Competencia

2.6.1'Haematology'and'Biochemistry'panels'

CRP! testing! was! undertaken! using! a! highly! sensitive! CRP! (hsCRP)! assay! undertaken! at! Greater! Glasgow! Biochemistry! Laboratories.! One! healthy! control! patient! (227)! was! recruited! from! NHS! Lanarkshire! where! local! Biochemistry! facilities! were! used.! CRP! results!are!a!standard!CRP!assay!and!unable!to!quantify!below!6mg/dl.!This!value!was! thus!excluded.!Haemoglobin!assay!was!undertaken!in!the!same!accredited!laboratories.!

2.6.2'Immunology5General''

All! Immunology! testing! was! undertaken! at! the! local! Immunology! Laboratory! at! Gartnavel!General!Hospital!serving!Greater!Glasgow.!This!laboratory!undergoes!regular! accreditation!by!the!Clinical!Pathology!Accreditation!regulatory!body.!

2.6.3'Immunology5Rheumatoid'Factor'

The! Siemens! Healthcare! Diagnostics! UK! NHLatexHRF! kit! was! used! to! quantify! RF.! ! The! manufacturer’s!guidance,!reagents!and!method!were!followed.!The!nephelometer!used! to!quantify!the!antigenHantibody!complex!was!the!Dada!Behring!model!BNII.!!

The! Latex! testing! principle! is! as! follows;! when! serum! containing! RF! is! mixed! with! polystyrene! beads! coated! in! human! immunoglobulin! and! sheep! antiHhuman! IgG! then! aggregation! (agglutination)! occurs.! This! scatters! a! beam! of! light! passed! through! the! sample! and! the! intensity! is! proportional! to! the! concentration! of! RF.! This! is! then! compared!against!a!reference!of!known!concentration.!

The! reference! range! suggested! by! the! manufacturer! and! on! the! basis! of! local! agreed! cutHoffs! at! the! Gartnavel! Immunology! Laboratory! were! determined! as! follows;! <15IU/ml=negative,! 15H34IU/ml=equivocal,! 35H350IU/ml=positive! and! >350IU/ml! as! strong!positive.!

2.6.4'Immunology5ANA'

ANA!slides!and!conjugate!were!manufactured!by!Menarini!Diagnostics.!The!analysis!was! carried! out! on! a! Zenit! processor! model! SP+.! The! technique! used! was! an! immunofluorescence!assay!and!HEPH2!slides.!The!principle!is!as!follows;!patient!serum!is! incubated!with!mitotic!human!epithelial!cells!(HepH2!cells)!and!if!ANA’s!are!present!then! binding! will! occur! forming! antigenHantibody! complex.! To! this! fluorescent! antibody! is! bound! which! is! visible! by! microscopy.! The! degree! of! and! pattern! of! binding! form! the! basis!of!the!result.!In!terms!of!reported!binding!avidity!then!1/40!and!1/80!is!considered! very! weak,! 1/160! and! 1/320! weak! positive,! 1/640! and! 1/1280! moderate! positive! and! 1/2560! strong! positive.! These! cutHoffs! have! been! determined! combining! the! manufacturer’s!guidance!and!those!of!the!laboratory!performing!these!tests.!

2.6.5'Immunology5CCP2'assay'

The!product!used!was!ELiATM!_{manufactured!and!distributed!by!Phadia!(Thermo!Fisher!} Scientific)! using! the! principle! of! ELISA.! Analysis! was! undertaken! using! the! Phadia! ImmunoCAP!250!automator.!

This!is!a!second!generation!CCP!antibody!test!using!a!mix!of!citrullinated!proteins!(exact! mix! proportions! undisclosed).! The! manufacturer! details! a! sensitivity! of! 87.8%! and! specificity! of! 96.7%! based! on! in! house! testing.! The! measured! range! is! 0.4U/ml! to! 340U/ml.!The!manufacturer!suggests!a!cutHoff!guide!of!<7U/ml!as!negative,!7H10U/ml!as! indeterminate!and!>10U/ml!as!positive.! In!general,!antiHCCP2!assays!have!a!sensitivity!of!55%!to!80%!and!a!specificity!of!90%!to! 98%!for!established!RA.!! 2.6.6'MicroRNA' Method!is!described!in!detail!in!Chapter!4.!

