Effect of Temperture nd Time on Fecl Hemoglobin Stbility in 5 Fecl Immunochemicl Test Methods nd One Guic Method Peter Ctomeris, PhD; Nncy N. Bxter, MD, PhD; Sheil C. Boss, PhD; Lwrence F. Pszt, MD, PhD; Lind Rbeneck, MD, MPH; Edwrd Rndell, PhD; Mrdie L. Serenity, MHSc, PMP; Rinku Sutrdhr, PhD; Jill Tinmouth, MD, PhD Accepted for publiction Mrch 3, 2017. Published s n Erly Online Relese October 2, 2017. From the Medicl nd Scientific Deprtment, LifeLbs, Toronto, Ontrio, Cnd (Drs Ctomeris nd Boss); the Division of Generl Surgery, St Michel s Hospitl, Toronto (Dr Bxter); the Deprtments of Generl Surgery (Dr Bxter), Rdition Oncology (Dr Pszt), nd Medicine (Drs Rbeneck nd Tinmouth), University of Toronto, Toronto; Prevention nd Cncer Control, Cncer Cre Ontrio, Toronto (Drs Rbeneck nd Tinmouth); the Division of Lbortory Medicine, Fculty of Medicine, Memoril University of Newfoundlnd, Helth Sciences Centre, St John s, Newfoundlnd, Cnd (Dr Rndell); Evlutive Clinicl Sciences (Drs Pszt nd Tinmouth nd Ms Serenity) nd Division of Gstroenterology (Dr Tinmouth), Sunnybrook Reserch Institute, Toronto; the Institute for Clinicl Evlutive Sciences, Toronto (Drs Bxter, Pszt, Sutrdhr, nd Tinmouth); the Institute for Helth Policy, Mngement nd Evlution (Drs Bxter, Pszt, nd Tinmouth); nd the Deprtment of Biosttistics (Dr Sutrdhr), Dll Ln School of Public Helth, University of Toronto, Toronto. Dr Boss is now retired. This study ws conducted with the support of Cncer Cre Ontrio (CCO) in collbortion with LifeLbs Medicl Lbortory Services. Dr Tinmouth ws led scientist for the ColonCncerCheck progrm, nd Dr Rbeneck ws vice president for Prevention nd Cncer Control t CCO. Drs Ctomeris nd Boss were both employees of LifeLbs Medicl Lbortory Services t the time of the study. The other uthors hve no relevnt finncil interest in the products or compnies described in this rticle. The opinions, results, nd conclusions reported in this rticle re those of the uthors nd re independent from the entities providing support. No endorsement by CCO or LifeLbs Medicl Lbortory Services is intended nor should be inferred. Reprints: Jill Tinmouth, MD, PhD, Sunnybrook Reserch Institute, 2075 Byview Ave, HG-40 Toronto, ON M4N 3M5, Cnd (emil: jill.tinmouth@sunnybrook.c). Context. Although promising for colorectl cncer screening, hemoglobin (Hb) stbility remins concern with fecl immunochemicl tests. This study implemented novel, stndrdized method to compre Hb stbility cross vrious fecl immunochemicl tests. The method cn be used to inform decisions when selecting kit for use in colorectl cncer screening. In so doing, this work ddressed criticl need for stndrdiztion in this field. Objective. To compre the stbility of Hb cross 5 different immunochemicl kits nd one guic kit. Design. The stbility of Hb ws nlyzed in collection devices inoculted with Hb-spiked feces nd (1) stored t vrious tempertures (frozen, refrigerted, mbient, nd elevted) for more thn 60 dys; (2) fter undergoing 3 controlled, freeze-thw cycles; nd (3) fter being trnsported by courier or postl services in uncontrolled temperture conditions from 3 loctions in Ontrio, Cnd, to centrl testing center. Results. The stbility of Hb vried with time nd temperture nd by kit. Lower Hb recoveries occurred with incresing temperture nd incresing time from smple collection to testing. Refrigertion provided the best stbility, lthough results vried cross kits (eg, from 4.2 dys to.60 dys before prespecified threshold [,70% probbility of the test results remining positive] ws reched). Freeze-thw stbility vried cross kits nd cycles (Hb recoveries: NS Plus [Alfres Phrm, Chuo-ku, Osk, Jpn], 91.7% to 95.4%; OC Din [Eiken Chemicl, Tito-ku, Tokyo, Jpn], 57.6% to 74.9%). Agreement regrding Hb levels before nd fter trnsporttion vried cross kits (from 57% to 100%). Conclusions. Importnt differences in Hb stbility were found cross the included fecl immunochemicl tests. These findings should inform prctice-bsed nd popultion-bsed colorectl cncer screening. (Arch Pthol Lb Med. 2018;142:75 82; doi: 10.5858/ rp.2016-0294-oa) Screening with guic-bsed fecl occult blood tests (gfobt), followed by colonoscopy for ptients who test positive, ws ssocited with reduction of 15% to 33% in colorectl cncer (CRC) relted mortlity in 3 lrge, rndomized clinicl trils. 1 3 Fecl testing for occult blood is the most commonly used screening method in CRC screening progrms worldwide. 4 Until recently, those progrms predominntly used kits bsed on the guic regent to detect the heme moiety of blood, relying on the inherent peroxidse ctivity of heme. Unfortuntely, there re importnt drwbcks to the guic method, 5 including low nlytic sensitivity, low nlytic specificity, nd low clinicl sensitivity. 6 Promising fecl immunochemicl test (FIT) methods hve been developed. These immunossys form n ntibodyntigen complex with the globin moiety of humn hemoglobin (Hb) to generte either n immunoturbidimetric signl, which cn be mesured (quntittive) or by lterl-flow immunochromtogrphic nlysis, bnd tht cn be detected visully (qulittive). 5 The FITs hve been shown to hve better nlytic sensitivity nd specificity thn gfobt hs, s well s better clinicl sensitivity, with miniml loss of clinicl specificity. In ddition, prticiption Arch Pthol Lb Med Vol 142, Jnury 2018 Hemoglobin Stbility in FIT nd gfobt Ctomeris et l 75
