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ISBN 978-952-62-1672-0 (Paperback)ISBN 978-952-62-1673-7 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1432

AC

TAK

yösti Vihanninjoki

OULU 2017

D 1432

Kyösti Vihanninjoki

THE HEIDELBERG RETINA TOMOGRAPH IN THE DIAGNOSIS OF GLAUCOMA

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;OULU UNIVERSITY HOSPITAL

ACTA UNIVERS ITAT I S OULUENS I SD M e d i c a 1 4 3 2

KYÖSTI VIHANNINJOKI

THE HEIDELBERG RETINA TOMOGRAPH IN THE DIAGNOSISOF GLAUCOMA

Academic dissertation to be presented with the assentof the Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defencein Auditorium 5 of Oulu University Hospital, on 13October 2017, at 12 noon

UNIVERSITY OF OULU, OULU 2017

Copyright © 2017Acta Univ. Oul. D 1432, 2017

Supervised byDocent Ville SaarelaProfessor P. Juhani Airaksinen

Reviewed byDocent Mika HarjuDocent Marko Määttä

ISBN 978-952-62-1672-0 (Paperback)ISBN 978-952-62-1673-7 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2017

OpponentProfessor Hannu Uusitalo

Vihanninjoki, Kyösti, The Heidelberg Retina Tomograph in the diagnosis ofglaucoma University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Oulu UniversityHospitalActa Univ. Oul. D 1432, 2017University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Glaucoma is a group of eye diseases characterized by a chronic, progressive optic neuropathy.During the disease process, the axon damage of the retinal ganglion cells leads to changes in theretinal nerve fiber layer, causing optic nerve head, and visual field defects typical of glaucoma.

The Heidelberg Retina Tomograph (HRT) is a confocal scanning laser imaging deviceacquiring and analysing three-dimensional data of the ocular fundus wit good accuracy andreproducibility..

Conventional planimetric measurements were compared to those taken with the HRT in a pilotstudy of 12 eyes with early glaucomatous optic disc, retinal nerve fiber layer and/or visual fieldabnormalities. The neuroretinal rim area measurements and cup-to-disc area ratio did not differstatistically from each other when using these two different methods.

The effect of four different reference levels on the HRT parameter measurement values wastested in two separate studies. In the first study there were 67 eyes, 40 of the eyes were healthy and27 eyes had glaucoma of different stages. Then, 279 eyes, 180 of which were non-glaucomatousand 99 glaucoma eyes, were included in another study. The flexible reference level gave the mostreliable HRT parameter measurement values in both non-glaucomatous and glaucomatous eyes.

The ability of the HRT parameters to separate between non-glaucomatous and glaucomatouseyes was tested in 77 eyes, 40 of the eyes were non-glaucomatous, 10 ocular hypertensives and 27eyes had different stages of glaucoma. The reference level dependent HRT parameters cup-to-discarea ratio, vertical linear cup-to-disc ratio, mean retinal nerve fiber layer thickness (RNFLt) andrim volume as well as the reference level non-dependent HRT parameter, cup shape measure(CSM), separated best between the clinical groups.

The best combination of the HRT and other structural and functional parameters in separatingbetween non-glaucomatous and glaucomatous eyes was studied in 55 eyes. There were 32 non-glaucomatous eyes and 23 eyes with ocular hypertension or glaucoma. CSM, RNFLt, togetherwith age- and lens coloration-corrected mean deviation of the B/Y perimetry showed gooddiscrimination (ROC area 0.91) between non-glaucomatous and glaucomatous eyes.

Keywords: confocal scanning laser ophthalmoscope, glaucoma, optic nerve head, retinalnerve fiber layer, visual field

Vihanninjoki, Kyösti, The Heidelberg Retina Tomograph glaukooman diagnostiikassaOulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Oulunyliopistollinen sairaalaActa Univ. Oul. D 1432, 2017Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Glaukooma koostuu joukosta hitaasti eteneviä näköhermon rappeumasairauksia. Sairausproses-sin aikana verkkokalvon gangliosolujen aksonivaurio johtaa muutoksiin verkkokalvon hermo-säiekerroksessa ja näköhermon päässä aiheuttaen glaukoomalle tyypillisiä näkökenttämuutoksia.

The Heidelberg Retina Tomograph (HRT) on konfokaali laserskanneritekniikkaan perustuvakuvantamislaite, joka tuottaa ja analysoi silmänpohjasta saatua kolmiulotteista mittaustietoa tar-kasti ja toistettavasti.

Tavanomaisen planimetrian antamia mittaustuloksia verrattiin HRT:n antamiin tuloksiin12:ssa silmässä, joissa oli todettu varhaisia glaukoomamuutoksia. Näköhermon pään hermoreu-nan (rim) pinta-ala ja keskuskuopan suhde papillan läpimittaan eivät poikenneet tilastollisestitoisistaan näitä kahta menetelmää käytettäessä.

Neljän eri referenssitason vaikutusta HRT-parametrien mittausarvoihin testattiin kahdessa eritutkimuksessa. Ensimmäisen tutkimusaineisto koostui yhteensä 67:stä silmästä, joista 40 oli ter-veitä ja 27:ssä eriasteisia glaukoomamuutoksia. Toisessa tutkimuksessa oli yhteensä 279 silmää,joista 180 oli terveitä ja 99:llä oli glaukooma. Papillomakulaarisäikeisiin tukeutuva, fleksiibelireferenssitaso antoi luotettavimmat HRT-parametrien mittaustulokset sekä terveissä että glau-koomasilmissä.

HRT-parametrien kykyä erottaa terveet silmät glaukomatoottisista testattiin yhteensä 77:ssäsilmässä, joista 40 oli terveitä, 10 oli korkeapaineisia ilman glaukoomamuutoksia, ja 27:ssä oliglaukoomamuutoksia. Referenssitasosta riippuvaiset HRT-parametrit, keskuskuopan suhdepapillan läpimittaan, vertikaali-lineaarinen keskuskuopan suhde papillan läpimittaan, keskimää-räinen verkkokalvon hermosäiekerroksen paksuus (RNFLt) ja `rim´:in tilavuus samoin kuinreferenssitasosta riippumaton keskuskuopan ´vinous´-mitta (CSM) erottelivat parhaiten nämäkliiniset ryhmät toisistaan.

Terveitä ja glaukoomasilmiä erottelevaa HRT:n ja muiden rakenteellisten ja toiminnallistenparametrien kombinaatiota etsittiin 55:n silmän aineistosta. Silmistä 32 oli terveitä ja 23 korkea-paineisia ja/tai glaukoomavaurioisia. CSM ja RNFLt, yhdessä iän ja mykiövärjäytymisen suh-teen korjatun sinikeltaperimetrian keskipoikkeaman kanssa osoittivat hyvää erottelukykyä (ROCarea 0.91) terveiden ja glaukoomasilmien välillä.

Asiasanat: glaukooma, konfokaali laserskanneri oftalmoskooppi, näköhermon pää,näkökenttä, verkkokalvon hermosäiekerros

To Professor P. Juhani Airaksinen (in mem.)

8

9

Acknowledgements

The studies for this thesis were carried out in the Department of Ophthalmology,

University of Oulu, and in the Department of Ophthalmology, University of

Heidelberg, during the years 1994-2002. The writing process of this doctoral

dissertation was performed under two supervisors.

First of all, I am deeply grateful to my first supervisor, Professor P. Juhani

Airaksinen, M.D., Ph.D. (†), for his enthusiastic and encouraging attitude in glaucoma

research. His wide experience in scientific field and extensive knowledge of glaucoma

was admirable. It has been an unique priviledge to be one of his numerous co-workers

in ophthalmologic research worldwide. He suggested the subject of this study to me

and showed great helpfulness through these studies up until his retirement, and even

after that.

The writing process of this compilation dissertation would not have been

accomplished without my second supervisor, Ville Saarela, M.D., PH.D., who picked

me up to finish this thesis. I will never forget about his absolute will to bring this work

to a conclusion. I wish to express my warmest gratitude to him for his patience and

ability to painstakingly inspire and support me during these years.

I wish to thank Professor Anja Tuulonen, M.D., Ph.D., my teacher and co-writer in

these studies, for her invaluable help and advice in writing scientific papers. Her energy

and exemplary organizational ability have guided me through numerous problem

situations.

I am very grateful to Professor Reinhard O.W. Burk, M.D., Ph.D., my co-writer,

particularly for his help in Study III, for providing knowledge and study material of

critical importance. Co-operation with his research group in the Department of

Ophthalmology, University of Heidelberg, has been essential.

I want to thank Professor Pait Teesalu, M.D., Ph.D., also my co-writer, for fruitful

and pleasant co-operation in preparing these studies. His study period and

accomplishment of academic thesis in our clinic was memorable.

I acknowledge Docent Mika Harju, M.D., Ph.D., and Docent Marko Määttä, M.D.,

Ph.D., for their careful review and advisable criticism of this dissertation manuscript.

I also want to thank Aura Falck, M.D., Ph.D., member of the follow-up group of

this study, for her valuable comments and all the support she has kindly addressed to

me and to my work. She receives all my admiration. Thanks go to Pasi Hägg, M.D.,

Ph.D., another member of the follow-up group, too.

Mr. Esa Läärä, M.Sc., is acknowledged for his special advice on the statistical

analyses as well as Anna Vuolteenaho, M.A., for revising the English of this thesis.

10

Mr. Heikki Nieminen (†), talented photographer in our Department of

Ophthalmology in 1980´s and 1990´s, deserves special thanks for his pioneering work

within glaucoma research. Thanks to his photographing skills numerous glaucoma

studies have been carried out.

I am very grateful to all my colleagues and all the staff in the Eye Clinic of the

Oulu University Hospital for their compassion and patience during these years. They

have made an important contribution to enable this work. They deserve everlasting

respect. My special thanks go to Professor Leila Laatikainen, M.D., Ph.D., Eila

Mustonen, M.D., Ph.D., Rauno Miettinen, M.D., Ph.D., Pentti Koskela, M.D., Ph.D.,

Jussi Kärnä, M.D., Ph.D., Kai von Dickhoff, M.D., Ph.D., Hannu Alanko, M.D., Pekka

Virtanen, M.D., Heli Hirvelä, M.D., Ph.D., Seppo Siik, M.D., Ph.D., Jouko Mattila,

M.D., Risto Salmi, M.D., Minna Virtanen, M.D., Juha Välimäki, M.D., Ph.D., Petri

Oksman, M.D., Harri Koskela, M.D., Ulla Lahtela, M.D. Sirje Ess, M.D., Tapani

Palosaari, M.D., Nina Hautala, M.D., Ph.D. and Pauli Hyytinen for all the support and

co-operation.

I acknowledge the personnel of Opti-Aika Järvelä, Kemin Silmälasi Oy, Näkötiimi

Oy and Opti-Silmä Oy for their patience and friendly co-operation when accomplishing

this thesis.

My sincerest thanks go to all my relatives and friends for their support and

encouragement during these years of the study.

I wish to express my deepest gratitude to my parents Paavo (†) and Mailis for their

care and love. They have showed unending sacrifices for all their children.

I want to express my warmest and loving thanks to my excellent sons Pyry and

Vesa, for making my life meaningful and bringing me plenty of happiness. They are the

joy of my life.

Finally, my loving thanks go to my dear companion, Tarja, my ´Honeysuckle´.

With love and unselfish care she has guided me back to real life from the ´Research

Wonderland´. Together with her I want to celebrate as well as to live our everyday life.

This study was financially supported by grants from Orbis Sensorius Oulu, Sigrid

Juselius Foundation, the Eye Foundation, the Eye and Tissue Bank Foundation, the De

Blindas Vänner – Sokeain Ystävät Foundation, Oulun Diakonissalaitos, the

Instrumentarium Science Foundation, Glaukooma Tukisäätiö Lux Foundation, EVO

funding of Oulu University Hospital and MSD (travel grant).

Oulu, at harvest time, 2017 KyöstiVihanninjoki

11

Abbreviations

AF autofluorescence

ANOVA analysis of variance

B/Y blue-on-yellow (visual field) CA cup area

CCT central corneal thickness

C/D cup-to-disc ratio

CDR cup-to-disc area ratio

CDRVer vertical linear cup-to-disc ratio

CDVer vertical cup diameter

CSM cup shape measure (Mom3)

CV cup volume (volume below reference plane)

DA disc area

dB decibel

FDT frequency doubling technology

GDx scanning laser polarimetry

GPS glaucoma probability score

HPeak minimum relative z coordinate of corrected contour line

HRT Heidelberg Retina Tomograph

HRTCALC HRT calculation program

HVar height variation along contour line

IOP intraocular pressure

LTI lens transmission index

MD mean deviation

MHC mean height contour

MRA Moorfields regression analysis

Mom3 third moment

OCT optical coherence tomography

OHT ocular hypertension

ONH optic nerve head

POAG primary open angle glaucoma

PSD pattern standard deviation

RA rim area

REFf HRT software 1.11 standard reference level (the flexible

reference level, SRP)

REFd HRT software 1.09 standard reference level

12

REFi individually determined reference level

REFm papillo-macular reference level

REFs curved surface reference level

RGC retinal ganglion cells

ROC receiver operator characteristics

RNFL retinal nerve fiber layer

RNFLc retinal nerve fiber layer cross section area

RNFLt mean retinal nerve fiber layer thickness

RV rim volume (volume above reference plane)

SAP standardized automated perimetry

SRP standard reference plane

SWAP short-wavelength automated perimetry

VF visual field

W/W white-on-white (visual field)

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List of original publications

This thesis is based on the following original articles, which are referred to in the

text by their Roman numerals:

I Vihanninjoki K, Tuulonen A, Burk ROW & Airaksinen PJ (1997) Comparison of optic disc measurements by Heidelberg Retina Tomograph and manual planimetric techniques. Acta Ophthalmol Scand 75: 512-515.

II Vihanninjoki K, Burk ROW, Teesalu P, Tuulonen A & Airaksinen PJ (2002) Optic Disc Biomorphometry with the Heidelberg Retina Tomograph at Different Reference Levels. Acta Ophthalmol Scand 80: 47-53.

III Burk ROW, Vihanninjoki K, Bartke T, Tuulonen A, Airaksinen PJ, Völcker H-E & König JM (2000) Development of the Standard Reference Plane for the Heidelberg Retina Tomograph (HRT). Graefe’s Arch Clin Exp Ophthalmol 238: 375-384.

IV Vihanninjoki K, Burk ROW, Teesalu P, Tuulonen A & Airaksinen PJ Identification of Non-glaucomatous and Glaucomatous Optic Discs with the Heidelberg Retina Tomograph. Manuscript.

V Vihanninjoki K, Teesalu P, Burk ROW, Läärä E, Tuulonen A & Airaksinen PJ (2000) Search for an Optimal Combination of Structural and Functional Parameters for the Diagnosis of Glaucoma. Multivariate Analysis of Confocal Scanning Laser Tomograph, Blue-on-yellow Visual Field and Retinal Nerve Fiber Layer Data. Graefe`s Arch Clin Exp Ophthalmol 238: 477-481.

