Microsoft Word - Aneequallah Baig 19 Original Article Epitheliotrophic Effect of Autologous Serum in Persistent Corneal Epithelial Defects Aneeq Ullah Baig Mirza, Naheed Ghani, Abdul Bari Khan Pak J Ophthalmol 2008, Vol. 24 No. 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . See end of article for authors affiliations …..……………………….. Correspondence to: Aneeq Ullah Baig Mirza Department of Ophthalmology IIMCT/ Railway Teaching Hospital, Rawalpindi Received for publication Purpose: To evaluate the efficacy and complications of autologous serum in cases of persistent corneal epithelial defect (PED). Material and Methods: Patients with PED unresponsive to conventional lubricant treatment were included in this clinical, prospective study which was conducted at IIMCT and Railways teaching Hospital. Patients were examined on day 1 and appropriate treatment including lubricant therapy was instituted. Subsequently, they were examined after 3 weeks and started on 20% autologous serum eye drops in cases of PED. All the other previously used lubricant therapy was stopped. Then, they were examined every 2 weeks for the next three visits. At each visit, in addition to routine eye examination some additional tests were performed i.e. Slitlamp examination for corneal epithelial defects, marginal tear strip evaluation, Schirmer’s test without anesthesia, tear film breakup time, fluorescein and rose Bengal staining and corneal sensitivity. The healing was defined as effective if occurred within 2 weeks of initiating serum therapy, partially effective if occurred within one month and ineffective if didn’t occur within one month. Results: A total of 17 eyes in 10 patients were studied. The local spectrum of indications was severe vernal limbitis with keratopathy (6 eyes, 35.3%), keratoconjunctivitis sicca (7 eyes, 41.1%), drug toxicity (1 eye, 5.9%), lime instillation (2 eyes, 11.8%) and trophic ulcer (1 eye, 5.9%). Healing in 9 eyes occurred within 2 weeks and in 3 eyes within further 2 weeks. In 5 eyes, healing didn’t occur within one month. All the patients were able to complete the follow-up of the study. Accordingly, healing was declared as effective in 9 out of 17 eyes (52.9%), partially effective in 3 out of 17 eyes (17.7%) and ineffective in 5 out of 17 eyes (29.4%). There were no adverse effects except for one red eye without discharge. Response rate was excellent in all except those with severe keratoconjunctivitis sicca (KCS) and below-normal Schirmer’s test value before serum therapy. The mean duration of PED before initiating serum therapy was compared between the effective and ineffective groups. Independent sample t-test was applied and the difference was found to be highly insignificant (p value 0.537). Conclusion: Autologous serum was considered effective in majority of the cases with PED recalcitrant to conventional lubricant therapy due to the presence of essential factors. Healing depended more upon the etiology than the duration of 20 March’ 2007 …..……………………….. PED. Due to scarcity of literature on serum therapy, further studies are required to establish the efficacy 21 orneal epithelial surface may be damaged in a variety of conditions like KCS, mechanical and chemical trauma, corneal infections, neurotrophic keratopathy, corneal dystrophies and long-term topical steroid therapy. Moreover, there may be defective regeneration of healthy corneal epithelium in cases of limbal stem cell deficiency and vernal limbitis. Various pharmacological lubricant preparations used to treat PED include hydroxypropylmethylcellu- lose (HPMC), polyvinyl alcohol, povidine and sodium hyaluronate. Recently, autologous serum1,2 and umbilical cord serum therapy3 have shown promising results in the treatment of PED. Surgical options for the management of dry eyes and PED include punctal occlusion, lateral and central tarsorrhaphy and amniotic membrane transplantation 4,5. Serum is the fluid component of blood minus its clotting factors and cellular components. The natural tears have optical, mechanical, antimicrobial and nutritional properties. They contain epidermal growth factor (EGF), fibronectin and vitamin A which help in proliferation, migration and differentiation of corneal epithelial cells6. EGF may also help in reepithelialization due to its