CGDpheno1 project protocol

Multi-system survey of mouse physiology in 72 inbred strains of mice (ANOVA-adjusted methodology)   (2009)

Center for Genome Dynamics (CGD)
With: Korstanje R, Svenson K, Churchill G, Yang H

See also: CGDpheno1 animal documentation


CGDpheno1_Protocol

Project protocol — Contents

General workflow and sampling

Procedure: Blood chemistry
Workflow and sampling
Equipment and supplies
Reagent and solutions

Definitions and calculations

Procedure: Complete blood count (CBC) with differential
Workflow and sampling
Equipment and supplies
Reagents and solutions
Definitions and calculations

Procedure: Bone mineral density and body composition
Workflow and sampling
Equipment and supplies
Reagents and solutions
Definitions and calculations

Statistical analysis and calculations: Mixed-effect model fitting (ANOVA adjustment)
Data
References

General Workflow
Tests
Age (wks)
Equipment used
Data collected
11
Bayer ADVIA 120
Complete differential cell count
Blood plasma chemistry
10, 12
Beckman CX5 Synchron Delta
Complete plasma chemistry
13
DXA PIXImus
Fat, lean, total composition; whole BMD

Acclimation to test conditions

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

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Blood Plasma Chemistry

Workflow and sampling

Steps
Procedure accomplished
Time used (hr)
# of Samples
Data collected
1
Mice are fasted: food and water removed for 4h
0.5
-
-
2
Sample tubes prepared for blood collection
0.5
80 samples/batch
-
3
Blood collected via retro-orbital vein
2.5
80 samples/batch
-
4
Post-blood collection clean-up
0.5
80 samples/batch
-
5
Blood samples centrifuged at 4°C
1.0
80 samples/batch
-
6
Plasma extracted (frozen when stored)
1.0
80 samples/batch
-
7
Beckman CX5 system calibrated
0.25
-
-
8
Reagents changed or replenished
0.25
-
-
9
System controls run
0.25
-
-
10
Plasma HDL test prepared
2.5
15 sectors (105 samples)/run
-
11
Plasma HDL measured automatically
1.0
15 sectors (105 samples)/run
-
12
Plasma CHOL, GLU and TG prepared
1.0
15 sectors (105 samples)/run
-
13
Plasma electrolytes, lipids, glucose, and proteins measured automatically
3.0
15 sectors (105 samples)/run
Na, Cl, K, CO2, CHOL, GLU, TG, HDL, NEFA, T4, TBIL, BUN
14
Final clean-up
0.5
-
-
15
Computer printed data are labeled, data files collected and stored on disks
0.5
-
-

Equipment and supplies

  • Refrigerated tabletop micro-centrifuge: Eppendorf Centrifuge, Model # 5415C.
  • Repeat pipettes 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.
  • Micro-hematocrit tubes (75µL capacity) coated with Heparin and equipped with a rubber bulb expunger
  • 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
    (4) 1 cc syringes with needles

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

  • 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
  • Sterile physiological (0.9%) saline solution
  • 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.
  • Reagent:                                          Reorder No.                
    Cholesterol (CHOL) Reagent                  467825
    Glucose (GLU) Reagent                         442640
    Triglycerides (TG) Reagent                    445850
  • 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 HDL 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.

Procedure: Blood plasma chemistry and electrolyte measurement using Beckman CX5 Synchron Delta Chemistry Autoanalyzer

I. Blood plasma collection
a. Blood samples are obtained from each animal at 10 and 12 weeks of age (after 4h fast from 0700 - 1100 h).
b. Approximately 200 µL of blood are obtained from mice via retro-orbital bleed using heparin-coated Hematocrit tubes. Remaining blood in the Hematocrit tube is flushed out using a rubber squeeze bulb assembly.

Investigator's note: In order to be able to bleed the mice the following week for another test, the same volume of blood taken is replaced subcutaneously with sterile physiological saline.

c. Blood samples from 12-wk old mice are collected into pre-labeled 1.5 mL Eppendorf tubes pre-loaded with 7.5 µL of the anticoagulant Heparin 1000U/mL, gently finger flicked and mixed or stirred, and momentarily stored in ice until all the samples are ready to be centrifuged. Blood samples from 10-wk old mice destined for electrolytes analysis are collected without pre-loading with heparin.
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 (for heparinized samples, or serum for non-heparinized samples) 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.

II. Using Beckman CX5 Synchron system to measure plasma glucose and lipids chemistries

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.

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.

Notes: 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.

For additional information

Definition and calculation

The average of the diluents analyzer value is subtracted from the analyzer value multiplied by two (dilution factor) to obtain the HDL (indirect) values.

