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Assessment of pulmonary function Whole body plethysmograph (Buxco

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Assessment of pulmonary function Whole body plethysmograph (Buxco

BLOOD PLASMA CHEMISTRY PROTOCOL

Prepared By: MAM

    Date Prepared: April 6, 2001

    Code: MP-004

    Reviewed By: KLS

    Revision #: 2.0

    Date Revised:

    0.0 Abstract: The Beckman Coulter Synchron CX5 (“Chemistry Analyzer”) is used to

    measure four blood plasma constituents for this PGA: HDL cholesterol (HDL), total

    cholesterol (CHOL), triglycerides (TG), and glucose (GLU).

1.0 Instrument Layout: A dedicated DOS-based desktop computer controls the

    programming of the Chemistry 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.

2.0 Reagents and Expendables.

    2.1 Expendables: The following supplies and quantities are used for each sample

    tested (for all four of the above chemistries): 1.5;l Eppendorf tube (1), 0.5ml

    Beckman Coulter Microtube? Tubecup (“sector cup”) (2), and 200;l pipette tips

    (3-4, depending on whether dilution is necessary).

    2.2 Reagents: The Chemistry Analyzer (or “CX5”) uses Beckman Coulter three-

    compartment reagent cartridges for HDLc, CHOL, TG, and GLU. Each cartridge

    contains enough reagent for 300 tests (approx. 104mL). In addition, in order to

    run the HDL Cholesterol test, HDL Cholesterol Separation Reagent (15;l per

    sample) is needed. The bottle from Beckman Coulter contains a volume of 34ml.

    If the dilution of plasma samples becomes necessary due to low plasma volume,

    use 0.9% saline solution for the dilution.

    Reagent: Reorder Number:

    Cholesterol (CHOL) Reagent 467825

    Glucose (GLU) Reagent 442640

    Triglycerides (TG) Reagent 445850

    2.3 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).

    2.4 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.

    3.0 Setup:

    3.1 Reagent Preparation: Using the DOS computer, check the number of runs

    available for each reagent cartridge using the following sequence: from the Main

    Menu, press <F2> (“REAGENT LOAD”), and then <F5> (“REAGENT

    STATUS”). Next, page down through the reagents to check the number of runs

    left in each cartridge (under “AVAIL”) for the four desired chemistries. Change

    the reagent cartridges as necessary, using the following procedure: from the Main

    Menu, press <F2> (“REAGENT LOAD”), and then page down through the

    reagents until the cursor is on the reagent that needs changing. Press <SELECT>

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    to highlight it, and then press <F1> (“AUTO LOAD”). When prompted to do so by the CX5, open the reagent door and remove the empty cartridge, taking care to slide it past the laser bar code reader (listen for the “beep”). Obtain the new

    reagent cartridge in the adjacent refrigerator, and load it past the bar code reader in the same manner into the CX5 when prompted to do so, after which the CX5 will check the levels in the new cartridge. After loading a new reagent cartridge, you must calibrate the CX5 for that reagent. Refer to calibration procedures below.

    3.2 Calibration: From the Main Menu, press <F3> (“CAL”), and then page down

    through the reagents until the cursor is over the reagent that needs to be calibrated. Press <SELECT> to highlight it, and then press <F1> (“CAL CUP

    ASSIGNMENT”). Enter the number of an available sector when prompted, and press <ENTER>. Place a single 0.5 ml sector cup in the first position (“cell”) of the previously assigned sector. Using the appropriate calibration reagent (described above in Section 2.3), squeeze several drops from the bottle into the sector cup, place the sector into the CX5, and press the green <START> button on the keyboard.

    3.3 Control Preparation: Controls are run using the same procedure used for

    samples described below. The Lipid Controls (Levels 1 and 2) used for HDLc must be reacted with the HDL Cholesterol Separation Reagent, spun down, and extracted in the same manner, whereas the Comprehensive Chemistry Control Serum Levels 1, 2 and 3 are used directly.

