Corneal Pachymetry & confocal microscopy

Dr. Ram Singh (Department of Ophthalmology)

S.P. Medical College, Bikaner

Pachymetry: - Literally meaning of this word– Thickness

• Measurement of corneal thickness is important factor in

deciding different kind of refractive surgeries.

• It is can be performed with optical pachometer or an ultra

sonic pachometer.

• Certain specular's microscopes are calibrated in such a

way so that they can measure corneal thickness while

focusing corneal endothelium.

OPTICAL PACHYMETRY :- This was the

original method to measure corneal

thickness.

• - It is used with slit lamp. Non Contact

Method

• - optical pachymentry has advantage -

• (1) It does not touch the cornea so does not

damage epithelium which sometimes

happen with contact methods eg. ultrasonic

pachymetry and specular microscopy.

• Major problem in clinical, practical use of

this instrument is repeatability of

measurements, particularly among

observers.

Major sources for these problems.

(1) Lack of small fixation target for the pt, which is located in a fixed position to the

instrument.

(2) Lack of known alignment of the pachymeter with the cornea in a reproducible

position so that the slit beam intersects the cornea of the same angle for

consistent thickness reading.

• Location on the cornea that is being measured can be identified visually.

• It is not easy to return to that specific point without auxiliary fixation devices.

• Errors in accuracy and precision inherent in this method are minimized when the

instrument is used by single observer & errors are <10µm, an acceptable error

for practical a clinical refractive keratotomy.

• Errors can increases up to 20µm or more when a multiple users and this is

unacceptable.

There are two criteria for measurement of

corneal

thickness.

(1) "Just touch" criterion: - Alignment is made

in such a way that an imaginary line extends

from the endothelial border of upper image

to the epithelial border of lower image.

(2) Overlap – Method: - imaginary extension of

the bright portion of endothelial image is

over lapped with bright portion of epithelial

image. Because bright portion is actually

produced by the finite width of the slit lamp

as it passes through each surface of cornea.

JUST TOUCH METHOD IS EASIER, POPULAR A PRACTICAL

AMONG MOST OBSERVERS.

• Five different studies using five different optical pachometers all showed mean central corneal thickness values 0.51 to 0.52 mm (S.D. 0.02 to 0.04mm)

• Kremer – Ultrasonic pachometer – (sound speed in cornea 1640m/sec.) – central average corneal thickness – 0.512+ 0.035mm in 175 eyes.

• Novak et al. compared optical (Haag streit 900 Ĉ mishima Hedbys attachments).

• Specular microscope (Pro-koster) and

• Ultrasonic (Accutome, 1630 ± 10m/sec.) Pachymetry in 93 pts in study – using mean value of 3 method corneal thickness measurements reading for each instrument on each eye.

• Optical – 0.554 ± 0.028mm

• Specular microscopy 0.551 ± 0.37mm

• Ultrasonic 0.542 ± 0.035mm.

• ULTRASONIC PACHYMETRY

• Developed by Henderson and Kremer in 1980

• Currently Preferred Method for corneal thickness measurement due to ease of use,

precision, portability and ability to measure corneal thickness eccentrically.

• Principle: - Instruments functions by measuring the amount of time (transit time)

needed for ultrasound pulse pass from the one end of Transducer to descemet's

membrane and back to the transducer.

• C→ speeds of ultra sound wave in cornea.

• Determined by density and compressibility of cornea.

• Cornea is made of 78% mater.

• Propagation Velocity → Water 1524m/sec.

→Steel – 6000m/sec.

• Thus it is imp. that propagation velocity of cornea be known accurately

because this variable can be set on many ultrasonic pachometer and

different setting will change than thickness of cornea.

• = Speed of sound in cornea: - Current standard is 1640 m/sec.

• Kremer selected 1640 m/sec., because that measurement gave corneal

thickness of 0.512 ± 0.035 in 175 Eyes.

• Components of ultrasonic pachometers • Probe handle with its transducer and tip

• Housing of instrument

• Accessories and convenience features.

• Pachometer probe handle: - it has piezoelectric crystal that emits an

ultrasonic beam of 20 MHz

• - All Probes are hand held.

• - Visualization of tip straight probe is sometimes difficult under the

operative micro scope in comparison to angled probe handle.

• Transducers: - Transducer sends the beam of ultrasound wave

through the probe tips into the cornea and receives them on return.

• Width of transducer beam is related to the size of the emitting

crystal and of the width and configuration of the probe through

which it passes.

# #:- A wide probe tip and wide transducer beam reduces the accuracy of

the corneal thickness reading at a single point.

• Transducer has limited lifetime approximately 150-200 cases. It

loses its accuracy and precision.

• The reading becomes increasingly variable on the calibration

block. The probe should be changed.