HDL (indirect) values = (sample analyzer value x 2) - average of diluents analyzer value

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Hematology

Workflow and sampling

Test
Procedure performed
Volume (µL)
Data collected
1
Body weight measurement
-
body weights
2
Blood collection; samples are kept at 24°C (for <3 h) until testing
200
-
3
Hematology samples are run on the ADVIA 120 Hematology Analyzer
200
Complete blood cell counts (CBC) with differential

Equipment and supplies

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.

Reagents and solutions

Bayer Diagnostics (Siemens Healthcare Diagnostics)
• ADVIA TESTpoint Hematology Controls
• ADVIA 120 autoRETIC Reagent T01-3622-01
• Perox Sheath T01-3633-01
• Perox 1 T01-3630-01
• Perox 2 T01-3631-01
• Perox 3 T01-3632-01
• RBC/PLT T01-3627-01
• HGB T01-3628-01
• BASO T01-3629-01
• AutoRETIC T01-3622-01
• EZ KLEEN T01-3624-01
• Defoamer T01-3625-04
• Sheath/Rinse T01-3664-01

  • 20% EDTA, Tetrasodium Salt (Fisher Lab item number BP121-500), anticoagulant

Procedure: Complete blood count (CBC) with differential using the Bayer ADVIA 120 Hematology Analyzer

Blood collection
a.    Blood samples are obtained from each animal at 10 weeks of age (non-fasted).
b.    Collection tubes (1.5 mL Eppendorf) are pre-loaded with 20 µL of 20% EDTA.
c.    A clean retro-orbital bleed is performed using EDTA-coated microhematocrit tubes.
d.    Blood collection tubes are filled to a final volume of 200 µL and then kept at room temperature for the duration of the test.
e.    The blood sample is checked for clots using a toothpick. (In the presence of a clot, the sample is discarded to avoid "aspiration failure" and lost data during sample run.)

Using Bayer ADVIA 120 system to conduct CBC with differential and reticulocytes count
a.    Blood samples are gently mixed well (by rolling the vial the between hands) and kept at ambient temperature.
b.    Samples are run according to manufacturer's instructions.
c.    As part of ADVIA daily routine maintenance, a control run is initially performed. This usually takes about 25 min to do. Since controls contain human blood cells in a preservative medium, proper safety precaution is exercised with the use of disposable gloves.
d.    As long as calibrated commercial controls are within established ranges, the PRIMER sample can be run first (to get the system "wet") in preparation for the test samples. Each run takes about 1 min, when done through open tube aspiration wand.
e.    To prevent "aspiration failure" or loosing a sample run, the blood sample is double-checked for clots so that no air gap is introduced while the sample is being aspirated.
f.     After each successful run, a lab screen pops up that can be hidden to return to Manual Sample ID Screen, and to review the result via the quality control (QC) button.
g.    After a hard copy of a successful run is automatically printed, the next sample is ready to be run.
h.    Values reported are those obtained directly from the ADVIA.
i.      No correction for the small dilution is made.
j.     For additional information.

Definitions and calculations

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

WBC differential percentages were computed for each cell type as follows:

  • % NEUT = [(NEUT count x 106) ÷ (total WBC count x 106)] x 100
  • % LYMPH = [(LYMPH count x 106) ÷ (total WBC count x 106)] x 100
  • % MONO = [(MONO count x 106) ÷ (total WBC count x 106)] x 100
  • % EOS = [(EOS count x 106) ÷ (total WBC count x 106)] x 100
  • % BASO = [(BASO count x 106) ÷ (total WBC count x 106)] x 100
  • HCT (hematocrit) = (RBC X MCV)÷10 representing the PCV (packed cell volume)
  • % Retic = [(Retic count x 109) ÷ (total RBC count x 109)] x 100

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Bone mineral density and body composition

Workflow and sampling

Step
Procedure accomplished
Age (wks)
Equipment/Reagent
Data Collected
1
Obtained body weight
13
top-loading scale
body weight
2
Mice anesthetized and measured for body length
13
needles and syringes, anesthesia
body length
4
DXA system prepared and calibrated
-
DXA system
-
5
Whole body scan
13
DXA system
whole body scan images
6
Analysis of whole body scan images
13
DXA system
bone mineral density (BMD), fat and lean tissue mass