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15

Contents

Abstract

Tiivistelmä

Acknowledgements 9 Abbreviations 11 List of original publications 13 Contents 15 1 Introduction 17 2 Review of the literature 19

2.1 Development of eye fundus observation and imaging ............................ 19 2.1.1 Ophthalmoscopy and biomicroscopy ........................................... 19 2.1.2 Eye fundus photography and photogrammetry............................. 19 2.1.3 Emerging eye fundus imaging devices in the 1990s ..................... 22 2.1.4 Measuring the optic disc and the RNFL ....................................... 23

2.2 Non-glaucomatous eye ............................................................................ 24 2.2.1 Structure of the non-glaucomatous optic nerve head and

the RNFL ...................................................................................... 24 2.2.2 Visual field in non-glaucomatous eye .......................................... 26

2.3 Glaucomatous eye ................................................................................... 27 2.3.1 Glaucoma...................................................................................... 27 2.3.2 Glaucomatous optic disc changes ................................................. 27 2.3.3 Glaucomatous RNFL changes ...................................................... 29 2.3.4 Glaucomatous visual field defects ................................................ 30

2.4 The confocal scanning laser tomographer ............................................... 32 2.4.1 General aspects of confocal scanning laser technology................ 32 2.4.2 Principles of use and technical characteristics of the HRT ........... 32 2.4.3 Definitions related to measurements with the HRT ...................... 39 2.4.4 Accuracy and reproducibility of the HRT .................................... 42

2.2 Earlier studies with the Heidelberg Retina Tomograph ........................... 43 2.2.1 Accuracy and reproducibility studies ........................................... 43 2.2.2 The HRT in separating healthy and glaucomatous eyes ............... 45

3 Purpose of the study 47 4 Material and methods 49

4.1 Subjects ................................................................................................... 49 4.2 The planimetric techniques ..................................................................... 50 4.3 HRT study protocol ................................................................................. 53

16

4.4 Automated perimetry .............................................................................. 60 4.5 Study protocol ......................................................................................... 60 4.6 Statistical methods .................................................................................. 61

5 Results of the study 63 5.1 Comparison of optic nerve head measurements using planimetric

techniques and the HRT (I) ..................................................................... 63 5.2 The influence of different reference levels on the HRT

measurement values (II) .......................................................................... 64 5.3 The development of the contour line based standard reference

plane (III) ................................................................................................ 66 5.4 The ability to separate clinical groups with the HRT (IV) ...................... 67 5.5 Logistic multivariate regression analysis of confocal scanning

laser tomograph, blue-on-yellow visual field and retinal nerve

fiber layer data (V) .................................................................................. 69 6 Discussion 73

6.1 Early glaucomatous optic disc, RNFL and visual field changes ............. 73 6.2 Accuracy, reproducibility and agreement between optic disc and

RNFL observation and evaluation methods ............................................ 74 6.3 The significance of the definition of the HRT reference level ................ 75 6.4 The significance of the HRT global parameters in optic disc

evaluation ................................................................................................ 77 6.5 Sensitivity and specificity of glaucoma imaging methods ...................... 77 6.6 Diagnostic accuracy of the HRT in screening studies ............................. 78 6.7 Clinical implications ............................................................................... 79 6.8 Further developments in the HRT ........................................................... 80

7 Summary and conclusions 83 References 85 Original publications 105

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1 Introduction

The definition of glaucoma refers to a progressive optic neuropathy causing

characteristic structural changes of the optic disc and the retinal nerve fiber layer

(RNFL) resulting in visual field defects, pathognomonic for glaucoma (Airaksinen

& Alanko 1983, Airaksinen et al. 1984, Jonas et al. 1989a, Jonas et al. 1989b,

Tuulonen & Airaksinen 1991, Airaksinen et al. 1992, Varma et al. 1992, Quigley

1999, Terminology and Guidelines of Glaucoma EGS 2014, AAO Glaucoma Panel

2016). Glaucoma is the second most common reason for blindness in the world

(Quigley & Broman 2006) and in Finland it is the second most common cause of

visual impairment in the age group of over 65 years (Finnish Register of Visual

Impairment 2014). Other most common causes for visual impairment worldvide

are age-related macular degeneration, corneal opacities, diabetic retinopathy and

congenital blindness, and in Finland age-related macular degeneration, inherited

retinopathies, diseases of visual pathways and diabetic retinopathy. There were

1271 patients with glaucoma diagnosis in the Finnish Register of Visual

Impairment in 2014. The early detectable glaucomatous optic disc and RNFL

abnormalities often precede the typical glaucomatous visual field defects

(Airaksinen et al. 1985a, Airaksinen et al. 1985b, Quigley et al. 1989, Sommer et al. 1991, Tuulonen & Airaksinen 1991, Quigley et al. 1992). However, any of these

three parameters may present the first clinical sign of glaucoma (Artes & Chauhan

2005, Strouthidis et al. 2006a, Strouthidis et al. 2006b). “Structure-function

dissociation” has been found in large glaucoma studies, too (Miglior et al. 2005b,

Heijl et al. 2009).

In earlier studies, with conventional optic disc and RNFL photography,

changes of the neuroretinal rim area (RA) (Airaksinen et al. 1985b, Airaksinen et al. 1992), the RNFL (Airaksinen & Drance 1985, Airaksinen & Nieminen 1985)

and the peripapillary RNFL contour line (Tuulonen et al. 1996) have been able to

separate between non-glaucomatous and glaucomatous eyes. However, the

qualitative and subjective nature of these assessing methods has shown the need for

more quantitative, accurate and reproducible methods (Lichter 1976, Varma et al. 1992, Caprioli et al. 1996, Coleman et al. 1996, Margo et al. 2002, Parrish et al. 2005). The eye fundus imaging devices emerging in the early 1990s have offered

the possibility to fulfill this deficiency, enabling the three-dimensional

measurements of the ONH.

As in manual planimetric techniques, the inner edge of the scleral ring, the

Elschnig’s ring, has been used as a reference level for manual optic disc

18

measurements in conventional optic disc stereophotogrammetry (Elschnig 1907,

Sommer et al. 1979a, Balaszi et al. 1984, Britton et al. 1987, Jonas et al. 1988b,

Jonas et al. 1988c, Jonas et al. 1988d, Gross & Drance 1995). This is because the

scleral ring forms a reference plane, which most probably does not change with

progressive glaucomatous damage (Airaksinen et al. 1992, Jonas et al. 1993). The

preservation of the papillo-macular bundles during the glaucomatous RNFL

atrophy suggests a favorable region for the definition of a stable reference plane for

the HRT measurements (Airaksinen & Drance 1985, Airaksinen & Nieminen 1985).

Confocal scanning laser tomographers, such as the HRT, have been reported as

instruments with accurate and reproducible three-dimensional measurements of the

optic nerve head (ONH) and central retina (Zinser et al. 1989, Burk et al. 1990,

Weinreb & Dreher (1990), Burk et al. 1991, Burk et al. 1992, Burk et al. 1993a,

Burk et al. 1993b, Weinreb 1993a, Chauhan et al. 1994, Rohrschneider et al. 1994,

Chauhan & Macdonald 1995, Chauhan & McCormick 1995, Vihanninjoki et al. 1995, Bartz-Schmidt et al. 1996a). However, depending on the definition of the

reference level, variation in the optic disc values measured with HRT at different

stages of glaucomatous damage has been published (Tuulonen et al. 1994). That is

why a well defined and stable reference level as well as a choice of clinically

meaningful HRT parameters are of utmost importance for the HRT, to become a

clinically useful tool in glaucoma diagnostics and follow-up (Zangwill et al. 1995,

Dichtl et al. 1996, Wollstein et al. 2000, Kesen et al. 2002, Correnti et al. 2003).

Static automated perimetry has been the standard for evaluating visual fields

in the diagnosis and follow-up of glaucoma (Enger & Sommer 1987, Heijl et al. 1987, Heijl et al. 1989, Drance 1991, Anderson et al. 2001, Viswanathan et al. 2003). However, it has been proposed that short-wavelength automated perimetry

(SWAP) may reveal visual field defects earlier (Johnson et al. 1993b, Sample et al. 1993b) and larger in extent than W/W perimetry (Sample et al. 1986, Sample &

Weinreb 1990, Sample & Weinreb 1992, Wild 2001). The predictability of

glaucoma with B/Y perimetry has been poor, however (van der Schoot et al. 2010).

Frequency-doubling technology (FDT) discriminates glaucomatous and non-

glaucomatous eyes with high sensitivity and specificity, too (Johnson & Samuels

1997, Burnstein et al. 2000).

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2 Review of the literature

2.1 Development of eye fundus observation and imaging

2.1.1 Ophthalmoscopy and biomicroscopy

In 1851, Hermann von Helmholtz presented the first usable direct ophthalmoscope,

Augenspiegel, in his monograph on ophthalmoscopy (von Helmholtz 1851).

Jacobson was the first to report of glaucomatous optic disc cup in 1853 (Duke-

Elder 1958), whereas von Graefe and Jaeger mistakenly described ONH swelling

in glaucoma (von Graefe 1855, Jaeger 1858). Direct ophthalmoscopy has been used

in glaucoma diagnostics, both in optic disc and RNFL assessment. Later, binocular

ophthalmoscopy allowed stereopsis and green light ophthalmoscopy improved the

RNFL evaluation (Vogt 1930). In 1916, Zeiss developed the slit-lamp

biomicroscope, an important invention in ophthalmology (Koeppe 1918). It was

perfected with a contact lens by Goldmann in 1938 and with a non-contact lens by

Hruby in 1942 (Goldmann 1938, Hruby 1942). Regardless of various classification

systems for glaucomatous changes, all these techniques are subjective methods

with high interobserver variation and without documentation of the findings.

2.1.2 Eye fundus photography and photogrammetry

In 1886, Jackman and Webster published the first photographs of a living human

eye fundus (Jackman & Webster 1886), and the first accurate, high-quality eye

fundus photographs were introduced in 1907 by Dimmer (Dimmer 1907). In 1929

Bedell published the first stereo atlas of fundus photographs (Bedell 1929).

Difficulty in obtaining high-quality images and magnification errors in the

measurements of eye fundus structures limited the clinical use of fundus

photography. Several attempts to resolve these photogrammetry problems were

introduced, first in model eyes. Johnson et al. noted that the poor reproducibility of

the cup volume (CV) measurements was partially a result of the difficulty in

specifying the top of the optic cup, because the topography of the rim varies

considerably about its circumference (Johnson 1994). A chart with concentric

circles for optic disc comparison was presented in 1960 by Colenbrander

(Colenbrander 1976) and in 1964 by Snydacker (Snydacker 1964). In 1967, Armaly

introduced the definition of cup-disc ratio in normal and glaucomatous eyes, a

20

method shown to be inaccurate (Leydhecker et al. 1979) with high interindividual

variation (Lichter 1976). Other two- or three-dimensional optic disc and RNFL

evaluation methods included flicker comparison method (Bengtsson & Krakau

1979), stereochronoscopy (Goldmann & Lotmar 1977), computerized image

processing methods (Nakatani & Suzuki 1981, Miszalok & Wollensack 1982),

photographic subtraction (Deininger 1970, Horiuchi 1971, Alanko et al. 1980,

Jaanio et al. 1980) and video-ophthalmograph in optic disc topographic

measurements (Mikelberg et al. 1984).

In their studies, Schwartz and Takamoto produced relative or absolute depth

and volume measurements of the optic cup, with an accuracy of 23 µm for depth

and 4 µm for horizontal and vertical measures (Schwartz & Takamoto 1978). They

calculated CVs based on the top surface of the ONH along the disc margin instead

of the cup margin, defining the space below this surface as the CV. Takamoto and

Schwartz also found that the shape of the volume profile curves is different in

normal eyes compared to glaucomatous eyes (Takamoto & Schwartz 1979). Cup

shape was quantified by calculating volume profile, which is a cross-sectional or

contour area extending from the top to the bottom of the optic disc. In 1970, Holm

and Krakau measured CVs by projecting illuminated multiple slits on the ONH

(Holm et al. 1972, Jönsas 1972).

Later, the development of eye fundus photography techniques made it possible

to achieve more accurate, reproducible and objective documentation of the optic

disc and the RNFL. The permanent recording of findings is of the utmost

importance in the follow-up of glaucoma progression. Stereophotogrammetrical

methods in medicine were introduced by Björn et al. (Björn et al. 1954). In 1964,

Donaldson presented simultaneous stereophotographs, showing better

reproducibility of cup depth measurements compared to the sequential techniques

used by Allen (Donaldson 1950, Allen 1964, Rosenthal et al. 1977). In 1969,

different stereoscopic photographic techniques were introduced: Crock and Parel

(stereoangiography), Forsius and Jönsas (Oulu project), Mikuni et al. (stereoscopic

contouring) and Tomlinson and Phillips (a prism stereoscope) (Jönsas 1972).

Portney measured the optic discs of normal and ocular hypertensive eyes by

stereophotogrammetry using sequential stereophotographs taken with the Zeiss

fundus stereocamera with the Allen stereoscopic separator and concluded that CV

asymmetry greater than 0.20 mm3 was a sign of glaucoma (Portney 1974). Using a

stereophotogrammetric technique, Carassa and associates quantitatively evaluated

and compared optic disc asymmetries between the two eyes of normal subjects and

between those of ocular hypertensive subjects. They found a significant asymmetry

21

in the normal and ocular hypertensive groups for cup area (CA) and CV (inferior

quadrant) (Carassa et al. 1995) Tomita et al. used stereophotogrammetry to

compute cup parameters in normal and open-angle glaucomatous eyes. There was

a significant difference in the parameters between normal and glaucomatous eyes

(Tomita et al. 1994). The techniques of optic disc stereophotographs and RNFL

photography were presented by Airaksinen and Nieminen (Airaksinen & Nieminen

1985). Flicker comparisons of serial disc photographs in the diagnosis of early

glaucoma were studied by Heijl & Bengtsson (Heijl & Bengtsson 1989).

In 1917, Vogt described the evaluation of the RNFL in red-free light. Green

light ophthalmoscopy made the nerve fiber bundles more visible (Vogt 1930). In

1965, Behrend and Wilson introduced monochromatic light into ophthalmic

photography: interference filters and black-and-white film made the RNFL visible

in greenblue/blue light at the wavelength of 549-477 nm (Behrend & Wilson 1965).

Blue light was reflected from the RNFL layer of the retina, discriminating normal

and degenerated areas. Monochromatic photography techniques were further

developed by Mizuno et al., Hoyt & Newman, Iwata et al. Delori & Gragoudas,

and Vannas et al. (Mizuno et al. 1968a, Mizuno et al. 1968b, Hoyt and Newman

1972, Iwata et al. 1975a, Iwata et al. 1975b, Delori & Gragoudas 1976, Vannas et al. 1977). The higher resolving power of low-sensitive black-and-white film in

contrast to that of color film was noticed by Durrey (Durrey et al. 1979) and Frisen

(Frisen 1980). A 30-degree angle fundus camera through a green filter and black-

and-white film was used by Airaksinen (Airaksinen et al. 1981a) and by Mustonen

and Nieminen (Mustonen & Nieminen 1982). A new wide-angle fundus camera

was used by Airaksinen (Airaksinen et al. 1982). Airaksinen et al. (Airaksinen et al. 1982) and Peli et al. (Peli et al. 1987) found it easier to detect RNFL defects

with a wide-angle fundus camera, using high-resolution, fine-grain, black-and-

white film with a blue narrow-band interference filter of 465 nm wavelength (a

Wratten #58). In 1983, Sommer et al. improved the RNFL visibility in photographs

by using a 566 nm short-pass cut-off filter (Sommer et al. 1983).

Cup shape can be estimated by calculating volume profile, which is a cross-

sectional or contour area extending from the top to the bottom of the optic disc.

Takamoto and Schwartz found that the shape of the volume profile curves of normal

eyes was different from that of glaucomatous eyes: the glaucomatous eyes had

larger cup areas and deeper cup depths when compared to normal eyes. The volume

profiles of ocular hypertensive eyes in the same study were between those of

normal and glaucomatous eyes (Takamoto & Schwartz 1979).

22

Ophthalmoscopy using a Goldmann contact lens and a slit lamp, with

magnification correcting formulas, was found to be a clinically practicable method

in measuring optic disc dimensions, diameters and areas, compared with planimetry

on photographs (Jonas & Papastathopoulos 1995). The simple ophthalmoscopic

estimation of the ONH and the neuroretinal rim size in glaucomatous patients

compared well with the more laborious planimetric techniques (Littmann 1988).

