British Journal ofOphthalmology, 1982, 66, 309-316

Atypical band-shaped calcific keratopathy withkeratocyte changesANTHONY J. DARK AND JOHN PROCTOR

From the Departments of Ophthalmology and Human Biology, Sheffield University

SUMMARY The clinical features of a patient with atypical band keratopathy are described.Histochemical and electron probe analyses indicate that the granular deposits in Bowman's layercontain calcium and phosphate. An unusual feature in this patient was the presence of severekeratocyte degeneration; its possible role in the pathogenesis of this condition is discussed.Exfoliation of the calcified Bowman's layer appears to have been the basis for severe attacks ofrecurrent ocular pain.

The mechanisms of ectopic comeal calcification,although the subject of much experimental workwhich has been reviewed by Obenberger and hiscoworkers,' 2 remain largely speculative. In mostpatients the clinical appearance of a horizontal band-shaped opacification of the Bowman layer isassociated with a variety of local and/or systemicdiseases. Calcification is often referred to as'secondary' in these cases to provide distinction fromthe much rarer hereditary or dystrophic variety inwhich no such associations are found.

Correspondence to Mr A. J. Dark, FRCS, Department ofOphthalmology, Royal Hallamshire Hospital, Glossop Road,Sheffield 510 2JF.

Fig. I Right eve. Annular opacitv in the pupillarv zone ofcornea. Jagged inner edge outlines central clear area.

We recently observed a patient with atypicalcalcific opacities which gave rise to recurrent attacksof irritation and pain. Biopsy indicated that thesefeatures were associated with histological and ultra-structural changes of the keratocytes. Since some ofthe clinical and cytological features appear to beunique, we have compiled the following clinico-pathological report.

Case history

A 41-year-old male Nigenran academic of the Yorubatribe was first seen in January 1980. He complained ofrecurrent episodes of redness and photobia with

Fig. 2 Left eye. Discoid opacitv with fissures. A grevishreticulum within the opacity imparts a mosaic appearance.

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Fig. 3 Corneal opacity in right eye is seen as blacksilhouette againstfundus glow.

severe pain which had affected the left eye almostweekly for the previous 4 months. Similaroccurrences had affected the right eye for a period of31/2 years, but they had ceased abruptly 6 months ago.No similar eye disorder was known to have occurredin any other members of his family. The patient'shealth had been unremarkable until the start of theseocular symptoms. There was no history to suggestcorneal exposure to chemical factors or of vitamin Dintoxication.

Fig. 4 Left eye. After removal of2 mm biopsy (arrowsmark site) and debridement ofmost ofthe remainder, only a

narrow rim ofthe original opacity remains.

Visual acuity was 6/9, N5 in each eye when mildmixed astigmatism had been corrected. Subepithelialdiscoid greyish-white lesions about 3-8 mm indiameter were present in the central zone of bothcorneas. On the right side the lesion was annular inform, a central clear area being outlined by a jaggedinner edge (Fig. 1). The left corneal opacity, whichwas somewhat larger, appeared as a grey disc in whichwhite lines formed a mosaic pattern (Fig. 2). Anirregular fissure running vertically almost divided thedisc. The temporal edge of the fissure was slightlyelevated, and the overlying epithelium stained withboth fluorescein and rose Bengal. At high magnifica-tion the lesions had a granular structure. Ophthal-moscopically the opacities were silhouetted in the redfundus glow (Fig. 3). Tear and meibomian secretionsand ocular tensions were normal. Sensation over thecorneal opacities was slightly diminished. No otherocular abnormalities were noted.The attacks of ocular pain affecting the left eye

became more severe and incapacitating. Since itseemed likely that the quiescent state of the rightcornea had been achieved by a natural exfoliation ofthe central part of the lesion over several years,biopsy and debridement of the left corneal lesionwere undertaken under retrobulbar anaesthesia. Theopaque material was removed with ease. Within 48hours the patient was symptom-free and the leftvision corrected to 6/9+. The slit-lamp appearancesfollowing this procedure (Fig. 4) now closelyresembled those seen on the right side. No furtherattacks have occurred over the past 9 months, visionand slit-lamp appearances remaining constant.

