Procedure for

Initial Standardization

Check the Optical Alignment and PMT Stability with BioSure CRBC's

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This generic procedure is used to monitor the optical alignment and PMT stability of a flow cytometer by using chicken erythrocytes that have been fixed in 0.2% v/v gluteraldehyde. The ovoid shape of the cells produces a unique scatter pattern which reflects the orientation of the cell as it passes through the laser beam. The separation (delta) between the high FSC peak and low FSC peak is maximized when the optics are correctly aligned. Daily monitoring of the FSC values for both peaks will reflect the alignment of the instrument. In addition, dust or dirt particles in the optical pathway can also be detected if a drop in the high FSC peak occurs. The procedure is also used to monitor the SSC and fluorescence parameters as the CRBC's fluoresce brightly over a wide emission spectrum (500 to 700 nm).

Initial Standardization Procedure using Lymphocytes and CRBC's

Ideally, the initial instrument setup using CRBC's should be performed in the presence of a qualified service technician. Instrument adjustments to FSC, SSC, FL1, FL2 and/or FL3 can be made at this time to achieve maximum separation of the CRBC's in each of the parameters. These optimized instrument settings establish target values for the CRBC's which are then used as a daily reference for the FSC, SSC, FL1, FL2 and/or FL3 parameters. Once target values have been established, instrument settings are recalled and the new daily values compared to the target reference values.

If a qualified instrument service technician is not available, the initial instrument setup can be performed as follows:

1) Align the instrumentation optics, if necessary.

2) Prepare a cell suspension of leukocytes from a venous blood sample collected from a control donor. Lyse the erythrocytes using the same lysing method which will be used on the laboratory samples. Fix the lysed whole blood sample in a 1% buffered paraformaldehyde solution.

3) Place the fixed sample on the cytometer. Set the instrument parameters as follows: FSC-linear, SSC-linear, FL1-log, FL2-log. Adjust the FSC amplifier gain to position the left edge of the lymphocyte population below mid-scale on the forward scatter display. Adjust the SSC to position the top of the granulocyte population close to the edge of the side scatter display. Record the FSC and SSC amplifier gain settings.

4) Set a gate around the lymphocytes and activate the gate. Change the display parameters to FL1 and FL2.

5) Adjust the high voltage on the Fluorescence 1 PMT to position the unstained lymphocytes in the first decade of the FL1 display. Adjust the high voltage on the Fluorescence 2 PMT to position the unstained lymphocytes in the first decade of the FL2 display. Repeat the PMT adjustment for any other parameters (FL3 for example), if desired. Record the PMT voltage settings.

6) Repeat Steps 1 - 5 at least nine times using venous blood collected from different control donors. Average the PMT settings for each of the parameters - SSC, FL1 and FL2. Record the average setting for each of the parameters. Since the FSC detector is a photodiode, only the amplifier setting will be recorded.

7) Prepare a fresh dilution of the CRBC's to achieve a flow rate of about 500 - 1000 events/second. Place the tube on the cytometer.

8) Adjust the FSC amplifier gain until both peaks of the CRBC population can be seen in the forward scatter histogram display. Once the gain has been established, this setting will be used as the FSC reference target value and should not be altered in subsequent daily monitorings. Record the FSC gain setting.

9) Calculate the difference in mean channels between the CRBC high scatter peak and the low scatter peak in the forward scatter display. Ideally, this difference should be at least 400 channels (out of 1024 channels), but separations of 500 channels or more are not uncommon. Record the values for the CRBC high scatter peak and the low scatter peak in the forward scatter display. These values will be used as target values for monitoring the FSC stability.

10) Proceed to the SSC histogram display. Adjust the SSC detector so that the main SSC peak is approximately in channel 50. Record the SSC PMT setting for the CRBC's. This value will be used as the target value for monitoring the SSC stability.

11) Proceed to the FL1 histogram display. Run the CRBC's using the averaged FL1 PMT setting established by the control donor lymphocytes. Record the mean peak value and C.V.'s of the CRBC's for the FL1 parameter. These values will be used as target values for monitoring the FL1 stability.

