WEBVTT

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Hello and welcome.

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I'm Heather Nelson.

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I'm an assistant professor

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at the University of Utah Health
within our clinical pathology department.

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And I'm also a medical director at R.P.

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laboratories in our chemistry section.

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Today, I have the delight of
talking to you about fecal cow protection.

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I'll start by describing
the clinical utility and talk about

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why we measure fecal cow protection.

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Then I'll discuss the analytical
considerations when it comes to measuring

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So the cow protectant is a calcium
and zinc

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binding protein of the S100 family.

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It's the predominant protein
in the site of solid neutrophils,

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accounting for up
to about 60% of the soluble protein.

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And it's also found in other leukocytes.

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When the gastrointestinal tract
becomes inflamed, neutrophils.

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Neutrophils migrate to the area
where they're activated and release

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cow protective.

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And biologically, cow protection
binds divalent metal ions,

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which is thought to to sequester them
from bacteria.

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Once cow protection is secreted
in the gut lumen,

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it will accumulate in the feces
where it's excreted from the body.

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Cow protecting is quite stable
for up to seven days at room temperature

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and resistant to bacterial degradation,
which are qualities

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that make it a great biomarker
for measurement.

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In the laboratory.

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When measured in feces, cow protection
correlates

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well with neutrophil migration
to the mucosal surface and gut lumen

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and is a hallmark of digestive
inflammatory pathology.

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Overall, fecal cow protection

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is a highly sensitive
biomarker of inflammation.

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But that said, it's not specific.

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Oftentimes, we're using fecal cow
protection to identify individuals

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with inflammatory bowel disease,
and that would include individuals

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with ulcerative colitis
and Crohn's disease.

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However, we know fecal cow
protection can be elevated

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in other conditions that might also
be associated with inflammation.

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In those could include individuals
with polyps,

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cancer, or diverticulitis.

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Here's a clinical use example
of how we might use fecal cow

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protect in a patient
comes in to their physician or the GI

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clinic complaining about complaining
about abdominal

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pain, gas, bloating, diarrhea.

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The physician might want to start
just by getting a basic understanding.

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Is this an inflammatory pathology
or is it non inflammatory?

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And to answer that question
they might order a fecal cow protect in.

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If the fecal cow protect and result
comes back

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lo less than 50 micrograms
per gram of stool.

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In this example it suggests
that there are non inflammatory causes

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of the patient's distress,
such as irritable bowel syndrome.

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If the fecal cow protection
comes back in a borderline range 50 to 150

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micrograms per gram in this example,
then the physician might start

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by excluding other causes of information,
such as a recent infection

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in said use,
and then consider repeat testing.

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If the cow protecting comes back

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elevated greater than 150 micrograms
per gram of stool.

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It's consistent with organic disease,
which suggests that the patient

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might have inflammatory bowel disease
or or colorectal cancer,

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and then they would proceed to endoscopy
with biopsy

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to try to identify the cause
of the patient's inflammation.

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So now I want to go through some studies

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that show you data
to support the use of fecal cow protected.

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In this study shown here,
the authors compared

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the cow protection
result measured in a random fecal sample

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to the gold standard,
which is endoscopy with biopsy.

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And what they looked
at was the the highest rating

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of inflammation
scored on the biopsy from physicians.

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And they plotted that on the x axis
against the cow protecting result.

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And what you can see

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is individuals with no inflammation
had a very low cow protecting.

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And you have a strong correlation
of increasing cow protecting

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with increasing severity of inflammation.

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So that suggests that cow

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protection correlates
well with disease activity.

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In a similar vein, individuals

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wanted to look
at how useful cow protection

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was for monitoring disease in individuals
that already had a diagnosis.

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In this study,
they looked at the symbol endoscopic score

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for Crohn's disease, which is a score
that's generated off of biopsy

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by looking at the ulcerative surface
of individuals with Crohn's disease.

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And so that can be rated from inactive,
or remission on a score of 0

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to 3, all the way
up to individuals with high activity,

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which would be anything greater
than 20 on the endoscopic score.

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They plotted that score against fecal cow
protecting again.

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And you can visualize a fit line here
of a positive correlation.

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Increasing cow protection
with increasing disease severity.

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They looked at 140 individuals
with Crohn's disease

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and 40 controls in the study,
and found that cow protect in was able

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to reliably distinguish those with active
disease from those without.

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In addition, another
study looked at the ability of fecal cow

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protecting to predict relapse
in individuals with both Crohn's disease

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as well as all sort of colitis
and they found that when they grouped

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individuals into groups based on their cal
protecting concentration.

