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Preanalytical issues in feces analysis
Andrea Padoan
Department of Laboratory Medicine
University-Hospital of Padova, Padova
Italy
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Presentation Outline
• The importance of laboratory stool samples analyses
• Specimens collection (how to & which bowel movement ?)
• Storage and handling
•Weighting
• Issues regarding faeces consistency
•Biological variation: might it be of aid ?
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Presentation Outline
• The importance of laboratory stool samples analyses
• Specimens collection (how to & which bowel movement ?)
• Storage and handling
•Weighting
• Issues regarding faeces consistency
•Biological variation: might it be of aid ?
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Stool Content • Food undigested contents and water
• Digestive enzymes (from pancreatic exocrine enzymes, such as trypsin, elastase, etc…)
• Intestinal Brush Border Enzymes (such as maltase, sucrase-isomaltase, lipases, peptidases, etc…)
• Inflammatory molecules (such as Calprotectin, lactoferrin)
• Hemoglobin
• Immunoglobulins (e.g. secretory Ig-A)
• Altered content of lipids or fibers
• Gut microbiota and/or Pathogens (direct identification of microorganisms or antigens, e.g. Helicobacter pylori antigen, or DNA)
• Cells (immune cells or gastrointestinal cells, etc…)
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The importance of stool samples in laboratory
Stool Samples
Microbiology Tests Fecal
Occult Blood Test
Malabsorption test
Exocrine Pancreatic function
Microbiota
Calprotectin
a-1-antitrypsin
Colorectal cancer
screening
Digestive tract
function
Pancreatic insufficient
Fecal transplantation and digestive
tract problems
Inflammatory bowel
disease diagnosis
Protein losing
enteropathy
Parasites and pathogens microorganism
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There are different (seven) types of stools
Lewis SJ et al. Scand J Gastroenterol. 1997;32(9):920-4. and http://www.gpnotebook.co.uk/simplepage.cfm?ID=x20100606160645260465
What should my stools look like?
Type 1 and 2: constipation Type 3 and 4: ideal stools Type 5 to 7: diarrhea, urgency or pathological conditions
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Question #1
Why faecal calprotectin is gaining attention as a
marker of intestinal inflammation:
1) Because of its antimicrobial activity
2) Because of its accuracy in diagnosing
inflammatory bowel disease (IBD)
3) Because of its ability in discriminating Chron
disease from Ulcerative colitis
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Faecal Calprotectin is gaining attention as a marker of intestinal inflammation
• Faecal calprotectin is a 24 kDa heterodimer composed of a S100A8 (light chain) and a S100A9 (heavy chain)
• Fecal calprotectin is produced locally, inside the inflammatory site, and released in the feces
• It presents antimicrobial activity • Produced mainly by Neutrophils and macrophages and secret
after stimulation.
• Recently, it has gained attention with the increase of gastrointestinal disease but also by the bunch of studies demonstrating good accuracy for diagnosing inflammatory bowel diseases (IBD).
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Faecal Calprotectin for IBD diagnosis or evaluation of disease activity
• IBD diagnosis: elevated Sensitivity (~ 90% for Adults and > 95% in children).
• In primary care GP can use faecal calprotectin to rule-out IBD (being irritable bowel disease underwent to differential diagnosis with IBD). In secondary care, gastroenterologist can use positive results to proceed to further investigations (e.g. colonoscopy).
• Distinguishing between active and inactive disease: Sensitivity of about ~ 80-85%. Laserna-Mendieta et al. Faecal calprotectin in inflammatory bowel diseases: a review focused on meta-analyses and routine usage limitations. Clin. Chem. Lab. Med. (2019).
