Immunoassay Testing

South San Francisco, CA (TOSOH) May 26, 2009 — Video script for Immunoassay Testing on the AIA 600 II Automated Immunoassay Analyzer has been posted to accompany web training videos for AIA 600 II.

AIA 600 II TRAINING VIDEO SCRIPT
Written by Bill Bevington, Technical Trainer

Reagents
There are three common reagents used on the 600 II. All assays use the same wash, diluent, and substrate. Sample diluting solutions, calibration/verification material, and calibrators are specific for each analyte. Refer to the analyte application manual for information about any of our reagents.

Substrate II is packaged with 2 sets per box. Each set consists of one bottle of lyophilized substrate and one 100 ml bottle of reconstituant. Add the entire contents of the bottle of reconstituant to the bottle of substrate. Once it has dissolved it is ready to use.

Once the substrate has been reconstituted it should be protected from light. It is stable for 3 days if kept at room temperature and 7 days if kept in the refrigerator after use. Do not mix one bottle of substrate with another. Do not add new substrate to substrate that is about to expire. Label with the date you reconstitute it so you will know when it will expire.

Diluent and Wash
Both are package with 4 100 ml bottles of concentrate per box. In the concentrate form they are stored in the refrigerator and are usable up to the expiration date. Once they are reconstituted they are stable for 30 days.

Diluent is made by adding 800 ml of Type 1 Reagent Grade water to the 1 liter reagent reservoir, then add 20 ml of diluent concentrate and then filling the reservoir up to the 1 liter mark on the reservoir. Wash is made the same way except that 40 ml of wash concentrate is used. Label with the expiration date.

Both wash and diluent need to be aged for 24 hours before use. The 600 II comes with 2 sets of reagent reservoirs so you can use one set to make up the wash and diluent ahead of the time they are needed.

Test Cups
Test cups are packaged in two different sizes. The 40 minute incubation tests are packed in boxes of 200. Each containing 10 trays of 20 cups each. The ST analytes are packaged in boxes of 100, containing 5 trays of 20 cups each. The cups are stable up to the expiration date on the front of the box if they are stored in the refrigerator at 2 – 8 degrees C. Refer to the analyte application manual for information about stability at room temperature.

Calibrators
Calibrators are packaged in two different size boxes, depending on whether they are two calibrator or six calibrator assays. Most calibrators are liquid but some are lyophilized and require the addition of one ml of type 1 reagent grade water. The two calibrator assays have bottles labeled 0 and P with P being the high calibrator. Six calibrator assays have the bottles labled one to six. Refer to the analyte application manual for information about reconstitution and stability.

Calbiration Verification Material
Calibration verification material is packaged with 2 sets per box. Each set contains one 2 ml bottle of calibration verification material labeled CVM and one 4 ml bottle of Sample diluting solution labeled SDS. Opened CVM should be used within 24 hours provided vials are kept tightly sealed and refrigerated at 2-8 degrees centigrade. CVM is a protein based serum containing the assigned concentration of the approximate upper linearity limit for the specific analytebeing tested. CVM is only required for two calibrator sandwich assays.

Sample Diluting Solution (SDS)
Packaged in boxes containing either 4 4 ml bottles or 4 100 ml bottles of SDS. For most assays SDS may be used directly from the bottle. See analyteapplication manual for complete information. Once opened, most SDS is stable at 2-8 degrees C for 90 days. However, some SDS is stable for shorterperiods, see the Analyte Application Manual for complete information. SDS is a protein based serum containing no detectable concentration of the specificanalyte being tested.

Standardization Cups (STD cups)
Packaged 200 cups per box. The boxes are labeled Detector. The cups are labeled STD. Stable indefinitely at room temperature. AIA-600 II uses one STD cup during daily maintenance for lamp check and substrate background reading.

Sample Treatment Cups (STC Cups)
Packaged 200 cups per box. Stable indefinitely at room temperature. The 600 II uses STC cups to perform sample dilutions and pretreatments.

Sample requirements
CKMB, Myoglobin, and Troponin use heparinized plasma. EDTA and citrated plasma should not be used.

All other assays require serum. Serum samples can be stored for up to 24 hours at 2-8 degrees centigrade. If analysis cannot be performed within 24 hours, the sample should be stored frozen at or below minus 20 degrees centigrade for up to 60 days.
Two assays can be run with urine specimens. C-Peptide and Beta-2 Microglobulin.

Location of Instrument Parts and Reagents
The waste container, and the wash and diluent reservoirs are located on the left side of the instrument. The waste container is a two-liter container. It has a level sensor and two tubings attached to the top of the cap. One is for the waste and one is attached to the vacuum connection. The 1-liter wash and diluentreservoirs also have level sensors to alert the operator when these fluids are low.

