Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
Workflow and sampling
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)
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.
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)
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)