Donahue9 project protocol

Blood chemistry of C57BL/6J-Chr#PWD/Ph/ForeJ mouse chromosome substitution strains   (2011)

Donahue L
With: Morgan J




Donahue9_Protocol

Project protocol — Contents
Workflow
Equipment and supplies
Reagents and solutions
Procedure: Blood chemistry and electrolyte measurement using Beckman CX5 Synchron Delta Chemistry Autoanalyzer
Data
References

Workflow and sampling

Workflow

Test
Procedure performed
Equipment
Data collected
1
 Body weight measurements
balance scale
bw
2
 Blood collection via retro-orbital bleed
heparinized hematocrit tubes
-
3
 Blood processed for plasma
centrifuge
-
4
 Blood chemistry
chemistry autoanalyzer system

Equipment and supplies

  • 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)
    • Dedicated DOS-based desktop computer for the analyzer
    • 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 (75 µL 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.

    For Synchron CX5 parts and all consumables: Beckman Customer Support

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 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

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

I. Blood serum collection
a.  Only previously fasted (4h) 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 1000U/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.

II. Using Beckman CX5 Synchron system to measure chemistries
a. In preparation for the auto-analyzer, bar-coded sectors with cups in place are loaded with 85 µL of completely thawed plasma.
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.
d. Function key F1 is used to deploy "Sample Program", Sample "type2" 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.


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.

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.

Data collected by investigator

  • Electrolytes
    • total Bilirubin (TBIL)
    • chloride (Cl)
    • magnesium (Mg)
    • phosphorus (P)
    • potassium (serum K)
    • sodium (Na)
    • dissolved-ionized carbon dioxide (CO2)
  • Enzymes and proteins
    • alkaline phosphatase (ALP)
    • alanine aminotransferase (serum ALT)
    • aspartate aminotransferase (AST)
    • lipase (LIP)
    • lactate dehydrogenase (LDH)
    • albumin (Alb)
    • serum creatinine
    • total protein (sTP)
    • blood urea nitrogen (BUN)