Svenson3 project protocol

Hematology and blood chemistry of C57BL/6J-Chr#PWD/Ph/ForeJ mouse chromosome substitution strains   (2006)

Svenson KL, Forejt J, Donahue L, Paigen B
With: Marion M

See also: Svenson3 animal documentation


Svenson3_Protocol

Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedure for blood hematology using ADVIA 120 system
Procedure for blood coagulation using Dade-Behring Blood Coagulation system
Procedure for blood chemistry using CX5 SYNCHRON Delta Chemistry Autoanalyzer system
Data
References

Workflow and sampling

Workflow

Test
Procedure performed
Volume (µL)
Age (wk)
Data collected
1
Body weight measurements
-
6, 7, 8
bw
2
Blood collection after 4h fast via retro-orbital bleed; samples are kept at 24°C (<3 hrs) until testing
~225
6
-
3
Plasma clotting factor measurements using Dade-Behring Blood Coagulation system
~125
6
AT III, factor VIII, fibrinogen
4
Hematology samples are run on the ADVIA 120 Hematology Analyzer
210
7
Blood cell counts (CBC) with differential
5
Blood collection after 4h fast via retro-orbital bleed; samples are kept at 24°C (<3 hrs) until testing
~270
8
-
6
Blood (plasma) chemistry using CX5 Synchron Delta Chemistry Autoanalyzer system
~160
8
CHOL, HDL, TG, FFA, GLU, T4, AST, TBIL, BUN

Equipment and supplies

• Blood coagulation system (BCS®, Siemens, Dade Behring) analyzer for clotting, chromogenic and immunologic coagulation assays.

For more product information


BCS® System

  • Bayer ADVIA 120 Hematology Analyzer (Bayer Diagnostic 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.

  • Refrigerated tabletop micro-centrifuge: Eppendorf Centrifuge, Model # 5415C
  • Repeat pipettors and regular Pipetman: 250 µL and 100 µL, respectively
  • Automated blood chemistry analyzer: Synchron CX5 Delta (Beckman Coulter, Inc., Fullerton, CA)
  • A dedicated DOS-based desktop computer controls the programming of this analyzer
  • A dedicated printer prints the results as they are measured, and an electronic file is simultaneously transferred to a second, Windows-based computer, which stores the data files
  • Beckman Synchron CX5 Delta blood chemistry analyzer system
  • Micro hematocrit tubes: heparinized, 75 mm, 75 µL volume size (Fisherbrand®)
  • Blood collection tubes: Microtainer® tube containing lyophilized K2EDTA (BD (Becton-Dickinson) Diagnostics, Franklin Lakes, NJ) or (Fisher Catalogue # 36-5974)
  • Toothpicks
  • BCS® System consumables: pipettors/syringes, SCS cleaner, sample rack, and tray
  • BCS® System reagents: actin FS, Owren’s Veronal buffer, calcium chloride, and wash solution
  • Micro-hematocrit tubes (75µL capacity) coated with Heparin and equipped with a rubber bulb expunger

  • Panels A-E illustrate the Beckman Synchron CX5 Delta. Panel B shows a closer look of area 1 in Figure A. Figure C presents a closer look of area 2 in Panel A. Panel D depicts a closer view of area 3, where ancillary reagents are refilled in Panel A. Panel E reveals the content of area 4, where samples are set up in trays for an automated run in Panel B.