• Probe tip: - it is interface between the cornea and

transducer.

• Material in the probe should not attenuate the ultrasound

beam and the geometric design of the probe tip should

facilitate its optimal transmission.

• Diameter of probe should be <2mm. to diminish the area

over which the ultrasound beam is spread (to allow) observer

to see exactly where the tip is placed on the cornea.

• Surface of tip should be smooth to avoid any injury to corneal

epithelium.

TYPES OF PROBE: - (1) Open, water filled type requires

frequent refilling.

(2) Solid tipped probes containing an

internal fluid

reservoir that is refilled

periodically.

(3) All solid tip probe with no internal

reservoir and no refilling.

• Each can provide accurate and

precise measurement, but

convenience and practicality vary.

(1) Open water filled tips: - Used Earlier

Inconvenient to use:-

• As fluid pulled out of the tip by surface tension & capillary action, air bubble enter

and give erroneons readings.

(2) The solid tipped probe – have replaceable couplet (glue or oil)

• and are more convenient.

• - Frequency of refilling varies from once a week to once a year, depending on

design.

• Tekner optha sonic pachometer has oil interface and has to changes once a year.

(3) All Solid tipped – No replaceable couplants • Tips are made of polystyrene.

• More convenient to handle a requires less maintenance eg. accutome corneometer, pach – pen, sonogaga and Humphreys Pachometers

- All pachomaters average a series of thickness measurements to give

the single, final read out display of corneal thickness.

- Instruments take 30-500 reading in a fraction of second.

There are two methods by which pachometer create an average

reading.

1. Pulse locked method: - Unit will record all readings that are within 5

to 10 0 of perpendicularity or within 5 to 10µ of each other, rejecting

those outside the range.

2. Fixed no. of consecutive reading must be within 5 to 100 of

perpendicularity on 5 to 10µ of each other, before there are

averaged. If the probe is not perpendicular or the readings are too

disparate, the series is rejected and must be began again.

Resolution of instrument is smallest unit

measurable by machine - 1 Clinical accuracy of most instrument 5 to 10 Ultrasonic corneal pachometer can measure thickness

range 200 to 2000 Most pachometers have a selected speed of 1640m/sec.

Some units allow adjustments of sound speed, so

operators can select faster or slower speed.

** Selection of faster speed will produced a thicker

corneal reading

Other methods for corneal thickness measurement.

High frequency ultrasound corneal pachymetry

70MHz is used frequency

This technique produces B-scan images in real time, by on the fly analog

processing, involving rectifying averaging reflected ultrasound waves.

Pachymetry using the ORBSCAN topography system

Orbscan technique results in pictorial representation of corneal

topography in true as opposed to derivative terms.

The creation of a surface of orbascan topographic measurement provides

the basis for the derivation of pachymetric & radius of curvature maps.

Pachymetry by laser Doppler interferometry (LDI)

Penta Cam – trade name of comprehensive anterior segment analyzer (five in one innovation)

It is 3-Dimensional (3D) rotating scheimpflug camera.

It can perform five functions in 2 sec.1. Scheimpflug image of anterior

segment

2. 3-D anterior chamber analyser

3. Pachymetry

4. Corneal topography

5. Cataract analyser

Pachymetry by pentacam is displayed as a color image over its entire area

from limbus to limbus.

Actual thickness can be measured individually by a mouse click at any

locations.

Thickness in the pupil centre

Thickness in the apex

Thinnest location

Corneal volume

Applications – 1. Preoperative planning for corneal refractive surgery

2. Glaucoma screening

3. IOP modification with regard to corneal thickness

4. Keratoconus detection & quantification.

CORNEAL CONFOCAL MICROSCOPY This unique method offers the ability to

examine objects at high magnification.

This revolutionary new tool permits real time

observation of living corneal (in vivo) in patients

at magnification ranging from 20X to 500X.

It also measure thickness of each layer by

using computerized scanning system providing

the total corneal thickness in studied area.

Beside endothelium examination also measure

endothelial cell count (density) which is

comparable to specular microscopy.

It offers the possibility to visualize structures

posterior to haze, scars or edema with in the

cornea.

Principles – In a normal microscope image

formation is composed of a single sharp image

in addition to superimposed blurry images.

Depth of field is inversely proportional to

magnitude of magnification.

Unique property of confocal microscope that it

eliminates the super imposed blurred images

that normally occurs with relatively high

magnifications it can exceed the final

resolution of the ordinary light microscope by >

50% of image sharpness.

This unique property is d/t its ability to project

intense illumination & capture its reflected light

through a narrow focal plane; blocking the out

of focus rays.

first study with clinical approach using this

instrument was done by Ichijima in 1992 to

document the changes in superficial o\epithelial cells

after extended wear rigid contact lenses.