Mnufcturer Alfres Phrm Corportion (Chuo-ku, Osk, Jpn) Eiken Chemicl Co, Ltd (Tito-ku, Tokyo, Jpn), Inc (Etontown, New Jersey) ulti med (Ahrensburg, Germny) Beckmn Coulter, Inc (Bre, Cliforni), Inc (Etontown, New Jersey) Tble 1. Kit Nme Chrcteristics of Kits Included in the Current Study Abbrevition Used in Article Quntittive or Qulittive Method Principle Wet or Dry Collection Device Positivity Threshold b,c Hemoglobin NS Plus Quntittive Immunochemicl Wet 20 NS-Plus OC-Sensor Din OC Din Quntittive Immunochemicl Wet 20 Hem-Screen FIT Qulittive Immunochemicl Dry 50 SPECIFIC FOB Advnced ulti med Qulittive Immunochemicl Wet 2 Hemoccult ICT Beckmn ICT Qulittive Immunochemicl Dry 1500 Hem-Screen Guic FOBT Guic Qulittive Guic Dry 600 Quntittive kits generte numeric result reflecting the concentrtion of hemoglobin (in microgrms of hemoglobin per grm of feces). A quntittive kit is considered positive if the concentrtion exceeds defined positivity threshold. Qulittive kits mesure the presence of hemoglobin reltive to the mnufcturer s preset positivity threshold, resulting in binry positive or negtive result. A numeric result is not provided. b Positivity threshold s stted by mnufcturer in microgrms of hemoglobin per grm of feces. c With the exception of the ulti med method, in-house studies confirmed s ccurte the mnufcturer s stted sensitivities of ech of the methods. For the ulti med, sensitivity of 100 lg hemoglobin/g of feces ws observed [dt not shown]. with screening is improved with FIT compred with gfobt, 7 10 likely becuse fewer smples (1 insted of 3) re required, nd there re no dietry restrictions. Humn Hb continues to degrde fter feces re pssed nd dely of smple testing cn cuse flse-negtive results in both gfobt 11 nd FIT methods. 12 FITs pper to be prticulrly unstble, s reported recently in n Itlin CRC screening progrm 13 ; furthermore, the wet collection methods used by some FIT kits my exhibit more Hb degrdtion thn dry collection methods. 11,14 Orgnized CRC screening progrms require Hb to remin stble within the collection devices for some time becuse the smple is typiclly collected t home nd returned, often by mil, to the lbortory for testing. The FIT mnufcturers recommendtions, s well s dt from studies of FIT stbility, 15,16 suggest shorter intervls between collection nd testing might be needed if FIT is used insted of gfobt. However, vrition in Hb stbility hs been reported cross kits. 17,18 It is possible tht cceptble times between smple collection nd testing could lso vry by FIT brnd. To dte, there re limited dt compring the Hb stbility in stndrdized fshion for vrious quntittive nd qulittive FITs, mking informed selection of FIT for use in orgnized CRC screening progrms difficult. 19 The im of the current study ws to exmine in stndrdized mnner the stbility of humn fecl Hb in the collection devices of 5 FIT kits nd 1 gfobt kit over time nd t severl different tempertures. MATERIALS AND METHODS Terminology The following terminology is used throughout. Kit refers to the pckge of components, including collection devices, required for the collection nd nlysis of Hb in fecl smples. Collection device is the component of kit to which the feces re dded nd stored until nlysis. Spiked feces is fecl smple to which Hb hs been dded in the lbortory. Inoculted device is collection device to which feces (spiked or nonspiked) hs been dded for nlysis of Hb. Overview Three in vitro studies were conducted from Februry to April 2012 to ssess Hb stbility. Study 1 ws controlled study of inoculted devices stored t vrious controlled tempertures (frozen, refrigerted, mbient, nd elevted) nd over time up to 60 dys. Study 2 ws controlled study of inoculted devices exposed to severl freeze-thw cycles. Study 3 ws field study in which inoculted devices were trnsported by courier or postl services under uncontrolled temperture conditions, which ws intended to simulte conditions tht inoculted devices would experience in the CRC screening progrm in Ontrio, Cnd. Approvl This study ws reviewed nd pproved by the Sunnybrook Reserch Institute (Toronto, Ontrio, Cnd) reserch ethics bord. Kits Tble 1 summrizes