anti-apoptotic properties7,8 . Serum also contains lysozyme, IgG and complement which may reduce the risk of infection. Autologous serum eye drops are non-allergenic with properties similar to the natural tears. They have also been used for cases of keratoconjunctivitis sicca9,10. Vitamin A is found in higher concentrations in serum as compared to tears. It may help in decreasing the squamous metaplasia in cases of KCS11. Poon et al conducted a clinical pilot study to compare the in-vitro toxicity of serum drops with unpreserved hypromellose (hydroxypropylmethyl- cellulose 0.3%) on corneal epithelial cell cultures12. Serum drops were found to have reduced toxicity compared with unpreserved hypromellose. The morphology and ATP levels of cultured cells exposed to serum were maintained better as compared to hypromellose. Both are found to be well established parameters of viability of the cells and used for evaluation of cellular toxicity13,14. The present study was conducted to evaluate the efficacy of autologous serum eye drops in cases of PED resistant to conventional lubricant therapy. MATERIAL AND METHODS All the patients with PED not responding to conventional lubricant dry eye therapy were included in this study. The study design was prospective. The case recording was done between Sept’ 2004 and Aug’ 2006. At the initial visit, detailed history was taken, specifically asking for previous topical treatment and duration. Eye examination included visual acuity, slit- lamp examination for corneal epithelial defects, marginal tear strip evaluation, Schirmer’s test without anesthesia, tear film breakup time (BUT), fluorescein and rose Bengal staining and corneal sensitivity. Appropriate lubricant treatment for dry eyes was instituted. The patients were subsequently examined after three weeks and started on autologous serum eye drops, if the corneal epithelial defects had not healed. At the same visit, previously used dry eye treatment was stopped to monitor the isolated effect of autologous serum. The protocol for autologous serum eye drops preparation was strictly followed in each case. In collaboration with pathology department of the hospital, patients’ blood was drawn under sterile conditions. It was centrifuged at 1500 revolutions per minute for five minutes2. Once the serum was separated, it was diluted with 0.9% normal saline to make a 20% preparation. This 20% preparation of serum was placed in multiple ultraviolet protected bottles under absolute sterile conditions. No preservative was added to the serum. In order to ensure sterility, patients were instructed to place all the bottles in the freezer compartment at -4°C. The bottle to be used the next day was to be put in the refrigerator (lower) compartment at +4°C, one night earlier. The frequency of instillation was adjusted at 2, 6 or 8 hourly, depending upon the severity of PED. Each bottle was to be discarded at the end of the day, in order to avoid any contamination. Then the patients were examined every two weeks for the next three visits. At each visit, the same protocol for eye examination was maintained as the initial one. The healing was graded as effective, partially effective or ineffective. If healing of all epithelial lesions occurred within two weeks of initiating serum therapy, it was declared effective. Healing between 2-4 weeks was declared partially effective and no healing within one month meant ineffective treatment. Mean duration of PED before serum therapy was compared between effective and ineffective groups and independent sample t-test applied. C 22 RESULTS This study comprised of 17 eyes belonging to 10 patients. All had persistent corneal epithelial defects in spite of previous lubricant therapy. There were 7 males and 3 females in the study. The ages ranged between 6 and 72 years (mean 34.2 years). All the patients aged 30 years or above had PED secondary to severe dry eyes or topical drug toxicity. In the patients below 30 years of age, PED was seen as a result of severe vernal limbitis with keratopathy, lime burns and trophic ulcer (Table 1). Table 1: Diagnosis in various age groups Age (Yrs) No. of eyes Diagnosis 12 2 Vernal limbitis with keratopathy 72 1 KCS 58 1 Drug toxicity 70 2 KCS 22 2 Lime instillation 6 1 Trophic ulcer 55 2 KCS 6 2 Vernal limbitis