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Causes of false positive results
Causes of false positive results in faecal calprotectin testing, that may potentially cause unnecessary colonoscopies:
• Infections (Viral or bacterial)
• Malignancy (Gastric or colorectal cancer)
• Intestinal lymphoma
• Eosinophilic colitis
• Autoimmune enteropathy
• Drugs (non-steroidal anti-inflammatory drugs and PPI)
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Cons of faecal calprotectin testing
• Fecal Calprotectin levels do not allow differentiation between Chron disease and ulcerative colitis
• Methods dependent cut-off
• Age-related reference ranges and cut-offs (e.g. paediatric or elderly patients)
• Poor standardization of the pre-analytical and analytical phases and standardization is needed
• Between-assays elevated differences
• There is no standard reference material or reference method listed on the Joint Committee for Traceability in Laboratory Medicine database
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Question #1
Why faecal calprotectin is gaining attention as a
marker of intestinal inflammation:
1) Because of its antimicrobial activity
2) Because of its accuracy in diagnosing
inflammatory bowel disease (IBD)
3) Because of its ability in discriminating Chron
disease from Ulcerative colitis
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Presentation Outline
• The importance of laboratory stool samples analyses
• Specimens collection (how to & which bowel movement ?)
• Storage and handling
•Weighting
• Issues regarding faeces consistency
•Biological variation: might it be of aid ?
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Faecal samples collection: possible issues
Urine contamination
should be avoided
A sufficient quantity (at least 1 g) should be
collected
1
2
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Which bowel movement should be collected ?
In a total of 287 stool samples of at least 2-3 g, collected from two different parts of the faeces and placed in two different plastic tubes, obtained from 18 IBD patients in two bowel movements for two days, Lasson et al. found a statistical significant correlation between faecal calprotectin levels, the time between two bowel movements (r = 0.5, p = 0.013) and the stool consistency (r = 0.68, p = 0.01).
The longer the time between bowel movements and the looser the stool consistency, the higher the concentrations of faecal calprotectin were found.
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Which bowel movements should be collected ?
Cohen’s kappa = 0.76 Cohen’s kappa = 0.76
Reliability study
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Sample heterogeneity : how many spot from a fecal sample?
Sampling effects
Is one spot sample
representative
enough? 0.1 g or lower/ 300 g or
higher
Is faecal sample
homogeneous?
Sampling from 10
different sites
within two separate
stool samples:
No significant
difference Dolwani et al., Aliment Pharmacol
Ther 2004;20:615-21
132 pairs of samples
collected from the
same bowel
movement (17
patients): a strong
correlation was found
being ICC = 0.79 and
95%CI 0.48-0.90.
Lasson et al. Journ
Crohn’s and Colitis
2015
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Within-stool variations at morning, afternoon and night in IBD patients with respect to the average
values of 3 faecal samples
“This is in keeping with the findings of Lasson et al., who found that FC sampling should be taken from the first bowel movement of the
morning given a significant positive correlation between the level of FC and the time between
bowel movements”. Du et al. J Clin Gastroenterol 2016
“…to be able to compare values over time, the patients should be recommended to
always take their samples with approximately the same time interval since the previous bowel movement”
Lasson A et al. J Crohns Colitis 9 (2015) 26-32.
Sample heterogeneity : how many spot from a fecal sample?
Du L et al. Within-Stool and Within-Day Sample Variability of Fecal Calprotectin in Patients With Inflammatory Bowel Disease: A Prospective Observational Study. J Clin Gastroenterol (2016)
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Presentation Outline
• The importance of laboratory stool samples analyses
• Specimens collection (how to & which bowel movement ?)
• Storage and handling
•Weighting
• Issues regarding faeces consistency
•Biological variation: might it be of aid ?
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Faecal samples storage Manufacturers’ claims for faecal
calprotectin
Laboratory
Analysis
2 days at RT and 48 hrs at 2-8 °C
1
10 days at RT and up to 4 days at 2-8°C
2 3 days at RT, 3 days at 2-8 °C
3
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Faecal samples storage: available evidences
In collected faeces, calprotectin was
initially claimed to be stable up to 7
days at room temperature* *Røseth AG 1992. Scand J Gastroenterol. 1992;27(9):793-8.
But soon after…
Measured calprotectin concentrations in large (10–20 g) stool samples (n=69) stored at -20 °C for 1 year were
stable and reproducible. At room temperature calprotectin in stool was stable for at least 3 days.
