This study suggested that the
severity of conjunctival changes in
CRF patients on regular
hemodialysis is related to the
presence of acute inflammation
but not to the presence or extent
of calcium deposition.

In patients with chronic renal failure (CRF) and on regular hemodialysis, conjunctival degeneration and corneoconjunctival calcifications are common. In these cases, the typical conjunctival changes consist of amorphous, white, crystalline, subepithelial precipitation in interpalpebral conjunctiva, goblet cell loss and squamous metaplasia.^1-5 The corneal changes resembled the limbus girdle of Vogt clinically and histopathologically, and they manifest as band keratopathy at advanced stages.⁴ The mechanisms of ectopic conjunctival and corneal calcification are largely speculative. There has been no consistent relationship to serum levels of calcium, phosphates or alkaline phosphatase.⁴ These changes may be related to the presence of antecedent tissue degeneration, which acts as a more calcifiable matrix and predisposes to calcium salt deposition.²
   The normal conjunctival epithelium is stratified and not keratinized, with goblet cells that produce mucin. The decreasing of number of conjunctival goblet cells, squamous metaplasia and other epithelial changes can be observed only by impression cytology. Metaplasia, which is the change from a mature type of epithelium to another type, refers to the decreased number of mucoproducing cells replaced by squamous cells in the conjunctival epithelium.
    Impression cytology was first introduced in oph­thal­mology in 1977 by Egbert et al.⁶ More recently, Tseng⁷ used impression cytology to study various ocular disorders and correlated their severity with squamous metaplasia and the density of goblet cells. In the course of time, impression cytology has proven useful for the diagnosis of external eye disorders.^8-10
    The aim of the present study was to evaluate the relationship between corneoconjunctival calcification and impression cytology findings in patients with CRF and on regular hemodialysis.

Materials and Methods
The local medical ethics committee approved the study, and all patients in the study gave informed consent. Fifty-seven patients on regular hemodialysis for CRF and 30 normal age- and sex-matched subjects were evaluated. One eye in each patient was studied. The eye having more prominent calcification was selected. Communication problems, insufficient impression cytology specimens, history of ocular surface disorders, current contact lens wear, topical medications within the previous 3 months, previous ocular surgery and trauma were the exclusion criteria. Twelve patients were excluded from the study. Controls were selected from patients who had not any ocular problem except refractive error or presbyopia.
     All patients underwent a full ocular examination. Biomicroscopic examinations were performed using direct and indirect illumination and scleral scatter techniques to study the difference in the incidence of limbal and corneal calcification. Grading of the calcification was done according to the system proposed by Porter and Crombie to establish the degree of calcification by comparing it with drawings depicting 5 stages of calcification intensity.⁵
    Impression cytology specimens were obtained after administration of topical anesthesia with proparacaine 0.5% (Alcaine, Alcon-Couvreur, Puurs, Belgium). Cellulose acetate filter paper (Durapore Membrane Filters, 0.22 µm QVPP, Millipore, Cork, Ireland) was prepared by cutting into 46-mm strips. The strips were gently applied to the eye for 7–10 seconds on the temporal bulbar conjunctiva and then removed with a peeling motion. The filter paper with adherent epithelial cells was immediately placed in a fixative solution prepared by mixing 70% ethyl alcohol, 37% formaldehyde and glacial acetic acid at a 20:1:1 volume ratio. After fixation, the specimens were stained with a combination of periodic acid–Schiff (PAS) stain and Gill’s modified Papanicolaou stain, as previously described; dehydrated in ascending grades of ethanol and then with xylene; and permanently mounted.⁷ The entire filter paper was initially examined  with a 10 objective lens, and cellular details were evaluated using a 40 objective. This change allowed us to analyze our specimens according to a staging system for squamous metaplasia previously described by Nel­son.^8-10 The slides were coded by a pathology technician and evaluated in a masked fashion by the same pathologist.
    Stage 0 is normal, with abundant goblet cells and sheets of small epithelial cells. In stage 1, the goblet cells are decreased, and epithelial cells with reduced nuclear/cytoplasmic ratio (1: 3) begin separating and enlarging. This continues through stage 2, a reduction of goblet cells and larger epithelial cells with a nuclear/cytoplasmic ratio of 1:4 to 1:5. By stage 3 the epithelial cells are markedly enlarged and separated and show early keratinization with folded edges and pyknotic nuclei (nuclear/cytoplasmic ratio of 1:6 or less); goblet cells disappear (Table I and Figure 1). In addition, specimens were examined for the presence or absence of the following cytologic features; PAS-positive mucous aggregates adherent to epithelial cells; long, PAS-positive mucous strands overlying the epithelium (Figure 2); and conjunctival epithelial cells infiltrated by >20 polymorphonuclear leukocytes per high power field (40) (Figure 3).

