Optical coherence tomography (OCT) has emerged as a fast, non-invasive, inexpensive, high-resolution imaging technique in multiple sclerosis (MS). Retinal layer quantification by OCT facilitates a ‘window’ into not only local retinal pathology but also global neurodegenerative processes, recognised to be the principal substrates of disability accumulation in MS. While OCT measures in MS have been demonstrated to reflect visual function, inflammatory activity outside of the visual pathways, disability measures including the prediction of disability progression, whole brain atrophy, and the differential neuroprotective effects of disease-modifying therapies, debate continues regarding the clinical utility of OCT in everyday practice. This review presents an overview of the evidence supporting OCT, with particular focus on its application in the MS clinic. We will also discuss the role of OCT in MS clinical trials to develop novel neuroprotective and potential remyelinating therapies.
FisnikuLKChardDTJacksonJS, et al. Gray matter atrophy is related to long-term disability in multiple sclerosis. Ann Neurol2008; 64(3): 247–254.
3.
FrieseMASchattlingBFuggerL.Mechanisms of neurodegeneration and axonal dysfunction in multiple sclerosis. Nat Rev Neurol2014; 10(4): 225–238.
4.
BalcerLJ.Clinical practice: Optic neuritis. N Engl J Med2006; 354(12): 1273–1280.
5.
ToussaintDPerierOVerstappenA, et al. Clinicopathological study of the visual pathways, eyes, and cerebral hemispheres in 32 cases of disseminated sclerosis. J Clin Neuroophthalmol1983; 3(3): 211–220.
6.
IkutaFZimmermanHM.Distribution of plaques in seventy autopsy cases of multiple sclerosis in the United States. Neurology1976; 26(6Pt.2): 26–28.
7.
GreenAJMcQuaidSHauserSL, et al. Ocular pathology in multiple sclerosis: Retinal atrophy and inflammation irrespective of disease duration. Brain2010; 133(Pt.6): 1591–1601.
8.
ShindlerKSVenturaEDuttM, et al. Inflammatory demyelination induces axonal injury and retinal ganglion cell apoptosis in experimental optic neuritis. Exp Eye Res2008; 87(3): 208–213.
9.
KerrisonJBFlynnTGreenWR.Retinal pathologic changes in multiple sclerosis. Retina1994; 14(5): 445–451.
10.
RatchfordJNSaidhaSSotirchosES, et al. Active MS is associated with accelerated retinal ganglion cell/inner plexiform layer thinning. Neurology2013; 80(1): 47–54.
11.
TalmanLSBiskerERSackelDJ, et al. Longitudinal study of vision and retinal nerve fiber layer thickness in multiple sclerosis. Ann Neurol2010; 67(6): 749–760.
12.
ParisiVManniGSpadaroM, et al. Correlation between morphological and functional retinal impairment in multiple sclerosis patients. Invest Ophthalmol Vis Sci1999; 40(11): 2520–2527.
SycSBWarnerCVHiremathGS, et al. Reproducibility of high-resolution optical coherence tomography in multiple sclerosis. Mult Scler2010; 16(7): 829–839.
15.
WarnerCVSycSBStankiewiczAM, et al. The impact of utilizing different optical coherence tomography devices for clinical purposes and in multiple sclerosis trials. PLoS ONE2011; 6(8): e22947.
16.
SaidhaSSycSBDurbinMK, et al. Visual dysfunction in multiple sclerosis correlates better with optical coherence tomography derived estimates of macular ganglion cell layer thickness than peripapillary retinal nerve fiber layer thickness. Mult Scler2011; 17(12): 1449–1463.
17.
OberwahrenbrockTWeinholdMMikolajczakJ, et al. Reliability of intra-retinal layer thickness estimates. PLoS ONE2015; 10(9): e0137316.
18.
SaidhaSSycSBIbrahimMA, et al. Primary retinal pathology in multiple sclerosis as detected by optical coherence tomography. Brain2011; 134(2): 518–533.
19.
BurkholderBMOsborneBLoguidiceMJ, et al. Macular volume determined by optical coherence tomography as a measure of neuronal loss in multiple sclerosis. Arch Neurol2009; 66(11): 1366–1372.
20.