Later Cavanagh used a tendon scanning confocal

microscope & examined various corneal diseases.

Confocal microscope uses white light or a focused

laser beam but clinical white light is safe becuase

laser having risk for damaging living tissue.

Procedure – After topical anaesthesia patient is

guided to the chin rest.

A clear visoelastic solution is applied to the cover tip of

the microscope to avoid corneal abrasion.

Machine is approximated until a bright image is seen &

the then the corneal scanning is done.

Once the epithelium is well focused, the zooming

examination of all corneal layers can be fulfilled in only

30 sec. examination is stored in computer software.

NORMAL CORNEA -

1. Epithelium – Superficial layers –

large surface cells arranged in

irregular polygonal mosaic.

These cells demonstrate hyper

reflective nuclei.

Basal epithelial cells – Immature

cells appear without nuclei

reflectivity or faint.

2. Bowman's Layer – Acellular hyper

reflective structure subepithelial

nerve plexus may be seen.

In normal cornea, vessels are not

present in epithelium & Bowman's

layer.

1. At the superficial epithelium, poorly demarcated roundish cells demonstrate hyperreflective nuclei (arrows) on confocal microscopy (original magnification 210).

Bowman’s layer is an acellular hyperreflective structure, where subepithelial nerve

plexus (arrows) may be identified easily (original magnification 210).

Basal epithelial cells appear hyporeflective and have hyperreflective borders

(original magnification 250).

3. Stroma – Hyper reflective keratocyte nuclei are scattered against a dark background.

Ketatocyte density is maximum (800 cells/mm2) immediately under Bowman's membrane and decreased (65 cells/mm2)sharply towards posterior cornea.

Nerves – Which may present branching images, are found in the stroma and are thicker than at the subepithelial level.

In normal eyes vessles are not found in stroma.

4. Descemet's Membrane – Acellular layer of moderate reflectivity : however nerve plexus is absent.

This layer is rather difficult to see under normal circumstances.

In the stroma, hyperreflective keratocyte nuclei (arrows) are scattered against a dark

background (original magnification 250).

5. Endothelium – Regular, hexaogonal, hyperreflective shape surrounded by hyporeflective borders and the absence of any nuclei reflection.Endothelial count with confocal & specular microscopy are comparable.Negative correlation b/w age & endothelial count.No vessels or nerves are present in this layer.The physiologic responses of the corneal to different stimuli may by analyzed by confocal microscopy.Activated keratocytes presenting as cells with increased reflectivity in the stroma, are seen when cellular metabolic activity is increased.Scar tissue, infection, inflammation – Hyperreflective images.

Vessels – Lumen – Hyporeflective Wall – Hyperreflective

Stroma – Hyper reflective keratocyte nuclei are scattered

against a dark background.

Ketatocyte density is maximum (800 cells/mm2)

immediately under Bowman's membrane and decreased

(65 cells/mm2)sharply towards posterior cornea.

Nerves – Which may present branching images, are

found in the stroma and are thicker than at the

subepithelial level.

In normal eyes vessles are not found in stroma.

Stromal scar appears hyperreflective on confocal microscopy (original

magnification 210).

Vessel lumen appears hyporeflective on confocal microscopy,

whereas vessel wall demonstrates well-delineated

hyperreflectivity (arrows) on each side of the lumen (original

magnification 210).

Cotton candy-like hyperreflective material may be found at the subepithelial level in

amyloidosis with corneal deposits (original magnification 210).

Cystic epithelial lesions are demonstrated in a patient with Fuchs’ dystrophy.

Horizontal field width 5 610 mm. (Reprinted from Ophthalmology

Hernandez- Quintela et al82, Copyright 1998, with permission of American

Academy of Ophthalmology.)

Hyperreflective deposits (arrows) are found in area devoid of epithelium in an

eye treated with topical ciprofloxacin (original magnification 240).

(Reprinted from Essepian et al60 with permission of Cornea.)

Confocal microscopy in a case of corneal lattice dystrophy disclosed hyperreflective, linear, and

branching images (black arrows) in the stroma. The white arrows indicate some hyperreflective

keratocytes (original magnification 210). (Reprinted from Graefes Arch Clin Exp Ophthalmol. Chiou

AG, Beuerman RW, Kaufman SC, Kaufman HE: Confocal microscopy in lattice corneal dystrophy.

237:697--701, 1999, with kind permission of Springer Science and Business Media.)

Highly reflective and irregular material (*) is found at the level of Bowman’s

layer region and anterior stroma in Reis-Bu¨ckler dystrophy. Bar 5 50 mm.

(Reprinted from Ophthalmology Werner et al,222 Copyright 1999 with

permission of American Academy of Ophthalmology.)

