34:33
woman one has a short time to pregnancy
1:34:34
and woman two has a
longer time to pregnancy.
1:34:37
Well, what have we done?
1:34:41
We have artificially fixed the association
1:34:43
between these perfluorinated compounds
1:34:48
and time to pregnancy.
1:34:51
How?
1:34:53
Well, a woman with a
short time to pregnancy
1:34:55
is guaranteed a lower body burden
1:34:59
and a woman with a
longer time to pregnancy
1:35:05
is guaranteed a higher body
burden of these compounds.
1:35:09
So without any kind of
causal mechanism at play,
1:35:15
we have guaranteed an association
1:35:21
between perfluorinated
compounds and time to pregnancy.
1:35:25
So the pharmacokinetics of
this exposure make it look like
1:35:31
for parous women who've had
a short time to pregnancy,
1:35:36
they're gonna have a lower body burden.
1:35:41
So a short time to
pregnancy is a good thing,
1:35:43
and they're gonna have lower
concentrations on average.
1:35:47
So what's that gonna do?
1:35:51
It's gonna introduce an association
1:35:53
whereby higher
concentrations are associated
1:35:58
with longer time to pregnancy.
1:36:00
And again, we would only
see this in parous women.
1:36:02
Why?
1:36:11
Because only parous women go
1:36:12
through this re-equilibration process.
1:36:14
Now, if you go back to
the previous slides,
1:36:19
recall that Whitworth only
observed the association
1:36:23
among parous women and not
among nulliparous women.
1:36:29
Based on that, you might
start to think that,
1:36:35
well, maybe the association they've seen
1:36:38
is really due to pharmacokinetics
1:36:41
and has nothing to do with
an actual causal mechanism
1:36:43
that has something to do with
the developmental toxicity
1:36:51
of these compounds.
1:36:55
So it's completely artificial.
1:36:56
This association is completely artificial
1:36:58
and it is due to the
pharmacokinetics of these compounds.
1:37:01
So again, it may not
be causal possibly due
1:37:07
to pharmacokinetics.
1:37:11
So longer time to pregnancy allows
1:37:12
for greater amounts of time
1:37:14
for these perfluorinated
concentrations to rise back
1:37:17
to baseline following birth.
1:37:20
So wrapping up some
conclusions about biomarkers,
1:37:24
exposure measurement needs
1:37:28
to be considered during
all phases of the study.
 1:37:30
So I started this lecture talking
1:37:33
about selecting your bio marker.
1:37:34
Which matrix are you gonna choose?
1:37:37
Is it gonna be urine?
1:37:40
Is it gonna be nails?
1:37:41
Is it gonna be hair, et cetera?
1:37:43
How many times do you need to sample it?
1:37:46
How many times do you need to collect it?
1:37:48
Then at the analysis phase,
how are you gonna analyze it?,
1:37:51
Are you gonna pool the data, perhaps?
1:37:56
Are you gonna pool the
exposure measurements together?
1:37:59
That's one technique.
1:38:04
And then further on in
the data analysis phase,
1:38:05
how are you gonna handle values
below a limit of detection?
1:38:09
How are you gonna handle
potential contamination?
1:38:13
How do you interpret the results?
1:38:16
So you need
1:38:17
to be thinking about all of
these issues about biomarkers.
1:38:18
You don't just sort of select
it and be done with it.
1:38:21
There are issues intrinsic to biomarkers
1:38:24
that carry throughout the entire study.
1:38:26
Biomarkers can be great
1:38:30
because they can reduce misclassification,
1:38:31
exposure misclassification compared
1:38:33
with other methods of exposure assessment.
1:38:36
So in environmental epi,
1:38:38
this is the biggest issue that we face
1:38:40
is really exposure, assessment
and misclassification.
1:38:43
Sometimes biomarkers are a lot
better at assessing exposure.
1:38:47
So if I ask you to tell me
about your fish consumption
1:38:51
during pregnancy and before,
1:38:55
if I want to assess methylmercury,
1:38:57
I would say that,
1:39:00
"Why don't you just
take a sample of my hair
1:39:02
"and get an objective measurement of it
1:39:04
"and not base the assessment
1:39:07
"of exposure on my recall?"
1:39:09
So that's a great example
1:39:12
where a biomarker is clearly
better than asking someone
1:39:14
to recall their diet.
 1:39:17
Nevertheless, you need to think carefully
1:39:20
about these biomarkers.
1:39:23
You need to think about
potential contamination.
1:39:25
You need to think
1:39:28
about whether you're gonna
have sufficient power
1:39:29
if you use these biomarkers.
1:39:32
We talked about the issue with BPA.
1:39:34
You also need to think about
how you interpret your results.
1:39:37
It is a biomarker.
1:39:40
It is subject to a whole
bunch of other processes,
1:39:41
physiological processes in the body.
1:39:45