The substrate compartment is located on the front left side of the instrument. The instrument does not have a level sensor for the substrate so the amount of substrate in the bottle can be measured using the ruler at the back of the substrate compartment.
The solid waste container is located on the front side of the instrument. The sample compartment is located on the left front of the instrument. The sample chain has 52 routine and 4 stat positions.

Both samples and test cups are placed on the chain . The samples in either sample cups or primary tubes are placed directly on the chain, the test cups are placed in test cup adapters following the samples. Up to five test cups can be loaded after the sample. If a sample requires more then 5 test cups, it can be put on with the additional test cups.

Tosoh sample cups, 13 x 75, 13 x 100, 16 x 75, and 16 x 100 tubes can be used on the chain.

The dead volume for a sample cup is 100 ul, for a 13mm tube it is 500 ul, and for a 16 ml tube it is 600 ul.

The sample chain can be advanced using the sample feed key on the keyboard.

The power switch, the external barcode scanner, the RS232 Port and the memory card slot are located on the right side of the instrument.

The external barcode scanner is also located on the right side of the instrument. The holder for the external barcode scanner is magnetic and can be moved.

The RS 232 port is the connection for the computer cable if your instrument is interfaced with a host computer. The memory card slot is where memory chips are inserted when software is changed.

Daily Maintenance
To start up the AIA-600 II turn on the main power switch located on the lower right side of the analyzer. The operator registration screen will appear. This screen allows you to change the operator registration if you want. To logon with a different operator name, press F1 (Correct). The Operator Name screen will appear. Use the arrow keys to select letters, press the enter key to accept the letter selection. Continue the process for up to 10 characters. Press the F5 key (Quit) once the operator name registration is complete. Use the up/down arrow keys to select the current operator and press the Enter key. The operator logon name will be used on result reports.

Press F2 (Skip) to bypass operator registration if you do not want to change the operator name.
The instrument will then initialize. During the initialization process, the AIA-600 II homes all components and checks the incubator and sampling station for remaining cups.

Once logon in completed, the Daily Maintenance 1 screen will be displayed. Physically inspect all materials listed except tips because the tip rack is not accessible at this time. Add, replace, or remove materials necessary.

Wash and Diluent solutions – make certain that the volume is sufficient for the number of tests to be run, check the expiration date, and replace if necessary. It is important to check the volume prior to the start of each run.

Substrate Compartment– remove the ethanol bottle used during shutdown and replace with a sufficient volume of Substrate II. Check the expiration date and replace if necessary.

Discard the following materials.
Waste cups and Tips – Empty the solid waste container.
Waste Solution – Empty the waste container if necessary. Check the volume prior to the start of each run.

When you have finished checking the reagents and emptying the waste, press F1 (OK) to continue.

The inventory screen will appear as an overlay to the Daily Maintenance 2 screen. The inventory screen displays the reagents and expendables previously entered into the system. Substrate volume must be entered in this screen. After entering the substrate volume, press the Inventory key on the keypad to move to the Daily Maintenance 2 screen.

The operator action items are highlighted. Follow the on screen instruction by placing a test cup adapter with an STD cup on the chain in position 2. Press the F1 (Start) key. The daily maintenance process will proceed automatically. The substrate background and lamp intensity are checked during this process. The results are printed automatically upon completion of daily maintenance.
The substrate background result printout contains the following information:

Substrate replacement – Complete (if substrate background passed) or Incomplete (if substrate background did not pass)
4MU Background – must be OK
Lamp Intensity Level – must be OK
SO – Sample off – Sample reading taken without light
S1 – Sample on – Sample reading taken with light R0 – Reference off – Reference reading without light
R1 – Reference on – Reference reading with light

The daily maintenance results should be retained as part of your maintenance record.

The Assay Monitor screen will be displayed after the completion of Daily Maintenance.

Reagent/Tip Registration
Press the Reagent/Tip Pause key on the keypad to display the reagent registration screen. The reagent/tip rack will move forward past a motor sensor to allow the operator to add or remove reagents and to add tips.

There are two ways of registering reagents. The first is to use the function keys F1-F5. The arrow keys move the cursor enabling the operator to inputanalyte, lot number and volume information. The alternate method is to scan the barcode labels on the reagent bottles which will automatically enter the type of reagent, the analyte, and the lot number. The operator must still enter the reagent volume.