    3.4 Sample Preparation: The chemistry tests are run on mouse plasma which has

    been extracted from mouse blood in the laboratory. The mice are fasted for four hours, beginning one hour after the start of their light cycle, prior to the collection of the blood for the chemistry tests. At the conclusion of the fast, approximately 225;l of blood (6-8 drops plus one full capillary tube) are collected from each mouse (depending on its size) using a retro-orbital bleed. The blood is collected into previously labeled 1.5 ml Eppendorf tubes containing 7.5µl of Heparin 1000u/ml and put on ice immediately. It is then spun down in a microcentrifuge at 14,000 rpm for 5 minutes. A minimum of 100µl of translucent blood plasma is collected from the upper layer using a 200µl Pipetman, leaving the blood cells behind in the Eppendorf tube which is discarded. This plasma is pipetted into a previously labeled 0.5ml Eppendorf tube and frozen for future analysis.

    3.5 HDL Cholesterol: [Procedure used by the Heart, Lung, Blood & Sleep Disorders Center at The Jackson Laboratory differs slightly from the Beckman protocol]. NoteThe following procedure uses six sectors (42 samples) as an example run which is a convenient size to run at a single time. If more or fewer samples need to be run, modify the procedure appropriately.

    1. Arrange 42 empty unmarked 1.5ml Eppendorf tubes in a 96-place rack.

    Orient the rack as 12 rows x 8 columns, and skip every other row, placing

    seven tubes per row.

    2. Record the plasma identifying information (mouse I.D. number) on a data

    sheet (lined paper), and assign a Beckman Coulter sector and cell number

    to each sample as in the following example. Be sure that the sector

    numbers assigned are actually available for imminent use.

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     Mouse I.D. # Sector Cell Date Test File Dilution Result

     Mickey 293 1 1 3/17/01 HDLc 345.cdf n/a 34 mg/dL

    Mickey 308 1 2 3/17/01 HDLc 346.cdf n/a 67 mg/dL

    Jerry 349-PW 1 3 3/17/01 HDLc 347.cdf n/a 85 mg/dL Jerry’s Uncle 1 4 3/17/01 HDLc 348.cdf n/a 57 mg/dL Speedy Gonzales 1 5 3/17/01 HDLc 349.cdf n/a 84 mg/dL Mrs. Speedy 1 6 4/05/01 CHOL 204.cdf 1:1 67 mg/dL Minnie 143 1 7 4/05/01 CHOL 205.cdf none 101 mg/dL

     Minnie’s Niece 2 1 4/05/01 CHOL 206.cdf none 56 mg/dL

    Mighty Mouse 2 2 5/21/01 GLU 1089.cdf 1:1 172 mg/dL

    Son of Mighty 2 3 5/21/01 GLU 1090.cdf 1:1 196 mg/dL

    Mighty’s Maid 2 4 5/21/01 GLU 1091.cdf none 168 mg/dL House Mouse 2 5 7/04/01 TG 867.cdf 1:2 89 mg/dL Deer Mouse 2 6 7/04/01 TG 868.cdf 1:2 78 mg/dL Soused Mouse 2 7 7/04/01 TG 869.cdf 1:2 69 mg/dL

3. Using a 250;l repeating pipettor, add 35;l of PEG (polyethylene glycol)

    to each open tube. Set the repeater for 35;l, to multi-dispense 7 times,

    which will load 245;l and fill one row of tubes (one sector's worth).

    Repeat this to complete the addition of PEG to the full rack. Using a 100;l

    Pipetman, put 35µl of plasma into the bottom of each tube. Change tips

    between each plasma sample. Close the caps.

    4. Label the caps with sector and cell number, i.e. 1-1 to 1-7 for Row One, 2-

    1 to 2-7 for Row Two, up through 6-1 to 6-7 for Row Six. 5. Vortex each Eppendorf tube for 10-15 seconds to mix solutions. 6. Let tubes sit for 15 minutes at room temperature.

    7. Place the tubes sequentially into a refrigerated microcentrifuge set at 4C

    with the hinges pointing out. Centrifuge for 10 minutes at 14,000 rpm. 8. Carefully remove the tubes from the centrifuge and replace them in the

    rack in the same six rows from which they were removed.

    9. Locate the six Beckman Coulter amber-colored sectors previously

    assigned on the data sheet, and place seven new empty 0.5ml Beckman

    Coulter Microtube? Tubecups into each sector, one in each cup holder.

    10. Each tube should have in the bottom a white pellet containing the

    undesired precipitate. Carefully draw off the supernatant, avoiding

    contact with the pellet, and transfer to the corresponding sector cup.