GENERAL INVESTIGATIONSAll studies to determine the aetiology of these lesionswere uninformative. Fasting levels of serum calcium,phosphate, alkaline phosphatase, glucose, and lipidswere normal on 3 occasions. The Kveim test andother investigations for sarcoid were negative. Therewas no radiological evidence of ectopic calcification.

METHODSA 2 mm trephine was used to biopsy the left cornealopacity. The disc, which included the anterior 1/6thof stroma, was placed in ice-cold 3% glutaraldehyde.Some of the material obtained by superficiallyscraping away the remainder of the opacity was fixedlikewise. After dehydration the specimens wereosmified and embedded in methacrylate. Afterstaining with uranyl acetate and lead citrate, sectionswere examined in a Phillips 301 electron microscope.Unstained sections were subjected to elementalanalysis with an Edax analysing electron probe.Orientational sections cut at 05 ,um were stained in1% toluidine blue for light microscopy. The

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Atypical band-shaped calcific keratopathy with keratocyte changes

Fig. 5 Corneal epithelium showsmild dysplasia with hydrops ofsome superficial squames. TheBowman layer contains intenselybasophilic granules. Some ofthesuperficial keratocytes arevacuolated. (Toluidine blue,x580).

remainder of the material was fixed in neutralformalin and subsequently embedded in JB4compound (Polysciences). Sections from thismaterial were subjected to routine histological stains,as well as certain histochemical procedures which willbe indicated alongside the results obtained.

Observations. The trephined biopsy specimen andcuretted material consisted of epithelium andsuperficial corneal stroma.

.- .... .~I

Fig. 6 Fissure in Bowman's layer, which may be traumatic(surgical) artefact, illustrates its friability. (Toluidine blue,x 467).

LIGHT MICROSCOPYIn toluidine blue preparations the epithelium showedmild dysplasic changes (Fig. 5). Surface squameswere often swollen and vacuolated. Nests of intenselybasophilic cells were seen in the intermediate layers.In many places the basal cells were separated fromBowman's layer by a stratum of vacuolatedamorphous material. Elsewhere the basal cells laydirectly on the Bowman layer or on the basementmembrane. The Bowman layer was studded withmyriads of intensely basophilic spherules, the largerof which were about 1 gm in diameter. Occasionalspherules were found in the adjacent stroma. Bothspherules and the basement membrane, wherepresent, gave positive reactions with the von Kossaand alizarin red methods for phosphate and calciumrespectively. The friability of Bowman's layer wasindicated by occasional vertical cracks withdisplacement of the fragments (Fig. 6). Beneath theBowman layer were patches of vacuolatedamorphous material similar to those on its anteriorsurface. Throughout the stroma keratocytes showeda variety of changes which are described in infra-structural detail below.

ELECTRON MICROSCOPYEpithelial cells which appeared dark blue in toluidineblue preparations were electron-dense and contained.numerous tonofibrils. In other areas paler cells wereundergoing hydropic degenerative changes, shown ascytoplasmic vesicles which were often prominentlyaligned under the cell membrane (Fig. 7). Similar

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46

Fig. 7 A band ofamorphousgranular debris isfound belowBowman's layer. A degeneratedkeratocvte (arrows) distended withclear vacuoles is seen below.(Electron micrograph, x 1470).