In granular dystrophy, reflective diffuse deposits (arrows) may be found

in the stroma. Bar 5 50 mm. (Reprinted from Ophthalmology Werner

et al222 Copyright 1999, with permission of American Academy of

Ophthalmology.)

Stromal intracellular hyperreflective material is the hallmark of fleck

dystrophy. In the mid-stroma a cluster of hyperreflective dots is enclosed in

a cyst-like structure. Calibration bar 5 50 mm. (Reprinted from Frueh and

Bo¨hnke62 with permission of Cornea.)

Stromal crystalline accumulation is associated with Schnyder’s dystrophy

and is readily revealed by confocal microscopy (image is 250 170 mm).

(Reprinted from Ophthalmology Vesaluoma et al215 Copyright 1999, with

permission of American Academy of Ophthalmology.)

Subbasal nerve plexus presenting beads in a case of cornea plana (image is

390 290 mm). (Reprinted from Vesaluoma et al217 with permission of

Investigative Ophthalmology and Visual Science.)

Confocal microscopy (original magnification 210). Areas of highly abnormal cells characterized by marked

epithelial-like appearance and loss of regularity in size and shape were found. Hyperreflective structures

were found within and adjacent to these abnormal areas. Relatively normal appearing endothelial cells

were also detected (upper right corner of the photograph). (Reprinted from Chiou et al33 with permission of

British Journal of Ophthalmology.)

Cornea guttae (arrows) appear as roundish hyporeflective images with an

occasional central highlight at the level of the endothelium. Cell

pleomorphism shown is this picture is also a common feature (original

magnification 210).

Epithelial cells at the level of the corneal endothelium is pathognomonic of

epithelial downgrowth (original magnification 230). (Reprinted from Journal

of Cataract and Refractive Surgery Chiou et al36 Copyright 1999, with

permission of ASCRS & ESCRS.)

Confocal microscopy in a case of fibrous retrocorneal membrane after

penetrating keratoplasty (original magnification 210). At the endothelial cell

layer, a hyperreflective fibrous-appearing layer was demonstrated at the

periphery of the graft. (Reprinted from Chiou et al30 with permission of

Cornea.)

Subepithelial extracellular deposits (D) may be found 3 months after

photorefractive keratectomy at the epithelial-stromal interface (image is 382

382 mm). N 5 stromal nerve. (Reprinted from Ophthalmology, Corbett et al40

Copyright 1996 with permission of American Academy of Ophthalmology.)

Linear structures may be detected in the stroma years after photorefractive

keratectomy. Bar indicates 50 mm. (Reprinted from Archives of

Ophthalmology, Frueh et al63, Copyright 1998 with permission of

American Medical Association.)

Confocal microscopy of the cornea performed 10 days after the surgery.

Hyperreflective interface debris could be detected (arrows). A superficial

stromal nerve was also visualized (arrow heads) (original magnification

210).

Acanthamoeba cysts (black arrows) and trophozoite (white arrows) may be

visualized by confocal microscopy (marker 5 100 mm). (Reprinted from

Pfister et al177 with permission of Cornea.)

At a depth of 115 mm from the anterior corneal surface, linear and

branching structures are detected in a case of aspergillus keratitis

(original magnification 135).

Confocal microscopy can also resolve the double-walled structure of the

acanthamoeba ectocyst surrounding theendocyst (white arrow). Several

pear-shaped cysts are shown by the black arrows (marker 5 100 mm).

(Reprinted from Pfister et al177 with permission of American Journal of

Ophthalmology.)

Acanthamoeba radial keratoneuritis presents as swollen nerve (arrowheads). A bright

irregular body on the nerve (black arrow) is consistent with an acanthamoeba

trophozoite. A stromal keratocytes is shown by the white arrow. Inset: normal human

stromal nerve (arrowheads) and stromal keratocyte (white arrow). Arrows 5

keratocytes (marker 5 100 mm). (Reprinted from Pfister DR et al177 with permission

of American Journal of Ophthalmology.)

Refractive objects showing eight consecutive spots arranged in a straight

row in a case of Borrelia keratitis (horizontal field width 5 275 mm).

(Reprinted from Linna et al125 with permission of Cornea.)

Microsporidial keratitis has been reported to demonstrate images of epithelial cells of the

corneal surface containing intracellular spores upon confocal microscopic examination. An

enlargement of the two cells outlined shows numerous small, discrete, high-contrast,

intracellular microsporidial spores (white arrows), and an aggregate of tightly packed

microsporidial spores (gray arrows) (marker 5 100 mm). (Reprinted from Shah et al194 with

permission of American Journal of Ophthalmology.)

Degenerated fine stromal hyperreflective dots may be detected in long-term

contact lens wearers. Bar550 mm. (Reprinted from Ophthalmology, Bohnke

and Masters,11 Copyright 1997 with permission of American Academy of

Ophthalmology.)

Thanks