    Discard the tubes containing the pellets.

    11. It is important to avoid introducing air bubbles into the sample, as

    these will interfere with the results or cause the CX5 to be unable to detect

    the sample. To minimize introduction of air bubbles into the sample,

    carefully position the pipette tip at the bottom of the sector cup and raise

    the tip up the side of the sector cup as the plunger is being depressed,

    taking care not to inject air into the sample once all the liquid has been

    expelled. Often one air bubble will remain in spite of these precautions

    this can be popped using the wooden sticks that come with the CX5 or a

    toothpick.

    12. CAUTION: THE CUPS IN EACH SECTOR ARE NUMBERED

    FROM RIGHT TO LEFT. BE SURE TO LOAD THE SAMPLES

    INTO THE CORRECTLY NUMBERED SECTOR CUPS.

    13. Occasionally, the precipitate will not form a pellet at the bottom of the

    tube, or the pellet will break apart easily. In these cases, there will be

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    opaque particles floating on or suspended in the plasma sample. The

    presence of these particles will often cause the HDL result to be

    suppressed by the Beckman Coulter CX5. To avoid this problem, try to

    position the pipette tip in such a way as to draw up only clear plasma,

    leaving the particles behind in the Eppendorf tube. Even when this

    procedure is attempted, it can still be quite difficult in certain cases to

    successfully separate the plasma from the suspended particles.

    3.6 Cholesterol, Glucose and Triglycerides:

    1. The levels of these three components are measured directly from the blood

    plasma without further handling of plasma as is required for HDL.

    2. Record the plasma identifying information (mouse I.D. number) on a data

    sheet (lined paper), and assign a Beckman Coulter sector and cell number

    to each sample as in the above example. Be sure that the sector numbers

    assigned are actually available for imminent use.

    3. Locate the six Beckman Coulter amber-colored sectors previously

    assigned on the data sheet, and place seven new empty 0.5ml Beckman

    Coulter Microtube? Tubecups (sector cups) into each sector, one in each

    cup holder.

    4. Using a 100;l Pipetman, pipette 35;l of .9NS into the bottom of each

    previously assigned Tubecup. Then add 35;l of plasma into each

    assigned cup. Change tips between each sample.

    5. Follow suggestions in Subsection 3.5, #11 to avoid introduction of air

    bubbles into the sample.

    6. CAUTION: THE CUPS IN EACH SECTOR ARE NUMBERED

    FROM RIGHT TO LEFT. BE SURE TO LOAD THE SAMPLES

    INTO THE CORRECTLY NUMBERED SECTOR CUPS.

    4.0 Run:

    4.1 The four chemistry tests used in the HLB PGA protocol cannot be run together

    and must be divided into two groups. The HDL test must be run separately

    because it utilizes a reagent to separate the two types of cholesterol from one

    another. The CHOL, GLU, and Tg tests can be run at the same time.

    4.2 The tests are run in the following order: 1) HDL, 2) CHOL & GLU (labelled as a

    CHOL level), 3) TG.

    4.3 The plasma used for the HDL test cannot be diluted but is calculated as a dilution

    factor of 2 because of the 35;l PEG and 35;l plasma. This should be removed

    first. If there is not enough plasma to run the other three tests, the plasma may be

    diluted in a 1:1 or 1:2 ratio.

    4.4 Using the DOS computer, be sure there are enough runs left in the reagent

    cartridge for the number of samples to be tested. Follow the procedure described

    in Section 3.1, Reagent Preparation. This is maintained by the Diagnostic Lab Staff.

    4.5 Program the CX5 to test the samples using the following procedure. From the

    Main Menu, press <F1> (“SAMPLE PROGRAM”), then <F2> (“PROGRAM

    BATCH”). Next, enter the sector numbers to program (e.g. “1-6”) where

    prompted, and press <ENTER>. The computer will respond with “Batch mode

    activated, (42) cups possible.” Enter number of cups in batch (e.g. “42”) where

    prompted, and press <ENTER>. Select the desired chemistries by scrolling

    through the list of possibilities, and press <SELECT> appropriately. Press <F8>

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    (“END BATCH”) to program the CX5. “PLEASE WAIT -- WRITING TO

    DISK” will be displayed as the information is recorded.