As planimetry is a two-dimensional method, it involves measuring the area of

a structure, as opposed to depth which is measured in photogrammetry, a three-

dimensional method. Typical planimetric parameters are cup diameter, disc

diameter, cup, disc and neuroretinal RA, thinnest portion of the neuroretinal rim

and cup-to-disc ratio. Planimetric measurement values are given in ‘machine units’,

which makes it difficult to compare the results given by different planimetric

methods (Sommer et al. 1979a, Sommer et al. 1979b, Balaszi et al. 1984, Britton et al. 1987, Jonas et al. 1988a). These methods showed significant interexamination,

intra- and interobserver variation because of interpretation problems in defining the

cup margin (Lichter 1976, Varma et al. 1992, Caprioli et al. 1996, Coleman et al. 1996, Margo et al. 2002, Parrish et al. 2005).

2.1.3 Emerging eye fundus imaging devices in the 1990s

The technical development of optical imaging devices has made it possible to

enhance the clinical examination and the ‘golden standard’ of optic disc and RNFL

photographs. These are subjective and qualitative methods with descriptive

documentation, sensitive to small pupil size and media opacities. In contrast, the

new, real-time, computerized, accurate and reproducible instruments produce

quantitative, three-dimensional information.

In the 1990s, several emerging optical imaging instruments were introduced:

confocal scanning laser ophthalmoscope (the HRT), scanning laser polarimetry (the

GDx) and optical coherence tomography (the OCT). Scanning retinal thickness

analyzer (the RTA) and raster tomography (the Glaucoma-Scope) were also

available. Each of them has different principles of function, with strengths and

limitations (Gundersen et al. 2000, Konno et al. 2001, Zangwill et al. 2001, Asrani et al. 2003).

The Heidelberg Retina Tomograph (HRT) uses a diode laser of 670 nm wavelength

to scan the eye fundus in the x and y directions at multiple focal planes. A three-

dimensional, color-coded topographic image is calculated from a series of optical

image sections. Three topographic images are combined and automatically aligned,

23

giving a single mean topography for detailed analysis (Wollstein et al. 1998, Iester et al. 1999).

The GDx Nerve Fiber Analyzer (GDx) techniques are based on the change in

polarization occurring when light illuminates birefringent tissue, such as the RNFL.

In the GDx a polarization modulated laser beam, 780 nm in wavelength, passes

through the RNFL, which changes the state of polarization (retardation). The

polarizing ability of the cornea and the lens is compensated in either fixed (GDx

FCC), variable corneal compensation (GDx VCC) or enhanced cornea

compensation (GDx ECC) (Weinreb et al. 2002, Greenfield et al. 2003, Brusini et al. 2005,). The change in polarization is measured, and the thicker the birefringent

structure, the greater the retardation of transmitted light.

In the OCT, a low coherence infrared light beam of 840 nm wavelength is used

in interferometry principles: a measurement beam is reflected from intraocular

tissues according to their distance, thickness and reflectivity, and the interference

information with the reference beam is presented in bi-dimensional images after

longitudinal scanning in transverse direction (Huang et al. 1991, Guedes et al. 2003). The modern spectral-domain OCT is faster in image acquisition and has

better resolution and reproducibility compared with the older time-domain OCT

devices (Huang et al. 1991, Chang et al. 2008).

2.1.4 Measuring the optic disc and the RNFL

The classic, ‘golden standard’ methods of the assessment of the ONH and the RNFL

have been ophthalmoscopy and eye fundus photography, stereophotography of

optic discs and RNFL photography. All of these techniques remain subjective in

nature, showing inter-observer and inter-examination variation (Lichter 1976,

Varma et al. 1992, Tuulonen et al. 1996, Azuara-Blanco et al. 2003). Both

planimetry and stereophotogrammetry have problems with reproducibility and

accuracy due to variation in interpretation and with correcting algorithms (Sommer et al. 1979a, Balaszi et al. 1984, Britton et al. 1987, Jonas et al. 1988a, Jonas et al. 1989a).

The technical progress of eye fundus imaging devices in the late 1980s and

during the 1990s made it possible to acquire precise three-dimensional data of the

optic disc in vivo. However, standardization of these emerging methods that would

allow clinically reasonable measurement results has been challenging (Frohn et al. 1990, Zinser et al. 1990, Tuulonen et al. 1994).

24

2.2 Non-glaucomatous eye

2.2.1 Structure of the non-glaucomatous optic nerve head and the RNFL

The optic disc or ONH is formed of the internal surface of the posterior scleral

opening through which the retinal ganglion cell axons leave the eye (Figure 1). The

peripapillary scleral ring of Elschnig forms the wall of the optic disc, separating the

intrapapillary from the parapapillary region. The clinically visible optic disc margin

may be formed of the innermost opening in Bruch´s membrane, Bruch´s membrane

opening (BMO), or of the dense connective tissue of Elschnig, Elschnig´s ring, or

of the combination of both. The clinical appearance depends on the pigmentation

of BMO and on the structure of the border tissue of Elschnig, joining the sclera to

Bruch´s membrane (Jonas et al. 1988d, Strouthidis et al. 2009). The exact borders

of BMO can be verified with the OCT. (Strouthidis et al. 2010).

The optic disc and the surface of the RNFL are visible in the assessment of the

eye fundus (ophthalmoscopy, slit-lamp biomicroscopy, photography) (Snydacker

1964). The prelamina and lamina cribrosa of the optic nerve consists of glial and

connective tissue and are typically injured in glaucoma. The ONH glial astrocytes

translate ONH stress into axon damage (Hernandez 2000). Morphologically the

retina consists of several cell layers. The retinal ganglion cells (RGC) receive the

visual information originating from photoreceptor cells via amacrine and bipolar

cells and transfer it to the optic nerve. The retinal ganglion cell axons form the

RNFL on the retinal surface. The retinal nerve fiber bundles are topographically

regularly arranged: temporally originating arcuate fibers, perifoveally originating

papillo-macular fibers and nasally originating radial fibers (Radius & Anderson

1979a). All of them enter the ONH, where the axons rotate 90 degrees through the

opening of the sclera. The width of the neuroretinal rim and the size of the optic

disc cup are dependent on the size of the optic disc itself and on the number of

nerve fibers passing through the scleral canal. (Radius & Anderson 1979b, Jonas et al. 1988b, Jonas et al. 1988c).

25

Fig. 1. Schematic presentation of the structure of the ONH (A), the RNFL (B) and VF (C)

with glaucomatous defect.

Clinically important optic disc features include optic disc size, optic disc form,

neuroretinal rim size and form, optic cup size and form, cup-to-disc ratio, optic disc

hemorrhages, and parapapillary atrophy (Radius & Andersson 1979b, Jonas et al. 1981, Jonas et al. 1988a). In non-glaucomatic eyes, the ONH neuroretinal rim has

a characteristic shape described by the ISNT rule: the rim is widest at the inferior

disc sector, followed by the superior, nasal, and the temporal region, where the rim

is narrowest (Jonas et al. 1988d).

Jonas et al. measured these optic disc dimensions in 457 normal eyes, using

Littmann’s method for correcting the magnification errors of photographs of the

central eye fundus. They found the following measures: mean optic disc diameter

horizontally 0.91-2.61 mm and vertically 0.96-2.91, mean optic disc surface 0.80-

5.54 mm², optic cup area 0-3.41 mm², mean horizontal cup diameter 0-2.08 mm

and vertical cup diameter 0-2.13 mm, neuroretinal RA (0.80-4.66 mm², the

narrowing of the neuroretinal rim followed the ISNT rule, horizontal cup-to-disc

ratio 0-0.87, vertical cup-to-disc ratio 0-0.85, the horizontal cup-to-disc ratio was

larger in 93.2% of the optic discs compared to the vertical cup-to-disc ratio (Jonas et al. 1988d).

The ONH is the internal surface of the posterior scleral foramen through which

the retinal ganglion cell axons leave the eye. This posterior scleral opening forms

a truncated cone with a narrow neck internally with a diameter of 1.5 to 2.0 mm

and a broad base externally with a diameter of 3.5 mm. The optic disc is divided

26

into the intrapapillary region containing the optic cup and the neuroretinal rim, and

the parapapillary region. The intrapapillary region is separated from the

parapapillary region by the peripapillary scleral ring of Elschnig forming the wall

of the optic disc (Radius & Anderson 1979b).

2.2.2 Visual field in non-glaucomatous eye

The normal VF ranges 90º-100º temporally, 60º nasally, 50º-60º superiorly and 70º-

75º inferiorly when the eye is fixed in a target. This is called the periferic VF. The

VF within 30º from fixation is called the central VF. The blind spot, corresponding

the ONH, locates 13º-18º temporally and 1º inferiorly from fixation. The light

sensitivity of the retina decreases from the fovea towards the periphery (Goldmann

1945).

The visual fields can be examined by conventional kinetic perimetry, such as

Goldmann perimetry, or static profile perimetry, such as Standardized Automated

Perimetry (SAP) or function-specific perimetry, such as short-wavelength

automated perimetry (SWAP) and frequency doubling perimetry (FDT). In

Goldmann perimetry, size III Goldmann stimulus is usually used within the 30º

central visual field. In automated perimetry VF data is summarized in global

summary indices: mean deviation (MD) represents the difference between observed

and expected mean sensitivity, pattern standard deviation (PSD) the standard

deviation of threshold’s distribution, and visual field index VFI expresses the

amount of VF capacity in percentages (Flammer 1986, Heijl et al. 1987, Bengtsson

& Heijl 2008). Variability of the static perimeter threshold is known to increase

with distance from fixation.

In the conventional W/W perimetry the deviation of the threshold values is

corrected for age. In B/Y perimetry, however, the lens-induced absorption of the

blue light may show large variability in subjects of similar age. We have previously

presented a procedure that provides an estimate of the absorption of blue light in

an individual lens by measuring the autofluorescence (AF) of the crystalline lens

(Siik et al. 1991, Siik et al. 1993). We have previously shown that the reference

level for correcting B/Y perimetry results can be determined more precisely using

fluorometry of the lens than with age alone. However, it was evident that the

variability was further decreased when both variables, LTI and age, were used

(Teesalu 1997a).

Static automated perimetry has been the standard for evaluating visual fields

in the diagnosis and follow-up of glaucoma (Enger & Sommer 1987, Heijl et al.

27

1987, Heijl et al. 1989a, Drance 1991, Anderson et al. 2001, Viswanathan et al. 2003). However, it has been proposed that short-wavelength automated perimetry

(SWAP) may reveal visual field defects earlier (Sample et al. 1993b, Johnson et al. 1993a, Johnson et al. 1993b) and larger in extent than W/W perimetry (Sample et al. 1986, Sample & Weinreb 1990, Sample & Weinreb 1992, Wild 2001). Also

frequency-doubling technology (FDT) discriminates glaucomatous and non-

glaucomatous eyes with high sensitivity and specificity (Johnson & Samuels 1997,

Burnstein et al. 2000).

2.3 Glaucomatous eye

2.3.1 Glaucoma

Glaucoma is a group of eye diseases characterized by a chronic, progressive optic

neuropathy. The degeneration of the RNFL and the optic disc results from the axon

damage of the RGC, causing RGC death. The reasons for glaucomatous process

are multifactorial: raised IOP causing mechanical or ischaemic injury, vascular,

systemic hypotension/vasospasm, immunological and inflammatory changes, as

well as blockage of neurotrophins have been presented as an etiology (Lichter &

Henderson 1977, Weinreb & Khaw 2004). This morphological damage may lead

to irreversible visual impairment and blindness if not treated. Based on anatomic

differences of the anterior segment of the eye, primary glaucomas are classified as

open angle glaucoma (OAG) and angle closure glaucoma. The known risk factors

for glaucoma are raised IOP, age, race, family history of glaucoma, exfoliation of

the lens, myopia, thin central cornea thickness (CCT) and type II diabetes (Armaly et al. 1980, Sommer et al. 1991, Mitchell 1996, Quigley et al. 1999, Kass et al. 2002, Burr et al. 2007, European Glaucoma Society 2008).

2.3.2 Glaucomatous optic disc changes

Glaucoma is not a “red disease”: no single test is able to define abnormality.

Glaucoma diagnosis is based on pre-test probability (risk-mapping and clinical

examination) fulfilled by post-test probability (glaucoma test for structure and

function (Garway-Heath & Friedman 2006). The recommendation for glaucoma

tests consists of measurement of IOP, gonioscopy, visual fields and imaging of the

eye fundus structures, photography and/or computerized imaging of the optic disc

28

and the RNFL) (Tuulonen et al. 2015). Glaucomatous findings in any one of the

diagnostic tests, changes of the ONH or the RNFL or defect in the VF may be the

first sign of the glaucomatous process (Artes & Chauhan 2005, Miglior et al. 2005b,

Strouthidis et al. 2006b). Therefore, a normal optic disc appearance and a normal

visual field do not rule out glaucoma (Tuulonen et al. 1993).

Open angle glaucoma is a chronic optic neuropathy that produces characteristic

visual field defects and ONH changes. It is generally thought that changes at the

ONH can be detected before the appearance of the earliest localized visual field

defects plotted with standard perimetry (Figure 2). In addition to the ONH, the

atrophy of ganglion cell axons can also be observed in the RNFL. In fact, a defect

in the RNFL may be the earliest sign of glaucoma, preceding changes in the ONH

configuration and the visual field (Hart et al. 1978, Sommer et al. 1991, Funk et al. 1993, Weinreb et al. 1995).

Some of early glaucomatous optic disc changes, such as the neuroretinal rim

losses, can be found both with conventional stereoscopic ONH photography (Hoyt et al. 1972, Cher & Robinson 1973, Hitchings & Spaeth 1976, Sommer et al. 1977,

Quigley et al. 1980, Airaksinen et al. 1981a, Jonas et al. 1981, Tuulonen &

Airaksinen 1991, Airaksinen et al. 1992, Caprioli 1994, Gordon & Kass 1999,

Leske et al. 1999) and with modern automated eye fundus imaging devices, such

as the HRT, the GDx and the OCT, based on different techniques (Mikelberg et al. 1995, Dichtl et al. 1996, Gundersen et al. 2000, Konno et al. 2001, Zangwill et al. 2001, Asrani et al. 2003, Miglior et al. 2005a). Some of the glaucomatous changes,

like optic disc hemorrhages, positional changes of blood vessel, optic disc pallor

and parapapillary atrophy are only found in ONH photographs (Drance et al. 1977,

Airaksinen et al. 1981a, Caprioli et al. 1989, Jonas et al. 1989d, Teng et al. 2010,

Hollands et al. 2013). Three-dimensional ONH parameters with glaucomatous

changes, such as rim volume (RV), CV, cup shape and RNFL thickness, are only

achieved with modern imaging techniques (Weinreb et al. 1989, Burk et al. 1990,

Burk et al. 1991, Burk et al. 1993a, Burk et al. 1993b). The qualitative nature of

ONH photography, with inter- and intra-observer variability in assessing the ONH

and the RNFL despite a variety of standardizing methods, weakens its diagnostic

and follow-up capability (Tielsch et al. 1988, Varma et al. 1992, Azuara-Blanco et al. 2003, Zeyen et al. 2003, Reus et al. 2010). The accuracy and reproducibility of

the automated imaging methods improves the observer independency in the studies

(Kruse et al. 1989, Dreher et al. 1991, Dreher & Weinreb 1991, Lusky et al. 1993,

Mikelberg et al. 1993, Chauhan et al. 1994, Owen et al. 2006).

