Jaxpheno4 project protocol

Hematological survey of 11 strains of mice   (2006)

Jackson Laboratory
With: Denegre J




Jaxpheno4_Protocol

Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
 Procedure
Data



Workflow and sampling

 Workflow

Steps
Accomplished procedure
Volume (µL)
Data recorded
1
Blood collection with EDTA anticoagulant
200
-
2
Blood samples are kept at 24°C until run (<6hrs)
-
-
3
Hematology samples are run on the ADVIA 120 Hematology Analyzer
200
CBC with differential and retic (CDR)

Equipment

Bayer ADVIA 120 Hematology Analyzer (Bayer Diagnostics Division (Siemens), Tarrytown, NY, USA).

Panel A shows the Bayer ADVIA 120 hematology analyzer system. Panel B shows the internal layout of the ADVIA, and the various reagents used in the system. Panel C shows the hematology setup, equipped with a computer and a printer. Panel D shows a typical computer screen-output.

More equipment information

ADVIA® 120 Hematology System Product Features

Reagents, supplies, solutions

Microhematocrit tubes: heparinized, 75mm, 75µL volume size (Fisherbrand®)

  

Blood collection tubes: Microtainer® tube containing lyophilized K2EDTA (BD (Becton-Dickinson) Diagnostics, Franklin Lakes, NJ) or (Fisher Catalogue # 36-5974)

Toothpicks

Local anesthetic: Tetracaine Hydrochloride Ophthalmic solution USP 0.5%

Acclimation to test conditions

Mice are obtained at 8 and 16 wks of age (DOB +/- 3 days) from production rooms and are acclimated to test conditions for at least half-hour.

Procedure: CBC with differential and reticulocytes count using Bayer ADVIA 120 Hematology Analyzer

Standard blood collection
a. A drop of local anesthetic (tetracaine) is applied on the eye before blood is drawn.
b. 200µL of blood sample is collected via a retro-orbital bleed using a heparin-coated capillary tube directly into BD Microtainer tube coated with lyophilized K2EDTA).
c. The freshly drawn blood is quickly and gently mixed with the anticoagulant using a toothpick. Vortexing or inverting the sample is not done in order to preserve the proper ratio of anticoagulant to blood and also to preserve the integrity of the Red Blood Cells.
d. Whole blood specimens are kept at room temperature until analysis, however, anticipated delay of greater than 6 hours required sample refrigeration, and subsequent return to room temperature before testing.

Blood cell count with differential and reticulocytes count using Bayer ADVIA 120 system

A minimum of 200µL of un-clotted whole blood is analyzed using the ADVIA 120 hematology system according to manufacturer's protocol.

Data collected by investigator

White blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), platelet count (Plt), mean platelet volume (MPV), reticulocytes hemoglobin content (CHr), reticulocytes count (#retic), reticulocytes percent (%retic) neutrophil count (#NEUT), lymphocyte count (#LYMPH), monocyte count (#MONO), eosinophil count (#EOS), basophil count (#BASO). neutrophil percent (%NEUT), lymphocyte percent (%LYMPH), monocyte percent (%MONO), eosinophil percent (%EOS), and basophil percent (%BASO).


This figure illustrates a close-up display of the computer screen result in panel D above.

Definitions & formulas

HCT (hematocrit) = (RBC X MCV)/10 representing the PCV (packed cell volume)