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Samples Short-term storage
*
Ref. = stools processed after 2 hrs from the collection
• 12% decrease already after 24 hrs.
• Statistical significant differences were obtained after 7 days at RT, but not at 4°C
• “Although sample storage at 4 °C did not prevent the initial fCal decrease, it was safer than RT for storing samples for a few days”
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Long-term storage of samples
W e e k 1 W e e k 2 W e e k 4 W e e k 8 W e e k 1 2
-4 0
-2 0
0
2 0
L o n g -te rm s to ra g e -2 0 ° C
% f
Ca
l w
ith
re
sp
ec
t to
Re
f.
W e e k 1 W e e k 2 W e e k 4 W e e k 8 W e e k 1 2
-4 0
-2 0
0
2 0
L o n g -te rm s to ra g e -8 0 ° C
% f
Ca
l w
ith
re
sp
ec
t to
Re
f.
Comparable Results
Ref. = stools processed after 2 hrs from the collection
Non significant variations Non significant variations
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Faecal samples storage: available evidences
Stability of faecal calprotectin in faeces at 4 different storage conditions (room temperature (8 days), refrigerator (2–8 °C; 10 days), freezer (−20 °C) (up to 1 year) and freeze-thaw cycli (4 cycles)) on three different samples was determined. Using internal laboratory criteria (defined as 30% (3 times CV%, including analytical and extraction variation)), no significant deviation in faecal calprotectin concentrations with respect to the initial value was determined, except for samples kept at room temperature, for which significant degradation was found after 3 days. These findings support the great consensus regarding the stability of faecal calprotectin at room tem- perature for 3 days
For faecal calprotectin testing,
store samples refrigerated at 2-8°C
or at RT for no more than 3 days
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Presentation Outline
• The importance of laboratory stool samples analyses
• Specimens collection (how to & which bowel movement ?)
• Storage and handling
•Weighting
• Issues regarding faeces consistency
•Biological variation: might it be of aid ?
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Standardization of samples weighting: do we really need that ?
Weighting: how to do that?
Manually • Time
consuming • Extraction
buffer should be added
Empty (universal) collection device
• Fast • Hygienic • Extraction
buffer should be added
Prefilled, method specific (dedicate) collection device
• Ready to use • Fast • Hygienic • Reduce time,
improve efficiency
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Is manually weighting feasible for routine examinations of faecal calprotectin ?
Department of Laboratory Medicine, University-Hospital of Padova, Italy
The example of an “Hub” Laboratory
~ 10,000 fCal tests / year
Manual weighting: ~ 2 minutes each sample = 333 hrs = 47 working days Using device: 10 sec each sample = 28 hrs = 4 working days
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Commercial devices for faecal stool weighting: how do they work?
Using commercial devices for stool weighting
After the recap, exceeding material is removed from the sampling pin by collar
of the device Sampling pin
Prefilled device
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Question #2
One aspect that affect the results of pre-filled
devices for faecal calprotectin testing is:
1) Samples Storage temperature
2) Stool consistency
3) Pipetting
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Question #3
Regarding liquid stools:
1) Using a sampling device is always recommended
2) Pipetting is generally recommended
3) Sample should be discarded
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Variability of three commercially prefilled devices for fCal testing
5 “normal” (not hard, not liquid) Stool Samples
Sample were carefully mixed
to avoid heterogeneity
Analysis with the same analytical method in duplicate (IDK Calprotectin ELISA, Immunediagnostik, AG, Germany)
Dilutions were adjusted to obtain similar fCal results
3 devices were prepared by trained personnel for
the each specimen
Experimental set-up
A
B
C
Vigorous mixing at 2500 rpm for 15 minutes x 2 times
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Results
All samples had fCal within in the method linearity range (100-600 mg/g)
Variability of three commercially available, prefilled devices for fCal testing
0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
E x tra c t io n V a r ia b ility
fC a l (m g /g )
S a m p le 1
S a m p le 2
S a m p le 3
S a m p le 4
S a m p le 5
Mean and SD
A B C
0
1 0
2 0
3 0
De
vic
e V
aria
bil
ity
(C
V%
)
1 6 %
2 6 %
1 4 %
Variance Decomposition of a total of 90 fCal results by ANOVA to derive «pure» within-device variability
The performance of B is worse than A and C
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Results – Visual Assessment
Variability of three commercially available, prefilled devices for fCal testing
Device sampling pin contain unremoved material
A
B
C
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Ratio between the feces quantity declared and really
collected by devices
It is important to note that the collection of different amount of feces positive for Hb can
give different analytical results that may result in different clinical recommendations
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Are device usable by patients ?