the chrcteristics of the kits included in the current study. The FIT kits were selected for inclusion if they were pproved for use by Helth Cnd nd if they were suitble for use in n orgnized, popultion-bsed CRC progrm (ie, they were not point-of-cre kits). The following FIT kits were included: (1) Hb NS-Plus, mnufctured by Alfres Phrm Corportion (Chuo-ku, Osk, Jpn); (2) OC-Sensor Din, mnufctured by Eiken Chemicl Co, Ltd (Tito-ku, Tokyo, Jpn); (3) Hem-Screen SPECIFIC, mnufctured by, Inc (Etontown, New Jersey); (4) FOB Advnced, mnufctured by ulti med (Ahrensburg, Germny); nd (5) Hemoccult ICT, mnufctured by Beckmn Coulter, Inc (Bre, Cliforni). The gfobt (Hem-Screen Guic, ) currently used in the Ontrio, Cnd, CRC screening progrm ws lso included. Preprtion of Spiked Feces For ll studies, fresh humn feces, without preservtives nd from multiple donors, were used. Feces were spiked with humn Hb from ethylenedimine tetr-cetic cid whole blood specimens. Initil Hb concentrtions were determined with the Sysmex XE- 2100 series utomted hemtology nlyzers (Sysmex Corportion, Kobe, Hyogo Prefecture, Jpn) using the sodium luryl sulfte method, clibrted to Interntionl Council for Stndrdiztion in Hemtology/World Helth Orgniztion interntionl hemoglobincynide stndrd (70600). Hemolystes were prepred by diluting whole blood 10-fold with deionized wter. The Hb ws trnsferred to the fecl mteril, nd homogeneous mixture ws obtined by stirring the feces with wooden sticks. Collection devices were inoculted with feces t room temperture within 30 minutes of spiking with Hb. Using the procedure described, 76 Arch Pthol Lb Med Vol 142, Jnury 2018 Hemoglobin Stbility in FIT nd gfobt Ctomeris et l
cceptble nd reproducible recoveries of Hb were obtined. When mesured with the OC Din kit, the rnge of Hb recovery ws 85.4% to 112.7% in 16 replictes. Men (SD) recovery ws 103.4% (9.7%). Definitions of Stbility To dte, there is no universl or stndrdized pproch to ssess Hb stbility. 19,20 Although some mnufcturers do provide estimtes of Hb stbility in their product inserts, those estimtes cnnot be relibly compred cross brnds becuse the methods to determine stbility were not described in the pckge inserts nd time frmes vried. For the purposes of our nlysis, we mesured Hb stbility using seprte, but stndrdized, pproches for the comprison of quntittive kits lone nd for comprison of ll kits included in the study. These 2 pproches re described below. For comprison of Hb stbility in the collection devices of the quntittive kits only, the durtion of stbility in study 1 ws defined s the time until the percentge of Hb recovery ws reduced (from 100%) by less thn 2 times the between-run precision, s determined from internl studies. The between-run precision ws pproximtely 13% nd 9% t concentrtions of 33 nd 100 lg Hb/g feces, respectively. Therefore, men recovery of 80% or more ws used s cutoff for stbility when compring the 2 quntittive kits. Becuse qulittive FIT nd gfobt test results re red in binry fshion, the bove pproch could not be used. To compre Hb stbility cross ll kits (both qulittive nd quntittive), we defined the durtion of stbility s the time tht elpsed until the probbility of the result remining positive dropped below prespecified threshold. For this nlysis, quntittive kit results were ctegorized s hving positive or negtive results reltive to the mnufcturer s suggested positivity threshold (20 lg Hb/g feces for both kits). Stbility t 3 prespecified thresholds (80% probbility of remining positive, 70%, nd 50%) ws mesured. The clinicl implictions of these thresholds re not known. However, the only other study 17 to estblish threshold for stbility used 50%, so our thresholds mtch or exceed those used by others. For ll 3 studies, results were defined s positive if the Hb concentrtion ws bove the mnufcturer s recommended positivity threshold (see Tble 1) for quntittive kits or if the visul bnd ws detected for qulittive kits. Study 1: Controlled Smple Stbility Study Preliminry in-house studies showed tht the nlytic sensitivities of the kits were 20 lg Hb/g of stool (NS Plus nd OC Din), 50 lg Hb/g of stool ( FIT), 100 lg Hb/g of stool (ulti med), 500 to 1000 lg Hb/g of stool (Beckmn ICT), nd 500 lg Hb/ g of stool ( Guic). Feces were spiked to produce Hb concentrtions from 25 to 3000 lg Hb/g of feces. As the nlytic sensitivities (which reflect the lower limit of Hb detection) vried cross kits, concentrtion rnges pproprite to the nlytic sensitivity of ech kit were selected. 