preanalytical issues in · 2019. 4. 30. · stool content •food undigested contents and water...

Preanalytical issues in feces analysis

Andrea Padoan

Department of Laboratory Medicine

University-Hospital of Padova, Padova

Italy

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 ?

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…)

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

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

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

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).

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).

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)

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

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





•Weighting



Faecal samples collection: possible issues

Urine contamination

should be avoided

A sufficient quantity (at least 1 g) should be

collected

1

2

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.

Which bowel movements should be collected ?

Cohen’s kappa = 0.76 Cohen’s kappa = 0.76

Reliability study

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

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)





•Weighting



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

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.

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”

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

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 temperature for 3 days

For faecal calprotectin testing,

store samples refrigerated at 2-8°C

or at RT for no more than 3 days





•Weighting



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

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

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

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

Question #3

Regarding liquid stools:

1) Using a sampling device is always recommended

2) Pipetting is generally recommended

3) Sample should be discarded

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

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

Results – Visual Assessment

Variability of three commercially available, prefilled devices for fCal testing

Device sampling pin contain unremoved material

A

B

C

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

Are device usable by patients ?

Patients prepared devices

Patients prepared devices





•Weighting



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

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

Liquid stools ? Manufacturers’ recommendations

Some manufacturers (not all) suggest pipetting a fix amount of

stool directly inside the extraction device

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 !!

Comparison of manual weighting and commercially prefilled devices

Hard stools represent also a potential issue

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

Question #3

Regarding liquid stools:

1) Using a sampling device is always recommended

2) Pipetting is generally recommended

3) Sample should be discarded





•Weighting



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

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

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

Thanks for the attention !

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preanalytical issues in · 2019. 4. 30. · stool content •food undigested contents and water...

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