29

The glaucomatous changes in the ONH and the RNFL can be verified

morphologically (Radius & Anderson 1979a, Jonas 1988a, Jonas et al. 1991,

Tuulonen et al. 2015). The loss of ganglion cell axons causes a decrease in the

amount of the neural rim structure, and this results in an increase in the optic CV

and changes in the optic cup and neural rim shape. The glaucomatous optic disc

changes reported in a wide variety of studies include: cupping of the optic disc

(Fuchs 1892, Elschnig 1907, Elliot 1922, Pickard 1923, Fingeret et al. 2005),

asymmetry between the patient’s two optic cups (Armaly 1967, Richardson 1968,

Fishman 1970), vertical ovality of the optic cup (Begg et al. 1972, Kirsch &

Andersson 1973, Read & Spaeth 1974), narrowing of the neural rim (Hoskins &

Gelber 1975, Chan et al. 1976), notching of the rim (Kirsch & Anderson 1973,

Spaeth et al. 1976, Hitchings & Spaeth 1976, Quigley 1982), diffuse loss of optic

nerve fibers (Spaeth et al. 1976, Hitchings & Spaeth 1976), pallor of the neural

tissue (Elliot 1922, Schwartz 1973), changes in the cup shape and blood vessel

position (Takamoto & Schwartz 1979, Varma et al. 1987) and optic disc

hemorrhages (Drance & Begg 1970, Airaksinen et al. 1981b, Airaksinen & Heijl

1983). Cup-to-disc ratio measurements from disc photographs were a weak

structural parameter to discriminate between non-glaucomatous and glaucomatous

eyes (Airaksinen et al. 1985b, Caprioli 1992, Bartz-Schmidt et al. 1996b, Feuer et al. 2002).

Airaksinen (Airaksinen et al. 1985b), Caprioli & Miller (Caprioli & Miller

1988) and Jonas et al. (Jonas et al. 1988b) showed in their studies that the neural

RA is diminished in glaucoma progression. Two different kinds of neural rim loss

patterns were found: localized and diffuse, and one or both may be identified in one

glaucomatous eye (Airaksinen et al. 1984, Jonas et al. 1993, Tuulonen 1993). In

early glaucoma, diffuse loss of axons was found to predominate, but a mixed

pattern of diffuse and localized loss was more frequent in the late stages of

glaucoma (Tuulonen & Airaksinen 1991). Airaksinen et al. (Airaksinen et al. 1985b)

found that in glaucoma, the rate of RA loss, over time was significantly more than

the age-related decline in non-glaucomatous eyes.

2.3.3 Glaucomatous RNFL changes

In 1972 Hoyt and Newman were the first to report of RNFL defects in glaucoma

(Hoyt & Newman 1972). Later, Hoyt et al. presented in a detailed report that the

first visible changes were thin slit-like defects in the arcuate area of the RNFL,

progressing further to wedge-shaped localized defects (Hoyt et al. 1973). The

30

RNFL defects seemed to precede the visual field defects by several years (Sommer et al. 1977, Sommer et al. 1979a, Quigley et al. 1982, Sommer et al. 1991). In

glaucoma, optic disc changes often follow the corresponding RNFL changes (Iwata et al. 1981).

The first visible glaucomatous RNFL changes are thin, slit-like defects in the

arcuate area of the RNFL (Hoyt & Newman 1972). Later, these changes progress

to wedge-shaped localized defects. (Hoyt et al. 1973). Optic disc changes have

frequently been found following RNFL defects (Iwata et al. 1982). In several

studies, the RNFL findings preceded the first visual field defects even by several

years (Sommer et al. 1977, Sommer et al. 1979a, Quigley et al. 1982). The RNFL

is best visualized with red-free light, using ophthalmoscope with green light, or

RNFL photography using a wide-angle fundus camera with high-resolution black-

and-white film with a blue narrow-band interference filter (Airaksinen et al. 1982,

Sommer et al. 1983, Peli et al. 1987). In their study on nerve fiber layer and color

fundus photography and on 1,344 eyes with elevated intraocular pressures (IOP),

Sommer et al. found that clinically detectable nerve fiber atrophy precedes the

onset of glaucomatous field loss (Sommer et al. 1991). Examiner experience and

the severity of optic nerve damage influenced the results. This was verified by

Quigley in his study (Quigley et al. 1992). Also relative RNFL height and visual

field mean defect discriminated well between normal and glaucomatous eyes

(Caprioli 1992). Cup-to-disc ratio measurements from disc photographs have been

found to be weak structural parameters to discriminate between non-glaucomatous

and glaucomatous eyes (Airaksinen & Alanko 1983, Caprioli 1992).

Several grading systems have been proposed to quantify the RNFL

abnormalities: a semiquantitative scoring system (Airaksinen et al. 1984), a four-

level grading system with three features: brightness of the reflexes, the RNFL

texture and the obscuring degree of retinal blood vessels by the RNFL (Quigley et al. 1993), and a visually supported grading system (Niessen et al. 1995).

2.3.4 Glaucomatous visual field defects

Landesberg was the first to describe typical glaucomatous arcuate visual field

defects (Landesberg 1869), later verified as “the Bjerrum scotoma”, by Bjerrum

(Bjerrum 1890). Rønne was the first to describe an extension of arcuate nerve fiber

bundle defect, the glaucomatous “nasal step” in the visual field (Rønne 1909). It

was rediscovered later by several investigators (Aulhorn & Harms 1967, Armaly

1971, LeBlanc & Becker 1971). The most common visual field defect in early

31

glaucoma besides the ‘nasal step’ is the isolated paracentral scotoma, reported in

several studies (Aulhorn & Harms 1960, Aulhorn & Harms 1967, Drance et al. 1967, Armaly 1971, Heijl 1976, Motolko et al. 1982 & Heijl 1989). Glaucoma

suspect arcuate scotoma starts from a blind spot and extends as an arch above or

below fixation temporally (Dyster-Aas et al. 1980, Schmied 1980). It finally results

in a central absolute defect (Heijl et al. 1987).

Fig. 2. The temporal relation of progression of the glaucomatous changes in the optic

disc and visual field (Sommer et al. 1991, Quigley et al. 1992).

Manual kinetic perimetry is insufficient in detecting early glaucomatous VF defects

(Aulhorn et al. 1966). The standardized automatic perimetry (SAP) is the standard

visual field test for measuring central visual function in glaucoma (Fankhauser et al. 1972, Heijl & Krakau 1975, Flammer et al. 1983).

Computerized automatic perimeters (Heijl 1977, Bebie & Fankhauser 1980,

Gloor 1980, Heijl & Drance 1980, Greve 1982) and relevant computer programs

(Anton et al. 1997, Flammer et al. 1983) improved the sensitivity and specificity

of visual field defect detection. The Swedish Interactive Testing Algorithm (SITA)

was developed as a test time reducing but accuracy maintaining and clinically

useful perimetric testing strategy by Bengtsson and Heijl (Bengtsson et al. 1997).

Short-wavelength automated, or blue-on-yellow, perimetry (SWAP) is believed to

detect early visual field defects even 3 years prior to white-on-white visual field

loss, because it specifically tests only one cell type, the small bistratified, RGC,

32

responsible for the blue-yellow opponency (Johnson et al. 1989, Hart et al. 1990,

Johnson et al. 1993a, Johnson et al. 1993b, Sample et al. 1993a, Sample et al. 1996,

Teesalu et al. 1998).

2.4 The confocal scanning laser tomographer

2.4.1 General aspects of confocal scanning laser technology

While laser scanning ophthalmoscopes, which were first developed in Boston and

later in Heidelberg, served predominantly the purpose of imaging, laser scanning

tomography provides additionally the feature of 3-D measurements of ocular

structures with good reproducibility. The laser tomographic scanner (LTS) in 1988

was the predecessor of the HRT, providing the three-dimensional structure of the

optic disc, precisely quantified with the technique of laser scanning tomography

(Kruse et al. 1989, Mikelberg et al. 1993, Weinreb et al. 1993, Janknecht & Funk

1994, Rohrschneider et al. 1994, Dannheim et al. 1995).

The main characteristics of the confocal scanning laser tomography technique

are: 1) sequential point-by-point imaging by a scanning laser beam, generally

requiring a computer system to acquire and display the image and 2) high spatial

resolution in three dimensions due to the confocal optical arrangement, introducing

the third dimension as a quantitative parameter, thus enabling formation of two-

dimensional optical section images and complete three-dimensional images and

subsequent three-dimensional measurements.

2.4.2 Principles of use and technical characteristics of the HRT

The HRT is a confocal scanning laser microscope designed for three-dimensional

imaging and three-dimensional measurements of the posterior segment of the

human eye in vivo. The system consists of a laser scanning camera, mounted on a

standard ophthalmic stand with chin rest, an operation panel, and a personal

computer system (The Heidelberg Retina Tomograph Operation Manual 1993).

The principle of function of scanning laser tomography consists of two-

dimensional optical section images of the retina and its structures, image planes

parallel to the retinal surface, and series of such section images showed in Figure

3. These images which make up a three-dimensional image of the structure are

recorded, digitized and displayed on the computer monitor.

33

Fig. 3. The principle of the technique used in confocal scanning laser tomography.

Dilatation of the patient’s pupil is not required for recording of the image data. A

pupil diameter of 1 mm was found to be sufficient to receive useful data. However,

the amount of detected light and, therefore, the signal-to-noise ratio increase with

increasing pupil diameter (Zangwill et al. 1997). The maximum irradiance at the

retina (0.5 mW/cm2) is approximately one hundredth of the irradiance deposit with

the viewing lamp of a fundus camera. The HRT system is a Class I laser product

and does not emit radiation hazardous to the human eye (The Heidelberg Retina

Tomograph Operation Manual 1993).

The three-dimensional image recorded is stored and analyzed using a standard

micro-computer. The topography image of the region examined is calculated and

presented on the computer monitor. The topography is analyzed quantitatively with

regard to the three-dimensional shape of the examined structure. Additionally,

changes in the topography over time can be detected and quantified for follow-up

analysis.

In a confocal imaging system, the illuminating light source, a diode laser beam

at 670 nm wavelength, is focused to a single point of the structure under

examination. The laser beam is deflected periodically in two dimensions by means

of scanning mirrors, so that a two-dimensional field of the object is scanned in a

time sequence. The light which is reflected from each point of the object is

deflected to a detector and registered. The detector is point-like, similarly to the

light source, and positioned optically conjugate to the focal plane (point-like: the

size of the diffraction limited focus spot of the laser beam). Such light which is

34

reflected from object structures at the focal plane is focused to the detector

diaphragm, passes the diaphragm and is registered by the detector. However, light

reflected or scattered from those parts of the object located outside the focal plane

is not focused to the detector diaphragm and can be registered only partially.

Suppression of reflected light increases rapidly with increasing focal distance.

Therefore, confocal imaging produces high optical resolution not only

perpendicular, but also parallel to the optical axis.

In consequence, the confocal image of a three-dimensional object contains only

information from a small domain at the focal plane. It may be considered as an

optical section through the object at the actual location of the focal plane. If a series

of confocal optical section images is recorded sequentially and the location of the

focal plane is changed along the optical axis between the individual image planes,

a volume of the object is scanned and this confocal image series forms a three-

dimensional image of the object that is spatially resolved in three dimensions. This

section-wise recording of three-dimensional images is called confocal scanning

laser tomography.

The z-profile width describes the highest achievable optical resolution parallel

to the optical axis (into the depth) when imaging the fundus with a confocal system.

For topography measurements, however, it is assumed that all reflected light

originates at one spatially curved surface, and the Z location of this surface at each

point in the (x,y) plane has to be determined. This is performed by searching the

maximum locations of the z-profiles measured at each point (x,y). The accuracy of

that measurement of the Z location depends on the width of the z-profile, on the

number of confocal section images recorded, and on the signal-to-noise ratio in the

image data. The Z location measurement accuracy achievable at the fundus is

typically some tens of microns. When the maximum positions of the z-profiles are

determined at each location in the (x,y) plane, the result is a matrix of numbers

Z(x,y). Each number Z represents the measured position along the optical axis or

height of the surface of the structure examined at the corresponding location (x,y),

as shown in Figure 4.

35

Fig. 4. The maximum positions of z-profiles at each location in the (x,y) plane are

determined.

This matrix of height measurement results in a topographic map of the examined

structure. It can be visualized on a computer monitor as an image Z (x,y) simply by

using each number of the matrix as a picture element (pixel), and by translating the

numerical value into a specific color. This visualized matrix of height measurement

results is called a topography image. The topography image is the basis for the

analysis and quantification of the spatial shape properties of the structure examined.

This includes planimetric and stereometric measurements and analysis of

topographic changes.

From the tomographical image series two images are calculated: the extended

focus image (summation image, the reflectivity image) and the topographic image.

The extended focus image is calculated as a sum of all individual images of the 32

focal planes, resulting in an image with an increased depth of focus which

approximately equals the scan depth along the z-axis. The grey values obtained are

color-coded for better structure differentiation. The topographical image displays

the depth and height information in color coding. Prominent structures are

represented in dark red colors, deeper structures in light yellow to white (The

Heidelberg Retina Tomograph Operation Manual 1993).

Given the optical characteristics of the human eye, the depth resolution is about

300 µm (Campbell et al. 1966). By calculating the center of gravity of the intensity

36

profile of the reflected light in 32 focal planes along the optical axis, a

reproducibility of the depth localization of less than 50 µm is achieved for each

individual pixel of the (x,y) plane (Weinreb & Dreher 1990).

In HRT software release 1.11 the contour line is automatically corrected for

rotation and different magnification between the topography images. The

correction of the rotation leads to a higher reproducibility of the stereometric

measurements, while the magnification correction ensures that the proper scaling

is always used. The quality control for acquired image series in release 1.11 also

requests a change of the focal plane setting if the required change is more than ±

0.5 diopters (The Heidelberg Retina Tomograph Operation Manual 1993).

The basic physical principles of confocal laser tomographic scanning are as

follows: The light source used in the HRT is a diode laser operating at 670 nm

wavelength. Single two-dimensional section images are recorded within 0.032

seconds at a repetition rate of 20 Hz and digitized in a frame of 256 x 256 pixels.

The value of each picture element is the measured height of the examined structure

at the corresponding location. Therefore, the topography image contains a total of

65,536 results of individual height measurements. A three-dimensional image of an

object volume is scanned and recorded as a series of 32 section images at 32 equally

spaced focal planes as shown in Figure 5. Total acquisition time for the complete

series is 1.6 seconds. The location of the three-dimensional scanning field along

the optical axis is adjusted by moving the focal plane of the living image. The total

depth extension of the image series into depth (the distance between the first and

the 32nd section image) can be adjusted between 0.5 mm and 4.0 mm in increments

of 0.5 mm; the spacing between each two subsequent images of a series can

therefore be changed between approximately 16 microns and 130 microns. For

recording an image series used for three-dimensional image formation, the operator

defines the focal planes between which the tomography is to be carried out. In the

case of an optic disk recording, the first focal plane is defined directly above the

first reflections of the retinal vessels. The last focal plane is selected in the region

at the bottom of the excavation, below the position of the maximum reflectivity of

the excavation (The Heidelberg Retina Tomograph Operation Manual 1993, The

Heidelberg Retina Tomograph Operation Manual 1994). The printouts of the non-

glaucomatous and glaucomatous ONH acquired with the HRT are shown in Figure

6. and Figure 7.

37

Fig. 5. The summary image of the HRT confocal scanning laser tomograph.

The acquired three-dimensional image data (256 x 256 x 32 = 2,097,152 three-

dimensionally defined pixels, voxels, per image) are stored automatically in an

image database, together with all relevant patient data and image acquisition

parameters. The value of each picture element represents an independent

measurement of height at the corresponding location. The height measurements are

calibrated by using the properties of the individual eye examined. The mean

standard deviation of the height measurement at each pixel is approximately 30

microns. This accuracy may be further increased by averaging of either arrays of

pixels within the same topography image or by averaging multiple topography

images received from multiple examinations.

38

Fig. 6. The printout of the ONH acquired with the HRT confocal scanning laser

tomograph (non-glaucomatous eye).

Fig. 7. The printout of the ONH acquired with the HRT confocal scanning laser

tomograph (glaucomatous eye).

39

2.4.3 Definitions related to measurements with the HRT

The contour line is defined as the outer limit of the optic disc. It is interactively

determined by the operator. For this, a contour line is created on the monitor

display using a trackball. The contour line is displayed simultaneously on the

extended focus and topographic image. The outer limit of the optic disk is the inner

edge of the Elschnig’s scleral ring (Jonas et al. 1988a). In case of doubt, the

topographical image on which the contour line is created simultaneously may be

taken for orientation purposes. In particular, the scleral ring of Elschnig is often

more distinctly recognizable in topographical images. Depending on the size of the

optic disk, the contour line consists of approximately 300 to 400 pixels. The

topography of the contour line is influenced by crossing blood vessels. Automatic

interpolation at the vascular crossing locations results in the topography of the

corrected contour line.

