FIT – Laboratory update

Sally C Benton

Consultant Clinical Biochemist, Berkshire and Surrey

Pathology Services (BSPS)

Director, Bowel Cancer Screening Southern Hub

Date: 26th February 2019

FIT laboratory challenges

PRE-ANALYTICAL

• Stool isn’t homogenous and has variable consistency

• “Pickers” from all manufacturers are different

• Inconsistent sampling techniques by patients

• Faecal Haemoglobin instability

• Impact of Hb variants?

February 2017…….

ANALYTICAL

• No assay standardisation – no primary reference

material/ method

• EQA scheme challenges

• No established IQC material

• How to verify/ validate Fit analysers for UKAS

Different methods

give different

results

Unable to confirm if

results are correct

Is a single cut-

off appropriate?

Quantitative FIT systems

SentiFIT

HM-JACKarc OC Sensor PLEDIA

NS Prime FOB

gold

Quantitative FIT systems

SentiFIT

HM-JACKarc OC Sensor PLEDIA

FOB

gold3 FIT systems recommended by NICE

Question:

Does the collar on the FIT devices stop excess sample going in to the

buffer as claimed by manufacturers?

Pre-analytical variation: Does the mass of

sample loaded affect faecal Hb concentration?

Conclusion:

• For all 4 manufacturer devices tested the collar appears effective in stopping excess

faeces entering buffer (at amounts of faeces added)

Further work:

• Repeat with more faeces added (ie overloading FIT devices)

• Look at impact of f-Hb result if multiple dips of probe into faeces

Does the collar on the FIT devices stop excess

sample going in to the buffer?

Piggott C, John C, Bruce H, Benton S. Does the mass of sample loaded affect faecal haemoglobin concentration using

the faecal immunochemical test? Ann ClinBiochem 2018; 55(6): 702-705

Detection of Hb by FITSlide from Maggie Carroll

Hb variants

Potential impact

= decreased

detection of Hb =

risk of false

negative

Slide from Maggie Carroll

Which Variants?

Variant Abbreviation Chain affected% present in the

samples tested

Normal Normal α2β2 HbA2 = 2.6

Cord Blood FA α2γ2 β (reduced) HbF = 62 / 78

Sickle SS α2β2S β-6(A3) GluVal 95 / 93

Sickle trait AS α2βSβ β 37 / 39

C AC α2β2C β-6(A3) GluLys 35 / 38

Sickle C CS α2βSβC β

HbS = 50 HbC = 47

HbS = 48 HbC = 45

D-Punjab AD α2β2D β-121(GH4) GluGln 36 /38

E AE α2β2E β-26(B8) GluLys 27 / 25

Hb J-Broussais AJ α2Jβ2 α90(FG2) LysAsn 22

Hb Q-India AQ α2Qβ2 α1 64(E13) Asp>His 17

Hb Manitoba AM α2Mβ2 α2 102(G9) Ser>Arg 9.7

β-Thalassemia Major BTM α2β20 β (reduced synthesis) HbF = 100

β-Thalassemia

carrierBTC α2β

+ β (reduced synthesis) HbA2 = 4.7 / 4.9

Slide from Maggie Carroll

Results:

• 17/ 20 Hb variants adequately detected by FIT

• 3/ 20 inadequately detected by FIT

• 1 = β -thalassaemia major (no beta-globin chain)

• 2 = cord blood samples (contains foetal Hb).

Conclusion:• Hb variants with a conformational mutation were adequately detected by

FIT

• There is a risk of false low/ negative result in variants with a missing α or β

globin chain

• Most of these patients would have regular transfusions so probably

not a common clinical problem

Do the Antibodies used in FIT systems

detect Haemoglobin variants?

Carroll M, John C, Mantio D, Djedovic N, Benton S An assessment of the effect of haemoglobin variants on

detection by faecal immunochemical tests. Ann Clin Biochem 2018. 55(6): 706-709

Specimen collection

• Hb is unstable in faeces

• Recommendation from all FIT suppliers and international

experts is to collect samples straight in to FIT tube NOT in

to a poo pot for lab to transfer in to FIT tube

• Laboratories have had to adapt sample collection

processes

• Some give GP’s FIT tubes to hand out

• Some offer a FIT postal service

Faecal Haemoglobin Stability

• 137 patients provided FIT device and remaining faeces in “poo pot”.

• 11 (8%) of patients had different interpretation

Mellen et al. Evaluation of sample stability for a quantitative faecal immunochemical

test and comparison of two sample collection approaches. Ann Clin Bio 2018

Assay standardisation

• Currently no primary reference material or method

• Impact

• different results on different platforms

Results from EQA material run on 4 different FIT analysers

IFCC FIT Working Group(International Federation of Clinical Chemistry & laboratory

medicine)

Chair

Sally C. Benton UK

Group Members

Marieke Fasa NL

Barcey Levy USA

Han Mo Chiu Taiwan

Josep-Maria Auge Spain

Erin Symonds Australia

Petr Kocna Czech Republic

Natasha Djedovic UK

Judith Strachan UK

Ingrid Zegers Belgium

Shizuka Takehara Japan

Samantha Jones UK

Corporate members

Maurizio Gramegna Italy Sentinel

Michael Zacherl Italy Sentinel

Hideyuki Hayashi Japan Eiken

Takuo Ichiyanagi Japan Eiken

Tsuyoshi Fukuda Japan Kyowa

Yasunobu Masuda Japan Kyowa

Mr Yosuke Doi Japan Alfresa

Dr Tetsuya Kosaka Japan Alfresa

Motohito Fujimura Japan Wako

Terms of Reference

• To attempt to standardize analysis of haemoglobin in faecal samples by immunochemistry