The regent rack has 11 positions. Positions 1 and 2 hold 30 ml bottles. These can be used for larger volumes of Sample Diluting Solutions, either from the 100 ml bottles or from pooling several small (4ml) bottles together. Positions 3 through 11 are for the 4 ml Sample Diluting Solutions or 5 ml pretreatment bottles.

The Reagent rack can be removed. Reagent registration information will be retained in memory until modified or deleted by the operator even when the analyzer is powered off.

The tip rack is also removable. The base of the tip rack holder is numbered. A full rack holds 96 tips. If a partial rack is used, fill all columns completely starting from the top right corner of the rack. Only black TOSOH brand tips can be used. The tips are conductive and function as the level sensor during sample aspiration. After loading the tips enter the number of tips in the rack.

After loading tips and reagents and updating the screen information, move the holder to the left past the sensor, and press the Reagent/Tip Pause key to remove the Reagent Registration window from display. The instrument will automatically move the Reagent/Tip rack holder into the home position.

Daily Operation
The Assay Monitor screen is used to add or edit assay schedules. Assay schedules can either be manually input (patients, calibrations, and controls) or downloaded from a laboratory computer system (patients and controls). The keyboard is used to manually input information.

It is possible to order a maximum of five tests per specimen. If more than five tests are needed, a second sample cup will be required. The additional tests are requested on the second sample cup.

Control and patient tests are ordered in the Assay Monitor Screen.
To order a control, press the CONTROL key and then the number of the control. For example, pressing CONTROL first and then the number 1 and then ENTER will order Control 1. To order a patient test, enter the sample ID number. You must then order the tests you want using the analyte table or by entering the analyte test identification code.

To use the analyte table, press the ANALYTE TABLE key. A window will pop up displaying a choice of analytes from which to choose. Press the down arrow to move the horizontal line just below the analyte you want to order. Press ENTER and the test will be ordered. To order using the test identification code, enter the test identification code and the test will be ordered. The repeat key may also be used to order patient tests. When the F5 (Repeat) is pressed, the analyzer will automatically sequence the sample ID number and will order the same tests that were ordered on the previous sample.

When you have finished ordering tests and are ready to start running them, press F1 (Work list) and a work list will print. The work list will have the sample ID number, the test cups that are needed for the tests that have been ordered, and a sample volume that includes the 100 ul dead volume for a sample cup. If you are using primary tubes, add 400 ul for a 13 mm tube or 500 ul for a 16 mm tube.

Using Automode
Automode 1 – Program the sample ID and allow the camera to automatically read the dot pattern on the test cups and assign the analytes.

Automode 2 – No specimen ID or analytes are programmed. Use this with barcoded tubes. The internal barcode reader will read the barcode to identify the sample and the camera will automatically read the dot pattern on the test cups and assign the analytes.
Load the samples on the sample/test cup chain with the corresponding reagent cups. Place the sample cup or tube on the chain and behind it place the number of sample cup adapters needed for the number of tests that were ordered. Place one test cup into each adapter. Press the Assay Start key to start the analyzer.

Loading the chain
It is possible to completely load the chain before starting the analyzer. To do that, use the SAMPLE FEED key to move the chain around. Pressing SAMPLE FEED once will advance the chain one position. Holding SAMPLE FEED until the chain has moved 3 positions will keep the chain moving until SAMPLE FEED is pressed again or until the chain reaches the home position. The chain will home automatically before sampling when ASSAY start is pressed.

Pausing Sampling
Once the instrument has begun sampling, sampling may be manually suspended by pressing the SAMPLE PAUSE key one time. The Sample Pause key will flash and become solid once sampling has been paused. All Assays in progress will be completed. Sampling may be restarted by pressing the ASSAY START/STOP key.

Additional methods of ordering analytes are:
If you are interfaced to a laboratory computer system, assay schedules can be downloaded to the AIA-600 II.
If you are interfaced to a laboratory computer system using host query as the interface method, the AIA-600 II will request assay schedules from the LIS when a barcode label is scanned. The specimens must be loaded according to the worklist.

Ordering Dilutions
Order the test that you want to run as a dilution. Use the left arrow key to move the cursor back over the test. Press F5 (DILUTION) to display the dilution menu. Select Option 3 (OTHER) using the keypad. Using the keypad, type in the desired dilution factor and press enter. The Assay Monitor screen will now display STC and the test name with an asterisk and the dilution factor you ordered.
Dilutions require a Sample Treatment Cup (STC) to be placed on the chain prior to the test cup for which the dilution is ordered. The maximum dilution factor for one STC cup is 51. Dilutions higher than 51 require two STC cups to be placed on the chain prior to the test cup. The highest possible dilution that the instrument can perform is 625.