ForooghianFSprouleMWestallC, et al. Electroretinographic abnormalities in multiple sclerosis: Possible role for retinal autoantibodies. Doc Ophthalmol2006; 113(2): 123–132.
21.
SaidhaSSotirchosESIbrahimMA, et al. Microcystic macular oedema, thickness of the inner nuclear layer of the retina, and disease characteristics in multiple sclerosis: A retrospective study. Lancet Neurol2012; 11(11): 963–972.
22.
SistoDTrojanoMVetrugnoM, et al. Subclinical visual involvement in multiple sclerosis: A study by MRI, VEPs, frequency-doubling perimetry, standard perimetry, and contrast sensitivity. Invest Ophthalmol Vis Sci2005; 46(4): 1264.
23.
SycSBSaidhaSNewsomeSD, et al. Optical coherence tomography segmentation reveals ganglion cell layer pathology after optic neuritis. Brain2012; 135(Pt.2): 521–533.
24.
SaidhaSAl-LouziORatchfordJN, et al. Optical coherence tomography reflects brain atrophy in multiple sclerosis: A four-year study. Ann Neurol2015; 78(5): 801–813.
25.
SeigoMASotirchosESNewsomeS, et al. In vivo assessment of retinal neuronal layers in multiple sclerosis with manual and automated optical coherence tomography segmentation techniques. J Neurol2012; 259(10): 2119–2130.
26.
WalterSDIshikawaHGalettaKM, et al. Ganglion cell loss in relation to visual disability in multiple sclerosis. Ophthalmology2012; 119(6): 1250–1257.
27.
KnierBLeppenetierGWetzlmairC, et al. Association of retinal architecture, intrathecal immunity, and clinical course in multiple sclerosis. JAMA Neurol2017; 74(7): 847–856.
28.
SaidhaSSotirchosESOhJ, et al. Relationships between retinal axonal and neuronal measures and global central nervous system pathology in multiple sclerosis. JAMA Neurol2013; 70(1): 34.
29.
GelfandJMNolanRSchwartzDM, et al. Microcystic macular oedema in multiple sclerosis is associated with disease severity. Brain2012; 135(Pt.6): 1786–1793.
Garcia-MartinEJarautaLViladesE, et al. Ability of swept-source optical coherence tomography to detect retinal and choroidal changes in patients with multiple sclerosis. J Ophthalmol2018; 2018: 7361212.
32.
PotsaidBBaumannBHuangD, et al. Ultrahigh speed 1050nm swept source/Fourier domain OCT retinal and anterior segment imaging at 100,000 to 400,000 axial scans per second. Opt Express2010; 18(19): 20029–20048.
33.
MurphyOCKwakyiOIftikharM, et al. Alterations in the retinal vasculature occur in multiple sclerosis and exhibit novel correlations with disability and visual function measures. Mult Scler. Epub ahead of print 16 May 2019. DOI: 10.1177/1352458519845116.
34.
LanzilloRCennamoGCriscuoloC, et al. Optical coherence tomography angiography retinal vascular network assessment in multiple sclerosis. Mult Scler2018; 24(13): 1706–1714.
35.
FeuchtNMaierMLepennetierG, et al. Optical coherence tomography angiography indicates associations of the retinal vascular network and disease activity in multiple sclerosis. Mult Scler2019; 25(2): 224–234.
36.
JakimovskiDGandhiSPaunkoskiI, et al. Hypertension and heart disease are associated with development of brain atrophy in multiple sclerosis: A 5-year longitudinal study. Eur J Neurol2019; 26(1): 87-e8.
37.
YazdanyarANewmanAB.The burden of cardiovascular disease in the elderly: Morbidity, mortality, and costs. Clin Geriatr Med2009; 25(4): 563–577, vii.
38.
FrederiksenJLPetreraJ.Serial visual evoked potentials in 90 untreated patients with acute optic neuritis. Surv Ophthalmol1999; 44(Suppl. 1): S54–S62.
39.
ThompsonAJBanwellBLBarkhofF, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol2018; 17(2): 162–173.
40.
KlistornerAFraserCGarrickR, et al. Correlation between full-field and multifocal VEPs in optic neuritis. Doc Ophthalmol2008; 116(1): 19–27.
41.
