Lyophilized Autologous Serum Eyedrops: Experimental and Comparative Study.

PURPOSE: To analyze the biological stability of autologous serum eyedrops after lyophilization. DESIGN: Prospective, comparative experimental study. METHODS: This was a comparative study with serum obtained from 12 healthy volunteers. The concentrations of different epitheliotropic factors (eg, transforming growth factor-ß [TGF-ß1], epidermal growth factor [EGF], platelet-derived growth factor AB [PDGF-AB], and albumin) were measured in fresh and lyophilized serum. The samples were studied after serum preparation (fresh serum) and immediately after saline solution reconstitution of lyophilized serum (0), 15, and 30 days later. The biological effects of both serum samples were also compared on conjunctival and corneal cell cultures. The pH, osmolarity, and serum density were also determined. RESULTS: No significant differences were found in the concentration of growth factors between fresh serum and re-dissolved serum samples after lyophilization. The concentration of growth factors remained stable during 1 month at 4°C in re-dissolved lyophilized form with saline solution. No differences were found related to osmolarity, pH, and density between fresh and lyophilized serum. In addition, no differences were found on the conjunctival and corneal cells proliferation and differentiation in cells cultures between either serum preparation. CONCLUSIONS: The properties of autologous serum remain after lyophilization. The lyophilized serum can be easily stored without temperature restrictions and easily reconstituted for preparation of eyedrops for standard clinical use.

Changes in Corneal Expression of MUC5AC after Autologous Serum Eyedrop Treatment in Patients with Limbal Stem Cell Deficiency.

Purpose: To evaluate the effect of autologous serum eyedrops treatment on corneal expression of the MUC5AC in patients with limbal deficiency. Methods: A prospective and comparative interventional case series study of 42 eyes of 21 patients was performed before and 8 weeks after treatment with autologous serum. All patients underwent a complete ophthalmic assessment of the tear film and ocular surface, corneal impression cytology (IC) and MUC5AC detection by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: Forty-one of the 42 eyes studied were available for both conventional cytology and MUC5AC analysis prior to and after treatment. Differences between outcomes obtained by impression cytology and MUC5AC detection were found in 9 of 82 samples (11%). We found changes in the corneal expression of MUC5AC after treatment in 19 of 41 eyes (46.3%): 18 of them (94.7%) changed from positive to negative expression, and 1 eye (5.3%) changed from negative to positive MUC5AC expression after autologous serum eyedrops. These changes were related with the corneal involvement prior to treatment (15 of them (78.9%) occurred in patients with slight corneal involvement), and with the improvement in the degree of squamous metaplasia after treatment (P = .001 and P = .003, respectively). Conclusions: The treatment significantly improved tear stability, squamous metaplasia, and subjective patient perception. Autologous serum eyedrops treatment diminished the corneal expression of MUC5AC mainly in patients with slight corneal involvement before treatment.

Modified lateral tarsal strip for involutional entropion and ectropion surgery.

PURPOSE: Our purpose was to assess our outcomes in involutional lower eyelid ectropion and entropion surgery after horizontal eyelid tightening by a modified tarsal strip technique. METHODS: A prospective study of 88 eyelids with ectropion and 96 with entropion was performed. Patients were randomly distributed into two groups: the control group included 90 eyelids (46 entropion and 44 ectropion) and the other group of 94 eyelids (50 entropion and 44 ectropion). The control group was treated with a conventional tarsal strip, whereas the second group underwent the same surgery except for a modified suture placement of the tarsal strip different in patients with ectropion and entropion. The mean follow-up was 5.4 years. Horizontal eyelid laxity, orbicularis muscle and lower eyelid retractors function were measured prior and after surgery. Recurrence rate was also evaluated. RESULTS: Recurrent entropion occurred in eight eyelids (17.4 %) treated with conventional tarsal strip, while only two eyelids (4 %) showed recurrence after surgery in the group treated with the modified technique. Recurrence ectropion was only found in one eyelid (2.3 %) in the control group. Pathological horizontal laxity was present in all patients at the baseline study. After surgery, the horizontal laxity improved in both groups, we found significant differences in the patients treated with the modified technique (P=0.04). CONCLUSIONS: The modified suture placement of the tarsal strip reported in this work improves the lower eyelid stability and prevents future recurrences after surgery. This modified technique is useful for the management of patients with entropion and moderate eyelid dysfunction retractors without other surgical procedures.

Stability of Growth Factors in Autologous Serum Eyedrops After Long-Term Storage.

PURPOSE: The purpose of this study is to assess the stability of the growth factors (GF) in autologous serum eyedrops under different storage conditions. METHODS: The concentration of epidermal growth factor (EGF), transforming growth factor-ß (TGF-ß1), platelet-derived growth factor AB (PDGF-AB), and albumin was measured in fresh and defrosted samples of autologous serum under different storage conditions. The fresh and defrosted samples were cooled at 4 °C, and they were studied immediately after preparation, or after defrosting, and after 1, 2, 3, and 4 weeks. The concentration of GF was also assessed after 1, 3, 6, and 9 months at -20 °C. We also investigated how the different storage conditions influence the biological effects of autologous serum on conjunctival and corneal cell cultures. RESULTS: The concentration of EGF, TGF-ß1, PDGF-AB, and albumin remained stable over the 4 weeks at 4 °C, both in fresh and in defrosted samples. Likewise, no statistically significant differences were found between the GF concentration in fresh samples and after 1, 3, 6, and 9 months of freezing at -20 °C. Moreover, no differences were found on the cell proliferation and differentiation between cultured cells with fresh or defrosted samples after 4 weeks at 4 °C or after 1, 3, 6, or 9 months at -20 °C. CONCLUSIONS: Long-term storage of autologous serum eyedrops at -20 °C does not affect the concentration of GF, simplifies clinical logistics, and reduces the frequency of blood extractions from the patients.

