Dade-Behring Blood Coagulation System
| Prepared by: | Heather R. Ellis |
| Date Prepared: | February 8, 2002 |
| Code: | MP-003 |
| Reviewed by: | Karen L. Svenson |
| Revision #: | 3.0 |
| Date Revised: | May 28, 2002 |
Calibrated commercial controls are run daily and must be within established ranges prior to analyzing samples.
| 0.0 | Abstract: This system measures coagulation capabilities of plasma. Using specific reagents, it can determine Fibrinogen, Prothrombin Time (PT), Partial Thrombin Time (PTT), Factor VIII Deficient Plasma, and Berichrom Anti-Thrombin III in plasma from blood collected using the anti-coagulant 3.8% Sodium Citrate. |
| 1.0 | Instrument Layout: This system consists of the analyzer and a MacIntosh computer containing the corresponding software located in Room 3160 GRB. |
| 2.0 | Reagents and Expendables. |
| 2.1 Expendables: Controls and reagents used on the BCS analyzer are purchased from the manufacturer of the analyzer, Dade Behring. Control N is reconstituted with 1ml. of sterile distilled water. Control P is reconstituted with 1ml. of sterile distilled water. New vials of each control should be prepared prior to running samples. Standard Human Plasma is also reconstituted with 1 ml. of sterile distilled water when being used to calibrate reagent curves. The above expendables should be disposed of as biohazardous waste. Reconstitute a 1 ml. vial of Thrombin Reagent with 1 ml. sterile distilled water (or according to manufacturer's instructions). Reconstitute a 4 ml. vial of Thromboplastin C+ with 4 ml. of sterile distilled water (or according to manufacturer's instructions). Reconstitute Clotting Factor-VIII with 1 ml. of sterile distilled water (or according to manufacturer?s instructions). Berichrom Anti-Thrombin III (ATIII) is a kit in which the Thrombin Reagent is reconstituted with 5 ml. of Buffer Solution and allowed to sit for one half hour prior to running controls and/or samples. The Substrate Reagent (also in kit) is reconstituted with 3 ml. of sterile distilled water. SCS cleaner is also used in running ATIII and is ready to use. Approximately 225 µl. of blood are collected via retro-orbital eye bleeding using plain hematocrit tubes and mixed with 30 µl. of 3.8% Sodium Citrate as an anti-coagulant. The anticoagulant is prepared by diluting. It is imperative that excellent skill is practiced in obtaining blood samples and each sample is checked for clots using a standard toothpick. Isopropyl alcohol purchased from any manufacturer is used to clean the waste water, disinfectant and distilled water containers when performing monthly maintenance cleaning. Terralin is the disinfectant used to clean and flush the lines. It is supplied in 20 ml. packets as a concentrate and is diluted/mixed with 1980 mls. of distilled water. Saline solution (NaCl) is also used and is prepared by diluting. | |
| 2.2 Reagents: Reagents purchased for use on the analyzer from Dade Behring are used "as is" and are the following: Actin FS, Owren?s Veronal Buffer, Calcium Chloride, and Washing Solution. | |
| 3.0 | Setup: Carefully remove the dust cover from the BCS analyzer and turn the machine on. Turn on the computer and monitor and then click on the BCS logo in the launcher window to start the program. Check the fluid levels in your wash solution vials and load them into the appropriate site on the analyzer. Reload the cooler racks into the reagent cooler. Wait until the incubator/cooler reach their respective temperatures (37°C/15°C). While waiting for the analyzer to reach optimum operating temperature, you may proceed with the daily maintenance. Click on System, then click on Syringe and click on Continue to clear the message. The next screen is usually set as a default, but be sure that the left and right syringes are checked. Click on distilled water; click on the number of rinse cycles and enter 1. Open the door on the left of the analyzer to view the valves and lines to check for leaks during this procedure. Click Close when the check is complete. (If leaks have been detected, then replace the appropriate parts according to manufacturer?s instructions.) Open the rotor waste bin and empty the container. Check the level in the liquid waste container and empty if needed. Restock the right rotor tray by lifting the lid and insert each rotor tray being careful not to touch any of the wells on the tray. Do not stock more than 10 trays in the right rotor bin. Clean the pipettors by clicking on the System window and selecting Park Position. Click on Park Now. When the transfer arm is in the parked position, open the Plexiglas shield and carefully wipe the exterior metal tip of both pipettors. Close the shield and click Done and Close. To check the functional capability of the pipettors, click on System and select Pipettor. Click on continue to clear the message. Click on the Left and Right boxes. Click on Washing intensity and select Check pipettor. Click on Start and quickly observe both pipettors to be sure that a straight steady flow of water is flushed through the pipettors. This procedure will alert you to any blockage or damage of the pipettors. If you observe any malfunction, then refer to the manufacturer?s manual for correction or replacement of the damaged pipettor. Click Close once the check is complete.