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So they looked at those with Cal
protecting less than 150 micrograms per

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gram in those with Cal protecting greater
than 150 micrograms per gram.

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They saw that individuals with a cow
protecting have greater

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than 150 micrograms
per gram, often went into,

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active disease before the end of the study
or before 12 months.

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So it was a great predictor of relapse.

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So you can see in the Crohn's disease
patients, about 50% of them with greater

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than 150 micrograms per gram protecting
relapse before the end of the study.

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And it was even higher in those

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with all sort of colitis,
with almost 80% having relapsed.

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In that same study, they also looked
at those individuals urethra

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sites, sedimentation rate
as well as their C-reactive protein.

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And neither of those were useful
predictors of relapse.

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They're just not specific enough
for intestinal inflammation.

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So what I've shown you in these studies

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is that fecal cow
protection has multiple uses.

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It can be used for the differential
diagnosis between IBD and IBS.

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So in the initial evaluation of a patient,

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it can also be used to estimate the degree

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of gastrointestinal inflammation
or the severity of disease.

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It's also useful for monitoring response
to therapy in individuals

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with a diagnosis,
as well as predict clinical relapse.

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In addition to these indications,
there are several qualities

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that make fecal cow protect in a useful
biomarker.

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One is that it can be quickly and easily
measured.

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It just takes a random stool
sample submitted to the laboratory

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and can have results back
within that same day.

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If there's an in-house test.

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It's also very inexpensive, inexpensive,

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especially when you start comparing it
to the alternative modality of endoscopy.

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It's noninvasive, and it has improved
sensitivity and specificity

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to more general biomarkers
of inflammation, such as CRP and ESR.

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Because of

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its, use in the data
that supports its use,

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the American Gastroenterology
Association recently added

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the use of fecal cow protection
to its, clinical recommendations

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for the management of individuals
with ulcerative colitis.

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You can see that
this was published in 2023,

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and it's the first time
that fecal cover testing was no longer

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considered experimental,
but it was advocated for

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in the management of individuals
with inflammatory bowel disease.

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Very similarly, they also included
fecal cow protection

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as a biomarker for the management
of individuals with Crohn's disease.

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The second form of inflammatory
bowel disease.

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With very similar recommendations,
you can see

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So now that you understand

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the clinical use of a fecal cow protected,
and let's talk about

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some of the analytical considerations
that are important to understand

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when you, try to measure how protected.

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So we measure cow protection
in a random stool sample or it's stable

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at room temperature for 3 to 7 days, both
which make it beneficial.

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It's quick easy question.

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And it's quite stable.

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With that comes some challenges.

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Working with stool in general

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a as a specimen type is challenging.

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One is that it's very heterogeneous.

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It can include undigested fibers
or food particles or mucus,

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all of which make it harder
to accurately sample that specimen.

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It's also very,
variable in its consistency.

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So shown here in
this image is the Bristol stool types.

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Stool can be rated on a scale of
1 to 7, one being where the soil is like

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rock hard solid lumps all the way to
seven, which is liquid stool.

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In addition to having these
variable consistencies, we know there's

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variable water content
across those stool types on the order

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of 65 to 75%.

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And we don't have a normalizing factor
for water.

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If you think about urine,
we have creatinine to normalize

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for concentration of the urine.

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And that just gives us an idea.

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Was it a really dilute sample
or a really concentrated sample.

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There's no analyte like that for stool.

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So we don't normalize it. Anything.

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We also know that there can be
substantial day to day

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variability within an individual.

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So even in healthy individuals
you can see CV's of up

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to 30 to 40% between measurements.

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So that means we're seeing large
differences in quantitation of cow

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protecting
even within in a healthy individual,

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that variability contributes
to a large reference change value

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for cow protect in we have a reference
change value of 87 to 115%.

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The reference change
value is a statistical measure

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to allow you to assess how significant

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differences
between two serial measurements are.

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It takes into account
analytical imprecision of the assay

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as well as biological variability,
and essentially tells you

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is the difference between results
statistically different?

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Is this statistically significant?

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And so, for example,

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if we have a reference change
value of 100%, which is right in the

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middle of our range for cow protection,
and a patient comes in one day,

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you measure their fecal cow protect in
and it's 50 micrograms per gram.

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We want to consider a subsequent measure
a significant change

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or significant increase, unless it was 100
micrograms per gram or more.

