## 4 Discussion  
Data from the present study show that individual pre-post-HSCT changes in tear protein levels, in particular TP, LACTO, TRANSF, and ZAG-2, estimated in the first six months postoperatively are correlated to the late onset of ocular GVHD. This finding may be of clinical impact for the early management of serious ocular complications following HSCT. To the best of our knowledge, the present paper is the first one dealing with individual changes of tear protein levels from baseline conditions before the conditioning protocol to conditions recorded in the interval 100-180 days after, thus introducing consistency in the time frame of comparison. Ocular GVHD represents a current diagnostic and therapeutic challenge, with increasing prevalence and severe involvement of the ocular surface with vision-threatening complications, and a dramatic reduction of the quality of life [28]. Therefore, an early diagnosis driving an appropriate and prompt treatment is important for avoiding the occurrence of severe complications [29]. Current diagnostic criteria of oGVHD include clinical parameters (such as the Schirmer test, the corneal damage evaluated by vital staining, conjunctival hyperemia, and the subjective discomfort symptoms) participating with different scoring points to a global score assessment [2, 25, 30, 31]. However, the well-known lack of causal correlation between clinical findings and subjective symptoms may hinder and delay the oGVHD recognition, and the search for biomarkers as objective diagnostic and monitoring tools is an urgent need. Data from the present study did not evidence statistically differences in ocular surface clinical parameters and subjective symptom scores in patients developing oGVHD as compared to those not developing oGVHD. Tears are good candidates as a body fluid to identify biomarkers for the management of patients suffering from not only ophthalmological diseases [32, 33, 34, 35, 36, 37], as they are close to the disease site, offer analytical advantages, and their sampling technique is relatively not invasive. Due to the extensive inflammatory processes underlying the GVHD onset and progression, several works have been addressed onto tear cytokine content, with the aim to differentiate patients with oGVHD from healthy or DED subjects [8, 10, 14, 15] or from patients without oGVHD [17], or without any control [16]. Apart from technical issues deriving from a large variability in pre-analytical and analytical procedures [38], all these investigations have been performed in patients only after HSCT and at different periods postoperatively. Similar attempts had been made on plasma cytokine panels for their potential to predict the development of GVHD [39, 40]. Tear proteomic-based studies were also performed to discriminate down or up regulated proteins, distinctive for oGVHD as compared to non oGVHD [18, 19], but again only in post-HSCT patients. Indeed, several proteomic approaches are currently used in the prediction or diagnosis of acute and/or chronic GVHD [41, 42, 43]. Interesting preliminary results are now available from the research setting, but without validation and impact on clinical decision making so far. The present paper introduces a relatively unexplored field for biomarker search in oGVHD, dealing with proteins mainly secreted by the disease target tissues of the ocular surface, and collected in tears. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Factor** &
\begin{tabular}{c} **Cut-Off** \\ **(mg/h)** \\ \end{tabular} & **OR 95\% CI** & \(p\) & **RR 95\% CI** & \(p\) \\ \hline \(\Delta\) pre-post-TP & \(\geq\!-\!1.86\) & 4.49 [1.9–10.5] & \(<\)0.001 & 2.1 [1.3–3.1] & \(<\)0.001 \\ \hline \(\Delta\) pre-post-LACTO & \(\geq\!-\!0.45\) & 3.08 [1.3–7.6] & \(<\)0.01 & 1.8 [1.1–3.1] & 0.01 \\ \hline \(\Delta\) pre-post-TRANSF & \(>\)0.34 & 15.7 [4.1–52.2] & 0.0001 & 3.8 [1.9–7.6] & 0.0001 \\ \hline \(\Delta\) pre-post-ZAG-2 & \(\geq\!-\!0.23\) & 11.1 [2.7–45.6] & \(<\)0.001 & 2.3 [1.5–3.4] & \(<\)0.001 \\ \hline \hline \end{tabular}
\end{table}