It is nice to see a few people finally speed-running this game.
I have had an incredibly bad streak of luck trying to re-attain a sub-25 run (I got one a few years ago but lost the original footage when I accidentally over-wrote the video file) as for AT LEAST my last 10 runs to satisfactorily make it to Stage 4, the pass was not in the right spot any of those times. Finally had my luck change this morning, though my play in stage 6 was quite sloppy. Nevertheless, I think I beat the record by a second. Will have that uploaded here soon.
Here are my rankings for RNG annoyances.
1 - Stage 4 passport. Nothing more annoying than to have a good run up to this point only to have the run end with the passport not being there.
2 - Stage 3 passport. Nearly as aggravating as the one above, except it is earlier in the run so not as big of a deal.
3 - Stage 3 end-boss enemy pattern. This can be a run-ending problem if you get really bad luck - especially if he spawns right in your path. If executed properly, you have about a 50/50 chance of getting a 3-spawn kill, and if you miss that, another 50/50 chance of getting the 4-spawn kill. After that you're probably dead. My run would have been about 6 seconds better with better luck here.
3 - Stage 6 glitchy ghost room enemy placement. While this won't usually end a run like a passport placement will, it can zap your life in a hurry and ultimately tack on 10-15 seconds if your path gets blocked several times, because not only do you lose time here, but you have to be careful afterwards AND spend extra time getting a med kit.
4 - Stage 6 first room enemy patterns. The fastest path to clear this first room is to stay on the bottom, but the enemies can take paths (as happened to me MULTIPLE times in my run) that cause you damage and slow you down.
5 - Stage 1 passport - only a minor annoyance since it comes so early in the run, but having 5 or 6 in a row sometimes be in the wrong spot can get annoying real quick.
There are other minor bits of RNG here and there that can affect the run by a second here or there, but those are the biggies. Everything else just comes down to execution - which, part of what makes this game so challenging to speed run is that there are a LOT of places that require perfect or near-perfect execution to pull off that if you mess them up can either outright end a run or can tack on significant time. My biggest challenge is always managing my life bars in Stage 1. The better you do earlier in the stage, the more aggressive you can be later in the stage.
I just watched the 4-year-old TAS run (23-something) and I see a few tricks in there that could probably save seconds here and there. There's quite a bit of stuff in that TAS, however, that seems impractical to incorporate into a run, and outright impossible stuff - mostly the speed at which the TAS can switch back and forth between characters and open the code-locked doors.
I think I could probably get down to a 24:30. I think a sub-24 may be theoretically humanly possible but would require ridiculous skill to pull off.