Topography and reflectivity image are shown in Figure 10. The determination

of a topography image is a two-step process. The originally acquired three-

dimensional image series, the 32 images of the series, are aligned to each other to

correct the shifts between the individual section images due to eye movements

during the acquisition time. Additionally, geometrical corrections are applied to the

images. The result of this first step is the processed image series, also referred to as

the aligned image series. In the second processing step, the topography of the

surface of the examined structure is determined from the processed image series.

The result of the second step is the topography image, which contains the

information on the three-dimensional shape of the structure examined and is used

for subsequent three-dimensional analysis. Along with the topography image, the

reflectivity image is always determined, which is the sum of the 32 two-

dimensional section images acquired. Its value at each picture element is the

maximal reflectivity of the examined structure at the corresponding location. The

topography image is a color-coded image in which the color codes height. The

reflectivity image is also a color-coded image, but in this case the color codes the

reflectivity at each point.

Mean topography images are typically determined from two or more individual

topography images received from multiple examinations of an eye during the same

day. They are useful for two reasons: to increase the accuracy of the height

measurements and to obtain an individual reproducibility measure. If n source

images are used to determine the mean topography, theoretically the accuracy of

the mean height at each location is a factor of √n better than the accuracy of the

40

individual height measurements. In practice, this is true for n≤3 and the increase in

accuracy is generally small for n>3 (Weinreb et al. 1993). Therefore, it is most

efficient to perform three examinations and determine the mean topography image

(The Heidelberg Retina Tomograph Operating Manual 1993, Chauhan et al. 1994).

During determination of the mean topography image out of a set of source

topography images, first the source images are aligned to each other with regard to

horizontal shift, vertical shift, rotation, tilt, and height offset, in order to normalize

the images and to compensate for different recording conditions. After the

alignment, at each location within the topography images (each picture element)

the mean and the standard deviation of the individual height measurements are

determined, resulting in two new images, the mean topography image and the

standard deviation image. The standard deviation image contains the

reproducibility of the height measurements at each location within the mean

topography image. It is an individual and spatially resolved reproducibility measure

for this set of examinations.

All stereometric measurements are done within a region of interest which is

enclosed by the contour line, i.e., the contour line provides the two-dimensional

boundary of the region of interest. To evaluate some stereometric parameters, e.g.

CV of the optic disc, also an upper limitation of the region of interest towards the

vitreous is required. It is derived from the height variation along the contour line.

For that purpose, the measured height along the contour line is first corrected for

artifacts due to crossing blood vessels which cause local elevations. The result is

the corrected contour line.

The curved surface is defined by the following conditions as shown in Figure

8:

1. the curved surface is bounded by the corrected contour line

2. the curved surface has the height of the corrected contour line in each point of

its boundary

3. the height in the center of the curved surface equals the mean height of the

corrected contour line

each section of the curved surface from its center to a boundary point is a

straight line

41

Fig. 8. The scheme of the curved surface used in the HRT images.

The measurement of the topography of an object is basically the measurement of

three spatial coordinates of individual points of this object. Since the spatial

coordinates have dimensions, the proper calibration of the measurement results

plays an important role. The measurement results which make up the topography

images of the HRT are absolutely scaled values. The laser scanning camera of the

HRT normally delivers a parallel laser beam. For absolute scaling, in ametropic

eyes the change of the laser beam divergence is translated into a shift of the focal

plane inside the individual eye examined. This translation depends on the refractive

system of the eye. The refractive system of an individual eye is determined mainly

by the curvature of the anterior surface of its cornea. In addition, a model for the

optical properties of the crystal lens is used. From these data, the focal length of

the eye is determined, which defines the exact translation from the change of the

laser beam divergence to the corresponding absolute shift of the focal plane inside

the eye. This is the absolute scaling of the height measurements. The absolute

scaling in all dimensions ensures that all measured spatial coordinate values are

independent from the refraction of the eye examined.

From the description of absolute scaling it is obvious that a measured change

of the absolute height at a certain location could be caused either by a real height

change of the examined structure at that location, or by a change in the refraction

of the eye (change of the focal length). In order not to depend on an independent,

precise refraction measurement, another approach can be used: If there is any

42

retinal structure with an absolute height location which can be assumed to be

constant over time, this structure can be used to cancel the influence of refraction

variations of the eye. This approach is used when two topography images recorded

at different times are compared to each other with the operation software of the

HRT. The mean height within a reference ring in both topography images is

determined. The outer diameter of the reference ring is 2.7 mm and its width is 0.15

mm. It is then assumed that the mean absolute height within the reference ring has

not changed between the two examinations. In consequence, any measured

difference of the mean height of the reference ring between the two examinations

is assumed to be caused by a change in the focal length (the refraction) of the

examined eye. The corresponding change of the refraction is determined and used

for the absolute scaling of the topography images. This procedure compensates for

any changes in the refraction of the examined eye over time, including those due to

accommodation of the eye during one or both of the examinations.

In the HRT Operation Software releases 1.08, 1.09 and 1.10 the standard

reference is located at the mean height of the peripapillary retinal surface (z= 0 in

the relative coordinate system). By default, a reference plane at a location 320 µm

posteriorly of the mean peripapillary retinal surface height is used. The HRT

operation software offers to use a reference plane either at a standard location

(standard reference) or at a user-defined location.

2.4.4 Accuracy and reproducibility of the HRT

The accuracy and reproducibility of confocal scanning laser tomography have been

determined in several studies by numerous investigators (Burk et al. 1990, Burk et al. 1991, Burk et al. 1992, Weinreb & Dreher 1990). Confocal scanning laser

tomography has been used to produce three-dimensional topographic

measurements of the optic disc structure with high reproducibility (Kruse et al. 1989, Burk 1993a, Burk 1993b, Weinreb et al. 1993b, Janknecht & Funk 1994,

Dannheim et al. 1995). The HRT (Heidelberg Engineering GmbH, Heidelberg,

Germany) has previously been reported as an instrument to obtain accurate and

reproducible measurements of the ONH (Mikelberg et al. 1993, Rohrschneider et al. 1994).

The range of standard deviations was found to be between 28.4 µm and 58.0

µm; the standard deviation over all measurements was 42.6 µm. The relative error

did not exceed 3.1% for the phakic model and was between 0.6 and 8.2% for the

aphakic model (model eye) (Weinreb & Dreher, 1990).

43

There are several factors decreasing the accuracy of laser scanning

tomographic results. Includid are improper eye adjustment, definition of scan

region and scan intensity, focusing, ametropy, height of the head, pupillary distance,

accommodation, transparency of the optical media, pupil width, fixation, palpebral

aperture and restlessness of head position (Owen et al. 2006). In young patients

there was no significant difference (p<0.01) in the measured ONH volume

parameters related to pupil dilation (Zangwill et al. 1997). However, slightly

inaccurate adjustment resulted in a coefficient of variation in volume data of 3.8%,

whereas an increased scanning range by a factor of three compared to the proper

scanning depth led to an increase of volume readings of 16% (Rohrschneider et al. 1990).

2.2 Earlier studies with the Heidelberg Retina Tomograph

2.2.1 Accuracy and reproducibility studies

The confocal scanning laser tomograph has been developed for obtaining accurate

and reproducible three-dimensional topographic measurements of the optic disc

and central eye fundus. The reproducibility of local height measurements in a mean

topography image of three topography images is about 20-30 µm for healthy and

glaucomatous eyes (Kruse et al. 1989, Weinreb et al. 1993, Janknecht et al. 1994,

Dannheim et al. 1995). The coefficients of variation of the stereometric parameters

are approximately 5% (Mikelberg et al. 1993, Rohrschneider et al. 1993,

Rohrschneider et al. 1994, Tomita et al. 1994). The data of reproducibility of local

height measurements and stereometric parameter values as well as of correlation of

the HRT stereometric parameters with visual field indices is presented in Table 1,

Table 2 and Table 3.

44

Table 1. Reproducibility of the HRT local height measurements (individual pixels).

Reference Software

version

Number of eyes SD (µm) of eyes

normal glaucoma normal glaucoma

Bathija et al. (1998) 2.01 49 50 16.8±6.4 20.8±7.5

Caprioli et al. (1998) 2.01 43 53 19 21

Zangwill et al. (1997) 1.1x 8 26

Chauhan et al. (1994) 1.1x 30 30 25.9 31.2

Rohrschneider et al. (1994) 1.0x 13 13 22±6 30±6

Lusky et al. (1993) 1.02 10 10 30.1±7.0 31.8±10.6

SD = standard deviation

Table 2. Reproducibility of the HRT stereometric parameter values.

Parameter SD of normal eyes SD of glaucoma eyes

Rim area (mm²) 0.04 0.06

Cup area (mm²) 0.04 0.06

Cup volume (mm³) 0.01 0.03

Max cup depth (mm) 0.03 0.04

Mean cup depth (mm) 0.01 0.02

Rohrschneider et al. (1994), Tomita et al. (1994)

45

Table 3. Correlation of stereometric parameters with visual field indices.

Reference Number of

subjects

Average MD Parameters Correlation

coefficient HRT Visual field

Brigatti & Caprioli (1995) 46 - 4.8 dB CSM MD -0.65

CSM CPSD 0.55

Eid et al. (1997) 125 -7.5 dB RNFLT MD 0.49

CDAR MD 0.41

Iester et al. (1997a) 294 -4.0 dB RA MD 0.44

RA CPSD -0.48

CSM MD -0.43

CSM CPSD 0.38

Iester et al. (1997b) 105 -3.9 dB infMHC supMD -0.53

infRV supMD 0.47

supCA infMD -0.44

supCSM infMD -0.46

Kono et al. (1997) 21 early supRA infMD 0.65

16 advanced tempRA MD 0.58

Lee et al. (1996) 81 RA MD 0.62

Teesalu et al. (1997c) 77 -3.5 dB CSM MD-B/Y, -W/W -0.65

RNFLT MD-W/W 0.62

Tsai et al. (1995) 34 -3.0 dB RA MD-B/Y 0.56

RA MD-W/W 0.47

supRNFLT MD-W/W 0.67

Caprioli et al. (1998) 53 -4.8 dB mean peri- MD -0.43

papillary

surface slope CPSD 0.43

MD = mean defect, CPSD = pattern standard deviation, W/W = white-on-white, B/Y = blue-on-yellow,

CSM = cup shape measure, RA = rim area, CA = cup area, CDAR = cup/disc area ratio, RV = rim volume,

RNFLT = retinal nerve fiber layer thickness, MHC = mean height contour, inf/sup = inferior/superior, temp

= temporal.

2.2.2 The HRT in separating healthy and glaucomatous eyes

A number of structural characteristics of the ONH and retina as well as

psychophysical functions were established by multiple regression analysis in a

study by Drance et al. (Drance et al. 1987). In another study by Drance et al. (Drance et al. 1991), a stepwise discriminant analysis found that the combination

of the vertical cup-disc-ratio, the diffuse nerve fiber layer score and the localized

nerve fiber layer score could correctly identify 98% of the normal and 84% of the

glaucoma patients.

46

Initial RA, change of RA per year in follow-up and standard deviation of the

mean defect of the initial visual field in the multivariate analysis model could

correctly classify 81% of the patients with 72% sensitivity and 87% specificity in

a study by Airaksinen et al. (Airaksinen et al. 1985a).

The sensitivity and specificity of direct ophthalmoscopic assessment of the

optic disc in screening of glaucoma was studied by Harper and Reeves in their

multivariate analysis (Harper & Reeves 2000). Several different linear regression

formulas were able to discriminate non-glaucomatous and glaucomatous eyes

(Larrosa et al. 2006). The precision of the diagnostic test is improved by using

linear discriminant functions taking into account several HRT stereometric ONH

parameters (Mikelberg et al. 1995, Iester et al. 1997a, Bathija et al. 1998, Mardin et al. 1999, Ferreras et al. 2008). Parameters that were useful in these tests included

CSM, volume above the reference level (RV), height variation along contour line,

mean RNFL thickness, RA and cup area.

47

3 Purpose of the study

The aim of the present study was to develop the HRT as a clinically practical tool

in glaucoma diagnostics: to compare the HRT results with a conventional ONH

assessment, the manual planimetric technique. The purpose was also to find the

most adequate reference level for the HRT parameter measurements, the most

useful HRT parameters and the best combination of all structural and functional

parameters to discriminate between different levels of glaucomatous damage.

In detail, the aim of the study was:

1. to compare the manual optic disc measurements to those taken with the

Heidelberg Retina Tomograph (I)

2. to assess the effect of different reference levels on the HRT topographic optic

disc measurement values (II and III)

3. to search for the best HRT parameters to separate healthy individuals from

patients with glaucoma (IV)

4. to evaluate, which of the selected HRT and other structural and functional

parameters, alone or in combination, separate best between non-glaucomatous

and glaucomatous eyes (V)

48

49

4 Material and methods

4.1 Subjects

For study I, 12 eyes of 12 patients (7 females and 5 males), with early glaucomatous

abnormalities, were selected, their ages ranging from 46 to 73 (mean 62) years. In

five eyes, typical early glaucomatous abnormalities in the optic disc, the RNFL and

the Humphrey 30-2 visual fields (MD < 5dB) were found. In four eyes the untreated

IOP was above 22 mmHg and in eight eyes below it.

Study II included one randomly chosen eye of each of the 67 healthy or

glaucomatous subjects (42 females and 25 males). The controls were 40 non-

glaucomatous subjects with a mean age of 57 (range 29 to 84) years. These subjects

had normal findings in the ocular examination with IOP ≤ 21 mmHg, normal optic

disc, normal RNFL, normal W/W visual field examined with the Humphrey 30-2

program. They had no family history of glaucoma, no ocular or neurologic disease,

no diabetes or other systemic diseases or medications that are known to have an

effect on visual field sensitivity or color vision. The eyes examined were not

excluded on the basis of visual acuity. Twenty-seven eyes of 27 patients with a

mean age of 62 (range 38 to 82) years were examined, in addition to the non-

glaucomatous group. There were 23 open angle glaucoma patients with elevated

IOP, glaucomatous optic disc damage and W/W visual field loss, not influenced by

other ocular or systemic disorders. Four patients had ocular hypertension (OHT)

with elevated IOP (IOP > 22 mmHg on three or more occasions), with normal W/W

visual fields, but abnormal RNFL and/or optic disc. They were added to the early

glaucoma group as “preperimetric” glaucoma patients (Horn et al 1997). The 27

glaucoma cases were divided into two groups for statistical analysis: early to

moderate glaucomas (n=19) and advanced glaucomas (n=8), by the Humphrey (30-

2) W/W visual fields, with MD better/same or worse than 10 dB, respectively.

In study III there were 180 normal eyes and 99 eyes with glaucoma examined

with ten-degree triple images in order to find the reference level for the HRT

measurements, taking into account interindividual variability and ensuring that the

automatic reference level determination for intrapapillary parameters remained

below the disc border height.

In study IV the material consisted of 77 eyes of 77 subjects (51 females and 26

males). There were 40 non-glaucomatous subjects with a mean age of 57 (range 29

to 84) years. The criteria for the normality of the eyes were the same as in study II.

50

In addition to the non-glaucomatous group, 37 ocular hypertensive or

glaucomatous patients with a mean age of 60 (range 30 to 82) years were examined.

There were 10 ocular hypertensives, with elevated IOP (IOP > 22 mmHg on three

or more occasions) with normal optic discs, normal RNFL and normal W/W visual

fields. There were 23 open angle glaucoma patients; the criteria for glaucoma were

the same as in study II. For statistical analysis, the glaucomatous eyes were divided

into three groups: early, moderate and advanced glaucomas, based on the

Humphrey W/W visual fields (program 30-2). In these groups, MD was better than

5 dB (n=9), between 5 and 10 dB (n=6) and worse than 10 dB (n=8), respectively.