Equipment and supplies

  • Top loading balance: Ohaus Portable Navigator Series Electronic, Model NV-210 (Ohaus Corp., Pine Brook, NJ) for measuring body weights.
  • DXA scanning by PIXImus: The Lunar PIXImus small animal DXA system (PIXImus™, Fitchburg, WI) was used to assess whole body areal (a)BMD and body composition at 16 weeks of age. This methodology has been validated in small animals (see Nagy, 2000; Nagy, 2001). 
  • Disposable specimen trays with sticky immobilizing tape: Lunar PIXImus Corporation Headquarters, 726 Heartland Trail, Madison, WI 53717, Phone: 800-445-8627, Fax: 608-826-7102.
  • Needles and syringes
  • Mouse densitometer dual energy X-ray absorptiometry (PIXImus small animal) DXA system (GE-Lunar, Madison, WI): The PIXImus mouse densitometer has been reconfigured with lower x-ray energy than in human DXA machines in order to achieve optimal contrast in small specimens. The Lunar PIXImus for rodents is a fully integrated densitometer designed for the estimation of bone mineral density (BMD) and body composition. The resolution of the PIXImus is 0.18 x 0.18 mm pixels with a usable scanning area of 80 x 65 mm, allowing for measurement of a single mouse or collections of isolated specimens. The PIXImus has been calibrated with a phantom utilizing known values, and QA is performed daily with this same phantom. The precision for BMD is less than 1% coefficients of variations (CV) for whole body, approximately 1.5% CV for specialized regions (Nagy et al, 2000). Correlation with pQCT values for 614 isolated spinal vertebrae is significant (p<0.001; r=.704). Assessment of accuracy for the PIXImus is done with a set of hydroxyapatite standards (0-2,000 mg), yielding a correlation of 0.999 between standards and PIXImus measurement of mineral. Full body scans and X-ray absorptiometry data are processed with manufacturer supplied software (Lunar PIXImus 2,vers. 2.1). For additional information: http://piximus.com/


Figure 1 A: Lunar PIXImus2 densitometer with integrated PC computer. Figure1 B: Close-up detail of the densitometer with specimen tray.

Reagents and solutions

  • Freshly prepared solution of tribromoethanol for anesthesia
  • Cleaning supplies and disinfectant

Procedures: Bone mineral density and body composition measurement using DXA PIXImus

I. Collecting image scans
PIXImus (small animal DXA system, PIXImus™, Fitchburg, WI) scanning of mice for BMC and body composition is both accurate and precise although body size must be considered when comparing inbred strains. Full body scans are obtained and X-ray absorptiometry data gathered and processed with manufacturer supplied software (version 1.43.036.008).

a. The PIXImus densitometer apparatus is initially calibrated with a "phantom mouse" according to manufacturer's protocol.
b. 13-wk old mice are first weighed and body weight recorded, anesthetized intraperitoneally with tribromoethanol at a dose of 0.2 mL/10g body weight, and then measured for body length (from the tip of the nose to the base of the tail).
c. Fully anesthetized mice are positioned dorso-ventral with the tail positioned away or alongside from the body, the front legs extended to the side, and the neck and spine are gently straightened.
d. Then each mouse is placed on the specimen sticky tray (body must be within blue line on the tray) under the PIXImus beam path (see Figure 1B above). The tail is placed alongside the body, the front legs are extended to the side, and the neck and spine are gently straightened.

e. Trays are positioned such that the area of the head is always oriented toward the left from the investigator's point of view, and the mice are position dorso-ventral- in order to scan the entire body and tail. The X-ray process to obtain a single full scan is approximately 5 minutes; data can be manipulated subsequently to obtain specific regions of interest (ROI's).
f.
After the whole body (excluding the head) is scanned, the isolated head alone is also scanned.
g. Disposable plastic trays, with sticky tape for immobilizing mice, can be saved and re-used after a thorough cleaning and disinfections.

Investigator's notes: To accommodate the large scope of this project, animals are phenotyped in “rounds” beginning every three weeks, over a period of approximately 18 mo. Every effort is made to include a complete strain set within each round. To correct for seasonal effects, changes in the equipment, and differences between different mouse rooms (A vs. B), C57BL/6J males and females are included in each round for each room. See CGDpheno2 for C57BL/6J controls over time.

II. Measurement acquisition and image scan analysis
Based on PIXImus validation studies (Nagy, 2000; Johnston, 2005) DXA- estimated measurements of fat tissue correlate well with measurements obtained from chemical extraction. This is made possible by developing software versions with equations that adequately correct raw DXA-estimated measurements.

a. Following the completion of an image scan the DXA system automatically implements specialized software to identify bone tissue from either fat tissue or from lean tissue based on the resulting X-ray densities at two distinct energy levels (Pietrobelli, 1996; Johnston, 2005).
b. Visually, following the completion of a scan, the mouse sample is then outlined with red and green colored circle and square to define specific regions of interests (ROIs).

d. By using the screen interactive display, F3 is first clicked to prompt measurement adjustments, and then clicked again for the second time to adjust ROI (region of interest).
e. The area to be analyzed is defined  (red box), and areas to be excluded from the calculations are defined (green). The arrow keys are used to adjust to the desired size, in addition to holding the control key down to enlarge or elongate the circle or square areas.
f. Once the desired ROI is achieved, the Enter key is clicked and resulting data measurement is displayed. By pressing F5 a hard copy of the image and the scan analysis result is printed.
g. To prompt the computer to finish the session, F8 or Esc key is clicked once, and then clicked again to return to the main menu screen where the next subject to be tested begin.
h. Acquired data is saved on the hard drive and on a zip or CD disk for later archiving.