changes were seen in the basal cells. The basementmembrane-hemidesmosome system was absent insome sections, the basal cells lying either directly onthe Bowman's layer or more commonly on the layerof vacuolated debris, which contained a variety offibrillar materials, including normal and 'curly'collagen (Fig. 8) as well as a variety of organelles.Similar material was also patchily distributed belowBowman's layer (Fig. 7). When the basal cells laydirectly on Bowman's layer, tufts of fibrils extendedupwards, producing a sawtooth appearance in the cellmembrane (Fig. 7).Bowman's layer, which was present in all sections

examined, contained numerous electron-dense

spherules which often showed a paler core and adenser rim, although sometimes the relative densitieswere reversed. The spherules consisted of tightlvpacked electron-dense granules which replaced orobliterated the random collagenous architecture ofthe Bowman's layer. Elemental analysis of thespherules by electron probe indicated that calciumand phosphorus were present in considerablequantities (Figs. 9A, B).

Degenerative changes in the keratocytes wereespecially severe in the vicinity of Bowman's layer;several stages of this process are suggested by theirvarying appearances (Figs. IOA, B, C). Firstly, thecytoplasm distends with vacuoles and electron-dense

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Atvpical band-shaped calcific keratopathv with keratocvte changes

E

Fiv. HeavilY calcifiedportionofBowman's laver showvs concentricring formations in some spherules.The hasement membrane (alsocalcified) is thickened andelectron-(lense. A laver containing'various preslumed collagen fibril.vseparates the hasemnent memnbranefromn the basal epithelial cell, E.(Electron micrograph, x 32 500).

Fig. 9A Elemnental anialvsis of one of

the spherlides in Bowmtan's layver shows

high concentraition ofcalcium (Ca) and

phosphorus (P) within the spherule, to

be compared with Fig. which

indicates lower concentraitions of these

elements in the adjacent stroma.

Copper (Cui) origjinating from the grid

holding the section is the mnaimn

component in both (A and

* K

.7t

lrv I

EtW

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Anthonv J. Dark and John Proctor

Fig. IOA Keratocvte showsnumerous vacuioles and granlularincllusions. (Electron micrograph.x 14255).

ei~~~~~I

inclusions. Secondlv, the cell membrane dis-integrates. Finally the cvtoplasm becomesincreasingly electron-lucent and occupied bvdegenerating organelles. the nucleus becomingpyknotic or disintegrating. Ultimatelv the spacesoccupied by dead keratocytes become emptv apartfrom tonofibrils and debris. 'Ghost' cells of this lattertype were especiallv common beneath the Bowmanlayer. Patches of vacuolated amorphous debris belowthe Bowman laver were the oniv other abnormalitv inthe stromal collagen.

Discussion

The corneal opacities described in this report do notappear to have been previouslv documented. Asuperficial mosaic opacitv, similar to that in ourpatient's left eye was noted bv Valerio3 in bothcorneas of a young man whose father and other familvmembers were afflicted with the primarv (hereditarv)form of band-shaped calcific opacitv.

Histochemical and electron micro-probe elementalanalysis of our biopsy material has indicated that thedarkly stained spherules found in Bowman's lavercontain calcium and phosphorus in considerablequantities, presumablv as a calcium phosphate. Aninteresting finding is the presence of widespreaddegenerative changes in the keratocvtes.The present case should be differentiated from

noncalcific band-shaped keratopathv in which sub-epithelial yellowish granules are the essential bio-microscopic features. Kloucek4 studied keratectomvspecimens from 2 related patients with the rareprimary form of this disorder. He observed that theyellow granules corresponded with electron-densespherules in the Bowman's layer and containeda nonlipid protein but no calcium. He also notedthat keratocytes in the superficial stroma hadproliferated and undergone a variety of degener-ative changes, similar-if less widespread-thanthose seen in our patient, leading him to consider thatthe spherules in Bowman's layer had been produced

314

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Atypical band-shaped calcific keratopathy with keratocyte changes

-~~~~~~~~~~~~~~~~~~~~a

Fig. IOB Keratocyte distended with electron-lucent vacuoles and granular debris. Nuclear membrane is present butplasmalemma has largely absorbed. (Electron micrograph, x4610).

by these cells, although the possibility that thesechanges were reactive rather than primary wasacknowledged.

