Drawing useable amounts of blood from dwarf mice. - Comparing notes on lab-animal phlebotomy. (Aug/01/2007 )
I'm working with Ames dwarf mice. Where normal mice weigh 20-30g, Ames dwarves weigh 8-15g. It's hard as hell to get blood out of these little suckers, even non-recovery. Therefore it takes longer to draw than it does for normal sized mice with which I'm comparing them. Therefore, there is increased hemolysis and perhaps other, subtler biases that call into question differences my tests find in this blood.
The following is what I've tried with the respective drawbacks:
Femoral vein, vena cava.
Drawbacks: vein collapses, having to keep waiting for it to re-inflate takes so long that blood clots in the syringe. Sometimes syringe gets clogged by the walls of the vein. Disappointing yields-- 200ul serum for normal mice, sometimes 25ul or less for dwarves. Slowest method of all those tried, at least in my hands.
Cardiac puncture (open chest).
Drawbacks: the heart collapses and waiting for it to re-inflate sometimes still leads to clotted/clogged syringes. Sometimes syringe pierces the heart through the other side. Sometimes syrings just gets into an 'unlucky' part of the heart and fails to draw any blood. Removing and reinserting the syringe becomes rapidly less effective. If syringe accidentally pops out, blood rushes out and that animal often becomes useless for the purposes of blood collection. Also, I have only two hands with which to hold the plunger, the barrel, and the heart. Usually okay yields for normal mice, but still sometimes as little as 25ul serum for dwarves. Sometimes blood bubbles or foams upon entering syring instead of drawing up in a solid column. Second slowest method.
Cardiac puncture (closed chest).
Drawbacks: high risk of missing or damaging the heart. When successful doesn't seem to have any advantage over open chest method.
Orbital sinus puncture.
Drawbacks: At first seemed fast and high-yield. Unfortunately, blood *much* more hemolyzed than than cardiac puncture.
Slitting heart and allowing blood to pool in ribcage, then drawing that with syringe.
Drawbacks: somehow, most of the blood ends up still inside the animal or on the table and the syringe ends up filled with foam and bubbles. As I write this, I just got the idea of filling up the rib cage but then decanting it directly into an SST tube without using the syringe. I'll try that when I have time and report back with what I find.
In small preliminary test (N=2), got excellent yield and no hemolysis at all. In the real deal, got crappy yields and hemolysis on every single sample. Blood sticks to fur, often misses the tube, and very annoyingly foams while still in the body. Cutting closer to the ears and further from the shoulders seemed to work better, but still hemolyzed.
Maybe the problem is the type of anaesthesia I'm using. We typically use RAC cocktail (ketamine/xylazine/acepromazine) and it seems to depress blood pressure. When I use isofluorane instead, I seem to get better results... but isofluorane is incompatible with some experiments we might wish to perform before the blood draw because the mice wake up too quickly when the anaesthesia is removed. Is ketamine known to cause problems with blood draws? Are there other comparably potent anaesthetics that are better?
This is driving me nuts! Many blood assays seem to require 100ul or more of serum, and I often can't run multiple assays on the same animal due to limited amounts of serum. Now, if cardiac puncture is the best I'll get, I guess I'll have to stick with that. But perhaps there is some method I haven't tried yet that produces maximal yield and minimal hemolysis? Perhaps there are some tricks to make this easier that nobody bothers publishing in papers? Does anybody reading this have any experience they'd like to share regarding drawing blood from dwarf mice or juvenile mice?
Finally, are there any books anybody could recommend about mouse surgical techniques in general and mouse phlebotomy in particular?
Thanks for reading this.
Have you try tail bleed?
The only reason I haven't is because I cannot imagine the tail vein yielding more blood than cardiac puncture. Furthermore, the tail vein would be susceptible to the same collapsing problems as the other veins, wouldn't it?