Supplementary Figure 1: Sphingolipid Metabolism.
Enzymes in bold were analysed for gene expression in the VKC and controls
Diagnosis of VKC and inclusion criteria for study recruitment:Patients with ocular itching, photophobia, presence of active tarsal/limbal papillae, bulbar congestion, Horner Tranta’s dots or superficial punctate keratitis were diagnosed as active VKC. The quiescent stage was diagnosed based on inactive or flat-topped papillae, a non -inflamed ocular surface with previous history of chronic itching. All cases were included after a complete ophthalmic examination and ruling out any other ocular or systemic morbidity or history of ocular surgery. Tear specimen and conjunctival imprints were collected in cases and controls, while blood sample was collected in a sub-set based on consent of the study participant. All cases with active VKC and age > 6 years were included in the study. Patients with history of previous ocular surgery, ocular co-morbidity other than VKC, any systemic disease other than allergic disorders; age < 6 years and quiet eye at presentation were excluded from the study. On basis of Bonini’s classification, patients with grade 1 and 2 (mild to moderate form of seasonal VKC) were considered as non-refractory (NR-VKC) and those with grade 3 and 4 (severe to very severe form of perennial VKC) as refractory (R-VKC). Patients with refractory VKC were further sub-grouped based on the duration and/ or use of topical steroid or immunomodulator, (C) those who were on topical steroid/immunomodulator <8 weeks; (D) those without steroid or immunomodulator, who presented as fresh case with a repeat episode; (E) those who were on steroid/immunomodulator > 8 weeks. Duration of 8 weeks was considered so that the maximum effect of topical immunomodulator could be assessed. All cases had active VKC at recruitment and were advised to defer use of any topical on the day of collection. Non refractory and fresh refractory cases (group D) were only on lubricants at presentation whereas refractory (groups C and E) were on dual acting mast cell stabilizer (Olopatadine 0.2% w/v, Alcon Laboratories, India) in addition to lubricants and topical steroid / immunomodulator (Fluoromethalone 0.1%w/v, Allergan India Private Limited/Tacrolimus ointment 0.03% w/w, Aurolab, India).
LC-MS/MS analysis of sphingolipids in tear: The sphingolipid standards were procured from Avanti Polar Lipids (Alabama, USA). Based on calibration of the LC column and linearity check, the following standards were finalized for the assay in pooled specimen of tear in cases and controls LC-ESI-MS/MS experiments were performed using a triple quadrupole tandem mass spectrometer (4000 Q-Trap, AB Sciex, Foster City, CA, USA) coupled with high performance liquid chromatography system (HPLC, Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA) that consisted of quaternary pump (G1311C), multi-sampler (G7167A), HIBAR (30 x 2.1mm, 2µm) column compartment (G1316A) with variable wavelength UV detector (G1314F) and online degasser. All the parameters of tandem mass spectrometer and HPLC were controlled by Analyst software, version 1.5.2 (AB Sciex, Foster City, CA, USA) and Open LAB control panel software (Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA), respectively.