*5.2. Adverse Events*

Clinical signs or adverse events were considered in four studies [27,29,30,33]. These covered all three of the large sample methods, that of retrobulbar, sublingual and facial vein sample. Regan et al. 2016 also studied submental sampling [33]. There was considerable heterogeneity in the types of adverse events reported which ranged from numbers of repeat attempts at sampling to instances of haemorrage from the site. Events also ranged in severity from mild, such as corneal opacity, to life-threatening. This finding implied that simple addition of incidences of event would provide a biased picture. Furthermore, some adverse events were clearly specific to the sample location, for example ocular lesions or ear canal hemorrhage rendering direct comparison non-meaningful. For this reason, results have been summarised narratively.

The number of punctures needed to obtain a sufficient blood sample was significantly less for retrobulbar bleeding (1.03 punctures), compared to facial vein (1.45) and sublingual (1.31), (*p* < 0.001 in all comparisons) [30]. Sublingual puncture caused haemorrhage from the nares in 3.33% of mice. Interestingly this rate was increased after the use of anaesthesia to 10.56% of mice [30].

Clinical signs following facial vein bleeding were evaluated in three studies [27,29,30]. Signs included inactivity after collection and being unsteady on release. These occurred at a frequency of 1/20 animals (5%) [27]. The more serious adverse effect of haemorrhage from the ear canal occurred at a rate of 2/20 (10%) [27]. These adverse events occurred when using needle, rather than lancet puncture [26]. Frolich et al. 2018 [29] similarly reported inactivity, ear and nose haemorrhage, as well as head tilt, convulsions, circling and corneal opacity, at rates of approximately 17–25%, but only with serial facial vein samples. Rates of haemorrhage from the ear canal were 2.78%, with an approximate doubling when anaesthesia was used (5.56%) in the Gjendal et al. 2020 study [30].

The most common adverse event reported with retrobulbar sampling was corneal opacity and periocular tissue prolapse occurring at a rate of 2/12 animals (17%) [29]. The incidence of corneal opacity was increased in serial RBB to 5/12 (42%) animals [29]. However, no ocular abnormalities were observed after use of this sampling route in the Gjendal et al. 2020 study [30].

In contrast to other studies Regan et al. 2016 [33], reported no adverse signs after retrobulbar, facial or submental bleeding in a cohort of 15 per group. Minor inflammation was noted at the point of capillary tube insertion in the retrobulbar group but this resolved quickly.
