This is based on opinion. Please don't list it on a work's trope example list.
Tear Jerker / Ctrl+Alt+Del
Say what you will about Ethan, but the circumstances of his death can really cause a severe Mood Whiplash in the flow of the comic, especially since the genre is slice-of-life comedy.
The blog post about the death of the creator's dog.
Lilah's miscarriage, hated as it was (and memetic as it still is today), is still rather sad.
Post-reboot, one of The Campaign mini-arcs is Best Served Cold. The party's Barbarian swore vengeance on one particular Evil Sorcerer Lich who killed his father. They managed to destroy the Lich, only to find out that the Barbarian's father was turned into the Lich's Soul Jar, thus he will have to destroy his own father. After a particularly heartwrenching fight, he managed to prevail, and the father's last words was how proud he was of his son.