2.7'Statistical'Analysis'

Descriptive! (univariable)! analysis! was! performed! including! calculation! of! mean! (and! standard! deviation),! median! (and! interHquartile! range! (IQR))! where! applicable.! Distribution!of!all!data!was!first!calculated!and!nonHparametric!testing!was!appropriate! for!the!majority!of!variables.!Spearman’s!correlation!coefficient!was!calculated!between! variables!to!test!the!strength!of!their!association!and!either!the!MannHWhitney!test!or! KruskalHWallis! test! were! chosen! when! comparing! distribution! of! variables! between! or! within!groups!unless!stated!otherwise.!Wilcoxon!signed!rank!test!was!used!as!the!nonH parametric!test!to!compare!two!related!results!at!subsequent!timepoints!(e.g.!baseline! with!3!or!6!month!disease!activity).!Power!calculations!were!not!performed!when!this! study! was! designed! for! the! reasons! stated! (lack! of! published! body! of! evidence).! Statistical!significance!was!assumed!where!pHvalue!was!less!than!0.05!(ie!less!than!5%! chance! the! result! is! non! random).! Bonferroni! correction! for! multiple! variables! was! applied!in!3.3.9.3.5.!

All!data!was!analysed!by!DB!and!outputs!produced!using!SPSS!version!19!for!Windows! software.!

2.7.2'Chapter'4'

DB,! using! SPSS! statistical! package! for! Windows! version! 19,! performed! all! statistical! analysis.!Analysis!of!descriptive!clinical!variable!was!presented!using!median!values!as! the! clinical! values! were! skewed.! Log! transformation! of! microRNA! relative! values! was! undertaken.!Mann!Whitney!U!test!was!used!to!compare!differences!between!the!clinical! variables! and! the! groupings.! Spearman’s! correlation! coefficient! was! used! to! test! associations! between! microRNA! copy! number! or! relative! expression! and! clinical/biochemical!variables.!!

Analysis!of!differentially!expressed!microRNA!was!performed!by!a!third!party!vendor,!as! referenced!in!Chapter!4.3.7.4,!and!statistical!method!referred!therein.!!

2.7.3'Chapter'5'

DB,! using! SPSS! statistical! package! for! Windows! version! 19,! performed! all! statistical! analysis.!Data!quality!was!highly!variable!and!distribution!curves!followed!nonHnormal!

distribution.!Log!transformation!was!performed!on!all!data!to!correct!for!skew!to!enable! parametric!testing.!A!number!of!analytes!remained!nonHnormally!distributed!after!visual! inspection!of!histograms!and!application!of!ShapiroHWilks!to!confirm!(EGF,!ILH6,!H10,!H13,! H17,!H5,!H4,!H21,!H23,!H7,!TNFa,!IFNg,!GMHCSF!and!MIP1Halpha).!Independent!tHtesting!was! used! with! the! Bonferroni! correction! method! to! allow! for! multiple! comparisons! for! parametric!data!or!Kruskal!Wallis!testing!with!pairwise!comparisons!for!non!parametric! results.!!

For! cross! sectional! analysis! the! null! hypothesis! was! considered! that! there! was! no! difference!between!the!four!study!groups.!

For! longitudinal! analysis! at! baseline,! three! and! six! months! in! the! biologic! resistant! group,!the!student’s!paired!t!test!was!used.!Significance!was!assumed!where!the!pHvalue! was!<0.05.! 2.7.4'Chapter'6' DB!scored!half!the!returned!questionnaires!and!the!other!half!by!Kelsey!Watt,!4th_!year! medical!student,!University!of!Glasgow!as!part!of!a!clinical!attachment.!!DB!using!SPSS! statistical!package!for!Windows,!version!19,!subsequently!carried!out!all!analyses.!HADS! items!were!scored!as!outlined!in!Chapter!1!with!a!cutHoff!of!greater!than!or!equal!to!8! applied! as! representing! ‘possible! depression! or! anxiety’! used! so! as! to! represent! a! categorical! variable.! Disability! was! determined! likewise! with! a! value! of! greater! than! 2.000!representing!‘severe!disability’.!

Again,!cohort!descriptive!data!was!analysed!using!nonHparametric!tests!as!the!data!did! not!follow!normal!distribution.!The!MannHWhitney!U!test!was!used!to!assess!differences! between! groups.! In! order! to! relate! variables! and! seek! any! relationship/causality,! bivariate!correlations!were!made!between!clinical!variables,!fatigue!and!disability!with! HADS! anxiety/depression! scores! and! Spearman’s! correlation! coefficient! used.! For! the! Brief! COPE! questionnaire! where! each! item! has! two! questions,! internal! consistency! coefficients!(ICC)!were!calculated!using!Cronbach’s!alpha!between!dimension!items.! Multivariable! analysis! was! performed! to! assess! the! independent! contributions! of! selected!clinical,!patient!related!outcome!variables!and!illness!perception!in!evaluating! mood! and! fatigue! as! dependent! continuous! variables.! Only! those! significant! bivariate! correlations! (where! p<0.05)! were! entered! into! regression! models.! Testing! for! coH linearity!was!performed.!

3.1'Chapter'3;'Results5'Clinical'Cohort'