Reagents and solutions

  • BCS® System consumables: pipettors/syringes, SCS cleaner, sample rack, and tray
  • BCS® System reagents: actin FS, Owren’s Veronal buffer, calcium chloride, and wash solution
  • Micro-hematocrit tubes (75µL capacity) coated with Heparin and equipped with a rubber bulb expunger
  • BCS® System calibrators-controls: Standard Human Plasma control N (normal) and control P (pathological), human and mouse fibrinogen, ATIII, and factor VIII
  • Heparin anticoagulant (Sigma (Sodium Salt) 50,000 U Cat. #H-3393)
  • Heparin 1000U/mL: 50mL of distilled autoclaved/sterile water + 50,000U Heparin Stock Solution
  • Polyethylene glycol (PEG) (Sigma, Cat. #P7181)
  • Expendables: (1) 1.5 µL Eppendorf tube, (2) 0.5 mL Beckman Coulter Microtube™ Tubecup (“sector cup”) (3) 100 µL and 250 µL pipette tips
  • Reagents: The Chemistry Analyzer (or “CX5”) uses Beckman Coulter three-compartment reagent cartridges for HDL, CHOL, TG, and GLU. Each cartridge contains enough reagents for 300 tests (approximately 104 mL). In addition, in order to run the HDL Cholesterol test, HDL Cholesterol Separation Reagent (15 µL per sample) is needed. The bottle from Beckman Coulter contains a volume of 34 mL. If the dilution of plasma samples becomes necessary due to low plasma volume, use 0.9% saline solution for the dilution.
  • Calibration Reagents: The two calibration reagents are “Synchron® Systems HDL Cholesterol Calibrator” (for HDL only), and “Synchron® Systems Multi Calibrator” (for CHOL, GLU, and TG).Controls: The controls for HDLc are Beckman Coulter™ Vigil™ Lipid Control 1 and Beckman Coulter™ Vigil™ Lipid Control 2. The controls for CHOL, GLU and TG are Synchron® Control Comprehensive Chemistry Control Serum Level 1, Level 2 and Level 3.

Acclimation to test conditions

In general all mice are brought into the procedure room and are tested within 1 h.

Procedure for blood coagulation using Dade-Behring Blood Coagulation System

I. System setup
a.
The analyzer is allowed to reach optimum operating temperatures (incubator at 37°C and cooler at 15°C) before proceeding to daily maintenance.
b. Daily maintenance include checking valves and lines for leaks, cleaning syringe/pipettors to assure patency, restocking rotor trays, and discarding liquid wastes.
c. Once the analyzer is operational, system's controls are conducted according to manufacturer's protocol.
d. System's calibrations are performed whenever new lots of reagents are used, and/or new standard curves are needed and calculated.
e. Approximately 20-30 min is required to conduct the start up procedure, daily maintenance, and control runs.

II. Plasma sample acquisition and preparation
a.
Approximately 225 µL of blood sample is collected from 6-wk old mice via retro-orbital eye bleeding using plain/un-heparinized hematocrit tubes.
b. The blood is collected into 1.5 mL Eppendorf tubes and is mixed with 30 µL of 3.8% Sodium Citrate using toothpicks to prevent clotting.
c. The blood samples are then centrifuged for 10 min. at maximum speed (14,000 RPM) to separate the plasma containing clotting factors.
d. The top plasma sample layers are carefully transferred into another 1.5 mL tubes and then placed in the appropriately sized analyzer loading racks. The bottom packed red cell layers are discarded.

III. Measurement of plasma clotting factors using the BCS® system
The BCS® System analyzer used for haemostasis assays is operated and calibrated using manufacturer- former Dade Behring (Siemens) protoco
a. Lanes 1 and 2 contain refrigerated racks that are loaded with reagents necessary to perform each test: Thrombin, thromboplastin C+, actin FS, thrombin in ATIII kit, and SCS cleaner.
b. Lanes 3 through 14 are loaded with the following reagents: Calcium chloride and Owren's Veronal buffer.
c. Lane 4 contains the essential quality controls: control N (normal control) and control P (pathological control). These controls are reconstituted with 1 mL of sterile distilled water, gently mixed, and allowed to incubate for 15 min at room temperature. These control vials are then loaded in appropriate racks with open slot showing barcode identifiers.
d. Test controls for mouse fibrinogen, human fibrinogen, ATIII.Mse, ATIII human, and F8x4 are performed before actual samples are run.
e. After the test controls are done, the plasma samples above (II.-d. including empty vials) are identified/labeled and their tops removed ready for automatic sampling using lanes 4 through 14.
f. After all the samples have been run once, the displayed results are examined thoroughly, such that repeat tests that are needed to be done are selected and performed again.
g. To obtain printed copy of the results, open <Job List> and scroll down to highlight desired test results, and then deploy <Print>.
h. Following the completion of the sample tests, a <Shift Change> is implemented before the system is closed. This is done by selecting <System> in the "Upper Menu" bar and then opening <Shift Change> to select <Disinfect Only>.
i. The disinfection process takes about 10-15 min during which the analyzer clean the lines and pipettors and gets the system ready for the next set of samples, or, if desired, for the system to shutdown.
j. Depending on the number of tests (i.e. 30 samples) and repeat tests to be performed, a typical set of measurements require about 45 to 60 min.

