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Hi all.
We are doing PK studies in mice (around 25 grams each).
Does anybody know if multiple sampling time points can be obtained from
the same mice in the same day?
I am aware that after a 0.05% b.w. blood withdrawal 1 week of recovery
is recomended for mice before a second collection. We are trying to
save cost and somebody suggested to use every mice for two time points.
In the first one 150 ul will be collected and two hours after that, a
terminal collection will be made. My guess is that after the first
withdrawal a lot of mechanism (hormonal, CNS, etc..) will be activated
and our second sample won't be reliable. Does that make sense?
Thaks very much for your help.
Jose M Valdivielso
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Dear Dr. Valdivielso:
We typically estimate blood volume to be ~6% of animal body weight
(e.g.,
~1.5 ml for a 25g mouse). Our veterinary staff recommends limiting acute
blood sampling to 15% of blood volume (0.9% of body weight or ~ 225 uL
for a
25 g mouse). We have found that we can take serial samples from the
saphenous vein of mice, collecting blood in 20 uL capillary tubes
(Unopette,
BD). Further blood loss is minimized by apply direct pressure for ~ 1
minute. A nice description of sampling via the saphenous vein may be
found
at the following URL:
http://www.uib.no/vivariet/mou_blood/Blood_coll_mice_.html
Sampling from the saphenous vein may be performed in conscious animals
with
little (apparent) animal discomfort / distress.
J. Balthasar
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Hi Jose:
We are doing multiple time points from the same mouse in the same day -
30-40 g mice. We're using an automated blood sampler, so the mouse is
not
restrained or in pain - he's free to move (actually run, rear, jump),
eat,
drink, sleep, etc. while samples are being withdrawn. We've not yet
measured adrenal hormone levels, although we've studied this
extensively in
rats and have found this technique to dramatically reduce these hormones
(catecholamines, corticosterone). In mice, we currently take very small
sample volumes (5 to 10 µL per time point) and we deliberately dilute
the
sample with 50 µL of saline so we can manipulate the fluid. This
requires
that you have a very good analytical method which may be too much to
expect
early in screening when you don't have the luxury of refining the
analytical protocol. We can take larger samples but decided to start
small
and see what was feasible. Total blood volume (tbv) of a mouse is ~
58.5
mL/kg and the safe limit normally imposed is 10% of tbv. So, for a 30 g
mouse, the tbv should be ~ 1.7 mL. If we take 12 x 10 µL samples in 24
hours, we're removing 7% of tbv but getting an entire PK curve. The
charts
never show you how much blood is wasted by the sampling method -- e.g.
the
amount of blood left in a syringe needle or the tip of the syringe
(we've
found up to 100 µL left behind in a syringe) so we think that it may
well
be possible to take more samples or more blood/sample since there is no
waste of blood due to the sampling method. We have to insert a catheter
into the mouse -- jugular vein is not too bad even with 25 g mice but
femoral vein is proving too small thus far.
Sincerely,
Candice Kissinger
Director of Research
BASi
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Jose,
Its my experience that you can collect two samples if the first is small
enough. We routinely do orbital sinus bleeds into a small capillary
tube
for the first collection time. The blood volume is around 2 mL, so it
is
not prohibitive to collect 150 ul at the first time point.
Jeffrey L. Larson, Ph.D.
Director of Toxicology and Pharmacokinetics
Tanox, Inc.
4888 Loop Central Drive
Houston, TX 77081-2225
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Dear all,
Further to the question which has been asked by Jose M.
Valdivielso, (a)is there any literature reference where 2
samplings can be taken from the mice species? (first one being
<10% total blood volume and second one being terminal bleeding)
(b) How siginificantly it will affect the pk and pd of a
compound?
(c) Should there be any minimum time gap b/w the first time
point(<10%TBV) and the second time point(terminal bleeding) during
a pk study?
(d) During the first sampling is anaesthesia is very much
important? (if yes which one and what dose would be ideal)?
Thanks in advance for your inputs,
Regards,
B.L.Suresh
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