A prospective, single-blinded, randomized, controlled crossover trial was conducted in patients with severe dry eye syndrome. Topical treatment with autologous serum eye drops (ASED) diluted at 20%, undiluted ASED and conventional preservative-free artificial tears (PFAT) were compared as a treatment for severe dry eye disease. The primary outcome measure was assessment of ocular symptoms using the Ocular Surface Disease Index (OSDI) questionnaire. Secondary outcomes were Schirmer 1 test, best-corrected visual acuity (BVCA), corneal fluorescein and conjunctival lissamine green staining using the Sjögren's International Collaborative Clinical Alliance Ocular Surface Staining (SICCA OSS) score, tear break up time (TBUT), conjunctival injection score (CIS) and Meibomian gland dysfunction (MGD) grading. Additionally, serum and tear cytokine analysis and microbiological cultures were performed.

This prospective, single-blinded, randomized, crossover clinical trial analyzed the difference in subjective symptoms and clinical signs between 20% and 100% autologous serum eye drops (ASED) versus preservative-free artificial tears (PFAT). After signing the informed consent form, each patient was randomized via RedCap. A 2-week washout was initiated prior to the baseline visit and the start of the first treatment. Patients were asked to discontinue current PFAT and/or ASED and replace them with the washout, in the form of preservative-free 3% trehalose and 0.15% hyaluronic acid (HA)-containing artificial tears. Concurrent use of necessary ocular anti-inflammatory therapy (cyclosporine, hydrocortisone) and moisturizing ocular ointment at night was allowed, provided it was maintained throughout the entire crossover study. Each patient received three 8-week treatments in a randomized sequence, being PFAT, AS 20% and AS 100%. After 8 weeks of treatment, the treatment effect was evaluated during a scheduled study visit. The patient was then instructed to start a new washout of 2 weeks to immediately follow with the next treatment. The same preservative-free artificial tears containing 3% trehalose and 0.15% hyaluronic acid (HA) were used during the PFAT treatment as during the washout. The total study duration was 30 weeks (Figure 1). The examining ophthalmologist (DR) was blinded to the type of eyedrops given to each patient in the trial. The minimum dosage for the eye drops (AS and PFAT) was eight times a day. If necessary, hourly application was allowed. In that case, the patient was asked to continue hourly application for all three treatments.

Inclusion Criteria:

The inclusion criteria for patients participating in this study are defined as follows: patients with severe Dry Eye Disease, including severe symptoms as evaluated with a standardized instrument (OSDI score > 33), associated with at least one of the following objective parameters:

A. Tear break-up time (tBUT) as a measure of tear film quality < 5 seconds B. Positive corneal and conjunctival staining quantified according to the SICCA OSS scale C. Schirmer 1 test score < 5 mm/5 min (without anesthesia)

Exclusion Criteria:

The exclusion criteria for patients participating in this study are defined as follows:

A. Inability to complete the study protocol, including study-specific procedures.

B. Inability to understand the Dutch-language ICF and/or unwillingness or inability to provide signed informed consent.

C. History of non-compliance with the proposed therapy D. Presence of known severe anemia based on medical history E. Hypersensitivity to the proposed treatment F. Pregnancy G. Age <18 years H. In the opinion of the investigator, the subject is not suitable for participation in the study

The examining ophthalmologist was blinded to the type of eyedrops given to each patient in the trial. Due to differences in color and viscosity of the different eye drops, complete blinding of the study patient was deemed impossible. All data were collected in REDCap by an unblinded study coordinator. The database was locked and the blinding lifted only after the Database Lock Approval Form had been signed by the principal investigator following completion of the entire study.