Amniotic membrane transplantation in acute toxic epidermal necrolysis: histopathologic changes and ocular surface features after 1-year follow-up.

PURPOSE: To evaluate ocular features and histopathologic changes in patients with toxic epidermal necrolysis (TEN) treated with amniotic membrane transplantation (AMT) in the acute phase. METHODS: A prospective study of 5 eyes (3 patients) with severe ocular involvement and 4 eyes (2 patients) with moderate involvement treated with AMT in the acute phase of TEN was performed. Patients underwent a complete ophthalmic assessment before the intervention and 3, 6, 9, and 12 months postoperatively. Tear film break-up time (BUT), Schirmer test, fluorescein_staining, corneal and conjunctival epithelial squamous metaplasia, ocular sequelae, and conjunctival retraction were measured. RESULTS: In patients with severe involvement, 2 eyes (40%) showed mild symblepharon and peripheral corneal neovascularization, and corneal epithelial defects, lid margin malposition, and trichiasis was found in 20% of the eyes; only 1 eye (20%) required surgery for structural defects. We did not find ocular sequelae after 1-year follow-up in the patients with moderate involvement. Only 1 eye (25%) developed mild symblepharon that did not require specific treatment. Amniotic membrane transplantation significantly improved the squamous metaplasia in corneal and conjunctival nonsecretory epithelial cells, and significantly improved the goblet cells density after 1-year follow-up. CONCLUSIONS: Amniotic membrane transplantation performed for acute TEN reduced inflammation and ocular surface scarring, improved dry eye and squamous metaplasia. and decreased ocular sequelae in the chronic stage. Although further and prospective studies are needed, AMT may be the optimal treatment for acute TEN with severe or moderate ocular involvement.

Ocular features and histopathologic changes during follow-up of toxic epidermal necrolysis.

OBJECTIVE: To evaluate ocular surface histopathologic changes and ocular sequelae in the follow-up of patients with toxic epidermal necrolysis (TEN). DESIGN: Prospective, consecutive, comparative, interventional case series. PARTICIPANTS: Eleven patients (22 eyes) with TEN that developed after drug treatment and 33 normal subjects as the control group. METHODS: Toxic epidermal necrolysis diagnosis was based on data obtained from medical records. Only patients with ocular involvement in the acute stage were included. Patients and controls underwent a complete ophthalmic assessment, including tear film evaluation and corneal and conjunctival impression cytology. These were performed at the beginning of the study (∼1 month after TEN) and 6 and 12 months later. Tear film production and stability were evaluated by break-up time, Schirmer's test with anesthesia, rose bengal, and fluorescein staining pattern. Conjunctival retraction was determined by studying the vanishing point in eye abduction of the lacunar folds. MAIN OUTCOME MEASURES: Break-up time, Schirmer's test, fluorescein and rose bengal stain, corneal and conjunctival epithelial squamous metaplasia, conjunctival retraction, and ocular sequelae. RESULTS: Mild or moderate ocular involvement was present in 73% of patients in the acute stage. Ocular involvement was not related to TEN severity, area of skin involvement, or drug etiology. One-year follow-up, ocular manifestations, slit-lamp findings, and epithelial cell squamous metaplasia were related to ocular involvement severity in the acute phase. We found pathologic conjunctival retraction in 86.4% of patients. Decreased levels of break-up time and Schirmer's test were found in patients with TEN. Conjunctival cytology showed a marked decrease in goblet cell density. No patients died, and there were no recurrences of TEN during the study. CONCLUSIONS: Ocular involvement in the acute stage was not related to TEN severity or etiology. Ocular sequelae were related to ocular involvement severity in the acute phase. Early ophthalmic assessment and frequent follow-up are helpful because ocular involvement represents the first long-term complication in patients with TEN. Impression cytology and measurement of conjunctival retraction can be useful tools in the assessment of dynamic ocular changes in patients with TEN.

Histopathologic limbus evolution after alkaline burns.

PURPOSE: To study the histopathologic evolution of the corneal limbus after alkaline burns according to the clinical severity and therapy used. METHODS: A prospective study of 15 eyes from 12 patients (9 men and 3 women) with moderate and severe alkaline burns was performed. All patients were divided into 2 groups in accordance with the clinical ocular severity and the therapy that was used: medical therapy, amniotic membrane transplantation (AMT), autologous limbal transplantation (ALT), and ALT combined with AMT (ALT + AMT). Biopsies were obtained from affected limbal areas immediately after the ocular burn and 9 months later. RESULTS: Limbal regeneration was limited to small areas in patients with moderate burns treated with medical therapy; in contrast, the limbal structure showed significant stromal and epithelial regeneration in patients with moderate burns treated with AMT. There was an important stromal regeneration with an incomplete reepithelialization in patients with severe burns treated with AMT. Patients treated with ALT showed a good reepithelialization with a defective stromal regeneration. Epithelial and stromal regeneration was notable in patients with severe burns treated with ALT + AMT. CONCLUSIONS: In patients with moderate alkaline burns, AMT improved both limbal stromal and epithelial regeneration more effectively than medical therapy. In patients with severe burns, the best reepithelialization and stromal regeneration were obtained with ALT + AMT.