Chem analysis bench protocol: v 5.0 2005


PM, RVS


Preparation of samples :

Prep 1.5 ml Eppendorf tubes with 7.5 ul of Heparin 1000u/ml. This is the anticoagulant being used when collecting blood for the Beckman instrument. Fast mice for 4 hours prior to obtaining blood sample.  Using Heparin coated Hematocrit tubes, obtain 8-9 drops of blood from a retro-orbital bleed. Stir sample with Eppendorf tube and flush out remaining blood in Hematocrit tube with bulb assembly instrument. Place in ice until you are ready to centrifuge. Centrifuge Eppendorf tubes for 5 minutes at 14,000 rpm. Pipette plasma into .5 ml Eppendorf tubes and freeze until you are ready to process it.

Recipe for Heparin 1000u/ml:
·  ·  Order Sigma Heparin (Sodium Salt) 50,000 u Cat. #H-3393 
·  ·  Dilute to 1000u/ml by using 50ml of DH20 with 50,000u Heparin Stock Solution 
·  ·  50,000u/50ml=1000u/ml 

If samples are frozen, allow them to defrost at room temperature for about 30 minutes before you begin.  Pipette 85 ul of plasma into individual cups per your run sheet.  This will be enough to run HDL, CHOL, GLU and TG data. 

Running Samples:

 

  1. Set up the sectors manually on the carousel. It only holds three sectors and will drop off one sector at a time until the lower carousel is filled to five. Be sure to pick up finished sectors as they come up. The Beckman will take a sector and then let you know it was run before. 
  2. Click F1 Sample Program - F2 Program Batch - message displayed will be "sector(s) to program:". 
  3. Key in the sectors you wish to program (e.g. 1-10) 
  4. Message displayed will be "Batch mode activated, 7 cups possible. Number of cups in batch:" (e.g. if you are running sectors 1-10, multiply 7 X 10=70 cups total). Maximum number of cups that can be run in a batch is 98.  Enter remaining sectors by pushing F2 Program Batch again.
  5. For HDL/ CHOL/GLU/TG: Leave the cursor in the PANEL field and type in "12". This activates a preprogrammed setting for CHOL/GLU/TG. Move the cursor over to the “HDLD” choice and click “Select”.  Hit ENTER to go to the SAMPLE TYPE.
  6. Enter "2" for plasma.  Then click F8 to set up the programmed batches. It takes a few minutes to set up the batches.
  7. Now you are ready to enter ID numbers and start running the Beckman. Push F1 (Program sectors) and enter the sector number you wish to bar code into the system. The cursor will sit on the ID field. Aim the bar code reader at the bar code lines, and the ID number will appear in the field after a beep from the bar code reader. Hit F8 to advance to the next cup in the sector. Continue this to cup 7. Push F8 and recheck ID numbers against Excel sheet. After ID numbers have been entered and the sector goes into the Beckman, no ID numbers or fields can be edited. Therefore, be sure the ID numbers, sample type, dilution factor and labs chosen are correct. 
  8. When you have six sectors programmed and ready to run, hit <prev screen> - then Master Screen - then START (green button). The Beckman will start sampling the first five sectors. You can continue to bar code in the remaining sectors while the Beckman is running. 
  9. On occasion, the computer will alarm that a reagent is low. Just hit <prev screen> to remove alarm and note. The next reagent cartridge will move into its place. 
  10. After first sector has completed its testing, the printer will start to print out the lab results. Make sure that the paper does not roll back into the printer. 
  11. When the results are complete, discard the cups from that sector and hit F5 (to clear sector) - enter sector number - hit ENTER. This will clear all information from that sector. When done, clear out all sectors used on the computer. Remove cups and return any sectors to their appropriate home.

Data Collection:

Data collection must be completed at the end of each run set. Problems have been encountered with the Beckman not sending data to the computer while simultaneously extracting data. Go to the computer on the right side of the Beckman. At the bottom of the screen, there should be at least two tabs to click on. One is D:\DawningFile\results and Synchron. Click on D:\DawningFile\results to view lab results for each cup run. You may see for example 1101.cdf 1KB CDF File 7/23/02 10:24AM. The HDL file and CHOL/GLU/TG file are collected in the manner that you put it into the Excel file.

For example:

  1. Run 20 samples HDL/CHOL/GLU/TG. 
  2. Click on D:\DawningFiles\results, and it will show the lab files 1100.cdf to 1119.cdf (for example, the numbering is determined by the equipment and changes for each run). 
  3. To collect files, highlight file 1100.cdf - hold down SHIFT key and highlight the last lab file. The entire set will be highlighted.
  4. Right click on highlighted set - click ADD TO ZIP - will see WINZIP screen. 
  5. Click "I AGREE" - under the ADD to ARCHIVE field 
  6. Click on D:\DawningFile\results\ 
  7. Key in:  YYYYMMDDHDLDCGT (HDL/CHOL/GLU/TG only file) 
  8. Click ADD. You will see WINZIP screen with YYYYMMDDHDLDCGT. 
  9. EXIT out of screen. This creates a zip file at the bottom of the lab files. 
  10. Right click on the highlighted set - click DELETE - click YES. 
  11. Bring up SYNCHRON screen. Highlight cdse zip files - right click on files and drag up to floppy disc. Release and click on COPY HERE. 
  12. Minimize SYNCHRON screen. 
  13. Now right click on highlighted cdse zip files and drag to HLBS file. 
  14. Select MOVE HERE. Minimize D:\DawningFile\results\ screen.