with keratopathy 11 2 Vernal limbitis with keratopathy 30 2 KCS The patients with KCS and trophic ulcer were already on artificial tears eye drops, with non-healing corneal epithelium, at the time of inclusion in the study. After a further 3 weeks trial of topical lubricant treatment and persistence of epithelial defects, all the eyes were put on autologous serum therapy. In nine eyes, total healing of corneal epithelium was seen within two weeks. Another three eyes healed in the following two weeks. Five eyes didn’t heal within one month. No patient skipped follow up visit. Therefore, healing was declared effective in 9 (52.9%), partially effective in 3 (17.7%) and ineffective in 5 (29.4%) out of 17 eyes, Table 2 and Fig. I. No adverse effects were noted except for one red eye without discharge, seen three days after initiation of serum therapy. Table 2: Percentage of healing pattern Healing pattern No. of eyes n (%) Effective 9 (52.9) Partially effective 3 (17.7) Ineffective 5 (29.4) Fig. I: Healing pattern percentage Results were better with good Schirmer’s test value. However, two eyes with total absence of tears (Schirmer’s value 0) also showed effective results. All the eyes with ineffective healing had below-normal Schirmer’s test value before serum therapy (Table 3). Response rate was excellent in all except those with KCS (Table 4). The mean duration of PED before initiating serum therapy was 106.2 days. In the effective group, it was 94.8 ± 67.1 days, while in the ineffective group, it was 117.6 ± 57.4 days. Independent sample t-test was applied between the effective and ineffective groups and the difference in the mean duration of PED before initiating serum therapy was found to be highly insignificant (p value 0.537). DISCUSSION Corneal epithelium may be damaged in a variety of clinical situations. Some of these which may result in non-healing epithelial defects include KCS, neurotrophic keratitis, exposure keratopathy, limbal stem cell failure, post-infectious corneal ulcers, topical drug toxicity, alkali burns, corneal dystrophies and diabetes mellitus15. Effective Partially effective Ineffective 23 Table 3: Summary of individual cases No. No. of eyes Duration of PED (days) Diagnosis Schirmer’s test value in mm Treatment (Serum eye drops) Healing Time (days) Effectivity Right Left 1 2 98 Vernal limbitis 26 29 QID 10 Both eyes Effective 2 1 112 KCS 0 - QID 14 R eye Effective 3 1 14 Drug toxicity - 0 TDS 7 L eye Effective 4 2 28 KCS 0 0 QID 28 R eye 60 L eye R-Partially effective L-Ineffective 5 2 14 Lime instillation 25 25 2 hourly 2 Both eyes Effective 6 1 168 Neurotrophic keratopathy 21 - QID 3 R eye Effective 7 2 168 KCS 7 0 2 hourly None Ineffective 8 2 168 Vernal limbitis 30 35 2 hourly 12 Both eyes Effective 9 2 168 Vernal limbitis 30 34 2 hourly 21 Both eyes Partially effective 10 2 112 KCS 0 0 2 hourly None Ineffective R = Right, L = Left Table 4. Response rate in different pathological conditions Diagnosis No. of eyes Effective and partially effective/Total Vernal limbitis with keratopathy 6 6/6 Severe KCS 7 2/7 Drug toxicity 1 1/1 Lime instillation 2 2/2 Neurotrophic keratopathy 1 1/1 Apart from persistent ocular discomfort, PED pose a potential threat to vision. Management of PED includes removal of etiological factors combined with promotion of epithelial healing. Any lid abnormality which may be responsible for PED such as chronic blepharitis, entropion, trichiasis or lagophthalmos should be corrected. Various preparations of ocular lubricants in the form of eye drops or gels are prescribed to promote epithelial healing. Non-healing lesions require additional strategies in the form of conservation of existing tears (punctal occlusion, tarsorhaphy), prevention of mechanical trauma (bandage contact lens, tarsorhaphy) and promotion of epithelial healing by using autologous serum eye drops and amniotic membrane4,5 or limbal stem cell trasplantation16,17. Seitz et al18 have recommended the use of autologous serum for PED secondary to neurotrophic keratopathy. Similarly, Das et al19 have recommended the use of autologous serum for delayed epithelial healing in eyes with lattice corneal dystrophy, undergoing phototherapeutic keratectomy. In our study, there were 7 males and 3 females. The mean age was 34.2 years. The