KlistornerAArvindHNguyenT, et al. Fellow eye changes in optic neuritis correlate with the risk of multiple sclerosis. Mult Scler2009; 15(8): 928–932.
42.
NarayananDChengHTangRA, et al. Multifocal visual evoked potentials and contrast sensitivity correlate with ganglion cell-inner plexiform layer thickness in multiple sclerosis. Clin Neurophysiol2019; 130(1): 180–188.
43.
DiMaggioGSantangeloRGuerrieriS, et al. Optical coherence tomography and visual evoked potentials: Which is more sensitive in multiple sclerosis. Mult Scler2014; 20(10): 1342–1347.
44.
Al-LouziOABhargavaPNewsomeSD, et al. Outer retinal changes following acute optic neuritis. Mult Scler2016; 22(3): 362–372.
45.
BermelRABalcerLJ.Optic neuritis and the evaluation of visual impairment in multiple sclerosis. Continuum2013; 19(4): 1074–1086.
46.
KupersmithMJGarvinMKWangJK, et al. Retinal ganglion cell layer thinning within one month of presentation for optic neuritis. Mult Scler2016; 22(5): 641–648.
CostelloFHodgeWPanYI, et al. Tracking retinal nerve fiber layer loss after optic neuritis: A prospective study using optical coherence tomography. Mult Scler2008; 14(7): 893–905.
49.
GabilondoIMartinez-LapiscinaEHFraga-PumarE, et al. Dynamics of retinal injury after acute optic neuritis. Ann Neurol2015; 77(3): 517–528.
50.
CostelloFCouplandSHodgeW, et al. Quantifying axonal loss after optic neuritis with optical coherence tomography. Ann Neurol2006; 59(6): 963–969.
51.
XuLTBermelRANowackiAS, et al. Optical coherence tomography for the detection of remote optic neuritis in multiple sclerosis. J Neuroimaging2016; 26(3): 283–288.
52.
Nolan-KenneyRCLiuMAkhandO, et al. Optimal intereye difference thresholds by optical coherence tomography in multiple sclerosis: An international study. Ann Neurol2019; 85: 618–629.
53.
XuSCKardonRHLeavittJA, et al. Optical coherence tomography is highly sensitive in detecting prior optic neuritis. Neurology2019; 92(6): e527–e535.
54.
FisherJBJacobsDAMarkowitzCE, et al. Relation of visual function to retinal nerve fiber layer thickness in multiple sclerosis. Ophthalmology2006; 113(2): 324–332.
55.
Sanchez-DalmauBMartinez-LapiscinaEHTorres-TorresR, et al. Early retinal atrophy predicts long-term visual impairment after acute optic neuritis. Mult Scler2018; 24(9): 1196–1204.
56.
CostelloF.Evaluating the use of optical coherence tomography in optic neuritis. Mult Scler Int2011; 2011: 148394.
57.
Optic Neuritis Study Group. Multiple sclerosis risk after optic neuritis: Final optic neuritis treatment trial follow-up. Arch Neurol2008; 65(6): 727–732.
58.
ZimmermannHGKnierBOberwahrenbrockT, et al. Association of retinal ganglion cell layer thickness with future disease activity in patients with clinically isolated syndrome. JAMA Neurol2018; 75(9): 1071.
59.
ZhangXYuHZhangY, et al. The application of RNFL thickness detection in early differential diagnosis among various types of idiopathic optic neuritis. J Clin Neurosci2018; 55: 82–85.
60.
JenkinsTMToosyAT.Optic neuritis: The eye as a window to the brain. Curr Opin Neurol2017; 30(1): 61–66.
61.
PengAQiuXZhangL, et al. Evaluation of the retinal nerve fiber layer in neuromyelitis optica spectrum disorders: A systematic review and meta-analysis. J Neurol Sci2017; 383: 108–113.
62.
NaismithRTTutlamNTXuJ, et al. Optical coherence tomography differs in neuromyelitis optica compared with multiple sclerosis. Neurology2009; 72(12): 1077–1082.
63.
OertelFCHavlaJRoca-FernandezA, et al. Retinal ganglion cell loss in neuromyelitis optica: A longitudinal study. J Neurol Neurosurg Psychiatry2018; 89(12): 1259–1265.
64.