(FIT)

• To identify all sources of pre-analytical variation and standardise if possible

• To establish external quality assurance and third party internal quality control programmes

• To determine impact of assay interference of Hb variants and other factors

First meeting held in

Athens in June 2017

2nd meeting held in

Barcelona in October

2017

3rd meeting held in Geel

in May 2018

4th meeting held in

Vienna in June 2018

Assay standardisation

progress

• Southern hub collaborating with Reference lab in Belgium

and all suppliers to identify a suitable reference material

• There will always be slight differences but we aim to align

results as much as we can between suppliers

• NICE FIT sub study

• collecting samples from patients using all 4 FIT

devices to see if same/ different diagnostic accuracy

with different FIT kits

EQA schemes update

• 2 schemes in the UK

• UKNEQAS

• WEQAS – starting in April

• Multiple schemes globally (>15)

• Varying approaches by EQA schemes

• Lyophilised samples

• Pre-loaded FIT devices

• Faecal like material sent to labs to load in to devices

• Analytical evaluation of 5 schemes being carried out by the Southern

Hub research team. 11 more schemes being contacted

• Challenge – ensuring EQA scheme assesses analytical accuracy

IQC material update

• No established 3rd party IQC material commercially

available

• Labs have to use manufacturers IQC

What should be the reporting threshold for

symptomatic FIT…..?

• Considerations;

Analytical performance characteristics

• Manufacturers recommendations

• Laboratory verification/ validation

NICE guidance

Diagnostic accuracy

• NPV and PPV/ clinical specificity and sensitivity

Impact on referral/ endoscopy capacity

Analytical performance characteristics

• Limit of detection (LOD)

• The lowest concentration at which f-Hb can be detected 95% of

the time

• The concentration where f-Hb can be reliably distinguished from a

blank sample

• Limit of quantification (LOQ)

• The lowest amount of f-Hb than can be reliably measured

according to pre-defined analytical perfomance characteristics

(CV <10%)

• Labs should not report a numerical value less than LOQ

• If f-Hb is greater than LOD but less than LOQ, lab could report

“Hb detected at very low concentrations”

Fraser CG & Benton SC. Detection capability of quantitative faecal immunochemical tests for haemoglobin (FIT)

and reporting of low faecal haemoglobin concentrations. Clin Chem Lab Med 2018.

What are labs reporting?

• Email sent on ACB mailbase for information on labs

offering symptomatic FIT

• 10 labs in England responded

• 9 labs use 10ug/g as threshold

• 1 lab uses 7ug/g

• Only 2 labs had worked out LOD and LOQ of their

instruments

NICE guidance: Evidence Base/

Diagnostic accuracy

Author Year Country

Patients

in study cut off (ug/g) NPV PPV Sensitivity Specificity

Number of

cancers

Cancers

missed

Mowat et al 2015 Scotland 755 10 99.5 14.2 89.3 79.1 28.0 3.0

Rodriguez-Alonso et al 2015 Spain 1003 10 99.9 12.8 96.7 79.8 30 1

Godber et al 2015 Scotland 484 10 100 100 11 0

Droste et al 2011 Netherlands 2145 10 92.4 86.4 79 6

McDonald et al 2012 Scotland 280 10 100 7.6 100 93.9 6 0

Cubiella et al 2014 Spain 787 20 97.8 35.3 87.6 77.4 97

Author Year Country

Patients

in study cut off (ug/g)

NPV for

CRC PPV Sensitivity

Specificit

y

Number of

cancers Cancers missed

Mowat et al 2015 Scotland 755 Undetectable (7ug/g) 100 6.4 100 43.4 28 0

Rodriguez-Alonso et al 2015 Spain 1003 Undetectable (0) 100 5.2 100 43.3 30 0

Widlak et al 2016 England 430 Undetectable (7ug/g) 99 44 84 93 24 3

10ug/g cut off – as recommended in NICE guideline

Cut off – “undetectable” Hb

Should we be reporting

down to limit of

detection??

Impact on endoscopy

• Threshold to use is a local decision

• Local lab, endoscopy services CCG to jointly decide

• Currently no published evidence to demonstrate impact of

symptomatic FIT on endoscopy

• Lower the concentration that is reported = more people

referred = pressure on endoscopy = more cancers/ pre-

cancers likely to be detected

FIT laboratory current status

PRE-ANALYTICAL

• Hb variants likely to have very minimal impact

• Collars on FIT tubes effective – more work on-going

• Samples should be collected straight in to FIT devices

• Symptomatic FIT testing is now available across much of

England

February 2019…….

ANALYTICAL

• Identification of a primary reference material/ method underway

• There are a number of EQA schemes now available. The most fit

for purpose still to be confirmed

• Guidance is available on how to verify/ validate FIT analysers

• Still no established IQC material

FIT laboratory current statusPRE-ANALYTICAL

• Hb variants likely to have very minimal impact

• Collars on FIT tubes effective – more work on-going

• Samples should be collected straight in to FIT devices

• Symptomatic FIT testing is now available across much of England

February 2019…….

ANALYTICAL

• Identification of a primary reference material/ method underway

• There are a number of EQA schemes now available. The most fit for purpose still to be

confirmed

• Guidance is available on how to verify/ validate FIT analysers

• Still no established IQC material

POST-ANALYTICAL

• What threshold should be actually be used…….?

• LOD/ LOQ??

Berkshire & Surrey Pathology Services A joint venture between Frimley Park Hospital, Royal Surrey County

Hospital, Royal Berkshire Hospital, Wexham Park Hospital and Ashford

and St. Peter’s Hospitals NHS Foundation Trusts