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| 4.0 | Run: Blood collected from mice is centrifuged for 10 min. at maximum speed, and plasma samples are transferred into Fisher Scientific brand 1.5ml. Eppendorf tubes and then placed in the appropriate sized loading racks. Click on the Loading icon and select the corresponding rack number by highlighting it (each rack is labeled with a number and is bar coded). Each position on the rack must be named and entered into the computer. Even empty positions must have a label entered. Once labeling is complete, the tops must be removed and/or cut off each tube. The sample rack(s) are then loaded onto the analyzer using lanes #4 through 14. Click on the Job List and highlight the appropriate tests to be performed on each sample. Run Mouse PT and Mouse PTT in the first test run; then run the Mouse Fibrinogen, F8 or ATIII. If a sample appears grossly hemolyzed or lipemic, then highlight the Mouse PT.570 and Mouse PTT.570 instead of the Mouse PT and Mouse PTT. Click on New and then click on the Samples Box to begin the tests. It has been found to be best if the PT and the PTT are run first, and then the Fibrinogen, ATIII and F8 are run as separate tests on the same samples. If any repeat tests need to be done, then they are performed after all tests have been run through once. For a repeat test, highlight the test to be repeated and then click on Repeat and then Samples/Box to clear the highlights. |
| 5.0 | Data Reduction: Once the analyzer has completed performing the appropriate tests and the results have been displayed, you may highlight the results you would like to print and select Print. You may also print out any graphs necessary for the corresponding results by double clicking on the value and then double clicking on the result and selecting Auto. This will allow you to view the graph; then select Print if you would like to print the graph. The results of the controls may also be printed out by clicking on the Control Journal and highlighting the controls desired to be printed and select Print. |
| 6.0 | Clean Up: After the analyzer has completed testing, you may begin shut down procedures. Eject the coolant racks and proceed directly with the reagent and sample racks by selecting Loading and then click on each rack and select Eject. If the analyzer is not going to be used for the remainder of the day, then continue with performing A Shift Change. To do this, click on System at the top of the screen and select Shift Change and click on Disinfect Only. The analyzer will proceed to disinfect the lines and pipettors. This procedure will last approximately 15 minutes. When this procedure is complete, close this screen and click on the File icon. Select Quit and then immediately click on Quit Now; otherwise the analyzer will proceed with disinfecting again. You can power down the analyzer and put the computer in "sleep" mode by clicking on Special and selecting Sleep, or you may shut down the computer by selecting Shut Down. If the analyzer is going to be used later in the day and you do not want to turn it off, then you will bypass the disinfecting process the File/Quit/Quit Now procedure and put the computer in "sleep" mode. Then at the end of the day after all tests have been performed, shut down the analyzer using the disinfecting procedure. Remove washing solutions and store in the refrigerator. Turn off the machine but do not cover. |
| 7.0 | 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 a biohazard waste material. |
| 8.0 | Time and Capacity: Calibrations only need to be performed when lot numbers are changed on any of the controls or reagents. You will need to load the appropriate reagents and controls used to perform the specified tests and load the vial of Standard Human Plasma to run the calibration. The amount of time required to run a calibration depends on the number of tests being run at one time. The start up procedure, daily maintenance and control runs usually take approximately a total of 20 to 30 minutes to complete. A typical set of approximately 30 samples usually requires anywhere from 45 to 60 minutes depending on the amount of lot tests being run and the number of repeated tests performed. |
| 9.0 | Protocols: Calibrations are performed according the instructions provided by the manufacturer of the analyzer, Dade Behring. Calibrations are only necessary when the lot numbers of reagents have been changed or new standard curves need to be calculated. This is accomplished by using the Standard Human Plasma, Control N, and Control P, along with the reagents used to perform the required tests: Actin FS, Thrombin Reagent, Thromboplastin C+, FVIII Deficient Plasma, ATIII Substrate Reagent, ATIII Thrombin Reagent, SCS Cleaner, Calcium Chloride, Owren?s Veronal Buffer, NaCl and washing solutions. Quality control is performed using Control N and Control P. Once the controls have been reconstituted and allowed to rest for 15 minutes, they are then loaded along with the reagents into the loading racks. Click on the Control Journal in the menu and highlight the tests requested: Mouse Fibrinogen; Mouse PT; Mouse PTT; Mouse PT.570; Mouse PTT.570; ATIII Mouse, F8x4, Human Fibrinogen; Human PT; Human PTT; Human PT.570; Human PTT.570 and ATIII Human. Click on New on the menu bar and click on controls. Print out the Control Journal Data by highlighting the data to be printed and select the Print key in the menu bar. To run plasma samples, load samples into the sample racks. Select Loading in the main menu and select the corresponding rack number. Enter a name for each position on the rack, even empty positions not containing samples must have a label. Click on the Jobs List and scroll down until you see the current entries. Highlight the tests requested. Remember to run PT and PTT first and then run the Fibrinogen, ATIII and F8. Once the tests are complete and values are shown, you can then highlight the information that you would like printed and click on Print. Upon completion of your sample run, perform a Shift Change before closing down the system. Select System in the Upper Menu bar and then click on Shift Change. Select Disinfect Only. This process takes about 10-15 minutes. Once this has been completed, you may close the window and Click on the File tab in the upper menu bar and select Quit. When the disinfecting only window pops up, click on this and then quickly click on Quit Now. This will bring you back to the start up menu. Click on System and select either Sleep Mode or Shut Down. |
| 10.0 | Notes: The assay procedures for Mouse PT, Mouse PTT, Mouse Fibrinogen, ATIII, Factor 8, Mouse PT 570, and Mouse PTT 570 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. |
Program for Genomic Applications (PGA)
Supported by the National Heart, Lung, and Blood Institute (NHLBI) (Grant # HL66611)