KaufholdFZimmermannHSchneiderE, et al. Correction: Optic neuritis is associated with inner nuclear layer thickening and microcystic macular edema independently of multiple sclerosis. PLoS ONE2013; 8(10): e71145.
65.
GelfandJMCreeBANolanR, et al. Microcystic inner nuclear layer abnormalities and neuromyelitis optica. JAMA Neurol2013; 70(5): 629.
66.
SotirchosESSaidhaSByraiahG, et al. In vivo identification of morphologic retinal abnormalities in neuromyelitis optica. Neurology2013; 80(15): 1406–1414.
67.
PacheFZimmermannHMikolajczakJ, et al. MOG-IgG in NMO and related disorders: A multicenter study of 50 patients. Part 4: Afferent visual system damage after optic neuritis in Mog-igg-seropositive versus Aqp4-igg-seropositive patients. J Neuroinflammation2016; 13(1): 282.
68.
Martinez-LapiscinaEHArnowSWilsonJA, et al. Retinal thickness measured with optical coherence tomography and risk of disability worsening in multiple sclerosis: A cohort study. Lancet Neurol2016; 15(6): 574–584.
69.
BalkLJCruz-HerranzAAlbrechtP, et al. Timing of retinal neuronal and axonal loss in MS: A longitudinal OCT study. J Neurol2016; 263(7): 1323–1331.
70.
GrahamECYouYYiannikasC, et al. Progressive loss of retinal ganglion cells and axons in nonoptic neuritis eyes in multiple sclerosis: A longitudinal optical coherence tomography study. Invest Ophthalmol Vis Sci2016; 57(4): 2311–2317.
71.
NarayananDChengHBonemKN, et al. Tracking changes over time in retinal nerve fiber layer and ganglion cell-inner plexiform layer thickness in multiple sclerosis. Mult Scler2014; 20(10): 1331–1341.
72.
SchipplingSBalkLJCostelloF, et al. Quality control for retinal OCT in multiple sclerosis: Validation of the OSCAR-IB criteria. Mult Scler2015; 21(2): 163–170.
73.
BehbehaniRAdnanHAl-HassanAA, et al. Predictors of retinal atrophy in multiple sclerosis: A longitudinal study using spectral domain optical coherence tomography with segmentation analysis. Mult Scler Relat Disord2018; 21: 56–62.
74.
WingesKMMurchisonCFBourdetteDN, et al. Longitudinal optical coherence tomography study of optic atrophy in secondary progressive multiple sclerosis: Results from a clinical trial cohort. Mult Scler2019; 25(1): 55–62.
75.
ChauhanDSMarshallJ.The interpretation of optical coherence tomography images of the retina. Invest Ophthalmol Vis Sci1999; 40(10): 2332–2342.
76.
JenkinsTMToosyAT.Optical coherence tomography should be part of the routine monitoring of patients with multiple sclerosis: No. Mult Scler2014; 20(10): 1299–1301.
77.
GaoLLiuYLiX, et al. Abnormal retinal nerve fiber layer thickness and macula lutea in patients with mild cognitive impairment and Alzheimer’s disease. Arch Gerontol Geriatr2015; 60(1): 162–167.
78.
RingelsteinMAlbrechtPSudmeyerM, et al. Subtle retinal pathology in amyotrophic lateral sclerosis. Ann Clin Transl Neurol2014; 1(4): 290–297.
79.
CalditoNGSaidhaSSotirchosES, et al. Brain and retinal atrophy in African-Americans versus Caucasian-Americans with multiple sclerosis: A longitudinal study. Brain2018; 141(11): 3115–3129.
80.
KimbroughDJSotirchosESWilsonJA, et al. Retinal damage and vision loss in African American multiple sclerosis patients. Ann Neurol2015; 77(2): 228–236.
81.
NaismithRTTrinkausKCrossAH.Phenotype and prognosis in African-Americans with multiple sclerosis: A retrospective chart review. Mult Scler2006; 12(6): 775–781.
82.
Lichtman-MikolSRazmjouSYarraguntlaK, et al. Racial differences in retinal neurodegeneration as a surrogate marker for cortical atrophy in multiple sclerosis. Mult Scler Relat Disord2019; 31: 141–147.
83.