Patients prepared devices
Patients prepared devices
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Presentation Outline
• The importance of laboratory stool samples analyses
• Specimens collection (how to & which bowel movement ?)
• Storage and handling
•Weighting
• Issues regarding faeces consistency
•Biological variation: might it be of aid ?
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Faeces consistency and sample collection devices
«Consistency of feces and the design of the sample collection device (pickers) can affect the amounts of collected materials» Rubeca T, Impact of preanalytical factors on fecal immunochemical tests: need for new strategies in comparison of methods. Int J Biol Markers. 2015;30(3):e269-74.
Comparison of sampling regions of three different
collection device for faecal calprotecin testing
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Faecal calprotectin: Comparison between manual weighting and commercial devices and liquid stool
FROM: Whitehead SJ, Ann Clin Biochem 2013; 50:53-61
Bias with respect to the manual method
Under-recovery of devices in comparison with the manual method, especially with liquid stools
Liquid faeces sample
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Liquid stools ? Manufacturers’ recommendations
Some manufacturers (not all) suggest pipetting a fix amount of
stool directly inside the extraction device
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Weighting or Pipetting in liquid stools ?
Fecal calprotectin result are reported in mg/g of wet faeces
Calibrators and controls: ng/mL
Patient’s samples are diluted more than 100-fold during the
pre-analytical step
Liquid stools
Wet faeces
Especially in liquid stools, the amount
of water in feces can varied
Can be pipetting equal to weighting ?
But …. with liquid faeces 1 ul is
considered equal to 1 ug, while density might be
different from 1.000 g/L !!
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Comparison of manual weighting and commercially prefilled devices
Hard stools represent also a potential issue
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Question #2
One aspect that affect the results of pre-filled
devices for faecal calprotectin testing is:
1) Samples Storage temperature
2) Stool consistency
3) Pipetting
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Question #3
Regarding liquid stools:
1) Using a sampling device is always recommended
2) Pipetting is generally recommended
3) Sample should be discarded
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Presentation Outline
• The importance of laboratory stool samples analyses
• Specimens collection (how to & which bowel movement ?)
• Storage and handling
•Weighting
• Issues regarding faeces consistency
•Biological variation: might it be of aid ?
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Faecal calprotectin biological variation: are faecal calprotectin levels comparable in serial stool samples measurement ?
Biological variation, available evidences
Einar Husebye, et al., Am J Gastroenterol 2001;96:2683–7
In patients with values < 50 mg/g CV = 37%
In patients with values > 50 mg/g CV = 63%
Bimodal distribution of inter-day variations
Intra- and inter-day variations
First bowel movement of the day: Intra-day CV = 40.8 % Inter-day CV = 52.0 %
Lasson et al. Journ Crohn’s and Colitis 2015
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Calprotectin biological variation: are faecal calprotectin levels comparable in serial stool samples measurement ?
Padoan, et al. Clin. Chem. Lab. Med. 56 (2018) 1926–1935
Non IBD patients
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Calprotectin BV data: can be used in the clinical setting ?
RCV can be a valuable alternative for monitoring disease activity
«Disease monitoring using fCal might benefit from the knowledge of the fCal reference change value …. because it is
independent from any specific threshold and allows the personalized monitoring of patients»
Method dependent cut-off? 50, 100, 150, 200 ?
Age dependent cut-off?
Padoan, et al. Clin. Chem. Lab. Med. 56 (2018) 1926–1935
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Thanks for the attention !
Hvala na pažnji!