Safety

The controls and standard human plasma used to calibrate and perform quality control have been derived from human plasma. Safety gloves and glasses should be worn during reconstituting and handling of these products. These products should also be disposed of in the appropriate manner and considered biohazard waste materials.

Investigator's notes: The assay procedures for Mouse Fibrinogen, ATIII, and Factor 8 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 for sample, Actin FS, and CaCl2 from 50 µL to 25 µL. 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 Control N and Control P. 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.

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

I. Standard blood collection
a.
Mice at 6-7.5, 12-13.5, and 18-19.5 months of age are fasted for 4 hrs (7:30 to 11:30 am) before they are bled.
b. Body weight measurements are recorded just before blood samples are drawn.
c. 270 µL of blood sample (about 11 drops plus one capillary tube) is collected via a retro-orbital bleed using a heparin-coated capillary tube directly into BD Microtainer tube coated with lyophilized K2EDTA.
d. 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.
e.
Whole blood specimens are kept at room temperature until analysis, however, anticipated delay of greater than 3 hrs required sample refrigeration, and subsequent return to room temperature before testing.

II. Blood cell count with differential and reticulocyte count using Bayer ADVIA 120 system
a.
Approximately 210 µL of un-clotted whole blood is transferred to a 2nd microtainer and is analyzed using the ADVIA 120 hematology system according to manufacturer's protocol.
b. For additional information see: Mouse Heart, Lung, Blood and Sleep Disorders Center (HLBS) Protocols.

Procedure for blood chemistry using CX5 SYNCHRON Delta Chemistry Autoanalyzer system

I. Blood plasma collection
a.
Only previously fasted (4 h) mice are used for the collection of blood.
b. Approximately 225 µL of blood (8-9 drops, depending on the size of the mouse) are obtained via retro-orbital bleed using heparin-coated Hematocrit tubes. Remaining blood in the Hematocrit tube is flushed out using a rubber squeeze bulb assembly.
c. Blood is collected into pre-labeled 1.5 mL Eppendorf tubes pre-loaded with 7.5 µL of the anticoagulant Heparin 1000 U/mL, gently finger flicked and mixed or stirred, and momentarily stored in ice until all the samples are ready to be centrifuged.
d. Blood samples are then centrifuged for 5 min at 14,000 RPM using a refrigerated table-top micro-centrifuge to separate the plasma.
e. The top plasma layer is pipetted (~100µL) into pre-labeled 0.5 mL Eppendorf tubes and frozen until ready to be assayed; remaining packed blood cell layer is discarded.
f. Previously frozen samples are defrosted at room temperature for about 30 min before measurements are done.

NOTES: Air bubbles are avoided and eliminated during sample loading in sector cups as they interfere with the colorimetric assay.


Panel F shows 2 empty sector cups. Panels G shows sector cups with/out hemolyzed sample, and Panel H shows sector cups without and with (red arrow) air bubbles. Panels I and J show consecutive sector trays identified with bar codes and seven sector cups contained within each tray.

II. Using Beckman CX5 Synchron system to measure plasma glucose and lipids
a.
In preparation for the auto-analyzer, bar-coded sectors with cups in place are loaded with 85 µL of completely thawed plasma (enough for the direct measurement of CHOL, GLU, and TG).
b. Up to five sectors are manually placed on the carousel to be run. Since each sector is bar-coded, the Beckman automatically detects a sector that has been run; regardless, finished sectors are removed immediately as soon as they come up.
c. The Beckman CX5 is operated according to manufacturer’s instructions in the measurement of plasma HDL, CHOL, GLU, and TG, which are run together.
d. Function key F1 is used to deploy “Sample Program”, Sample type “2” is for plasma

-Function key F2 is used to deploy “Program Batch/Sector(s)” and “sector(s) to program:” (i.e. sectors 1-5 is programmed). When "Batch mode activated, 7 cups possible. Number of cups in batch:" message is displayed, the total cups for this batch is then (7 x 5) = 35 cups (the maximum number of cups that can be ran in a batch is 98). To program remaining sectors, F2 Program Batch is deployed again.
-Panel “12” is preprogrammed for CHOL/GLU/TG and “HDLD” is manually “Selected” from the screen menu. Once the correct chemistries are selected, they are “ENTERed” to bring about “SAMPLE TYPE”, wherein “2” is given to denote plasma (not serum).
-Function key F8 is used to set up the programmed batches and to advance to the next cup/sample (from cup #1 up to cup #7) in a given sector. By selecting F8 ID numbers can be recorded and reviewed against an Excel reference sheet.