CostelloFVan StavernGP.Should optical coherence tomography be used to manage patients with multiple sclerosis. J Neuroophthalmol2012; 32(4): 363–371.
84.
LangACarassAAl-LouziO, et al. Combined registration and motion correction of longitudinal retinal OCT data. Proc SPIE Int Soc Opt Eng2016; 9784: 97840X.
85.
TewariePBalkLCostelloF, et al. The OSCAR-IB consensus criteria for retinal OCT quality assessment. PLoS ONE2012; 7(4): e34823.
86.
Cruz-HerranzABalkLJOberwahrenbrockT, et al. The APOSTEL recommendations for reporting quantitative optical coherence tomography studies. Neurology2016; 86(24): 2303–2309.
87.
ChilinskaAEjmaMTurno-KrecickaA, et al. Analysis of retinal nerve fibre layer, visual evoked potentials and relative afferent pupillary defect in multiple sclerosis patients. Clin Neurophysiol2016; 127(1): 821–826.
88.
García-MartínEPueyoVFernándezJ, et al. Effect of treatment in loss of retinal nerve fiber layer in multiple sclerosis patients. Arch Soc Esp Oftalmol2010; 85(6): 209–214.
89.
PulRSaadatMMorbiducciF, et al. Longitudinal time-domain optic coherence study of retinal nerve fiber layer in IFNβ-treated and untreated multiple sclerosis patients. Exp Ther Med2016; 12(1): 190–200.
90.
ButtonJAl-LouziOLangA, et al. Disease-modifying therapies modulate retinal atrophy in multiple sclerosis: A retrospective study. Neurology2017; 88(6): 525–532.
91.
TalmageGDCoppesOJMJavedA, et al. Natalizumab stabilizes physical, cognitive, MRI, and OCT markers of disease activity: A prospective, non-randomized pilot study. PLoS ONE2017; 12(4): e0173299.
92.
KnierBSchmidtPAlyL, et al. Retinal inner nuclear layer volume reflects response to immunotherapy in multiple sclerosis. Brain2016; 139(11): 2855–2863.
93.
KnierBBertheleABuckD, et al. Optical coherence tomography indicates disease activity prior to clinical onset of central nervous system demyelination. Mult Scler2016; 22(7): 893–900.
94.
KapposLRadueE-WO’ConnorP, et al. A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med2010; 362(5): 387–401.
95.
ZarbinMAJampolLMJagerRD, et al. Ophthalmic evaluations in clinical studies of fingolimod (FTY720) in multiple sclerosis. Ophthalmology2013; 120(7): 1432–1439.
96.
CohenJABarkhofFComiG, et al. Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis. NEJM2010; 362(5): 402–415.
97.
KapposLBar-OrACreeBAC, et al. Siponimod versus placebo in secondary progressive multiple sclerosis (EXPAND): A double-blind, randomised, phase 3 study. Lancet2018; 391(10127): 1263–1273.
98.
NolanRGelfandJMGreenAJ.Fingolimod treatment in multiple sclerosis leads to increased macular volume. Neurology2013; 80(2): 139–144.
JainNBhattiMT.Fingolimod-associated macular edema: Incidence, detection, and management. Neurology2012; 78(9): 672–680.
102.
MinukABelliveauMJAlmeidaDRP, et al. Fingolimod-associated macular edema: Resolution by sub-tenon injection of triamcinolone with continued fingolimod use. JAMA Ophthalmol2013; 131(6): 802–804.
103.
ThooSCugatiSLeeA, et al. Successful treatment of fingolimod-associated macular edema with intravitreal triamcinolone with continued fingolimod use. Mult Scler2015; 21(2): 249–251.
104.
ChuiJHerkesGKChangA.Management of fingolimod-associated macular edema. JAMA Ophthalmol2013; 131(5): 694–696.
105.
CutterGKapposL.Clinical trials in multiple sclerosis. Handb Clin Neurol2014; 122: 445–453.
106.
ConnickPKolappanMCrawleyC, et al. Autologous mesenchymal stem cells for the treatment of secondary progressive multiple sclerosis: An open-label phase 2a proof-of-concept study. Lancet Neurol2012; 11(2): 150–156.
107.
