Collins1 project protocol

Hematological parameters in 8 inbred founder strains and 130+ emerging lines (pre-CC) of the Collaborative Cross   (2012)

Collins FS
With: Kelada SP, Aylor DL, Peck BE, Ryan JF, Tavarez U, Buus RJ, Miller DR, Chesler EJ, Threadgill DW, Churchill GA, de Villena FP

Collins1 animal documentation

Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure for conducting complete blood cell count (CBC)
Definitions
Data
References

Workflow and sampling

Workflow

Step	Procedure performed	Equipment	Data collected
1	Mice acclimated for at least a week	-	-
2	Mice bled via retro-orbital sinus	blood collection kit	-
3	Blood samples analyzed	Hematology analyzer	WBC, RBC, neutrophil, lymphocyte, monocyte, eosinophil, basophil, and platelet counts; RBC and platelet parameters

Equipment and supplies

• Blood collection kit: sample tube, hematocrit tube etc.
• HEMAVET Multispecies Hematology analyzer (Drew Scientific Inc., Waterbury, CT)

Reagents and solutions

• Anticoagulants: EDTA

Acclimation to test conditions

Mice are acclimated to the facility for a minimum of 1-wk before testing.

Procedure for conducting complete blood cell count (CBC)

a. Whole blood samples for CBC are collected by retroorbital sinus puncture into tubes containing EDTA as an anticoagulant.
b. Blood samples are gently mixed with the index finger to assure proper mixture of anticoagulant.
c. About 20 µL blood is drawn automatically into pre-calibrated and pre-configured HEMAVET Multispecies Hematology analyzer, according to manufacturer's instruction, to initiate testing.
d. After 2 min sample cycle, results are automatically printed, including lymphocyte count and percentage (LYM, LYM %), monocyte count and percentage (MONO, MONO %), neutrophil count and percentages (NEUT, NEUT %), eosinophil count and percentage (EOS, EOS %), and basophil count and percentage (BASO, BASO %), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean platelet volume (MPV), hematocrit (HCT), hemoglobin (HGB), platelet count (PLT), red blood cell count (RBC), and white blood cell count (WBC) (see image below, right panel).

A. HEMAVET Multispecies Hematology analyzer, B. Sample probe and holder, C. Display screen and keypad, D. Species selection keys, and E. Printed result.

Definitions

Collaborative Cross (CC): is an emerging mouse recombinant inbred (RI) panel that was designed to extend the available mouse genetic reference populations (Philip et al. 2012) by combining the genomes of eight genetically and phenotypically diverse strains through randomized breeding design that systematically outcrosses eight founder strains, followed by inbreeding to obtain new recombinant inbred strains.

Founder strains: five common inbred (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ) and three wild-derived inbred (CAST/EiJ, PWK/PhJ, WSB/EiJ) mice

Data collected by investigator

blood cell counts

• white blood cell count (WBC; units per volume x 10³)
• lymphocyte count (LYM; units per volume x 10³)
• lymphocyte differential (LYM; percent of viable WBC)
• neutrophil count (NEUT; units per volume x 10³)
• neutrophil differential (NEUT; percent of viable WBC)
• monocyte count (MONO; units per volume x 10³)
• monocyte differential (MONO; percent of viable WBC)
• eosinophil count (EOS; units per volume x 10³)
• eosinophil differential (EOS; percent of viable WBC)
• basophil count (BASO; units per volume x 10³)
• basophil differential (BASO; percent of viable WBC)
• red blood cell count (RBC; units per volume x 10⁶)

blood erythrocyte and platelet parameters

• RBC corpuscular distribution width (RDW)
• mean RBC corpuscular volume (MCV)
• hemoglobin (HGB)
• platelet count (PLT; units per volume x 10³)
• mean platelet volume (MPV)