## 2 Materials and Methods  
This single-center retrospective study included adult patients who received a first allogeneic H3 CT between January 2010 and April 2020 at the Hematology Unit of the IRCCS AOU di Bologna, Policlinico di Sant'Orsola (Bologna, Italy) and underwent subsequent ocular surface examinations at the Ophthalmology Unit of the same hospital. The study was performed in accordance with the principles of the Declaration of Helsinki and was approved by the local Ethics Committee (Comitato Etico di Area Vasta Emilia Centro della Regione Emilia-Romagna). Exclusion criteria were: survival <100 days after transplantation, presence of other ocular surface disorders or any systemic disease potentially affecting the ocular surface at the time of H3 CT, missing ophthalmological data after H3 CT.  
Data collected on charts and related to H3 CT included the source of stem cells (bone marrow, peripheral blood or cord blood), type of donor (matched related--MRD or matched unrelated donors--MUD), conditioning regimen (myeloablative or reduced-intensity regimen based on patient age, previous treatments, comorbidities and status of malignancy). For the statistical analysis, patients were divided into two groups according to the underlying disease: chronic lymphoproliferative disorders (LPDs) including Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and stem cell malignancies (SCMs) including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML). All patients had undergone GVHD prophylaxis with either cyclosporine and methotrexate or cyclosporine and mycophenolate moefetil. Demographical and hematological data including age, sex, primary hematological disorder, age and sex of donor, presence of sex and HLA mismatch were recorded for each patient. Grading of acute GVHD was performed in a 0 to IV scale according to the Glucksberg classification [22]. The severity of chronic GVHD was scored using the NIH 2014 criteria [23]. Data retrieved on ocular surface parameters included those collected before H3 CT 7 to 9 days before the beginning of the conditioning regimen, and those collected in the first subsequent ophthalmological examination performed 100 to 180 days post-H3 CT. Data included subjective ocular discomfort symptoms scored by using the ocular surface disease index (OSDI) validated questionnaire [24], tear film break-up time (TBUT), corneal staining, and Schirmer test. Tear film break-up time was evaluated after administration of 2 uL of 2% fluorescein dye and measuring the time interval between the last complete blink and the first appearance of a dry spot or disruption in the tear film. Corneal staining was graded using the National Eye Institute score (NEI Grading 2014). Schirmer test was performed without anesthesia using test strips kept into the temporal lower conjunctival sac for 5 min with closed eyes. The diagnosis of ocular GVHD was based on the International Consensus Criteria on Chronic Ocular GVHD Group, which assign a 0-3 scoring point to Schirmer test, corneal fluorescein staining and OSDI, and 0-2 scoring point to conjunctival injection [25]. However, these criteria were introduced only in 2013, and we did not routinely score conjunctival injection before then. Thus, we employed modified criteria without conjunctival injection, and reduced by 1 point the aggregate score required for reaching the diagnosis: in the presence of systemic GVHD, score \(\geq\) 5 indicated ocular GVHD (oGVHD); in the absence of systemic GVHD, score \(\geq\) 7 indicated oGVHD [6]  
**Tear sampling procedure. ** A minimum amount of 10 uL of unstimulated tears was collected using a micropipette with sterile tips, avoiding any reflex tearing [26], from each subject in both eyes. Subjects were requested to position the head slightly inclined in such a way that tears are gathered at the most outer side of the lower fornix, avoiding tear drainage through the naso-lacrimal duct.