EsfahaniMRHarandiZAMovasatM, et al. Memantine for axonal loss of optic neuritis. Graefes Arch Clin Exp Ophthalmol2012; 250(6): 863–869.
108.
SuhsKWHeinKSattlerMB, et al. A randomized, double-blind, phase 2 study of erythropoietin in optic neuritis. Ann Neurol2012; 72(2): 199–210.
109.
LlufriuSSepulvedaMBlancoY, et al. Randomized placebo-controlled phase II trial of autologous mesenchymal stem cells in multiple sclerosis. PLoS ONE2014; 9(12): e113936.
110.
WaubantEMaghziA-HRevirajanN, et al. A randomized controlled phase II trial of riluzole in early multiple sclerosis. Ann Clin Transl Neurol2014; 1(5): 340–347.
111.
SalariMJanghorbaniMEtemadifarM, et al. Effects of vitamin D on retinal nerve fiber layer in vitamin D deficient patients with optic neuritis: Preliminary findings of a randomized, placebo-controlled trial. J Res Med Sci2015; 20(4): 372–378.
112.
ShayegannejadVShahzamaniSDehghaniA, et al. A double-blind, placebo-controlled trial of adding erythropoietin to intravenous methylprednisolone for the treatment of unilateral acute optic neuritis of unknown or demyelinative origin. Graefes Arch Clin Exp Ophthalmol2015; 253(5): 797–801.
113.
CadavidDBalcerLGalettaS, et al. Safety and efficacy of opicinumab in acute optic neuritis (RENEW): A randomised, placebo-controlled, phase 2 trial. Lancet Neurol2017; 16(3): 189–199.
114.
RaftopoulosRHickmanSJToosyA, et al. Phenytoin for neuroprotection in patients with acute optic neuritis: A randomised, placebo-controlled, phase 2 trial. Lancet Neurol2016; 15(3): 259–269.
115.
McKeeJBElstonJEvangelouN, et al. Amiloride clinical trial in optic neuritis (ACTION) protocol: A randomised, double blind, placebo controlled trial. BMJ Open2015; 5(11): e009200.
116.
GreenAJGelfandJMCreeBA, et al. Clemastine fumarate as a remyelinating therapy for multiple sclerosis (ReBUILD): A randomised, controlled, double-blind, crossover trial. Lancet2017; 390(10111): 2481–2489.
117.
SpainRPowersKMurchisonC, et al. Lipoic acid in secondary progressive MS: A randomized controlled pilot trial. Neurol Neuroimmunol Neuroinflamm20174(5): e374.
118.
FoxRJCoffeyCSConwitR, et al. Phase 2 trial of ibudilast in progressive multiple sclerosis. N Engl J Med2018; 379(9): 846–855.
119.
TourbahAGoutOVighettoA, et al. MD1003 (High-dose pharmaceutical-grade Biotin) for the treatment of chronic visual loss related to optic neuritis in multiple sclerosis: A randomized, double-blind, placebo-controlled study. CNS Drugs2018; 32(7): 661–672.
120.
KoduahPBrandtAUPiperSK, et al. Vitamin D supplementation in multiple sclerosis: Primary efficacy endpoint and safety of a randomized, controlled, double-blind phase II trial (EVIDIMS). In: Proceedings of the 34Th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS), vol. 228159, Berlin, 10–12 October 2018. Basel: ECTRIMS.
121.
CiampiE. Efficacy, safety and tolerability of IB-MS versus placebo in patients with progressive forms of MS: Preliminary results from a phase II, randomized, placebo-controlled, double blind, proof of concept study. In: Proceedings of the 34Th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS), vol. 232042, Berlin, 10–12 October 2018. Basel: ECTRIMS.
122.
ChatawayJ.MS-SMART Trial: A multi-arm phase 2b randomised, double blind, parallel group, placebo-controlled clinical trial comparing the efficacy of three neuroprotective drugs in secondary progressive multiple sclerosis [NCT01910259]. In: Proceedings of the 34Th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS), vol. 232077, Berlin, 10–12 October 2018. Basel: ECTRIMS.
123.
DiemRMolnarFBeisseF, et al. Treatment of optic neuritis with erythropoietin (TONE): A randomised, double-blind, placebo-controlled trial-study protocol. BMJ Open2016; 6(3): e010956.