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So 50 to 100 is a pretty, big change.

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And that's a lot
related to not the imprecision

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of the assay itself,
but the imprecision of the matrix.

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It's really hard to sample
a heterogeneous matrix.

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In order

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to measure cow protection, you first
need to extract it from the stool

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and get it into a buffer suitable
for analysis.

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There are a couple ways we can do it.

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The gold standard
method for extraction is what we called

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the manual or manual weighing process.

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In this process,
the individual must weigh out

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a specified amount of stool,
generally 100mg of stool

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and record the weight
that they, weighed out.

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They then dilute it into the buffer
for analysis and vigorously vortex

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to help get that protein out of the stool.

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It also then often includes

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a second dilution step to get it
in the appropriate concentration range,

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and then centrifugation
before that can go on to the assay.

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Now there are newer extraction devices
that have been released

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that are supposed to help speed up
the extraction process and make it easier.

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What these extraction devices are
are tubes pre-filled with the buffer

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for the assay, and they include this wand
that has precision grooves

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that were designed to deliver an accurate
and precise amount of stool every time.

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So what the individual does, they'll mix
a stool sample to make it homogenous.

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They'll dip the precision
one into the stool,

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and then they'll feed it
through this rubber stopper.

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And that rubber stopper is designed to 
remove any excess stool.

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They'll then vortex
it and it's ready for the assay.

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What I'm

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showing here is a few different devices
that are on the market,

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and you can see that they're designed
to live, to deliver anywhere

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from ten milligrams up to 50mg of stool,
depending on the device used.

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So in general,
a little bit lower than what we're doing

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with the manual extraction.

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And so here's a comparison
of some of the pros

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and cons to each, extraction method.

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For the manual method
it does use more stool

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typically, which is better
for heterogeneous stool samples.

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When you have a lot of heterogeneity,
the more you can sample,

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the better representation
of that heterogeneity you'll get.

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It's also used for liquid stool samples.

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So this is an important note.

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You cannot use the extraction device
for liquid samples

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because the liquid does not adhere
to the grooves appropriately.

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It does
require a lot more time and effort.

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So you have to sit at this scale,
try to get the precise weight.

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There's some extra dilution
and vortex and step.

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So it does take time.

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When we look at the extraction device,
it generally uses less stool.

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So this makes it better
for homogenous samples

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because we have less representation
with the less stool that we're capturing.

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It's a lot more efficient.

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It's a quick dip and go, but
there are differences in extract stability

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depending on the device you use, so it's
just important to be aware of that.

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The other note on the device
is that you can only extract

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stool samples
that are Bristol stool type 2 to 6.

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So you can't do any real hard clumps
because you can't pierce them

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well with the stick
and you can't do any liquid samples.

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So oftentimes you'll have to have,
a backup manual procedure

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if you want to be able to process
all stool submitted to the lab.

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So there

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are two different formats of assays
available.

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They're both immunoassay based.

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However they can be,
a plant based slicer, or they can be bead

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based for the plate
B, Ally's plate based lysis.

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There are several
commercially available assays.

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Because they're in a plate format.

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It typically requires batch testing.

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You want to fill up your plate
before you run it.

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A caveat is that

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if you run the plate
and there's a sample that is elevated,

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you then have to dilute it
and run it on a second plate.

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So turnaround
times can really start to bump up.

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Once you do that,

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however, the steps can be performed manual
with a plate based assay.

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So you don't need any kind of fancy
equipment, you just need some pipettes

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and a plate reader.

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So this offers a low cost point for labs.

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Thinking of bringing the assay in

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in the

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bead based format, we have the amino acid
sandwich that occurs on a bead.

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There are again
several commercial available assays.

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The nice thing about these B based assays
is that they're random access.

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You can put a sample on and get a result
back within 20 to 30 minutes.

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They generally use
CME luminescence fluorescence

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or immuno turbid imagery
as far as their immunoassay readout.

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And the A benefit here is that dilutions
can be performed on the instrument.

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So you're not waiting three hours
for your lizer to finish

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to find out that you have to dilute it
and put it on another plate.

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The instrument
will automatically dilute it.

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So you're significantly
reducing your turnaround times.

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The downside or maybe not,
depending on what you have in

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your lab, is that it does
require an immunoassay analyzer.

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So you'll need to have a instrument
from that specific vendor

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to be able to, perform these assays.

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So it could potentially
have a higher cost point.

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And with that,
I thank you for your attention

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and hope you found this presentation
informative.

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Thank you.