Four ocular hypertensive patients with normal W/W visual fields but abnormal

RNFL and/or optic discs were added to the early glaucoma group as ‘preperimetric’

glaucoma patients, as in study II.

Fifty-five subjects (36 females and 19 males) were selected for study V. There

were 32 non-glaucomatous subjects with a mean age of 54 (range 29 to 83) years,

and 23 patients with different stages of glaucoma with a mean age of 59 (range 39

to 82) years. As in studies II and IV, four ocular hypertensive patients were included

in the glaucoma group. The criteria for normal, hypertensive, ‘preperimetric’ and

glaucomatous eyes were the same as in studies II and IV. The number of subjects

in studies I – V, non-glaucomatous and glaucomatous with different stages of

glaucoma, are shown in table 4.

Table 4. The number of non-glaucomatous and glaucomatous subjects, with different

stages of glaucoma, in studies I – V.

Study Number of subjects Non-glaucomatous Glaucomatous

Ocular hypertensive Early Moderate Advanced

I 12 12*

II 67 40 19** 8

III 279 180 99***

IV 77 40 14 9 6 8

V 55 32 23***

*Early, **Ocular hypertensive, Early and Moderate, *** Early, Moderate and Advanced

4.2 The planimetric techniques

Planimetry expresses the areas (x- and y-axes) of a structure, leaving the third

dimension, depth (z-axis), to another technique, photogrammetry. In planimetry,

only one-dimensional and area measurements are provided. However, these may

be related to topography or pallor. Planimetric linear measurements include disc

51

diameter, cup diameter, cup-to-disc ratio, and the thinnest portion of the

neuroretinal rim. Area measurements include area of the cup, disc, and neuroretinal

rim (Airaksinen et al. 1985b, Jonas et al. 1988d).

Planimetry expresses results in ‘machine units’ and ignores any magnification

factors in either the fundus camera or the eye. Algorithms that correct for

magnification in the eye have been developed by Littmann (Littmann 1988). These

convert the machine units of planimetric analysis into absolute units (millimeters

or square millimeters), thus allowing both comparison among individuals and

extrapolation of results from normal distributions. However, there is inter-

individual and inter-examination variation in planimetric studies (Jonas et al. 1988a,

Sommer et al. 1979a, Balaszi et al. 1984). A pair of stereophotographs and a

photograph of the ONH used in the planimetric techniques are shown in Figure 9.

and Figure 10.

The correlation between actual measured dimensions of the scleral canal

relative to the photographic results is an important consideration. Quigley et al. have reported that histologic measurements of the disc, which may be altered by

fixation, range between 1.75 and 1.89 mm in diameter (Quigley et al. 1999). Most

photographic results tend to be lower than the histologic ones; Jonas et al., however,

reported a photographic measurement of 1.92 mm (Jonas et al. 1988d).

52

Fig. 9. A pair of stereophotographs of the ONH used in the planimetric techniques.

53

Fig. 10. A photograph of the ONH used in the planimetric techniques.

Comparison of planimetry with automated devices has demonstrated a high degree

of correlation. Comparing planimetry with photography, Airaksinen et al. showed

the correlation coefficients 0.61 for the neuroretinal RA, 0.77 for the cup area, and

0.76 for the disc area (DA) (Airaksinen et al. 1985b). Mikelberg et al. compared

the results obtained using the Rodenstock ONH analyzer with ones obtained by

planimetry; the correlation coefficients for each of the parameters were as follows:

vertical cup-disc ratio 0.67, horizontal cup-disc ratio 0.63, neuroretinal RA 0.72,

and DA 0.89 (Mikelberg et al. 1986). In a similar study, Varma et al. compared

planimetry with the Rodenstock video-ophthalmograph and the Topcon image

analyzer. The correlation coefficients between planimetry and the others were 0.56

and 0.46 for the DA, 0.78 and 0.79 for the RA, and 0.66 and 0.86 for the ratio of

RA to DA, respectively (Varma et al. 1992).

4.3 HRT study protocol

In this study (studies I and III-V) the HRT, (Heidelberg Engineering, Gmbh,

Heidelberg, Germany) with software versions 1.10 and 1.11 was used to acquire

and evaluate topographic measurements of the optic disc. Dilatation of the pupil

54

was not used for image acquisition (Zangwill et al. 1997). For the optic disc

structure measurements we used the mean image of three scans (Weinreb et al. 1993). In this study we used a 10-degree picture angle and the tilted, relative

coordinate system in all measurements (HRT Operation Software Release 1.08

1993, HRT Operation Software Release 1.11 1994). Optic disc contour line was

manually marked around the disc at the inner edge of the scleral ring (Elschnig’s

ring) by an experienced glaucoma specialist. The HRT global parameters used in

this study are defined in Table 5.

Table 5. The definitions of the HRT global parameters used in this study.

Parameter Abbreviation Definition

The disc area DA The total area of the parts within the contour line.

The cup area CA The total area of those parts within the contour line that are

located below the reference plane.

The rim area RA The difference between disc area and cup area.

The cup/disc area ratio CDR CA/DA, basis reference plane.

The vertical cup diameter CDVer The vertical cup diameter

The vertical linear cup/disc

area ratio

CDRVer The vertical CA/DA

The cup volume CV The total volume of those parts within the contour line that

are located below the reference plane.

The rim volume RV The total volume of those parts within the contour line that

are located above the reference plane.

The mean retinal nerve fiber

thickness

RNFLt Reference height minus the mean height of contour.

The retinal nerve fiber RNFLc The RNFLT times the length of the layer cross-section area

contour line.

The height variation along Hvar The difference between the most elevated and the the

contour line most depressed point of the corrected contour

line.

Cup shape measure CSM Third central moment (skewness) of thefrequency

distribution of depth values relative to the “curved surface”

of the parts located inside the contour line and within the

measured segment. Only structures located below “curved

surface” (positive depth values) contribute.

The parameter CSM describes the overall shape of the ONH. Values are typically

negative in normal eyes (flat cup where small depth values are most frequent) and

positive in glaucomatous eyes (high slopes at the cup boundary, deep cup, high

depth values most frequent).

55

Fig. 11. The HRT software version 1.09 standard reference level (REFd) in non-

glaucomatous and glaucomatous eye.

56

The reference levels used in the present study are defined as follows: The HRT

software version 1.09 standard reference level (REFd) is defined at location 0.320

mm posteriorly of the mean height of the retinal surface (at z= 0.320 mm in the

relative coordinate system) (Figure 11).

Fig. 12. The HRT individual reference level (REFi) in non-glaucomatous and

glaucomatous eye.

The individually determined reference level (REFi) is obtained by the following

method: the x-y cursor in the intensity image, with the best scleral ring (Elschnig’s

ring) appearance out of the 32 intensity images with the tilted and relative

coordinate system, is placed at the Elschnig’s ring. This is just outside the contour

line at the point where the line gives an artifact “notch”. The z coordinate of that

point gives the value of the reference level (Figure 12).

57

Fig. 13. The HRT papillo-macular reference level (REFm) in non-glaucomatous and

glaucomatous eye.

The papillo-macular reference level (REFm) is defined as the level parallel to the

reference ring (corresponding to the retinal surface) at the mean height of the

corrected contour line in the segment between 0 degrees and +1 degrees (Figure

13).

58

Fig. 14. The schema of the REFf reference level used in the HRT images in non-

glaucomatous (left) and in glaucomatous (right) eye.

59

Fig. 15. The HRT software version 1.11 standard reference level (REFf) in non-

glaucomatous and glaucomatous eye.

The HRT software version 1.11 standard reference level (REFf) is the level parallel

to the reference ring and located 50 µm posteriorly of the mean contour line height

in the segment between -10 and - 4 degrees (Figure 15).

60

4.4 Automated perimetry

Humphrey Field Analyzer (Model 610; Humphrey Instruments, San Leandro, CA)

was used to obtain both W/W and B/Y visual fields on a modified 30-2 program.

Details of the B/Y test procedure have been provided in several previous

publications (Hart et al. 1990, Sample et al. 1993b). B/Y perimetry was performed

with a 100 cd/m2 yellow background and a size V blue (440 nm) stimulus.

W/W visual field MD was obtained using the statistical package provided by

Humphrey Instruments. The calculation of B/Y visual field MD was based on

the results of non-glaucomatous subjects enrolled in this study. The 24-2 test data

was used to obtain precise model of the normal B/Y visual field. In non-

glaucomatous subjects, the W/W perimetry was carried out during the first visit. To

achieve data for program 24-2 test, we subtracted respective peripheral location

values from program 30-2 data.

4.5 Study protocol

The clinical protocol was the same for each subject included in this study: best

corrected visual acuity, slit lamp biomicroscopic examination, determination of the

refractive error and keratometric values, stereophotographs of the ONH, RNFL

photographs and ONH planimetry, as well as Humphrey 30-2 visual field

examination (Humphrey Field Analyzer, Model 610, Humphrey Instruments, San

Leandro, CA, USA). The AF of the lens was measured using our fluorometer. A

lens transmission index (LTI) was calculated from the ratio between the heights of

the posterior and anterior AF peaks.

By using red-free or green light it is possible to discriminate normal and

degenerated RNFL areas, because greenblue light is reflected from the RNFL layer

in the retina (Behrendt & Wilson 1965). A wide-angle fundus camera with a blue

narrow-band interference filter and high-resolution black-and-white film has been

used to acquire RNFL photographs (Airaksinen & Nieminen 1985). The semi

quantitative scoring method with the total localized score, the total diffuse score

and the total overall score were calculated (Airaksinen et al. 1984). The HRT

images were taken through undilated pupils.

The present study followed the tenets of the Declaration of Helsinki. An

informed consent was obtained from all subjects prior to their inclusion in the study.

This study was approved by the Ethical Committee of the Medical Faculty of the

University of Oulu.

61

4.6 Statistical methods

The HRTCALC Utility Version 1.05 (Heidelberg Engineering GmbH, Heidelberg,

Germany) was used to collect and calculate the HRT parameter values from the

mean image topographic measurements. HRTCALC is a DOS program that scans

the HRT database and automatically determines the stereometric parameters for the

topography images or mean images present. HRTCALC creates an ASCII data file

containing the results of the parameter determination. The data was analyzed as an

ASCII data file in the SPSS 6.1.3S for Windows (SPSS Inc., Chicago, Illinois,

USA).

The statistical analyses used in these studies are as follows. In study I,

Wilcoxon Signed-Rank test was used to test the means of the parameters, the level

of statistical significance was set at p� 0.05. In study II and study IV, one-way

ANOVA with Duncan’s multiple range test was used in the statistical analysis

between the HRT parameter values. The HRTCALC Utility Version 1.05 was used

to collect and calculate the HRT parameter values from the mean topography

images. In study III, two-sided t-test for unequal variances and two-sided

asymptotic U-test of Mann-Whitney-Wilcoxon was used to analyze the contour-

line segment height readings. Means of regression analysis was used to analyze the

relation between the average SD of the mean topography image and the variability

of the contour-line segment height. Spearman rank correlation coefficients were

determined to compare the height variability of the contour-line segment (-10º to -

4º) and the mean topography image SD, both in non-glaucomatous and

glaucomatous eyes. In study V, stepwise logistic regression analysis was used, with

the default criteria of the program for selecting covariates (P� 0.050 for entry and

P� 0.1 for removal) at each step. The area under the receiver operating

characteristics (ROC) curve was the summary measure of the discrimination power.

62

63

5 Results of the study

5.1 Comparison of optic nerve head measurements using planimetric techniques and the HRT (I)

No statistically significant differences were found between the mean neuroretinal

RA and cup-to-disc area ratio (CDR) with these two techniques. However, the mean

optic disc area measured larger values with manual (planimetric) techniques than

with the HRT (a statistically significant difference). The results of study I are

presented in detail in Table 6. In this study the appearance of the neuroretinal RA

as defined by HRT corresponded well to the clinical definitions as shown in Figure

16.

Table 6. Optic disc parameters measured with manual planimetric techniques and with

the Heidelberg Retina Tomograph (HRT) (mean ± SD).

Parameter Manual HRT P-value

Optic disc area (mm²): Mean±SD 2.10±0.56 1.97±0.57 <0.05

Range 1.23-3.07 0.99-3.05

Neuroretinal rim area (mm²): Mean±SD 1.43±0.46 1.33±0.41 NS*

Range 0.98-2.63 0.88-2.23

Cup/Disc area ratio: Mean±SD 0.31±0.14 0.31±0.13 NS*

Range 0.01-0.51 0.11-0.61

*NS = not significant

64

Fig. 16. The appearance of the ONH rim area as defined by the HRT (left) and by ONH

photograph (right).

5.2 The influence of different reference levels on the HRT measurement values (II)

The results of study II show the RNFL thickness values with the reference level

REFi and REFm to be statistically significantly smaller than with reference levels

REFd and REFf in the normals and in the early-to-moderate glaucoma groups. The

results of study II are presented in detail in Table 7. The RNFL thickness measured

with REFi level was statistically significantly smaller than that measured with the

REFm level, too (Figure 17 a and b). The same finding was observed with the RA

values, but the differences were not statistically significant, except between rim RA

values measured with reference levels REFi and REFf in the normals (Figure 17 d

and e).

In advanced glaucoma, RNFL thickness values with the reference level REFd were

statistically significantly larger compared to measurements with all other reference

levels. With the REFi level the HRT gave the smallest RNFL thickness values

(Figure 17 c). Similar findings could be seen with RA values, but the difference

was statistically significant only between reference levels REFd and REFi (Figure

17 f). Again, for RV measurements, we noticed the same kind of differences in all

clinical groups as were seen for RNFL thickness and RA values, but here the

differences of measurement values with the tested reference levels were not as

65

frequently statistically significant as with the RNFL thickness values (Figure 17 g,

h and i).

Fig. 17. Boxplot presentation of the RNFLt, RA and RV in non-glaucomatous eyes (A, D

and G) and in eyes with early and moderate (B, E and H) or advanced glaucoma (C,F

and I) using the reference levels REFd, REFi, REFm and REFf (median with 10th, 25th,

75th and 90th percentile). For abbreviations see Table 3 and Table 5.

66

Table 7. Mean ± standard deviation of three HRT reference level dependent optic disc

parameters (RNFL thickness, rim area and rim volume) with four different reference

levels (REFd1 , REFi2 , REFm3 and REFf4).

Parameter/

reference levels

Normals

n=40

Early and moderate

glaucomas

n=19

Advanced

glaucomas

n=8

RNFL thickness (RNFLt) (mm)

REFd 0.25 ± 0.05 0.18 ± 0.05 0.17 ± 0.06

REFi 0.18 ± 0.06 0.10 ± 0.06 0.01 ± 0.06

REFm 0.21 ± 0.07 0.14 ± 0.06 0.04 ± 0.07

REFf 0.26 ± 0.07 0.18 ± 0.0 0.08 ± 0.06

ANOVA REFi < REFd,

REFm, REFf

REFi < REFd,

REFm, REFf

REFd < REFi,

REFm, REFf

REFm < REFd, REFf REFm < REFd, REFf REFi < REFf

Rim area (RA) (mm²)

REFd 1.43 ± 0.35 1.17 ± 0.43 0.83 ± 0.30

REFi 1.29 ± 0.31 0.91 ± 0.34 0.42 ± 0.28

REFm 1.37 ± 0.32 1.04 ± 0.38 0.52 ± 0.33

REFf 1.47 ± 0.33 1.18 ± 0.40 0.65 ± 0.29

ANOVA REFi < REFf NS REFi < REFd

Rim volume (RV) (mm3)

REFd 0.38 ± 0.13 0.24 ± 0.13 0.15 ± 0.08

REFi 0.28 ± 0.11 0.15 ± 0.10 0.05 ± 0.05

REFm 0.33 ± 0.13 0.19 ± 0.12 0.06 ± 0.07

REFf 0.40 ± 0.14 0.25 ± 0.14 0.08 ± 0.06

ANOVA REFi < REFd REFi < REFd, REFf REFi, REFm, REFf <

REFd REFi, REFm < REFf

ANOVA: p < 0.05 1 REFd = HRT software version 1.09 standard reference level 2 REFi = individual reference level 3 REFm = papillo-macular reference level 4 REFf = HRT software version 1.11 standard reference level

5.3 The development of the contour line based standard reference

plane (III)

The results in study III show that the average optic disc surface inclination angle

was -7° ± 3° below the horizontal meridian (0°). The 6° wide contour-line segment

for the “flexible” standard reference plane (SRP) was chosen according to the

67

average surface inclination angle (-10° to -4°). The reproducibility of the SRP-

segment height measurements was 16.0 ± 10.8 µm for normal eyes and 23.4 ± 18.0

µm for glaucoma eyes.

