This version was published on 1 November 2008
Ann Clin Biochem 2008;
45:604-605
doi:10.1258/acb.2008.008024
© 2008 Association for Clinical Biochemistry
Effect of delay in sampling on haemoglobin determined by faecal immunochemical tests
Louise F Brown1 and
Callum G Fraser2
1 Department of Biochemical Medicine, Ninewells Hospital and Medical School;
2 Scottish Bowel Screening Centre Laboratory, Kings Cross, Dundee, UK
Corresponding author: Louise F Brown. Email: l.brown3{at}nhs.net
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Abstract
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Background: Faeces must be sampled directly onto guaiac-based faecal occult
blood test (FOBT) cards since analysis of specimens collected
in traditional faeces containers is inappropriate because degradation
of haemoglobin continues after faeces have been passed. Newer
faecal immunochemical tests (FIT) are replacing FOBT, but it
is likely that the practice of obtaining specimens in traditional
faeces collection containers for later analysis will continue.
The aim of this study was to assess the effect of delay in stool
sampling on FIT.
Methods: Five specimens of faeces from healthy volunteers, all qualitatively FIT negative, were supplemented with whole blood haemolysate to three different FIT positive concentrations. Each sample was analysed daily after 1–14 days delay using a quantitative latex immunoturbidimetric-based FIT and also after five and ten days delay using a qualitative FIT.
Results: Haemoglobin concentrations fell each day, the rate being generally proportional to the original haemoglobin concentration. After eight days delay, no sample had a haemoglobin concentration >100 ng/mL and, after nine days, no sample had a haemoglobin concentration >50 ng/mL. After five days delay, five of the 15 supplemented faeces with initially positive qualitative FIT had negative FIT; after 10 days, none had positive FIT.
Conclusion: False-negative results will occur if sampling of fresh faeces into or onto FIT collection devices is delayed. Laboratories that undertake FIT analyses on faeces collected into traditional containers are likely to miss significant neoplasia. FIT collection devices must be used for sampling fresh faeces.
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Introduction
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There is evidence that degradation of haemoglobin continues
after faeces have been passed.
1 This has relevance for traditional
guaiac-based faecal occult blood tests (FOBT), which detect
the pseudoperoxidase activity of haem. Using samples from volunteers
spiked with whole blood haemolysate, Young
et al.
2 examined
the effect of delayed versus immediate application of faecal
samples onto FOBT, and showed that false-negative results would
occur when sampling of faeces onto the test card was delayed:
it was emphasized that FOBT should be prepared immediately after
passing of faeces and transport of moist samples avoided. However,
clinical laboratories do not follow these recommendations and
still receive most samples for FOBT in the traditional faecal
collection containers.
3
It has been suggested that FOBT be ceased in all clinical settings except screening programmes and also proposed that FOBT be replaced by faecal immunochemical tests (FIT), as they undoubtedly have many advantages and fewer problems in both performance and interpretation.4 These are based upon a different principle to FOBT, in that FIT use monoclonal or polyclonal antibodies and detect intact human haemoglobin or very early degradation products. Since current practices might continue and FIT analyses performed in laboratories, wards, clinics and general practices on samples of faeces collected in the traditional faecal containers, the effect of delay in sampling on faecal haemoglobin concentration, as assessed with both quantitative and qualitative FIT, was investigated.
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Methods
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Five fresh specimens of faeces from apparently healthy volunteers
were tested with
hema-screen SPECIFIC (Immunostics Inc., Ocean,
NJ, USA, supplied by Alpha Labs Ltd., Eastleigh, Hants, UK).
After thorough mixing, one portion of each was saved; another
was supplemented with whole blood lysate prepared by freezing
and thawing a specimen of venous blood collected into K
2-EDTA
vacutainers (Becton Dickinson, Oxford, UK) until the qualitative
FIT result was weak-positive. Then, approximately twice and
four times this amount of lysate was added to portions of each
to give medium- and strong-positive FIT results. All were stored
at between 18–21°C.
Samples were taken from the native and supplemented specimens after 24 hours and then at the same time each day for a further 13 days (14 days in total) into hema-screen SPECIFIC sample preparation tubes; these tubes have an integral sampling ‘stick’ on the lid to facilitate the safe transfer of a small amount of faeces into the tube containing phosphate-buffered saline with 0.5% sodium azide, which stabilizes the haemoglobin present.
The samples were then assayed using two methods. First, haemoglobin concentration was quantitatively measured in the material in the hema-screen SPECIFIC sample preparation tube using latex turbidimetry with FOB Gold reagents, calibrators and controls on a SENTiFOB fully automated photometric analyser (Sentinel Diagnostics, Milan, Italy). Secondly, after five and 10 days delay, qualitative FIT were also performed using hema-screen SPECIFIC test cassettes, which use a sandwich dye conjugate immunoassay using immunochromatography.