18 Finl Hb concentrtions of 25, 50, 100, nd 200 lg Hb/g of feces were used for the NS Plus, OC Din, nd FIT kits; 100, 200, 500, 1125, nd 1500 lg Hb/g of feces were used for the ulti med kit; nd 500, 1125, nd 1500 lg Hb/g of feces were used for the Beckmn ICT nd Guic kits. Multiple inoculted devices were prepred for ech kit (33 sets per kit) using feces spiked with ech of the Hb concentrtions specified bove for tht prticulr kit. The first set ws nlyzed immeditely t dy 0 (bseline). The other 32 sets were stored under the following temperture conditions: frozen, 208 to 158C ( 48 to 58F) (8 sets); refrigerted, 28C to88c (368F to468f) (8 sets); mbient, 208C to228c (688F to728f) (8 sets); nd elevted, 458C (1138F) (8 sets). For ech temperture condition, set ws removed nd nlyzed t 4, 7, 10, 14, 21, 28, 45, nd 60 dys fter bseline. Ech inoculted device ws tested only once during this study. We clculted the medin percentge of Hb recovery t ech time point in ech of the temperture conditions for ech of the 2 quntittive kits. We then exmined Hb stbility in 2 wys: comprison of the inoculted devices from the quntittive kits only, nd comprison of the inoculted devices from ll kits, both quntittive nd qulittive. For the comprison of Hb stbility in collection devices of the quntittive kits only, the percentge of Hb recovery compred with the bseline were first clculted for ech temperture condition nd t ech time point. As noted bove, recovery of 80% or more ws used s cutoff for stbility. Under ech of the 4 temperture conditions, the reltionship between the percentge of Hb recovery nd time ws first modeled using liner regression model. Qudrtic nd cubic terms were included in the regression if they improved the model fit bsed on likelihood rtio tests. 21 A generlized estimting eqution pproch ws used to ccount for ny repeted mesurements tken on the sme smple. 22 Once the estimtes of the regression coefficients were obtined, those vlues were then used to predict the time in dys nd the 95% CI tht Hb recovery would drop below 80% compred with the bseline. For the comprison of Hb stbility in the collection devices cross ll kits, results for both the quntittive nd qulittive kits were expressed s binry results (negtive/positive) only. As noted bove, stbility ws defined s the time required for the probbility of results remining positive to drop below 3 prespecified thresholds. Ech threshold ws modeled seprtely, strting with the 80% threshold. Under ech of the 4 temperture conditions, the reltionship between positive result (yes/no) nd time ws modeled using logistic regression model. As bove, qudrtic nd cubic terms were included where pproprite, nd generlized estimting eqution pproch ws used. Once the estimtes of the regression coefficients were clculted, those vlues were used to predict time in dys nd the 95% CI when the probbility of results remining positive dropped below 80%. These nlyses were repeted for the other 2 probbility thresholds, specificlly, the time t which the probbility of results remining positive dropped below 70% nd below 50%. Study 2: Controlled Freeze-Thw Study In rel-world screening progrms, ptient smples my be exposed to vrible tempertures nd conditions, including freezing nd thwing, during the trnsport of the smples from the ptient to the testing lbortory. This study ws designed to determine the effect of freezing nd thwing on smples in the collection devices. The stbility of Hb during freezing nd thwing ws studied in the NS Plus, OC Din, nd ulti med collection devices. (Kits with single-use collection devices could not be used for this study.) Fecl smples were spiked to produce smples with Hb concentrtions rnging from 25 to 1200 lg Hb/g of feces, in keeping with the rnges for these 3 kits used in study 1. Inoculted devices were prepred with smples t Hb concentrtions pproprite to the nlytic sensitivity of ech type of kit, s described in study 1. Inoculted devices (NS Plus, n ¼ 12; OC Din, n ¼ 12; ulti med, n ¼ 12) were nlyzed immeditely (bseline), nd the sme devices were then nlyzed fter ech of 3 freeze-thw cycles. Frozen storge conditions were 208C to 158C ( 48Fto58F). Smples were thwed nd refrozen fter nlysis t 4, 7, nd 10 dys fter the initil storge in the freezer. Inoculted devices were kept frozen for 4, 3, nd 3 dys between thwing, testing, nd refreezing for cycles 1, 2, nd 3, respectively. The number of cycles nd durtion were selected becuse they were likely to reflect nticipted rel-world conditions. For the nlysis, the medin percentge of Hb recovered compred with bseline vlues ws clculted for NS Plus nd OC Din fter ech of the freeze-thw cycles. For ulti med, the percentge of inoculted devices remining positive fter ech cycle ws clculted. Study 3: Field Study A totl of 30 fecl smples were freshly spiked with Hb t the testing loction to chieve finl Hb concentrtions of 0 lg Hb/g of feces (n ¼ 6), 30 lg Hb/g of feces (n ¼ 3), 60 lg Hb/g of feces (n ¼ 6), 125 lg Hb/g of feces (n ¼ 3), 500 lg Hb/g of feces (n ¼ 6), nd 2000 Arch Pthol Lb Med Vol 142, Jnury 2018 Hemoglobin Stbility in FIT nd gfobt Ctomeris et l 77
