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Peters LL, Barker JE
Hematology and clotting time survey in 43 inbred strains of mice
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Hematology parameters:
complete blood count with white cell differentials,
reticulocyte and red blood cell indices,
platelet count and indices
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Conditions: Mice were shipped from JAX production facility
at 8 wks of age and fed
LabDiet 5K52 (6% fat)
during the acclimation period.
Sample collection: Blood (250 uL) was collected into an eppendorf
tube containing 20 uL 20% EDTA via retro-orbital bleed. This procedure
minimizes clot formation. Values reported are those obtained directly
from the ADVIA; no correction for the small dilution was made.
Equipment: ADVIA 120 Hematology System (Bayer Diagnostics
Division, Tarrytown, NY, USA)
Reagents and expendables: all from Bayer Diagnostics
Division.
Controls: calibrated commercial controls were run before
each use and were within established ranges prior to
analyzing samples.
Loading and running: Samples were run according to
manufacturer's suggested protocol.
More details may be found at
http://pga.jax.org/protocol_006.html
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Coagulation parameters: fibrinogen, prothrombin time, partial thromboplastin time
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See Peters, et al., 2002 for more details.
Conditions: Mice were shipped from JAX production facility
at 8 wks of age and fed
LabDiet 5K52 (6% fat)
during the acclimation period.
Sample collection: Mice were bled from the retro-orbital
sinus using an uncoated microhematocrit tube. 275 uL of
blood was mixed into an microcentrifuge tube containing 30
uL of 3.8% sodium citrate in phosphate-buffered saline (PBS;
10mM NaCl, 155 mM KCl, 10 mM glucose, 1 mM MgCl2, 2.5 mM
KHPO4, pH 7.4). The standard ratio of whole blood to
anticoagulant was used in all cases (9:1 (vol/vol) whole
blood : 3.8% citrate). For each mouse, the collection
process was completed within 5 - 20 s. Each sample was
checked for clots using a standard wooden toothpick.
Samples were centrifuged at 15,000 rpm for 10 min at room
temperature to separate plasma. The plasma was centrifuged
again at 15,000 rpm for 10 min at room temperature to remove
any remaining cellular debris (this was an important step
for establishing reliable baselines). Plasma samples were
transferred to Fisher Scientific brand 1.5 mL
microcentrifuge tubes and then placed in the appropriate
sized loading racks. Samples were analyzed immediately.
Equipment: Dade-Behring Blood Coagulation System (BCS) (Deerfield, IL, USA).
Reagents: Controls and reagents were purchased from Dade Behring.
Reagents and standards designed for use in human clinical testing were used in all procedures (see below).
Fresh vials of normal (N) and pathological (P) controls were
prepared prior to running samples. These controls were
reconstituted with 1 mL of sterile distilled water, gently
swirled to mix, and allowed to sit for at least 15 min
before using.
Modified protocol: The assay procedures for mouse samples
were modified from the corresponding standard human assay
procedures in order to lower the sample volume requirements.
The Dade-Behring BCS protocol for each test was modified by
duplicating and renaming the original assay procedure file.
The pipetting cycle page of the newly created assay
procedure was then modified by changing the volume from 50
uL to 25 uL for the following: sample, Actin FS, and CaCl2.
A corresponding assay definition file was also made by
duplicating the original and renaming it accordingly. The
new test procedures were validated using Dade-Behring normal
and pathological controls. The values obtained when run on
"Mouse Assay" procedures vs. the standard "Human Assay"
procedures were not statistically significant for any of the
tests and are available upon request
(Luanne Peters) .
Load and run samples: according to manufacturer's procedure.
Safety: Some controls are derived from human plasma. Safety
gloves and glasses should be worn during reconstitution and
handling of these products. These products should be
disposed of in the appropriate manner and considered a
biohazard waste material.
More details may be found at
http://pga.jax.org/protocol_003.html.
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References
Peters LL, Cheever EM, Ellis HR, Magnani PA, Svenson KL, Von Smith R, Bogue MA. Large-scale, high-throughput screening for coagulation and hematologic phenotypes in mice.
Physiol Genomics. 2002 Dec 3;11(3):185-93.
PubMed 12419856
MGI
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Test age:
10wks
