Full back Adam Henley definitely misses the re-match against the Lions with a hamstring strain with the young defender unlikely to return before the end of the season.
Henley's Sunday replacement, Bradley Orr, is also struggling with the rib injury that forced him off at the Den. The defender will given as long as it takes to prove his fitness, but it is unlikely that he'll feature.
There's also a fitness concern over David Bentley with the midfielder having picked up a knee injury in Sunday's match.
"Adam has done his hamstring again," confirmed Appleton. "It is a grade two and quite a bad one.
"So it is probably the end of his season which is really disappointing because he's a young player that we rate very highly at the club.
"Brad is still very sore. We're still not sure if there is damage to the ribs because of swelling around the muscle area of the ribs, so we'll probably know more today.
"In terms of him being ready for Wednesday looks highly unlikely.
"Also David Bentley has a lot of swelling on his knee so the next 24 hours are going to be really critical as well to see if he is going to be ready to play in the game."
Despite those injury concerns, the good news is that Leon Best and Joshua King came through Sunday's match well. Best reporting no ill-effects after his first 90 minutes for Rovers, whilst King will be all the better for his outing.
"I think the game will have done them both the world of good, I think with Leon he is obviously a little bit sore and stiff after being out for so long," added Appleton. "Luckily there were no ill-effects of the injury.
"With Josh it was probably more of a psychological one. He had to get that game out of his system. He looked at times as if he was only playing about 70%, it looked like he was playing within himself. Not at one point did he open himself up and show his pace.
"So from a psychological point of view that game will have done him the world of good and hopefully he'll feel confident that if he needs to sprint and if he needs to get in behind their back four then he'll do that.
"There are also going to be two or three younger players on the bench, so hopefully it will be a good occasion for them as well."
Supporters are advised to get their tickets in advance of Wednesday's match as large queues are expected in the run-up to the 7.30pm kick-off.
Tickets are priced at £18 for Adults, £12 for Seniors/Students and £6 for Juniors under 16.
Book online today at www.onerovers.co.uk
and collect your tickets from the Blackburn End colleciton point nice and early on the day of the game.