factors responsible for PED were KCS, severe vernal limbitis with keratopathy, topical drug toxicity, alkali burns and neurotrophic keratopathy. The commonest cause of PED in older age group was KCS. The patients with dry eyes and neurotrophic keratopathy were already on artificial tears treatment at the time of presentation. The mean duration of PED before initiating serum 24 treatment was 106.2 days. In the effective and ineffective groups, it was 94.9 and 117.6 days respectively. Independent sample t-test depicted the difference to be highly insignificant (0.537). In 9 eyes out of 17, PED healed within 2 weeks of initiating serum therapy and the treatment was declared effective. Out of these, 2 had a Schirmer’s test value of 0 before serum therapy. Five eyes didn’t heal even after one month and were included in the ineffective group. Four out of these had a pre-serum Schirmer’s test value of 0. Results tend to be poorer in cases with total absence of tears. All the cases healed within one month except those with KCS. Only two eyes with KCS healed within one month out of a total of 7 (Table 4). In the study of autologous serum for PED by AL Young et al2, there were ten patients with a gender ratio of 7M : 3F which resembled our study. The mean age was 36.8 years (range 17-73). Treatment was effective in 6 eyes out of 10(60%) as compared to 52.9% in our study. Case series of Tsubota et al20 depicted healing of 43.8% within 2 weeks. In AL Young series, 2 eyes out of 10 (20%) did not heal after one month while another 20% defaulted follow-up. In our study, the treatment was in-effective (poor healing after one month) in 29.4% of eyes and there were no defaulters. No adverse effects were reported in their study as compared to a single case of red eye in ours. There was no significant difference in the mean duration of PED before initiating serum therapy in effective and ineffective groups. However, healing depended more upon the etiology in our study (Table 4). Patients with KCS exhibited poorer healing. AL Young et al2 observed that delayed onset of autologous serum treatment might be associated with tendency of poorer healing. In the prospective study of Poon et al12, success was defined as closure of epithelial defects beyond one month. Seven eyes out of 15 healed within one month (46.7%) as compared to 70.6% in ours. A concentration of 50 or 100% serum was used in their study. In our cases, 20% serum eye drops were used. In their study, three patients developed microbial infections that required cessation of serum therapy. Alvarado Valero et al21 studied the effect of 20% autologous serum on PED and squamous metaplasia in 17 eyes. The epithelial defects healed within 2 weeks in 6 eyes (35.2%). This figure was much less than our study (52.9%) with the same concentration of serum. The duration of PED before serum therapy was 36 days. Using impression cytology technique, they observed involution of squamous metaplasia in 6 of 7 eyes, 28 days after initiation of serum eye drops. de Souza et al22 studied autologous serum therapy for PED in 70 eyes. Out of these, 45 had corneal epithelial defects secondary to penetrating keratoplasty. A complete corneal re-epithelialization was achieved in 57 of 70 eyes (81%) after 3 to 45 (mean 15±12) days. They also concluded that eyes with accompanying deep stromal defects were not good candidates. Tears contain nourishing, antimicrobial, mecha- nical and optical properties6. Serum contains essential components in comparable concentrations to tears. These include EGF (epidermal growth factor), TGFβ (transforming growth factor β), PDGF-AB (platelet derived growth factor), neuropeptides (Substance P), insulin-like growth factor, fibronectin and vitamin A. The growth factors, fibronectin and vitamins support proliferation, migration and differentiation of corneal and conjunctival epithelium. The TGF-β family are fibrogenic cytokines responsible for fibroblast activation in wound healing23. The natural substitutes used as tears are superior to artificial tears because their pH, osmolality and biomechanical properties resemble natural tears. Secondly, they contain essential nutrients, like growth factors and vitamins. Thirdly, bacteriostatic components are present such as IgG, lysozyme and complement. Fourthly, there