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Results
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The five native faeces had haemoglobin concentrations of 14.4,
14.4, 14.9, 30.1 and 33.2 ng/mL: all were negative by qualitative
FIT analysis. The supplemented faeces had haemoglobin concentrations
between 101.6 ng/mL and 2089.2 ng/mL, and all were positive
by qualitative FIT. Weak-positive samples had mean haemoglobin
concentration of 314.7 ng/mL, medium-positive had 561.6 ng/mL
and strong-positive had 1430.5 ng/mL.
The haemoglobin concentration in the faeces fell with time. The rate of decrease was variable from one sample to the other, but generally proportional to the amount of haemoglobin as shown in Figure 1 for the series of faeces from one of the volunteers: all showed similar patterns. Mean haemoglobin (% of original concentration) in supplemented faeces (SEM) fell from 100% to 82.5% (5.7%), 60.3% (5.3%), 55.7% (6.0%), 46.5% (5.0%), 20.0% (3.1%), 14.2% (2.4%), 12.9% (2.6%), 8.1% (1.7%), 5.7% (2.0%), 7.3% (2.3%), 6.9% (1.9%), 5.7% (1.4%), 4.5% (1.3%) and 3.2% (0.9%) after 1–14 days delay.
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Figure 1 Haemoglobin concentration (ng/mL) in native faeces and faeces supplemented with whole blood lysate to give weak, medium and strong qualitative faecal immunochemical test results
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Faecal specimens with haemoglobin near to the cut-off concentration
became negative after short periods of delay. For example, the
haemoglobin concentration in the weak-positive samples fell
below 50 ng/mL after three to six days. After eight days, no
sample had a haemoglobin concentration >100 ng/mL, a commonly
used cut-off concentration for the declaration of positive or
negative results in quantitative FIT analysis.
5 After nine days,
no sample had a haemoglobin concentration >50 ng/mL, which
is the usual cut-off concentration of commercially available
qualitative FIT such as that used in the Scottish Bowel Screening
Programme.
6
To verify these results with a different FIT, the samples were assayed with the qualitative FIT after five days, when five of the 15 supplemented faeces now had negative FIT, and after 10 days, when none had positive FIT.
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Discussion
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In conclusion, false-negative results for faecal haemoglobin
will occur if sampling of fresh faeces into the tubes or onto
the cards of FIT collection devices is delayed. Clinical laboratories
and other sites that undertake FIT analyses on faeces collected
into traditional containers (e.g. ‘pots’ with integral
spoons in the lids) are likely to miss significant neoplasia.
This will lead to adverse outcomes for patients since the earlier
the diagnosis of colorectal (bowel) cancer is made, the better
the prognosis. The actual collection devices supplied with the
particular FIT must be used for sampling fresh faeces.
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ACKNOWLEDGEMENTS
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We thank Sentinel Diagnostics for the loan of the SENTiFOB analyser
and Alpha Labs Ltd. for the loan of pipettes. NHS Tayside has
a consultancy agreement with Immunostics Inc.
(Accepted May 6, 2008)
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REFERENCES
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- Rose IS, Young GP, St John DJB, Deacon MC, Blake D, Henderson RW. Effect of ingestion of hemoproteins on fecal excretion of hemes and porphyrins. Clin Chem 1989;35:2290–6[Abstract/Free Full Text]
- Young GP, Sinatra MA, St John DJB. Influence of delay in stool sampling on fecal occult blood sensitivity. Clin Chem 1996;42:1107–8[Free Full Text]
- Fraser CG. Faecal Occult Blood Tests – Eliminate or Improve. See www.acbscot.org.uk/science/FOBt.html (last accessed 15 January 2008)
- Fraser CG. Faecal occult blood tests – eliminate, enhance, or update. Ann Clin Biochem 2008;45:117–21[Abstract/Free Full Text]
- Chen LS, Liao CS, Chang SH, Lai HC, Chen TH. Cost-effectiveness analysis for determining optimal cut-off of immunochemical faecal occult blood test for population-based colorectal cancer screening (KCIS 16). J Med Screen 2007;14:191–9[Abstract/Free Full Text]
- Fraser CG, Mathew CM, Mowat NAG, Wilson JA, Carey FA, Steele RJC. Evaluation of a card collection-based faecal immunochemical test in screening for colorectal cancer using a two-tier reflex approach. Gut 2007;56:1415–8[Abstract/Free Full Text]
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Abstract
Introduction