  

  

Figure 1    Cytologic views of representative features of squamous metaplasia of the conjunctival epithelium. (A) Grade 0, impression cytology from normal subject. Abundant PAS positivity, oval-plump goblet cells (arrow). Sheets of small, round, nonsecretory epithelial cells with nuclear­/cytoplasmic ratio of 1:1 to 1:2. (B) Grade 1, slight loss of goblet cells. (C) Grade 2, goblet cells are reduced. (D) Grade 3, marked goblet cell loss. Note the cellular cohesion and pyknosis (PAS-hematoxylin, 40).

 

Figure 2    Cytology from conjunctiva of CRF patient demonstrating loss of goblet cells, epithelial squamous metaplasia (nuclear/cytoplasmic­ ratio <1:3 and pyknotic nuclei), and PAS-positive, coiled mucous strands (PAS-hematoxylin, 40).

 

Figure 3    Acute inflammatory cells intercalated with epithelial cells on the conjunctiva of a CRF patient (PAS-hematoxylin, 40).

 

Results
The patients included 24 males and 21 females ranging in age from 21 to 74 years (mean, 46.6±16.9); the controls included 18 males and 12 females, ranging in age from 20 to 65 (mean, 43.9±18.4). Both groups were similar regarding age and sex characteristics (p>0.05). The duration of CRF ranged from 7 to 152 months (mean, 45.4±34.9), and the hemodialysis duration was 1–146 months (mean, 371±34.4).
     The results of cytologic analysis are reported in Table II. In the study group, impression cytology showed grade 0 changes in 4 (9%), grade 1 in 23 (51%), grade 2 in 14 (31%) and grade 3 in 4 cases (9%). In the control group there were grade 0 changes in 21 (70%) and grade 1 changes in 9 cases (30%). The difference between the subjects and controls was statistically significant (p<0.001).
    Corneoconjunctival calcification grades are presented in Table II. In the CRF group there were grade 0 calcifications in 9 (20%), grade 1 in 11 (25%), grade 2 in 15 (33%) and grade ≥3 and over in 10 subjects (22%). In the control group, 14 subjects (47%) had grade 0 changes, 9 (30%) grade 1 changes and 7 (23%) grade 2 changes. The difference between the patients and controls with respect to calcification grades was statistically significant (p=0.01).
    No correlation between squamous metaplasia of the conjunctiva and corneoconjunctival calcification was found (r_s=.21, p=0.12).
    Acute inflammatory cells that infiltrated epithelial cells were observed in 11 patients (25%) with CRF. A strong correlation was noted between the inflammatory infiltrate and extent of squamous metaplasia of the conjunctiva in CRF specimens (r_s=.46, p=0.001). All those patients had acute ocular inflammation symptoms, such as redness, burning and foreign body sensation to different degrees. In addition, mucous strands were observed in 15 patients (33%) in the CRF group, while none were found in the controls. There was a strong correlation between the presence of squamous metaplasia and acute inflammatory cells with PAS-positive mucous strands (p=0.001) but no correlation with corneoconjunctival calcification. There was no correlation between PAS-positive mucous aggregates and conjunctival epithelial changes.
    The degree of ocular involvement (squamous metaplasia of the conjunctiva or calcium deposits) did not correlate with the patient’s sex (p=0.41, 0.52) or age (p=0.87, 0.91), duration of renal disease (p=0.71, 0.88) or duration of hemodialysis (p=0.20, 0.58) in CRF patients.

References

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From the Departments of Pathology, Ophthalmology and Internal Medicine, School of Medicine, Kahramanmaras Sutcu Imam University, and Department of Internal Medicine, Kahramanmaras State Hospital, Kahramanmaras, Turkey.

Dr. Bakaris is Assistant Professor, Department of Pathology, School of Medicine, Kahramanmaras Sutcu Imam University.

Dr. Isik is Physician, Department of Internal Medicine, Kahramanmaras State Hospital, Internal Medicine Department, Kahramanmaras, Turkey.

Supported by the Nefro Dialysis Center.

Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

Accepted for publication June 24, 2004.