lg Hb/g of feces (n ¼ 6). Two btches of inoculted devices using those smples were prepred for ech of the 6 kits. One btch of inoculted devices (set A, 30/kit for 180 inoculted devices) ws nlyzed t the testing loction to obtin bseline vlues. The second btch of inoculted devices (set B, n ¼ 180) ws pckged for trnsporttion to 3 distribution centers (Thunder By, Ottw, nd London; ll in Ontrio, Cnd). The pckges were prepred without ny temperture control, tht is, no cooling pcks, but simply with cushioning mteril to prevent excessive contct within the pckge. The study ws rbitrrily designed to hve 10 pckges for ech center. Ech pckge contined 6 inoculted devices (1 for ech of the 6 kits), for totl of 60 devices. Ech center received the sme distribution of Hb concentrtions. For ech center, thermometers were dded to 9 of the 10 pckges to determine minimum nd mximum tempertures during trnsport nd continuous temperture dt logger ws plced in the 10th pckge. The btches of pckges were delivered from the testing loction to the 3 distribution centers by courier. From ech of the distribution centers, the 10 pckges were individully miled bck to the testing loction, vi Cnd Post regulr mil. Upon receipt t the testing loction, the temperture monitors were retrieved, the trnsporttion tempertures were recorded, nd the inoculted devices were nlyzed. This study ws performed in Februry nd Mrch 2012. Two nlyses were performed. In the first, the percentge of kits in which the results of the before-trnsporttion nd ftertrnsporttion tests (set B) greed with the expected result (bsed on the mount of Hb spiked into ech of the initil 30 fecl smples, set A) ws clculted. In the second, the percentge of kits in which the result fter trnsporttion greed with the result from before trnsporttion (sme kits, from set B) ws clculted. Tble 2. Results From the Liner Regression Models Predicting the Stbility of NS Plus nd OC Din Kits t Vrious Tempertures Temperture Period of Stbility, d (95% CI) NS Plus b OC Din c Frozen 9.9 (8.0 12.9) 4.5 (1.6 6.9) 208C to 158C ( 48F to58f) Refrigerted.60 d 37.4 (32.3 46.5) 28C to88c (368F 468F) Ambient 6.0 (4.2 7.7) 4.6 (3.3 5.7) 208C to228c (688F 728F) Elevted 0.5 (0 1.5) 0 e 458C (1138F) Stbility is defined using the number of dys until the percentge of hemoglobin recovery ws predicted to drop below 80% of the concentrtion t dy 0. b NS Plus, Alfres Phrm Corportion (Chuo-ku, Osk, Jpn). c OC Din, Eiken Chemicl Co., Ltd. (Tito-ku, Tokyo, Jpn). d Throughout the 60-dy mesurement period, the men predicted hemoglobin recovery ws more thn 80%. e Throughout the 60-dy mesurement period, the men predicted hemoglobin recovery ws less thn 80%. RESULTS Study 1: Controlled Smple Stbility Study The medin percentge of Hb recoveries in the collection devices of the 2 quntittive kits over time nd t vrying tempertures re shown in the Figure, nd b. Inoculted devices stored under refrigerted conditions hd the gretest smple stbility for both NS Plus nd OC Din. Freezing the devices produced n initil decrese of 20% in Hb recovery, but then Hb recovery remined stble over time therefter for tht temperture condition. There were decresing recoveries with incresing temperture nd incresing time for the other 2 temperture conditions. Across ll temperture conditions, there ws tendency towrd better stbility for NS Plus. Results from the modeling compring the 2 quntittive kits re shown in Tble 2. Hemoglobin stbility ws similr, lthough there ws tendency towrd longer stbility for NS Plus t most tempertures. At mbient tempertures for both devices, the predicted stbility, using time to less thn 80% Hb recovery s the stndrd, ws less thn week. Results compring ll 6 kits re shown in Tble 3. The Hb stbility in the 2 quntittive kits NS Plus nd OC Din ws poor compred with the devices of the qulittive FIT nd guic kits in the frozen condition when n 80% threshold ws used to define stbility. The observed differences between quntittive nd qulittive kits were reduced for the 70% nd 50% thresholds. Agin, most kits performed well under refrigerted conditions, regrdless of the threshold used, with the exception of Beckmn ICT nd ulti med kits. Under mbient conditions, FITs tended to hve the strongest performnce, lthough the quntittive kits lso performed resonbly well, prticulrly t the 70% nd 50% thresholds. The Guic lso performed well, wheres the ulti med nd Beckmn ICT kits performed quite poorly t mbient temperture. None of the kit collection devices performed well t high (458C) tempertures, with the exception of the Guic (19.8 dys t 80% threshold). Study 2: Controlled Freeze-Thw Study As illustrted in Tble 