Note: Since ID sample numbers or field identifiers are un-editable once Beckman CX5 is in operation, relevant information, including sample type (i.e. plasma vs serum), dilution factor, and other information must be precisely entered.
- As soon as six sectors are programmed, the <prev screen> is activated first, and then the Master Screen, and then last START (green) button. The Beckman automatically starts sampling the first five programmed sectors. Additional sectors can be programmed while the Beckman is operating.
-In the event that the Beckman alarm is activated because of reagent volume getting low, <prev screen> is activated to turn off the alarm and make the necessary notations for the record. The next available reagent cartridge is automatically installed.
-When all the data is safely recorded, Function key F5 is used to clear all the information regarding a sector after ENTERing the number of the sector to be deleted.

e. Once a sector is finished running, the results are automatically printed, removed from the printer, and then labeled accordingly. Otherwise, the printed paper is checked and guarded from rolling back into the printer and disrupting the data recording.
f. Used Eppendorf tubes, pipette tips, sector cups, and reagent cartridges are discarded into biohazard waste containers, and any spilled liquids are cleaned.
g. For additional information: JAX-PGA Protocols.

Data collected by investigator

  • blood clinical chemistry
    • total bilirubin (plasma TBIL)
    • aspartate aminotransferase (plasma AST, SGOT)
    • glucose (plasma GLU, 4h fast)
    • blood urea nitrogen (plasma BUN)
  • clotting factors
    • antithrombin III (AT III) anticlotting factor, percent of normal human value
    • clotting factor VIII, percent of normal human value
    • blood fibrinogen
  • blood cell counts
    • total red blood cell (RBC, erythrocyte) count (units per volume x 106)
    • total white blood cell (WBC, leukocyte) count (units per volume x 103)
  • blood cells differentials
    • percent neutrophils (percent of total WBC)
    • percent lymphocytes (percent of total WBC)
    • percent monocytes (percent of total WBC)
    • percent eosinophils (percent of total WBC)
    • percent basophils (percent of total WBC)
    • percent large unstained cells (percent of total WBC)
    • platelet (PLT) count (units per volume x 103)
    • reticulocyte (Retic) count (units per volume x 109)
    • percent reticulocytes (percent of total RBC)
  • blood erythrocyte parameters
    • RBC corpuscular distribution width (RDW)
    • mean RBC corpuscular volume (MCV)
    • RBC corpuscular hemoglobin concentration mean (CHCM)
    • hemoglobin concentration distribution width (HDW)
    • mean RBC corpuscular hemoglobin content (MCH)
    • mean RBC corpuscular hemoglobin concentration (MCHC)
  • blood hematocrit (HCT)
  • blood hemoglobin
    • hemoglobin (HGB)
    • hemoglobin (HGB)
    • calculated hemoglobin (HGB)
  • blood mean platelet volume (MPV)
  • blood — lipids
    • high density lipoprotein cholesterol (plasma HDL)
    • total cholesterol (plasma CHOL
    • free fatty acids (plasma FFA)
    • triglycerides (plasma TG)


References

    Lippi G, Salvagno GL, Solero GP, Franchini M, Guidi GC. Stability of blood cell counts, hematologic parameters and reticulocytes indexes on the Advia A120 hematologic analyzer. J Lab Clin Med. 2005 Dec;146(6):333-40. PubMed 16310516

    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

    Svenson KL, Von Smith R, Magnani PA, Suetin HR, Paigen B, Naggert JK, Li R, Churchill GA, Peters LL. Multiple trait measurements in 43 inbred mouse strains capture the phenotypic diversity characteristic of human populations. J Appl Physiol. 2007 Jun;102(6):2369-78. Epub 2007 Feb 22. PubMed 17317875