are no preservatives in natural substitutes24. Nistor and Nistor25 observed improvement in fluorescein and rose Bengal scores, non-allergenicity and non- immunogenicity with the use of autologous serum for PED. Autologous serum can induce faster epithelial healing than artificial tears, which leads to a decrease in keratocyte apoptosis and migration of fibroblasts and myofibroblasts in the wound site, a decrease in migration of inflammatory cells and consequently, inhibition of cytokine release26. This could help in improving the long-term refractive results in eyes undergoing LASIK. The cellular morphology and ATP levels are better preserved in the cells exposed to serum drops as compared to unpreserved hypro- mellose12. In the study of Schrader et al27 combination of serum eye drops and hydrogel bandage contact lenses were used in cases of PED, which had not responded well to previous amniotic membrane transplantation or keratoplasty. Five of six eyes healed after a treatment period of 14.2± 8.9 days. In one eye, the PED reduced in size and took 90 days to resolve 25 completely. Random control clinical trial between autologous serum and umbilical cord serum for PED has shown that umbilical cord serum leads to faster healing of PED compared to autologous serum3. Liu et al24 observed that using a longer clotting time resulted in an increased concentration of all the epitheliotrophic factors in the serum. Moreover, the density of epithelial microvilli was greater when a longer clotting time was adopted. They concluded that clotting time, centrifugation and diluents have a significant impact on the composition and epitheliotrophic effects of serum. A clotting time of ≥120 min, a sharp centrifugation (3000xg for 15 min) and dilution with BSS (balanced salt solution) enhance the ability of serum to help proliferation, migration and differentiation of corneal epithelial cells. Sauer et al28 have concluded that if serum drops are applied by trained personnel, the absence of contamination can be ensured up to the fourth day. The additional application of prophylactic antibiotic drops can help avoid infection even if refrigerated non-preserved serum is used up to 7 days. We ensured sterility by preparing the serum under absolute sterile conditions, storing the serum bottles in the freezer at -4°C, shifting the bottle to be used into the refrigerator compartment at 4-8°C and discarding that bottle by the end of the day. CONCLUSION We consider autologous serum to be effective in majority of cases with PED recalcitrant to conventional lubricant therapy due to the presence of essential factors. Healing depended more upon the etiology than the duration of PED before serum therapy, in our study. Author’s affiliation Aneeq Ullah Baig Mirza Assistant Professor Ophthalmology IIMCT / Railway Teaching Hospital Rawalpindi Naheed Ghani (Clinical Ophthalmologist) IIMCT / Railway Teaching Hospital Rawalpindi, Pakistan. Abdul Bari khan Associate Professor of (Microbiology) IIMCT / Railway Teaching Hospital Rawalpindi, Pakistan. REFERENCE 1. Geerling G, Hartwig D. Autologous serum-eye-drops for ocular surface disorders. A literature review and recommendations for their application. Ophthalmology. 2002; 99: 949-59. 2. Young AL, Cheng ACO, Ng HK, et al. The use of autologous serum tears in persistent corneal epithelial defects. Eye 2004; 18: 609-14. 3. Vajpayee RB, Mukerji N, Tandon R, et al. Evaluation of umbilical cord serum therapy for persistent corneal epithelial defects. Br J Ophthalmol. 2003; 87: 1312-6. 4. Hanada K, Shimazaki J, Shimmura S, et al. Multilayered amniotic membrane transplantation for severe ulceration of the cornea and sclera. Am J Ophthalmol. 2001; 131: 324-31. 5. Kruse FE, Rohrschneider K, Volcker HE. Multilayer amniotic membrane transplantation for ocular surface disorder. Ophthalmology. 1999; 106: 1504-10. 6. Geerling G, Maclennan S, Hartwig D. Autologous serum eye drops for ocular surface disorders. Br J Ophthalmol. 2004; 88: 1467-74. 7. Collins MK, Perkins GR, Rodriguez Tarduchy G, et al. Growth factors as survival factors: regulation of apoptosis. Bioessays. 1994; 16: 133-8. 8. Rodeck U, Jost M, Kari C, et al. EGF-R dependent regulation of keratinocyte survival. J Cell Sci 1997; 110: 113-21. 