4, of the 3 kits tested, the NS Plus kit provided the most consistent nd cceptble recovery fter up to 3 freeze-thw cycles. Recovery with the OC Din kit declined stedily through the 3 freeze-thw exposures. For the ulti med kit, the only qulittive test tht could be included in this study, significnt reduction in recovery ws observed even fter one cycle. Study 3: Field Study During trnsporttion from the 3 distribution centers, inoculted devices were exposed to verge tempertures of 18.48C, 18.88C, nd 17.08C. The lowest nd highest tempertures encountered were 148C nd þ268c, respectively. The number of freeze-thw cycles encountered during trnsport vried depending on the distribution center to which the devices were sent (one center hd none, one center hd 3, nd one center hd 1). Totl trnsporttion times vried from 5 to 12 dys. Tble 5 summrizes the 2 nlyses of greement in the field study. Both NS Plus nd OC Din exhibited good greement before nd fter trnsporttion nd with the expected result. FIT nd ulti med hd good greement before trnsporttion, in keeping with their known nlytic sensitivities, which should be ble to detect the lower Hb concentrtions in the inoculted devices. However, the greement for both ws poor between the before nd fter trnsporttion tests, indicting degrdtion of Hb (ulti med more thn FIT). The Beckmn ICT nd Guic hd poor greement before trnsporttion, in keeping with their known poorer nlytic sensitivities. However, greement ws good between the 78 Arch Pthol Lb Med Vol 142, Jnury 2018 Hemoglobin Stbility in FIT nd gfobt Ctomeris et l
The medin percentge of hemoglobin recovery compred with bseline (time 0 of NS Plus [] nd OC Din [b]) over time t different tempertures. NS-Plus is mnufctured by Alfres Phrm Corportion (Chuoku, Osk, Jpn), nd OC-Sensor Din is mnufctured by Eiken Chemicl Co, Ltd (Tito-ku, Tokyo, Jpn). before nd fter trnsporttion tests, suggesting tht Hb remined stble in the collection devices of those kits during trnsporttion. DISCUSSION Using stndrdized nd rigorous methodology, we found importnt vritions in Hb stbility cross 5 commercilly vilble FIT kits suitble for use in orgnized CRC screening progrms nd 1 gfobt kit currently in use in the Ontrio, Cnd, progrm. The FIT ppered to be the best-performing qulittive kit, wheres, mong the quntittive kits, NS Plus tended to perform slightly better thn OC Din. As tempertures incresed bove freezing, Hb recoveries decresed over time for ll kits. Freezing smples resulted in smll initil loss of recovery in the collection devices of the quntittive kits. However, Hb concentrtion ws then mintined cross ll time points. Some loss of recovery during the trnsport process ws observed in the field study. The current study did not find uniformly better stbility in FIT kits using dry collection devices ( FIT nd Beckmn ICT) compred with wet collection devices. Although the lck of Hb stbility over time, prticulrly t higher tempertures, is well documented, 15,16,18,23,24 nd sesonl vrition in positivity rtes hs been previously reported, 13,25 there re only few reports compring the Hb stbility of different FIT kits. 17,18 Interestingly, one of those reports 17 lso compred NS Plus nd n OC product nd, similr to our results, found tht the NS Plus tended to exhibit slightly better stbility. To our knowledge, FIT hs not been compred with other FITs for stbility. In the current study, we report decrese in Hb concentrtion of bout 1.4%/d nd bout 2.1%/d t mbient temperture (208C 228C) for the NS Plus nd the OC Din, respectively. One other study 18 hs lso reported Hb instbility for FITs t mbient tempertures, rnging from 1.7%/d to 7.8%/d, depending on the kit. Other studies hve shown importnt differences in other performnce chrc- Arch Pthol Lb Med Vol 142, Jnury 2018 Hemoglobin Stbility in FIT nd gfobt Ctomeris et l 79
Tble 3. Temperture Results From the Logistic Regression Models Predicting Stbility of Fecl Immunochemicl Tests (FITs) nd Guic Kits t Vrious Tempertures Threshold, % NS Plus OC Din Period of Stbility, d (95% CI),b FIT FOB Advnced Beckmn ICT Guic Frozen 80 0 0.60 57.1.60.60 d (NA) c (NA) c (NA) (NA) (NA) 70.60 13.9.60.60.60.60 d (NA) (NA) (0.3 NA) (2.9 NA) (11.9 NA) 50.60.60.60.50.60.60 d (NA) (NA) (NA) (56.2 NA) (NA) MSS, d None stted None stted None stted None stted None stted None stted Refrigerted 80.60 50 56.6 1.4 1.8 26.8 (NA) (NA) (NA) (NA 7.1) (NA 59.8) (0.3 NA) 70.60.60.60 4.2 9.6.60 (NA) (NA) (5.7 NA) (NA 13.8) (NA) (24.0 NA) 50.60.60.60 10.3.60.60 (NA) (49.1 NA) (48.2 NA) (4.0 NA) (6.4 NA) (NA) MSS, d 7 30 None stted 3 4 None stted None stted Ambient 80 1.4 3.2 8.5 0 1.2.60 (NA 71.6) (NA 26.3) (NA) (NA 1.2) (NA) (NA) 70 12.1 13 25.3 0.5 4.9.60 (NA) (NA 37.5) (NA) (NA 3.4) (NA) (7.3 NA) 50.60 28.31.60 