If you have ordered a dilution, make sure you have registered the dilution solution for that analyte and that it is the reagent rack.

Ordering Panels
The AIA 600 II is able to have up to 10 panels created. A panel is a predefined combination of tests, using up to 5 cups that can be ordered instead of ordering each test separately.

To create a panel, press SPECIAL MENU, and then press 5 to select PANEL SET. Use the analyte test code or analyte table to order the tests you want included in the panel.

To order tests using a panel, in the Assay Monitor screen, type in the sample number, press enter, press F2 (Panel) to select panel. The panel screen will be displayed. Use the down arrow to move the line under the panel you want to order and press enter. That panel will then be ordered.

STAT Programming
STAT programming is available when the AIA-600 II is sampling. Pressing the STAT key on the keypad brings up the STAT programming window. The instrument will continue to sample the specimen it is sampling at the time you press STAT. When it is finished, the chain will move around until the 4 STAT positions on the chain are in position. When the chain stops moving the STAT programming window will be exposed. Tests are ordered as usual. After the sample ID has been programmed and tests have been requested, press the ASSA START key to begin priority sampling. Once the STAT sample has been processed, the analyzer will resume processing routine samples. Do not remove any specimens or test cups from the sample chain while the analyzer is processing the STAT. It is possible to run only one stat specimen at a time with up to 3 tests ordered on that specimen.

Results
The AIA 600 II can be set to automatically print test results as soon as a test is completed or to only print results when requested. Additional copies of test results can be requested. The AIA-600 II can hold 500 test results in memory.

The printed result has the following information:
Each sample has a sequence number, a chain position number, the sample ID, the date and time the results were completed, and the operator name. Each result is listed with the name of the analyte, the result, the units, the dilution factor if the result is from a dilution, up to two flags, and the fluorescent rate.

Results can be printed, retransmitted to the LIS if the instrument is interfaced with a LIS, recalculated, or erased. To perform any of these functions:

Press RESULT REVIEW to get to the result review screen.

Select the results that you want to print, retransmit, recalculate or erase by using the arrow key to move to the first result and pressing F2 AREA SELECT. Move the cursor to the last result and press AREA SELECT again. The selected area will be backlit.
Press F4 EXECUTE and a small pop-up window will display 4 options: 1 Print Out, 2 RS232 Out, 3 Recalculate, 4 Data Erase.
Use the keypad to choose the number of the option you want, press ENTER. A small pop-up window will appear to confirm your selection. After the confirmation box is displayed, press ENTER for the action to occur.

Display modes
The RESULT REVIEW screen can be set to display results in 5 different ways. There are five options:
1. ALL: Every patient sample, whether they are complete or incomplete (meaning they have flags), control result, and pending calibration will be displayed.
2. PATIENT: Only patient sample results will be displayed
3. CONTROL: Only control results will be displayed.
4. CALIBRATION: Only pending calibrations will be displayed.
5. INCOMPLETE: All flagged results will be displayed.
To select the display mode, go to the RESULT REVIEW screen and press F5 DISPLAY MODE. A pop up window will be displayed with all 5 options. Use the keypad to select the option you want.

Shutdown
Shut down at the end of the day is a very easy procedure that only takes a few minutes. This procedure must be performed at least once a day even if the instrument operates 24 hours a day.

To perform the shutdown procedure:
1. Remove and dispose of all used reagent cups and sample cups.
2. Replace the substrate solution with Ethanol. Refrigerate the substrate if testing has been completed.
3. Press SPECIAL MENU
4. Choose option 6, SHUT DOWN
5. Press F1 to begin the shutdown process
6. Turn off the power when prompted onscreen.

Calibration
Calibration is required for the following reasons:
1. Every 30, 60, or 90 days, depending on the analyte calibration curve stability.
2. After changing reagent pack lot number
3. Following a major service, such as incubator temperature adjustment or periodic maintenance.
4. If QC is out of range and instrument problems have been ruled out.

Calibration Flags – There are two calibration related flags that can occur.
1. CV (Calibration Void) – Valid calibration period of 30, 60, or 90 days has expired. Results displayed are calculated on the expired curve. Once the new calibration has been accepted and results recalculated the CV flag will disappear and results can be printed without the flag.
2. NC (No Calibration) – No calibration curve exists in the system memory for the lot number. No result is printed. Rates are stored in the memory until a calibration for this lot has been performed and accepted. Once the calibration has been accepted and the results recalculated, a result will appear and the NC flag will disappear.
How to calibrate:
3. Verify or correct the calibrator lot number and concentrations of each calibrator.
4. Order the calibration
5. Load the calibrators and the reagent cups
Verifying or correcting the calibrator lot number and concentrations: Press SPECIAL MENU, press F2 ASSA SPECIFICATIONS. Press the down arrow until the cursor is at the first analyte to be calibrated. Press the right arrow once to move the cursor to the calibrator lot number. Verify that the calibrator lot number is the same as the lot number of the calibrator that you are using. If it is not, you will have to enter the correct lot number. To enter the lot number, scan the lot number barcode label with the external barcode reader. Do the same with the calibrator concentrations.