It is improbable that keratocyte degeneration seenin our case is merely a sequel to recurrent erosions ofthe epithelium and Bowman's layer, since the mainbiopsy was taken towards the upper periphery of thelesion, where such occurrences are rare. Moreover,Rice and his co-workers' observed only inconstantkeratocyte injury even in long-standing cases of Reis-Bucklers's dystrophy, in spite of recurrent epithelialbreakdown and disintegration of Bowman's layer.Nor does it appear that calcification of the Bowman'slayer would in itself account for such severe changesin the keratocytes, since no gross abnormality of thekeratocytes was noted either by Koseki6 in hiselectron microscopic study of the calcified anteriorstroma in a patient with band keratopathy anduveitis, or by Ticho and his coworkers' in theirexamination under light microscopy of a keratectomyspecimen from a patient with primary calcific band-shaped keratopathy. Artefactual change may beexcluded on the grounds that cytological preservationof the specimen was otherwise adequate and that thechanges were seen throughout the biopsy. Keratocyte

degeneration in this case may have been a response toa precursory unknown noxious stimulus, but there isno other clinical or ultrastructural evidence tosupport this contention. Thus, disease of the cornealkeratocytes of the anterior stroma cannot beexcluded as the primary disorder in our patient. Itmay be further hypothesised that such a breakdownof the superficial keratocyte system, liberatinglysosomal enzymes including phosphatases, couldproduce a local increase in phosphate which,combined with calcium, has impregnated theBowman's layer.

Dehiscence of this layer probably played a role inthe natural exfoliation of the opacity, causingrecurrent erosions. These attacks stopped spon-taneously on the right side, presumably when most ofthe opacity had been eroded, and they have ceased onthe left side following superficial keratectomy. Wefound no ultrastructural correlation with the greyishbars outlining the polygons seen in the opacityclinically. This is in contrast with the observationsmade by Tripathi and Bron,8 who in their case ofsecondary anterior crocodile shagreen of Vogt foundcalcified folds of the Bowman layer, correspondingwith the mosaic.

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Fig. IOC A 'ghost' keratocvte inwhich a few organelles andtonofibrils remain. (Electronmicrograph, x8415).

Mr R. Cousins, the Charles Clifford Dental Hospital. Sheffield. was

responsible for the photomicrographs.This work was supported by a grant from the British National

Committee for the Prevention of Blindness.

References

1 Obenberger J. Cejkova J, Brettschneider 1. Experimental cornealcalcification. A studv of acid mucopolvsaccharides. OphthalmicRes 1970( 1: 175-186.

2 Obenberger J. Ocumpaugh DE. Cubberiv MG. Experimentalcorneal calcification in animals treated with dihvdrotachvsterol.Invest Ophthalmol Visual Sci 1969; 8: 467-74.

3 Valerio M. Di una nova rara forma di distrofia corneale ereditaria.Arch Ottal 1942; 19: 76-93.

4 Kloucek F. Familial hand-shaped keratopathv and spheroidaldegeneration. Clinical and electron microscopic studv. Alhrechtvon Graefv Arch Klin Ophthalmol 1977; 205: 47-59.

5 Rice NSC. Ashton N. Jav B. Blach RK. Reis-Bulcklers' dystrophy.Br J Ophthalmol 1968; 52: 577-603.

6 Koseki T. Electron microscopic findings of juvenile hand-shapedkeratopathy. Folia OphthalmolJpn 1978;29: 19(5-13.

7 Ticho U. Lahav M. Ivrv Y. Familial hand-shaped keratopathv. JPediatr Ophthalmol 1979; 16: 183-5.

8 Tripathi RC. Bron AJ. Secondarv anterior crocodile shagreen ofVogt. Br J Ophthalmol 1975; 59: 59-63.

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