To assure that the automatic reference level determination for intrapapillary

parameters remains below the disc border height, the SRP level was defined at an

50 µm offset (> 2 SD of average segment height reproducibility in glaucoma) added

to the individual height position of the 6° contour line segment.

5.4 The ability to separate clinical groups with the HRT (IV)

In study IV we used independent sample t-test to separate between normal and

glaucomatous groups (i.e., groups 1, normals, and 3 – 5, early, moderate and

advanced, combined) in the preliminary observation of the HRT parameter values

(Table 7.). More detailed analysis with one-way ANOVA and Duncan’s multiple

range test was performed with variables showing a statistically highly significant

difference between normals and glaucomas.

For further analysis the following reference level independent HRT parameters

were selected: cup shape measure (CSM) and the height variation along contour

line (Hvar). The following reference level dependent variables were also included:

cup area (CA), rim area (RA), cup/disc ratio (CDR), vertical cup diameter (CDVer),

vertical linear cup/disc ratio (CDRVer), mean RNFL thickness (RNFLt), RNFL

cross section area (RNFLc), cup volume (CV) and rim volume (RV). The group

means and standard deviations of these selected HRT parameters and their

respective clinical groups are presented in Table 8. According to the presented HRT

measurement values the ANOVA showed that there was a statistically highly

significant difference among the clinical groups for all selected HRT variables.

Finally, we performed a more detailed analysis of the clinical group differences

with Duncan’s multiple range test. The HRT parameters CSM, CDR and CDRVer

gave the best overall separation: the advanced glaucoma group was statistically

significantly different from all the other clinical groups. The early and moderate

glaucoma groups showed statistically significant differences from the normals and

the ocular hypertensives, but not from other glaucomas. Based on the measurement

values of the variables RNFLt and RV, the normals were different from all the three

glaucoma groups, the ocular hypertensives from moderate and advanced glaucoma,

and advanced glaucoma from all other clinical groups. The measurement values of

the most useful HRT parameters (CSM, CDR, CDRVer, RNFLt and RV) in the

clinical groups are presented in box plot form in Figure 18.

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In our study population, the glaucomatous eyes (clinical groups 3-5 combined)

showed larger size of the optic disc (2.05 ± 0.40 mm²) compared to that of the

normal controls (1.86 ± 0.42 mm²).

Table 8. Mean ± standard deviation (SD) of the HRT parameters in the clinical groups.

Parameter Normals

n=40

Ocular

hypertensives

n=10

Early

glaucomas

n=13

Moderate

glaucomas

n=6

Advanced

glaucomas

n=8

Reference level non-dependent

parameters:

Cup shape measure (CSM) -0,22 ± 0.07 -0.20 ± 0.05 -0.11 ± 0.06 -0.10 ± 0.08 0.01 ± 0.06*

Height variation along

contour line (mm) (HVar)

0.41 ± 0.08 0.42 ± 0.12 0.35 ± 0.08 0.31 ± 0.13 0.24 ± 0.09**

Reference level dependent

parameters: thickness

describing parameters

Mean retinal nerve fiber

layer thickness (mm)

(RNFLt)

0.26 ± 0.07 0.25 ± 0.08 0.20 ± 0.04 0.16 ± 0.08 0.08 ± 0.06*

Retinal nerve fiber layer

cross section area (mm²)

(RNFLc)

1.25 ± 0.30 1.21 ± 0.44 1.02 ± 0.26 0.80 ± 0.38 0.37 ± 0.27*

Area describing parameters:

Cup area(mm²) (CA) 0.39 ± 0.31 0.56 ± 0.40 0.95 ± 0.32 1.02 ± 0.59 1.19 ± 0.34*

Rim area (mm²) (RA) 1.47 ± 0.33 1.38 ± 0.33 1.18 ± 0.42 1.17 ± 0.40 0.65 ± 0.29*

Cup/disc area ratio (CDR) 0.19 ± 0.14 0.28 ± 0.17 0.45 ± 0.12 0.45 ± 0.23 0.65 ± 0.14*

Vertical linear cup/disc area

ratio (CDRVer)

0.31 ± 0.22 0.40 ± 0.19 0.64 ± 0.11 0.63 ± 0.26 0.85 ± 0.09*

Vertical cup diameter (mm)

(CDVer)

0.50 ± 0.36 0.65 ± 0.34 1.08 ± 0.19 1.08 ± 0.47 1.30 ± 0.21*

Volume describing parameters

Cup volume (mm³) (CV) 0.09 ± 0.11 0.14 ± 0.14 0.29 ± 0.20 0.24 ± 0.16 0.31 ± 0.15*

Rim volume (mm³) (RV) 0.40 ± 0.14 0.37 ± 0.14 0.25 ± 0.14 0.23 ± 0.14 0.08 ± 0.06*

* p<0.00001, ** p<0.0001

69

Fig. 18. Boxplot presentation of HRT parameters (CSM, CDR, CDRVer, RNFLt and RV)

that provide the best separation between the clinical groups (Nor, OHT, GE, GM, GA)

(median with 10th, 25th, 75th and 90th percentile).

5.5 Logistic multivariate regression analysis of confocal scanning laser tomograph, blue-on-yellow visual field and retinal nerve

fiber layer data (V)

In study V we performed logistic multiple regression analyses to compare the

separating value of selected diagnostic methods and to test the sensitivity and

specificity of the model to correctly identify non-glaucomatous eyes (n = 32) and

glaucomatous eyes (n = 23).

70

First we selected all the global HRT parameters, W/W and B/Y visual fields

(age- and lens-adjusted) with the RNFL total diffuse, total localized and total

overall score values as covariates in the logistic model. The RNFL total overall

score gave the best separation between non-glaucomatous and glaucomatous eyes

(ROC area 0.99) with these parameters in the model.

However, the RNFL scoring is a subjective method in optic disc and RNFL

assessment. That is why we left the RNFL total diffuse, total localized and total

overall score out of the regression model in order to see better the discriminatory

ability of the other parameters. The visual field parameters (W/W and B/Y) were

also left out of the model to avoid their influence on the results. With all the other

parameters in the model, by the first step, CDR was selected (ROC area 0.90 for

CDR only). In the next step, RNFLt was selected, improving discrimination

marginally (ROC area 0.92 for CDR and RNFLt jointly in the model).

Because some of the global HRT parameter values (e.g. CDR, CDVer, CDRVer,

CV) are dependent of the optic disc size, and at the same time in our study

population, the areas of non-glaucomatous discs are somewhat smaller than the

areas of glaucomatous discs, we wanted to remove the effect of different disc sizes.

That is why we performed logistic regression analysis in a subset of data (22 non-

glaucomatous and 22 glaucomatous eyes, the subjects being matched for disc area).

With these data and with the HRT and B/Y visual field parameters in the model, the

stepwise procedure did not choose CDR or other disc size dependent HRT

parameters to the logistic model, i.e., the disc size dependent HRT parameters could

not separate glaucomatous from non-glaucomatous subjects in this restricted subset.

The best discriminating single parameter was RV (ROC area 0.92), and adding B/Y

visual field improved the discrimination (ROC area 0.96) for RV together with B/Y

visual field MD (Figure 21).

Therefore, in the next phase, we performed logistic multiple regression

analysis again in the original study population (32 non-glaucomatous and 23

glaucomatous eyes) with all the other above-mentioned HRT and B/Y visual field

parameters in the regression model, but we excluded all the optic disc size

dependent HRT parameters as well as the RNFL total diffuse, total localized and

total overall score. At the first step, cup shape measure (CSM) was selected (ROC

area 0.88) (Figure 19). At the second step, the mean RNFL thickness (RNFLt) was

added to the model, and the discrimination was somewhat improved (ROC area

0.91) (Figure 20). At the third step, age- and lens-corrected B/Y visual field MD

was added to the model, but it did not improve the result further (ROC area 0.91)

71

(Figure 21). If CSM was dropped, the discrimination provided by RNFLt and age-

and lens-corrected B/Y visual field MD jointly was still high (ROC area 0.93).

Fig. 19. The ROC curve. The area under the curve was 0.88 for CSM.

Fig. 20. The ROC curve. The area under the curve was 0.91 for CSM and RNFLt.

72

Fig. 21. The ROC curve. The area under the curve was 0.91 for CSM, RNFLt and age-

and lens-corrected B/Y visual field MD.

73

6 Discussion

6.1 Early glaucomatous optic disc, RNFL and visual field changes

The diagnosis of glaucoma is quite logical if the following structural and functional

changes characteristic of glaucoma are present: progressive, diffuse and/or

localized, loss of the ONH neuroretinal rim, changes in the RNFL and

corresponding visual field defects. The glaucomatous loss of ganglion cell axons

causes morphologically detectable tissue damage in both ONH and the RNFL.

Depending on the structure examined, different techniques for observation give the

most feasible results. In the present study the difference between moderate and

advanced glaucoma was set at 10 dB MD. Patients with the VF defects only barely

separated by this limit may have very similar glaucomatous damage compared to

patients on the upper and lower regions of the limits.

In this study, the HRT measurements showed clinically meaningful results and

the appearance of the neuroretinal RA assessed with the HRT corresponded well to

the conventional techniques. No statistically significant differences between the

mean neuroretinal RA and cup-to-disc area ratio was found when comparing the

ONH measurements using planimetric techniques and the HRT. However, the mean

optic disc area measurement values were larger with planimetric techniques than

with the HRT (study I). Among the global HRT parameters, the reference level non-

dependent CSM and the reference level dependent CDR, CDRVer, RNFLt and RV

discriminated best between non-glaucomatous and glaucomatous eyes (study IV).

In study IV the glaucomatous eyes showed larger optic discs compared to normal

controls. However, the difference was statistically not significant, possibly due to

the limited sample size of the study. Depending on the study population different

HRT parameter values are accurate to diagnose glaucoma (Mikelberg et al. 1995,

Bathija et al. 1998). Using logistic regression analysis method in order to find the

best combination of parameters discriminating the clinical groups, the RNFL total

overall score separated best between non-glaucomatous and glaucomatous eyes

when all the structural and functional parameters were included in the model. When

subjective assessing methods were left out of the regression model, in the first step

CDR gave a ROC area of 0.90, and in the next step CDR and RNFLt jointly gave

a ROC area of 0.92. Furthermore, the regression model did not choose the optic

disc size dependent HRT parameters in the model. This would suggest that the size

of the optic disc is taken into account when diagnosing glaucomatous damage with

74

the HRT. When tested in a subset of data, subjects were matched for disc area. By

leaving these optic disc size dependent HRT parameters as well as the subjective

RNFL scoring parameters out of the regression model, but using all the other

parameters in the original study population, the stepwise procedure selected the

CSM with ROC area 0.90 in the first step. The RNFLt with a ROC area of 0.94 was

chosen in the second step, and finally, the age- and lens corrected B/Y visual field

MD with ROC area 0.95 in the third step, in the model. This would suggest that the

diagnostics of glaucoma is improved when the optic disc, the RNFL and visual

fields all are included in the diagnostic panel (Tuulonen et al. 2015).

6.2 Accuracy, reproducibility and agreement between optic disc and RNFL observation and evaluation methods

Optic disc photography is a reliable method for documenting glaucomatous optic

disc abnormalities and their progression (Airaksinen et al. 1984, Balazsi et al. 1984). Several planimetric techniques have been used in the evaluation of the ONH

(Littmann 1988, Britton et al. 1987, Jonas et al. 1989a, Sommer et al. 1979a,

Balaszi et al. 1984). These methods have shown interobserver variation caused by

differences in image interpretation and the use of various ‘machine units’ in

measurements, corrected by magnification algorithms. In their study among

European general ophthalmologists Reus et al. (Reus et al. 2010) found accuracy

of 80.5% for detecting glaucoma in stereoscopic ONH photographs. In the same

study, Finnish ophthalmologists showed specificity of 93.2% and sensitivity of 69.3%

for detecting glaucomatous damage.

In earlier studies, the reproducibility of the HRT optic disc topography

measurements has shown standard deviations of pixel height measurements of 20-

30 µm; variability was slightly higher in glaucomatous eyes compared to non-

glaucomatous eyes (Kruse et al. 1989, Weinreb et al. 1993b, Janknecht & Funk

1994, Dannheim et al. 1995). The coefficient of variation of the HRT stereometric

topographic parameters has been reported to be between 2 and 10% (Mikelberg et al. 1993, Rohrschneider et al. 1994, Zangwill et al. 2001). Interobserver agreement

in interpreting HRT printouts in order to separate non-glaucomatous and

glaucomatous eyes has been reported with kappas between 0.67 and 0.73 for

diagnostic agreement (Sanchez-Galeana 2001).

The inter- and intra-operator agreement in the RNFL measurements with the

GDxVCC has shown coefficient of variation between 3.5% and 10% in non-

glaucomatous and glaucomatous eyes (Greenfield 2003, Zangwill 2001).

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Interobserver agreement in discriminating clinical groups by interpreting the GDx

Nerve Fiber Analyzer printouts has been reported with kappas between 0.55 and

0.66 (Sanchez-Galeana 2001), and kappas of 0.42 and 0.48 for non-glaucomatous

and glaucomatous eyes, respectively. Reproducibility studies of the RNFL

thickness measurements with the OCT have showed coefficients of variation of

approximately 10% (Blumenthal et al. 2000, Carpineto et al. 2003). The

interobserver reproducibility for separating non-glaucomatous and glaucomatous

eyes with the OCT printouts has been reported with kappas between 0.51 and 0.73

(Sanchez-Galeana 2001). Both with GDx and OCT, variability has been found in

the RNFL thickness measurements (Iacono et al. 2006, Vizzeri et al. 2009).

6.3 The significance of the definition of the HRT reference level

The values of the HRT stereometric parameters depend on the definition of

reference plane, the instability of which results in measurement variability (Poli et al. 2008). More than half of the variability of the important HRT parameters RA,

RV, and RNFL could be explained by the variation of the standard reference height

(Breusegem et al. 2008). The interobserver variability is minimized by choosing

the most stable and most easily predictable reference level for HRT measurements,

also in follow-up studies (Tan & Hitchings 2003, Tan et al. 2004, Strouthidis et al. 2005a).

The definition of the reference level is of fundamental importance for ONH

stereoscopic parameter measurements. An ideal reference level in optic disc

assessing should ensure the accuracy and reproducibility of measurements also in

follow-up, taking into account the variation in morphology of individual ONHs.

The reference level should be clinically relevant, easy to apply and it should keep

its stability over time. As the reference level definitions always remain arbitrary,

depending on defining the method and individual anatomy of the ONH, no

reference plane is capable of measuring the stereometric parameters exactly.

The ‘curved surface’ was the first reference plane tested for the quantitative

assessment of the HRT (software version 1.0) stereoscopic parameter values. The

curved surface represents the mean height of the corrected contour line in the center,

connected by straight lines to its boundary points in the corrected contour line.

