| This project is part of the Heart, Lung, Blood and Sleep Disorders Consortium. This page describes protocols used for hematology parameters (complete blood count with white cell differentials, reticulocyte and red blood cell indices, platelet count and indices), and coagulation parameters (fibrinogen, prothrombin time, partial thromboplastin time). These protocols were used in MPD projects Peters1, Peters2 and Peters3. |
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 |
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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