3.2.60.60 (4.6 NA) (0.8 NA) (12.4 NA) (0.7 9.4) (5.1 NA) (NA) MSS, d 3 15 30 None stted 14 21 Elevted 80 2.4 0 d 0 0 0.42 19.8 (NA 8.5) (NA 1.8) c (NA 0.4) c (NA 25.0) (NA) 70 5.1 0 d 0 0.6 1.2 52.3 (NA 10.7) (NA 3.4) c (NA 0.9) (NA 2.9) (NA) 50 9.3 0 d 2.3 2.2 2.7.60 (1.7 14.5) (NA 8.1) (1.7 NA) (0.4 4.4) (29.9 NA) MSS, d None stted None stted None stted None stted None stted None stted Abbrevitions: MSS, mnufcturer s stted stbility t ech temperture (none of the mnufcturers described the methods used to mesure stbility); NA, not vilble. Results from the regression models under ech temperture condition for ll kits in which stbility ws defined by the number of dys (nd 95% CI) until the probbility of remining positive ws predicted to drop below thresholds of 80%, 70%, nd 50%. b Hemoglobin NS-Plus, Alfres Phrm Corportion (Chuo-ku, Osk, Jpn); OC-Sensor Din, Eiken Chemicl Co, Ltd (Tito-ku, Tokyo, Jpn); Hem-Screen SPECIFIC,, Inc (Etontown, New Jersey); FOB Advnced, ulti med (Ahrensburg, Germny); Hemoccult ICT; Beckmn Coulter, Inc (Bre, Cliforni); Hem-Screen Guic,, Inc (Etontown, New Jersey). c Throughout the 60-dy mesurement period,,80%,,70%,,50% were positive. d Unble to crete model. teristics for CRC screening cross FIT kits. 26 29 In sum, these studies highlight key messge: not ll FIT kits re the sme; s such, selection of FITs for use in orgnized screenings should include crefully considertion of dt on performnce nd qulity control. There re few dt on the effect of freezing on Hb stbility in FIT collection devices, lthough study from the Netherlnds reported reduced FIT detection rtes in the winter. 25 We noted drop in Hb concentrtion in the quntittive collection devices with freezing, but, once frozen, the recovery for the quntittive kits ws reltively constnt for up to 60 dys. However, the effect of sequentil freezing nd thwing vried cross the 3 kits we evluted, with the NS Plus performing best. Our findings suggest tht the process of freezing nd thwing (which my occur during kit trnsport to the lbortory), nd not necessrily the time frozen, my decrese Hb recovery. Our field study ws conducted outside controlled lbortory setting, nd s result, there ws considerble vrition in importnt prmeters, such s temperture, trnsporttion time, freezing, nd thwing. Although there my be limittions to this rel-world pproch in Tble 4. Effect of Freeze-Thw on Recovery of Hemoglobin in Vrious Fecl Immunochemicl Test Collection Devices Quntittive Kits Medin Recovery, % (95% CI) Qulittive Kits Smples Remining Positive, No. (95% CI) Freeze-Thw Cycle NS Plus OC Din b FOB Advnced c 0 100 100 6 1 91.7 (68.5 99.4) 74.9 (53.6 89.5) 4 (67) 2 95.1 (67.8 107.3) 66.8 (53.0 79.7) 1 (17) 3 95.4 (64.4 106.7) 57.6 (42.8 71.2) 1 (0) Hemoglobin NS-Plus, Alfres Phrm Corp (Chuo-ku, Osk, Jpn). b OC-Sensor Din, Eiken Chemicl Co, Ltd (Tito-ku, Tokyo, Jpn). c FOB Advnced, ulti med (Ahrensburg, Germny). 80 Arch Pthol Lb Med Vol 142, Jnury 2018 Hemoglobin Stbility in FIT nd gfobt Ctomeris et l
Tble 5. Agreement Between Before-Trnsporttion nd After-Trnsporttion Results nd Agreement With Expected Results in the Field Study Smple Hb Concentrtion Expected Result, lg Hb/g Smples Producing Negtive Results for Unspiked generlizbility, importnt lessons bout Hb stbility hve been lerned from other similr studies. 13,25 Although there ws some loss of recovery during the trnsport process in this field study, in spite of long trnsit times of up to 12 dys nd vritions in temperture conditions ( 148C toþ268c), the 2 quntittive kits performed better (Hb remined stble in their collection devices cross the full rnge of Hb concentrtions tested) thn the other kits. A number of studies hve exmined the potentil clinicl significnce of exposure to higher tempertures 13 nd delyed smple returns. 15,16 Unfortuntely, we were not ble to perform the field study during the summer when tempertures re expected to be hotter. Nevertheless, dt from the current study suggest tht the collection devices of the quntittive kits nd some devices of the qulittive kits pper resonbly robust nd my be suitble for postl trnsport in climtes similr to tht in Cnd, t lest in winter. In wet collection devices, such s those used by some FIT kits, the rte of Hb degrdtion hs been reported to be greter thn tht in dry collection devices, perhps becuse of bcteril degrdtion of Hb in moist environments. 