Unfortunately, it was unable to correctly separate between neuroretinal rim and

optic cup structures (Burk et al. 1990). The HRT (software version 1.08, 1.09 and

1.10) default ‘fixed 320 µm offset’ reference level (Burk et al. 1993a) is

independent of observer and automatically set in the image, but has the

76

disadvantage of measuring clinically inaccurate values in advanced glaucoma eyes.

This reference level was defined 320 µm posteriorly to the reference ring positioned

on the RNFL surface around the ONH. Erroneously high neuroretinal rim values

and optic CV measurements that are too low are obtained because the peripapillary

glaucomatous RNFL damage shifts the reference ring level (Tuulonen et al. 1994).

To avoid this inadequacy, the basis of reference level definition had to be more

individual and stable. In order to achieve these requirements, the inner border of

the scleral ring, the Elschnig’s ring, as well as the papillo-macular region of the

RNFL bundles were chosen as ‘landmarks’ for the more individually determined

reference level (Tuulonen et al. 1994, Vihanninjoki et al. 1994). This choice is

based on the findings of earlier studies showing that the scleral ring remains

constant and that the papillo-macular bundles are best preserved during glaucoma

progression (Airaksinen et al. 1984, Sommer et al. 1977, Sommer et al. 1979a,

Drance et al. 1977, Drance 1978, Drance et al. 1981). However, glaucomatous

changes may also affect the papillo-macular bundles (Chen et al. 2001). Another

arbitrary reference level, the papillo-macular 0-1º reference level, was proposed

based on the same assumptions (Airaksinen 1994a, Airaksinen 1994b, Airaksinen et al. 1995). Applying a wider contour-line segment, still respecting the most stable

region of the disc margin, and taking the interindividual variability of ONH

topography better into account, a flexible reference plane was established as the

SRP for the HRT (software version 1.11). The SRP is located 50 µm posteriorly of

the mean contour line height in the segment between -10 and -4 degrees. Further

studies were needed to show the validity of this reference plane.

An alternative reference plane, assisted with the OCT measurements, was

defined as being located posteriorly from the average height of the disc margin by

the amount of mean RNFL thickness measured with the OCT (Park & Caprioli

2002). This reference plane may be particularly useful in detecting early

glaucomatous changes in eyes with tilted discs, such as in myopic glaucoma.

Another, experimental reference plane, is positioned to ensure that it always lies

entirely below the circumference of the contour line. The distance of the reference

plane is beneath LOW5%, the lowest 5% region of the contour line, where RA

variability is the lowest (Tan & Hitchings 2003). This experimental reference plane

presented good correspondence between the appearance of the neuroretinal rim in

optic disc images and the rim defined by the reference plane (Tan et al. 2004).

The Moorfields reference plane is a combination of the SRP used in the

baseline images and the height difference between the SRP and the reference ring

of the 320 reference plane, which is kept constant (Poli et al. 2008). This reference

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plane showed significantly lower variability and had better discrimination between

RA change and measurement variability in a longitudinal HRT image series (Poli et al. 2008).

6.4 The significance of the HRT global parameters in optic disc evaluation

The HRT has improved eye fundus assessment by moving from conventional,

subjective ophthalmoscopic and photographic methods to a more objective and

reproducible method. In addition, a more adequate interpretation of the results by

means of normative databases respecting racial variability, Moorfield regression

analysis (MRA) (Wollstein et al. 1998, Reus et al. 2007) and the glaucoma

probability score (GPS) (Swindale et al. 2000, Coops et al. 2006, Taibbi et al. 2009)

is achieved. The ability of the HRT stereometric parameters, both reference level

dependent and non-dependent, to discriminate between non-glaucomatous and

various stages of glaucomatous eyes has been shown in several studies (Brigatti &

Caprioli 1995, Zangwill et al. 1995, Dichtl et al. 1996, Uchida et al. 1996, Iester et al. 1997d, Wollstein et al. 1998). However, measurement accuracy and

reproducibility is imperative in the diagnosis as well as the follow-up of glaucoma.

The HRT techniques have enabled three-dimensional measurements,

determination of the volume of the neuroretinal rim and optic cup, as well as the

assessment of the optic cup shape, the parameter values optic disc which could

previously only be estimated with conventional photographic methods (Zinser et al. 1989, Burk et al. 1990, Burk et al. 1993a).

6.5 Sensitivity and specificity of glaucoma imaging methods

The sensitivity and specificity of direct ophthalmoscopic assessment of the optic

disc in the screening of glaucoma was studied by Harper and Reeves in their

multivariate analysis (Harper & Reeves 2000). The specificity for finding

glaucomatous damage in RNFL photographs has been between 83% and 97%

(Quigley et al. 1980, Airaksinen et al. 1984, Wang et al. 1994).

In their multivariate analysis study on 96 OHT patients with a minimum of 5

years of follow-up Airaksinen et al. found the factors best separating stable OHT

patients from the ones who developed glaucoma to be initial RA, change of the rim

area/year of follow-up and SD of the MD of the initial visual field. This model

correctly classified 81% of the patients with 72% sensitivity and 87% specificity.

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IOP variables, RNFL score and peripapillary atrophy were poor predictors

(Airaksinen et al. 1991).

Using the Rodenstock ONH analyzer, Damms and Dannheim, in terms of

separation characteristics of the different disc parameters, found the maximum and

vertical cup-to-disc ratio (C/D) to have the best diagnostic power (ROC area 0.97

and 0.96 respectively), followed by superior C/D (ROC area 0.92), vertical, total

and temporal RA (ROC area 0.86, 0.79 and 0.70, respectively) and cup CV (ROC

area 0.55) (Damm & Dannheim 1993). In a study with the scanning laser

ophthalmoscope of Rodenstock, Chihara et al. found the optic disc and cup area

smaller when obtained with the SLO than those in conventional photographs, the

coefficient of variation of the optic disc parameters ranging from 4.2% to 9.1%.

The correlation between the optic disc measures studied with the SLO and MD of

the visual field was statistically significant (Chihara et al. 1993).

In glaucoma studies the HRT has shown sensitivities of 63-85%, specificities

of 90% and ROC area of 0.86–0.96 (Bowd et al. 2002, Greaney et al. 2002,

Zangwill et al. 2001).

Glaucoma studies with the GDx have shown sensitivities of 32–89%,

specificities of 86–96% and ROC area of 0.84–0.94 (Bowd et al. 2001, Greaney et al. 2002, Medeiros & Susanna 2003, Yamada et al. 2000, Zangwill et al. 2001).

The ability to detect glaucoma with the OCT has shown specificities of 68–

88%, specificity of 71–96% and ROC area of 0.87–0.94 (Bowd et al. 2001,

Greaney et al. 2002, Guedes et al. 2003, Kanamori et al. 2003, Soliman et al. 2002b,

Zangwill et al. 2001).

6.6 Diagnostic accuracy of the HRT in screening studies

The conventional screening tests for POAG have consisted of ophthalmoscopy,

tonometry and perimetry. However, approximately 50% of persons over 40 years

of age with POAG were not detected with these screening methods in the United

Kingdom (Crick et al. 1994). Several studies have confirmed this problem

(Sommer et al. 1991, Wong et al. 2006).

Burk et al. found the scanning laser ophthalmoscopic measurement values of

the shape of the ONH to be similar to the measurements of computed

stereophotogrammetry (Burk et al. 1993b). In addition, in a study by Cooper et al., measurements of the RNFL produced by scanning laser ophthalmoscopy and red-

free photographs were comparable (Cooper et al. 1992). In several HRT studies

even one HRT parameter has been able to separate between non-glaucomatous and

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glaucomatous eyes (Burk et al. 1991, Vihanninjoki et al. 1994, Dichtl et al. 1996,

Iester 1997d, Wollstein et al. 1998). However, a combination of HRT parameters

most often gives the best accuracy and reproducibility in diagnosing glaucoma

(Mikelberg et al. 1995, Broadway et al. 1998, Ferreras et al. 2008).

The HRT with MRA has been reported as a good screening method in the

diagnostics of glaucoma, even compared to SAP and frequency doubling perimetry

(Robin et al. 2005).

The effectiveness of the modern eye fundus imaging devices, including the

HRT, as screening methods has been only poorly investigated (Kass et al. 2000,

Polo et al. 2006, Burr et al. 2007, Tuulonen et al. 2015, Burr et al. 2012). The

results in the studies with modern, digitalized ONH assessing methods are quite

variable in diagnosing glaucoma (Caprioli et al. 1996, Polo et al. 2006, Coleman et al. 1996, Hadwin et al. 2013). Evidence-based recommendations in glaucoma

diagnosis and follow-up of progression emphasized the lack of randomized

multicenter studies of glaucoma screening and diagnostics. The high risk of

research bias of the published studies and the variability of the measurement values

in the assessment of eye structures and function, depending on the method, observer,

patient or the degree of disease indicate that there is a need for further evidence

(Tuulonen et al. 2015). Although the new eye fundus imaging devices can minimize

the inter- and intraobserver variability of the results, the interpretation of the digital

printouts and detection of artifacts is still challenging. The conventional

photographs of the ONH and the RNFL remain necessary (Chauhan et al. 2013).

6.7 Clinical implications

The criteria of glaucoma diagnosis have been debated for decades. The consistency

of the definition of glaucoma influences the estimation of the prevalence rates, risk

factors and strategies of treatment. In a literature review of 182 articles from the

American Journal of Ophthalmology, Ophthalmology and Archives of

Ophthalmology for the years 1980, 1985, 1990 and 1995, Bathija et al. found that

36% of the articles used both optic disc and visual field criteria for glaucoma, 13%

used optic disc or visual field criteria, 26% used only visual field criteria, 20% used

only IOP, and 5% used only optic disc criteria. In the 1990s, specific descriptions

of the optic disc (34% of articles) and visual field (34% of articles) were favored

(Bathija et al. 1998). This progress may have depended on the availability of the

glaucoma imaging techniques in the 1990s. Still, according to several public health

80

studies, approximately 50% of the POAG subjects are not detected (Sommer et al. 1991, Klein et al. 1992, Weih et al. 2001, Wong et al. 2004).

In a prospective, longitudinal study, Caprioli et al. (Caprioli et al. 1996)

evaluated change, progressive glaucomatous damage, in the optic nerve and nerve

fiber layer by four techniques: 1. qualitative evaluation of stereoscopic color optic

disk photographs, 2. qualitative evaluation of monochromatic nerve fiber layer

photographs, 3. manual stereoplanimetric measurements of disk RA, and 4.

computerized measurement of peripapillary nerve fiber layer height. Of the patients

followed up for 3.3±1.0 years, 15% progressed by qualitative optic disk evaluation,

7.2% by qualitative nerve fiber layer evaluation, 3.6% by stereoplanimetry, and

13.2% by measurement of nerve fiber layer height. Visual field deterioration was

detected in 5.2% and correlated best with qualitative optic disk and nerve fiber layer

evaluations. Evaluation by stereoplanimetry and nerve fiber layer height

measurement detected change in eyes with primarily diffuse structural damage, a

pattern not well detected by qualitative methods.

Based on the results of this thesis, SRP was developed to improve the

diagnostic accuracy of the HRT. However, the results of the multivariate analysis

show that the diagnosis of glaucoma should not be based on the results of the HRT

alone. Evaluating the structural and functional parameters gives the most reliable

basis for the diagnosis of glaucoma.

The clinical HRT studies have maintained the HRT flexible reference level,

introduced in the software version 1.11, as still appropriate in glaucoma diagnostics.

This applies despite though the increased variability in RA measurement with the

standard reference level compared to the 320 reference plane (Strouthidis et al. 2005a). However, in the follow-up of glaucomatous progression, the Moorfields

regression analysis (MRA), using the standard reference level in the baseline

studies and the 320 reference plane in follow-up, has shown its adaptability (Poli et al. 2008). The Glaucoma Probability Score (GPS), an automated HRT image

analyzing method independent of the contour line and the reference level, has

facilitated the interpretation of the HRT topography images especially in

progression studies (Swindale et al. 2000, Coops et al. 2006, Taibbi et al. 2009).

6.8 Further developments in the HRT

The progress from the HRT I to HRT II and III includes both technical

improvements with automated functions as well as development of the software

from the versions 1.0, 1.09 and 1.11 used in the HRT I. New features such as

81

averaging of the scans, serial scans, fine focus, scan depth, correction of scaling

errors and automated drawing of the contour line minimize the effect of the operator

on the measurement values (Strouthidis & Garway-Heath 2008). These methods

decrease the variability of HRT measurement and improve its repeatability. The

imaging head and light source of the HRT I was changed in the HRT II and III,

making these two later devices compatible with each other. Unlike the HRT III

software, the HRT II software cannot analyze the images acquired with the HRT I.

However, technical differences in image acquisition make the HRT I and HTRT III

image analyses uncomparable per se. Use of larger normative databases,

considering ethnicity, enables clinically reasonable analyses of the images acquired.

The development of image alignment techniques, such as applying a subpixel

method (Burk & Rendon 2001) or face recognition technique (Capel 2004), has

improved image quality and decreased measurement variability, manifesting as

lower topography standard deviation (TSD) (Bergin et al. 2008). The Moorfields

regression analysis (MRA) and Glaucoma Probability Score (GPS) as well as a

number of HRT RA progression strategies represent tools for clinical classification,

especially in the follow-up of glaucoma progression. The combination of HRT and

OCT techniques might provide more accurate parameters for discriminating non-

glaucomatous and glaucomatous eyes in follow-up studies as well.

Despite the development of HRT methodology the reference plane described

in article III still remains the SRP definition for the HRT in the diagnosis of

glaucoma.

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83

7 Summary and conclusions

The main results and conclusions of this study:

1. In this study population, the measurements of the mean neuroretinal rim area

and cup-to-disc area ratio as defined by the HRT corresponded well to the

clinical definitions in the optic disc photographs.

2. As the HRT standard reference level was defined at a 50 µm offset (� 2 SD of

average segment height reproducibility in glaucoma) added to the individual

height position of the 6º contour line segment (-10º to -4º), the automatic

reference level determination for intrapapillary parameters remains below the

disc border height.

3. The HRT parameters CSM, CDR, CDRVer, RNFLt and RV were most

powerful in separating non-glaucomatous and glaucomatous eyes.

4. Multiple logistic regression analysis of the HRT, B/Y visual field and the RNFL

data gave the best result (ROC area 0.91) when CSM, RNFL thickness and the

age- and lens-corrected B/Y visual field MD were added to the model.

84

85

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Original publications

I Vihanninjoki K, Tuulonen A, Burk ROW & Airaksinen PJ (1997) Comparison of optic disc measurements by Heidelberg Retina Tomograph and manual planimetric techniques. Acta Ophthalmol Scand 75: 512-515.

II Vihanninjoki K, Burk ROW, Teesalu P, Tuulonen A & Airaksinen PJ (2002) Optic Disc Biomorphometry with the Heidelberg Retina Tomograph at Different Reference Levels. Acta Ophthalmol Scand 80: 47-53.

III Burk ROW, Vihanninjoki K, Bartke T, Tuulonen A, Airaksinen PJ, Völcker H-E & König JM (2000) Development of the Standard Reference Plane for the Heidelberg Retina Tomograph (HRT). Graefe’s Arch Clin Exp Ophthalmol 238: 375-384.

IV Vihanninjoki K, Burk ROW, Teesalu P, Tuulonen A & Airaksinen PJ Identification of Non-glaucomatous and Glaucomatous Optic Discs with the Heidelberg Retina Tomograph. Manuscript.

V Vihanninjoki K, Teesalu P, Burk ROW, Läärä E, Tuulonen A & Airaksinen PJ (2000) Search for an Optimal Combination of Structural and Functional Parameters for the Diagnosis of Glaucoma. Multivariate Analysis of Confocal Scanning Laser Tomograph, Blue-on-yellow Visual Field and Retinal Nerve Fiber Layer Data. Graefe`s Arch Clin Exp Ophthalmol 238: 477-481.

Reprinted with permission from John Wiley and Sons Ltd (I, II) and Springer (III,

V).

Original publications are not included in the electronic version of the dissertation.

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