Ordering the calibration:
1. Press ASSAY MONITOR to select the assay mode.
2. Verify the cursor is under the SAMPLE ID position.
3. Press CALIB to select the calibration mode. The word Calibrator 1 will be displayed on the screen.
4. Type in the analyte code number or use the ANALYTE TABLE to select the analyte and press enter. The screen will now display the calibrators required to calibrate that analyte, each calibrator will be ordered in triplicate.
5. Print a worklist by pressing F1 WORKLIST.

Loading the calibration
1. Load calibrators and reagent cups according to the calibration worklist. Press ASSAY START to start the calibration.

Accepting Calibrations
Sandwich Assays
1. The 0 Calibrator has a rate less than or equal to 3 nm/sec.
2. The precision of the replicates is within 10% of each other.
3. The slope of the graph is correct. Look at previous calibrations of the lot number and analyte.
4. The rates of the calibrators should increase as the calibrator concentration increases.

Competitive Assays
1. The rates of the calibrators should decrease as the concentration increases.
2. The precision of the replicates is within 10% of each other.
3. The slope of the graph is correct. Look at previous calibrations of the lot number and analyte.

To accept calibrations
1. Press CALIB REVIEW
2. Press F1 NEXT CALIB if the calibration on the screen is not the one you want to review. The calibration data will be displayed one assay at a time, in the order performed. Each data point, the assigned value, and the rate will be displayed.
3. Review the data according to the calibration acceptance criteria. The instrument will automatically highlight ACCEPT for every data point unless it is flagged. If you want to reject any data points move the cursor to the point to be rejected, move the cursor to the right and REJECT will be highlighted. Press the CALCULATE key to display a graph.
4. Press ACCEPT. A window will be displayed allowing you to choose which position the calibration will be stored. The AIA-600 II will store two calibration curves simultaneously for different pack lots of the same analyte. Position the cursor over the calibration curve that is void, or for a lot number that is no longer in use. Always position the same lot number of test cups over the same lot previously calibrated. Once ENTER is pressed, a printout of the graph and rates will occur provided no assays are being processed. This is the only time that all the rates will be printed. Calibrations may be reprinted at a later time, however only the mean value of the rates will print.

Maintenance
Maintenance is essential to the reliable performance of the AIA-600 II. Proper maintenance of your AIA-600 II is one of the most important aspects of a complete quality assurance program.
Weekly Maintenance – Cleaning the Wash Probe
1. Open the two metal covers to expose the incubation area and the wash probe.
2. Remove the wash probe assembly by loosening the metal thumbscrew. Turn the screw counter-clockwise to loosen it. Unscrew and lift the entire assembly towards you, as far as possible.
3. Place a paper towel under the area in which you are working to protect the analyzer from spills. Gently scrub the probe tip with a brush (such as a toothbrush) dipped in a de-proteinizing solution. Rinse with a small amount of DI water.
4. Reinstall the wash probe assembly. Tighten the metal thumbscrew finger tight.

Monthly Maintenance
1. Clean Sample Area with Ethanol – Open the sample/reagent cup compartment. Using cotton balls soaked with ethanol, wipe up any noticeable spills on the white platform inside the chain. Using cotton applicator sticks and cotton balls soaked with Ethanol clean the sample/reagent chain.
2. Clean diluent and wash reservoirs with 1:5 dilution of bleach
a. Remove and empty the reservoirs from the AIA-600 II.
b. Wash the reservoirs with a 1:5 dilution of bleach.
c. Thoroughly rinse the reservoirs with Reagent Grade Type 1 water.
d. Refill the reservoirs with fresh diluent or wash solution.

Changing the Printer Paper
1. After removing the old roll of paper, open the box containing the new roll of paper. Place the new roll on the spindle with the front of the paper coming off the bottom of the roll. The red Tosoh lettering will be along the left side of the paper.

2. Feed the leading edge of the paper into the paper feed slot. The paper will load automatically into the printer. Use the Paper Feed key to advance the paper.

3. If the position of the paper needs to be adjusted, move the paper release lever to the release position and manually adjust the paper. Then move the release lever back to the On position.