12) Proceed to the FL2 histogram display. Run the CRBC's using the averaged FL2 PMT setting established by the control donor lymphocytes. Record the mean peak value and C.V. of the CRBC's for the FL2 parameter. These values will be used as target values for monitoring the FL2 stability.

13) Repeat for FL3 or other parameters, if desired.

Target reference values have now been established for the FSC, SSC, FL1 and FL2 parameters using CRBC's. Subsequent daily monitoring of the parameters will use these values as baseline references. If daily values of greater than 10% variation from the reference are recorded, realignment or service of the instrument may be necessary.

Initial Standardization Procedure using Lymphocytes and CRBC's

Ideally, the initial instrument setup using CRBC's should be performed in the presence of a qualified service technician. Instrument adjustments to FSC, SSC, FL1, FL2 and/or FL3 can be made at this time to achieve maximum separation of the CRBC's in each of the parameters. These optimized instrument settings establish target values for the CRBC's which are then used as a daily reference for the FSC, SSC, FL1, FL2 and/or FL3 parameters. Once target values have been established, instrument settings are recalled and the new daily values compared to the target reference values.

If a qualified instrument service technician is not available, the initial instrument setup can be performed as follows:
1)	Align the instrumentation optics, if necessary.
2)	Prepare a cell suspension of leukocytes from a venous blood sample collected from a control donor. Lyse the erythrocytes using the same lysing method which will be used on the laboratory samples. Fix the lysed whole blood sample in a 1% buffered paraformaldehyde solution.
3)	Place the fixed sample on the cytometer. Set the instrument parameters as follows: FSC-linear, SSC-linear, FL1-log, FL2-log. Adjust the FSC amplifier gain to position the left edge of the lymphocyte population below mid-scale on the forward scatter display. Adjust the SSC to position the top of the granulocyte population close to the edge of the side scatter display. Record the FSC and SSC amplifier gain settings.
4)	Set a gate around the lymphocytes and activate the gate. Change the display parameters to FL1 and FL2.
5)	Adjust the high voltage on the Fluorescence 1 PMT to position the unstained lymphocytes in the first decade of the FL1 display. Adjust the high voltage on the Fluorescence 2 PMT to position the unstained lymphocytes in the first decade of the FL2 display. Repeat the PMT adjustment for any other parameters (FL3 for example), if desired. Record the PMT voltage settings.
6)	Repeat Steps 1 - 5 at least nine times using venous blood collected from different control donors. Average the PMT settings for each of the parameters - SSC, FL1 and FL2. Record the average setting for each of the parameters. Since the FSC detector is a photodiode, only the amplifier setting will be recorded.
7)	Prepare a fresh dilution of the CRBC's to achieve a flow rate of about 500 - 1000 events/second. Place the tube on the cytometer.

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8)	Adjust the FSC amplifier gain until both peaks of the CRBC population can be seen in the forward scatter histogram display. Once the gain has been established, this setting will be used as the FSC reference target value and should not be altered in subsequent daily monitorings. Record the FSC gain setting.
9)	Calculate the difference in mean channels between the CRBC high scatter peak and the low scatter peak in the forward scatter display. Ideally, this difference should be at least 400 channels (out of 1024 channels), but separations of 500 channels or more are not uncommon. Record the values for the CRBC high scatter peak and the low scatter peak in the forward scatter display. These values will be used as target values for monitoring the FSC stability.
10)	Proceed to the SSC histogram display. Adjust the SSC detector so that the main SSC peak is approximately in channel 50. Record the SSC PMT setting for the CRBC's. This value will be used as the target value for monitoring the SSC stability.
11)	Proceed to the FL1 histogram display. Run the CRBC's using the averaged FL1 PMT setting established by the control donor lymphocytes. Record the mean peak value and C.V.'s of the CRBC's for the FL1 parameter. These values will be used as target values for monitoring the FL1 stability.
12)	Proceed to the FL2 histogram display. Run the CRBC's using the averaged FL2 PMT setting established by the control donor lymphocytes. Record the mean peak value and C.V. of the CRBC's for the FL2 parameter. These values will be used as target values for monitoring the FL2 stability.
13)	Repeat for FL3 or other parameters, if desired.