Critical appraisal:Church TR, Wandell M, Lofton-Day C, Mongin SJ, Burger M, Payne SR, et al 2014 2

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Risk of bias assessment: diagnostic accuracy study

Patient Selection
Prior tests and any referral filters
Patients scheduled for colonoscopy at

one of the participating clinical centres were approached about
volunteering for the study

Condition that defined entry into study
asymptomatic individuals, aged 50 years and

older, meeting average risk screening guidelines

Setting
multicentre US and German study, sponsored by Epigenomics AG
Was a diagnostic case-control design avoided?
Yes
Consecutive or random sample?
Yes
Did the study avoid inappropriate exclusions?
Yes
Reasons
To increase accrual,

approximately 6 months after study initiation the CSSC revised
the initial eligibility criteria to no longer exclude subjects with
flexible sigmoidoscopy more than 5 years before enrolment.

If comparing more than one index test was the design fully paired or paired randomly?
Not applicable
If a paired randomised design was used, was allocation to groups concealed and was the generation of allocation sequence adequate?
Not applicable
What is the risk that the selection of participants introduced bias?
Low
Comments
Index test 1
Describe index test and how it was conducted and interpreted
methylated SEPT9 DNA (mSEPT9). Briefly, free circulating DNA

from 3.5 ml of plasma, obtained through pooling of two aliquots of subject plasma each containing ∼2 ml, was captured on
magnetic beads, purified and concentrated. To ensure only
methylated cytosine was replicated, the unmethylated cytosine
in the purified DNA was then chemically converted to uracil
using sodium bisulfite. DNA was then captured again on magnetic beads, purified through washing steps and eluted in a final
volume of 55 μl. The resulting bisulfite converted DNA was
amplified in a duplex PCR comprised of oligonucleotides for an
internal control assay, actin β for determining the validity of the
result and oligonucleotides for the target analyte, methylated
Septin9. Two separate PCR replicates were prepared with the
bisulfite converted DNA eluate from each subject sample by
adding 15 ml of eluate to each of two wells of a 96 well PCR
plate. Prior to adding subject DNA to the PCR plate, 25 ml of a
mixture of PCR buffer, oligonucleotides and polymerase were
added to the entire 96 well plate. Real time PCR was performed
on an LC480 device (Roche Diagnostics, Indianapolis, Indiana,
USA). The remaining ∼25 ml of bisulfite converted DNA material was frozen at −80°C and stored.

Were the index test results interpreted without knowledge of the results of the reference standard?
Unclear
If a threshold was used, was it pre-specified?
Yes
If two tests are being compared, have they been assessed independently / blind to each other?
Not applicable
What is the risk that the conduct or interpretation of the index test introduced bias?
Unclear
Comments
Index test 2
Describe index test and how it was conducted and interpreted, if applicable
Were the index test results interpreted without knowledge of the results of the reference standard?
Not applicable
If a threshold was used, was it pre-specified?
Not applicable
What is the risk that the conduct or interpretation of the index test introduced bias?
Not applicable
Comments
Reference Standard
Describe the reference standard and how it was conducted and interpreted
Is the reference standard likely to correctly classify the target condition?
Yes
Were the reference standard results interpreted without knowedge of the results of the index test/s?
Unclear
Was the reference test standard independent of the index test?
(i.e. the index test did not form part of the reference standard)
Yes
What is the risk that the reference standard, its conduct or interpretation introduced bias?
Unclear
Comments
Flow and timing
Describe any patients who did not receive the index test(s) and/or reference standard or who were excluded from the 2x2 table
Nine withdrew but

their baseline data were used in the calculation of weights; 12
German subjects withdrew and their data were redacted in
accordance with German law. 56 didn't meet eligibility criteria; 6 non-CR cancer; 747 incomplete data at time of batch selection; 64 omitted by data error.

Describe the time interval and any interventions between index test(s) and reference standard
The time between sample collection and colonoscopy averaged 14 days (IQR 6–16 days)
If a predictive test (the reference standard is a later event that the test aims to predict) were any subsequent interventions between test and later event blind to test result?
Not applicable
Was there an appropriate interval between index test(s) and reference standard?
Yes
Did either all participants or a random sample of participants receive a reference standard test?
Yes
Did all patients receive the same reference standard irrespective of index test result?
Yes
Were all test results including unclear results reported?
Yes
Were all patients included in the analysis?
Yes
What is the risk that the patient flow introduced bias?
Low
Comments
Overall risk of bias
At risk of bias Additional comments: Please replace this text and include any additional comments in regards to your risk of bias rating


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Article
Church TR, Wandell M, Lofton-Day C, Mongin SJ, Burger M, Payne SR, et al. Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut 2014 Feb;63(2):317-25 Available from: http://www.ncbi.nlm.nih.gov/pubmed/23408352.
Assigned to
User:Victoria.freeman
Topic area
Guidelines:Colorectal cancer
Clinical question
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