11,14 The current study shows Hb stbility in the collection devices of the FIT nd Guic kits, which use dry collection device, ws, in generl, longer thn tht of the other FIT kits. By contrst, Beckmn ICT, which lso uses dry collection device, underperformed reltive to some of the other kits using wet collection devices. These findings suggest tht clims of superior smple storge cpbility my not be generlizble cross ll dry collection devices. There re strengths nd weknesses to the current study. Becuse few dt compring Hb stbility in collection devices cross FIT kits re vilble, we ttempted to ddress tht issue with novel, trnsprent, nd esily reproducible methodology. To compre cross kits, we estblished definition of stbility: for quntittive kits, time until Hb concentrtion decresed to less thn 80%, vlue selected bsed on internl studies of between-run precision; nd for qulittive kits, time until the probbility of remining positive dropped below 3 prespecified thresholds. This N NS Plus OC Din FIT FOB Advnced Beckmn ICT Guic Before After Before After Before After Before After Before After Before After 6 6 6 6 6 5 5 6 5 6 6 6 6 Smples, No. Smples Producing Positive Results for Spiked Smples, No. 30 3 2 3 2 1 3 2 2 2 0 0 0 0 60 6 6 6 6 6 6 2 4 2 1 1 0 0 125 3 3 3 3 3 3 1 3 0 1 1 0 0 500 6 6 6 6 6 6 6 6 1 6 6 5 5 2000 6 6 6 6 6 6 6 6 5 6 6 6 6 Agreement with expected result, % Agreement between before nd fter, % 97 100 97 93 97 73 90 50 67 67 57 57.100 97 76 56 100 100 Abbrevition: Hb, hemoglobin. Hemoglobin NS-Plus, Alfres Phrm Corportion (Chuo-ku, Osk, Jpn); OC-Sensor Din, Eiken Chemicl Co, Ltd (Tito-ku, Tokyo, Jpn); Hem-Screen SPECIFIC,, Inc (Etontown, New Jersey); FOB Advnced, ulti med (Ahrensburg, Germny); Hemoccult ICT, Beckmn Coulter, Inc (Bre, Cliforni); Hem-Screen Guic,, Inc (Etontown, New Jersey). pproch is similr to tht used by nother study from the United Kingdom, 17 lthough tht study used lrger drop in Hb (50% from bseline) to define loss of stbility. A second study from Frnce 18 simply reported the percentge of chnge in Hb concentrtion by dy becuse the uthors felt they could not use regression techniques becuse of differences mong kits in the distribution of the dt. The clinicl significnce of different thresholds is not known nd likely vries with the Hb concentrtion of the fecl smple nd the nlytic sensitivity of the prticulr FIT, but use of threshold llows common reference with which to compre kits. Unlike the 2 other studies mentioned, we used regression methods pproprite to the distribution of the dt, llowing us to obtin robust summry mesures of stbility over time; in ddition, the use of generlized estimting equtions ccounted for multiple mesures on the sme kit. One of the chllenges we fced in developing this methodology ws tht the kits hd significntly different nlytic sensitivities. We ddressed tht issue by selecting fecl Hb concentrtions pproprite to the reported nlytic sensitivity of ech kit. It ws observed tht some of the unspiked smples gve unexpected positive results with the nd ulti med kits, which my indicte flse-positive results in some of those smples. A second limittion of this study ws tht the dt for the smples in these kits my reflect the stbility of n interferent rther thn tht of Hb. A third limittion ws tht in August 2012, the OC Din buffer ws updted; therefore, our results my not be generlizble to the kit currently in use. A fourth potentil limittion ws tht the design of the study offered no opportunity to correlte decreses in Hb concentrtions with chnges in polyp or cncer detection rtes; therefore, we cnnot comment on the clinicl impct of the observed declines in Hb stbility from the different kits. Dt from recent studies hve shown tht there cn lso be importnt differences in clinicl outcomes cross FIT kits, even when using the sme positivity threshold. 26 The use of stndrdized methodology to compre Hb stbility is strength of this study. Although the clinicl impct of the differences we report re not known, it my be resonble Arch Pthol Lb Med Vol 142, Jnury 2018 Hemoglobin Stbility in FIT nd gfobt Ctomeris et l 81
to ssume tht kits tht performed reltively poorly in our study my be likely to lso do so in the rel world. Our pproch confers benefit when compring products for Hb stbility becuse the reported stbilities in mnufcturers inserts lmost certinly use vrious methodologies. In so doing, our study ddresses recent cll for stndrdized methodology to mesure Hb stbility in FIT collection devices. 19 In summry, the current study reports considerble vrition in the performnce of FITs for Hb stbility. We found tht the FIT ws the best performer mong the qulittive kits. Both quntittive kits performed well, lthough the NS Plus ppered to hve slight edge over the OC Din in our study. It is not known whether tht difference will be mintined now tht OC Din hs been updted with new buffer. Becuse not ll FIT methods re the sme, the current study provides importnt performnce dt on the FIT methods evluted tht will ssist CRC screening progrms nd providers in FIT selection. 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