    4.6 If the status of the CX5 is “WAITING,” press the green <START> button on the

    keyboard to begin testing the samples.

    4.7 If the CX5 is already operating, press the “LOAD” button on the CX5 itself to

    load the samples.

    4.8 Load three of the programmed sectors into the CX5 and wait while they are

    automatically loaded onto the “Sample Wheel.” Load new sectors onto the wheel

    as space becomes available and remove sectors from the machine as the testing of

    those sectors is completed. It is important to monitor the progress of the CX5

    closely in order to promptly troubleshoot any problems arising.

    4.9 Clear the sectors from the computer so that they may be re-programmed for future

    tests. From the Main Menu, press <F1> (“SAMPLE PROGRAM”), then <F5>

    (“CLEAR SECTORS”), and then enter the sector numbers to clear (e.g. “1-6”)

    when prompted.

    4.10 Remove and discard the sector cups once all desired tests have been run.

    5.0 Clean Up: Discard the Eppendorf tubes, pipette tips, sector cups, and reagent cartridges into biohazard waste containers. Clean up any spilled liquids.

    6.0 Data Reduction

    6.1 The results will be printed on the dedicated dot-matrix printer as the CX5 finishes

    the test(s) per sample. Carefully monitor the activity of the printer to ensure

    uninterrupted feeding of the continuous-feed paper, and to collect the printed

    results in an accordion-folded stack, which will prevent the output paper from

    getting dragged into the printer with the paper supply, causing the printer to shut

    down. If this does happen, the results can be re-printed only up until the time

    when the sectors containing that data are “cleared” as described above in Section

    4.9.

    6.2 An electronic copy of the data will be sent at the same time it is printed to the

    Windows-based computer, and stored in the following folder: D:\Dawning

    Files\results. The files should be transferred as soon as the run is finished into the

    following folder within that one: D:\Dawning Files\results\H L B S\[test]\[set#],

    where [test] equals “HDL Cholesterol,” “Cholesterol & Glucose,” or thth“Triglycerides,” and [set#] equals “4 set,” “5 set,” etc. One folder should be

    used for each “run” where a “run” is defined as the maximum number of files for

    which tests were run sequentially and did not re-use the same numbered sector.

    Thus, once an individual sector is re-used for more samples (whether they are the

    same test or not), those files should be stored in a new folder as part of a different

    “run.” One the run is completed, its files should be condensed into a Zip file ththlabeled “4_set.zip,” “5_set.zip,” etc. A copy of each zip file should be stored

    both in the folder labeled “zipped files” and on a 3.5”disk to be transferred to the

    data analyst.

    7.0 Safety: No known safety concerns.

    8.0 Time and Capacity: (The following is a rough estimate of time required for one technician to process 80 samples, although the work is much more efficient if several people work together for some of the tasks.)

    Capacity (max.) Task Time required

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    (hrs.)

    Remove food and HO from mice .5 n/a 2

    Set-up for eye-bleeding .5 n/a

    Eye-bleed mice 2.5 variable

    Clean-up from eye-bleeding .5 n/a

    Centrifuge blood samples 1.0 prop. to centrifuge

    capacity

    Extract plasma 1.0 n/a

    Calibrate CX5 .25 n/a

    Change reagents .25 n/a

    Run controls .25 n/a

    Prepare samples for HDL test 2.5 15 sectors (105

    samples)/run

    Run HDL test 1.0 15 sectors(105

    samples)/run

    Prepare samples for other 3 tests 1.0 15 sectors(105

    samples)/run

    Run other 3 tests 3.0 15 sectors(105

    samples)/run

    Clean-up .5 n/a

    Collect, label and file data .5 n/a

    9.0 Protocols: Mutagenized mice are bled for these chemistries at 8 weeks of age after consuming a regular chow diet, and again at 13 weeks of age after consuming a high fat, high cholesterol “atherogenic” diet.

    10.0 Notes: Be sure to plan the sequence and timing of all tasks carefully. There are numerous procedures within this protocol that may not be stopped in the middle, such as the HDL Cholesterol sample preparation, followed immediately by the HDL test. The

    reagent cartridges in the CX5 need to be changed regularly which interrupts the run. The cartridge needs to be changed, calibrations done, controls run, and then the run of samples can be resumed. Plan well and good luck!

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