9. Fox RI, Chan R, Michelson J, et al. Beneficial effect of artificial tears made with autologous serum in patients with keratoconjunctivitis sicca. Arthritis Rheum. 1984; 29: 577-83. 10. Tseng SCG, Tsubota K. Important concepts for treating ocular surface and tear disorders. Am J Ophthalmol. 1997; 124: 825-35. 11. Tsubota K, Goto E, Fujita H, et al. Treatment of dry eye by autologous serum application in Sjogren's syndrome. Br J Ophthalmol. 1999; 83: 390-5. 12. Poon AC, Geerling G, Dart JKG, et al. Autologous serum eyedrops for dry eyes and epithelial defects: clinical and in vitro toxicity studies. Br J Ophthalmol. 2001; 85: 1188-97. 13. Pasternak AS, Miller WM. First-order toxicity assays for eye irritation using cell lines: parameters that affect in vitro evaluation. Fundam Appl Toxicol. 1995; 25: 253-63. 14. Wang XM. A new microcellular cytotoxicity test based on calcein AM release. Human Immunol 1993; 37: 264-70. 15. Albert DM, Jakobiec FA. Principles and practice of ophthalmology. W.B. Saunders Co. Philadelphia, 2000. 16. Kenyon KR, Tseng SCG. Limbal autograft transplantation for ocular surface disorders. Ophthalmology. 1989; 96: 709-23. 17. Kenyon KR. Limbal autograft transplantation for chemical and thermal burns. Dev Ophthalmol 1989; 18: 53-8. 18. Seitz B, Gruterich M, Cursiefen C, et al. Conservative and surgical treatment of neurotrophic keratopathy. Ophthalmology. 2005; 102: 15-26. 19. Das S, Langenbucher A, Seitz B. Delayed healing of corneal epithelium after phototherapeutic keratectomy for lattice dystrophy. Cornea. 2005; 24: 283-7. 20. Tsubota K, Goto E, Shimmura S, et al. Treatment of persistent corneal epithelial defect by autologous serum application. Ophthalmology. 1999; 106: 1984-9. 21. Alvarado Valero MC, Martinez Toldos JJ, et al. Treatment of persistent epithelial defects using autologous serum application. Arch Soc Esp Oftalmol. 2004; 79: 537-42. 22. Ferreira de Souza R, Kruse FE, et al. Autologous serum for otherwise therapy resistant corneal epithelial defects- Prospective report on the first 70 eyes. Klin Monatsbl Augenheilkd. 2001; 218: 720-6. 26 23. Haber M, Cao Z, Panjwani N, et al. Effects of growth factors (EGF, PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing. Vet Ophthalmol. 2003; 6: 211-7. 24. Liu L, Hartwig D, Harloff S, et al. An optimised protocol for the production of autologous serum eye drops. Graefes Arch Clin Exp Ophthalmol. 2005; 243: 706-14. 25. Nistor M, Nistor C. Autologous serum utilization in patients with lacrimal hyposecretion and persistent epithelial defects of cornea- clinical study. Oftalmologia. 2005; 49: 30-3. 26. Esquenazi S, He J, Bazan HE, et al. Use of autologous serum in corneal epithelial defects post-lamellar surgery. Cornea. 2005; 24: 992-7. 27. Schrader S, Wedel T, Moll R, et al. Combination of serum eye drops with hydrogel bandage contact lenses in the treatment of persistent epithelial defects. Graefes Arch Clin Exp Ophthalmol. 2006; 244: 1345-9. 28. Sauer R, Bluthner K, Seitz B. Sterility of non-preserved autologous serum drops for treatment of persistent corneal epithelial defects. Ophthalmology. 2004; 101: 705-9. Picture Quiz Answer Bietti's Corneoretinal Dystrophy Bietti's Corneoretinal Dystrophy, also called Crystalline Retinopathy, is a congenital disease with autosomal recessive type of inheritance involving the cornea and retina. The patients present usually during their third decade with mild to moderate decrease in visual acuity and some visual field loss. Night vision is markedly affected too. On examination, vision can range from 6/6 to hand motion. Color vision is abnormal suggesting tritan deficiency. The cornea has sparkling crystals mostly at the peripheries. The fundus also has small shiny crystals all over posterior pole, which has RPE atrophy. Electron microscope crystal analysis reveals ultra structural resemblance to lipids within fibroblasts. Fluorescein angiography shows transmitted hyperfluorescence in the area of crystals. Adjacent areas with no crystals show confluent loss of RPE and choriocapillaris. ERG is normal with white light but abnormal with blue light. EOG is usually subnormal. Visual fields are affected only in advanced stages, the earliest defects being pericentral scotomas. Unfortunately, like many other retinal conditions, there is no known treatment up till now.