Safety

For safety, gloves must be worn and radiation safety guidelines are strictly adhered to, such that technicians must be 6 feet away from the PIXImus machine during scanning.

Definitions and calculations

ROI = Region of interest

Bone area measurement is generated by outlining or specifying the limits or dimensions of the entire skeletal bone regions of the body (yellow outline in figure below), excluding the head (green), as regions of interest (ROI, red) following a full body X-ray scan.

Bone mineral content (BMC) is generated from PIXImus density scans which are assessed for accuracy using a set of 0.0 mg to 2,000 mg of hydroxyapatite standards. According to the DXA system, bone mineral content (measured as the attenuation of the X-ray by the bones being scanned) is divided by the area (also measured by the machine) of the site being scanned to obtain bone mineral density (BMD):

BMC = Bone mineral content (g)

BMD = Bone mineral density = BMC ÷ Area (g/cm2)

Fat tissue mass = all tissues with low density (x-ray scan)

Lean tissue mass = Total body tissue mass - Fat tissue mass

% Fat = (Fat tissue mass ÷ Total body tissue mass ) x 100

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Statistical analysis and calculations: Mixed-effect model fitting (ANOVA adjustment)

Mixed-effect model fitting (ANOVA adjustment)
Phenotypes were measured over an 18-month period. Each round may reveal different seasonal or machinery effects. To adjust the round effect, a mixed-effect model was implemented. Different room environments (room A vs B) can also cause measurement variance. However the number of strains measured in each room at each round is not large enough; therefore, only the round effect is corrected for. In this mixed-effect model, phenotype is explained by sex and strain effect (fitted terms), and by round effect (random term). After the initial mixed model fitting, observations outside of 1.5*IQR (interquantile range) were removed. The mixed model was refit and the mean estimators and standard error (SE) were computed.

 

Data collected by investigators

  • blood — clinical chemistry
    • total bilirubin (plasma TBIL)
    • chloride (plasma Cl)
    • potassium (plasma K)
    • sodium (plasma Na)
    • dissolved-ionized carbon dioxide (CO2)
    • glucose (plasma GLU, 4h fast)
    • blood urea nitrogen (BUN)
  • blood — hematology — cell counts
    • red blood cell count (RBC; per volume x 106)
    • white blood cell count (WBC; per volume x 103)
    • eosinophil differential (percent of total WBC)
    • lymphocyte differential (percent of total WBC)
    • monocyte differential (percent of total WBC)
    • neutrophil differential (percent of total WBC)
    • platelet count (PLT; units per volume x 103)
    • reticulocyte differential (percent of total RBC)
  • blood — hematology — erythrocyte parameters
    • RBC corpuscular distribution width (RDW)
    • mean RBC corpuscular volume (MCV)
    • hemoglobin concentration distribution width (HDW)
    • mean RBC corpuscular hemoglobin content (MCH)
    • mean RBC corpuscular hemoglobin concentration (MCHC)
  • hematocrit (HCT)
  • measured hemoglobin (HGB)
  • mean platelet volume (MPV)
  • blood — lipids
    • HDL cholesterol (plasma HDL)
    • total cholesterol (plasma CHOL)
    • non-esterified free fatty acids (plasma NEFA, FFA)
    • triglycerides (plasma TG)
  • body composition without head
    • percent fat
    • calculated lean tissue mass
    • calculated total tissue mass
  • body length (tip of nose to base of tail)
  • body weight
  • whole body bone mineral density
  • thyroxine (plasma T4)


References

    Nagy TR, Clair AL. Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition of mice. Obes Res. 2000 Aug;8(5):392-8. PubMed 10968731

    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

    Pietrobelli A, Formica C, Wang Z, Heymsfield SB. Dual-energy X-ray absorptiometry body composition model: review of physical concepts. Am J Physiol. 1996 Dec;271(6 Pt 1):E941-51. PubMed 8997211

    Pietrobelli A, Wang Z, Formica C, Heymsfield SB. Dual-energy X-ray absorptiometry: fat estimation errors due to variation in soft tissue hydration. Am J Physiol. 1998 May;274(5 Pt 1):E808-16. PubMed 9612238