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CRBCs

CRBCs

Chicken Red Blood Cells

Ideal for initial standardization and monitoring of the cytometer's optical and fluorescence parameters and PMT stability. Makes peak assessment a snap.

 
2.0 mL Catalog No.  1004$91.00
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6.0 mL Catalog No.  1005$165.00
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Save Money - Order in Bulk
Custom Order CRBCs
Catalog No. 1018

BioSure® Chicken Red Blood Cell (CRBCs) cytometry control is a red cell standard for use in calibration and set-up of flow cytometers (FACS, EPICS). CRBCs are prepared from chicken erythrocytes that have been processed to remove leukocytes and are fixed with glutaraldehyde. CRBCs are a useful cytometry control because these processed but unstained cells fluoresce at about the intensity as cells stained for immunofluorescence.

FORM

BioSure® CRBCs are supplied in 2.0 mL (P/N 1004) or 6.0 mL (P/N 1005) volumes. The cell count per milliliter is at least 2.5 X (10)7 CRBCs. The control is guaranteed to have a CV of less than or equal to 10% on FL2.

STABILITY

CRBCs are stable at 2o - 8o C until the expiration date denoted on each vial label. Freshly diluted (1:10) preparations are stable for at least 72 hours at room temperature (25o - 30o C), although longer stability has been observed when diluted material is stored at 2o - 8o C.

PREPARATION OF WORKING SOLUTION

The recommended working solution is a 1:10 dilution of the stock solution in particulate-free physiological saline or isotonic buffered saline. This provides an adequate cell density and sufficient working solution for several days.

1. Gently mix the stock CRBCs, and add 2 drops into a test tube (13 X 75 mm).

2. Add 0.9 mL of diluent, mix and evaluate.

Typical CRBC Graph

NOTE: The flow cell of some cytometers is susceptible to clogging. Filtering the stained preparation through a 30 - 50 mesh nylon screen will eliminate large nuclear aggregates.

CRBC Bulk Custom Orders

Cytometry Control

Product Number 1018

Custom order Chicken Red Blood Cells are prepared quarterly and are produced specifically for customers who require larger volumes for their manufacturing. Custom order CRBCs are dispensed in one container only, and the minimum purchase volume is 40 milliliters. Pricing for larger volumes will be given upon request.

Specifications

Volume: Minimum 40 mL - 500 mL per container

Concentration: 2.5 X 107 CRBCs per milliliter

Storage

Stable at 2o - 8o C in unopened container until expiration date noted on each container.

Ordering

Orders must be received before March 31, June 30, September 30, and December 31, for delivery in the subsequent month.

  • Initial Standardization
  • Daily Monitoring
  • Sensitivity Assessment
  • Alignment & Linearity
  • Biological Safety Data
  • Material Safety Data Sheet

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 CRBCs fluoresce brightly over a wide emission spectrum (500 to 700 nm).

Initial standardization Procedure using Lymphocytes and CRBCs

Ideally, the initial instrument setup using CRBCs 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 CRBCs in each of the parameters. These optimized instrument settings establish target values for the CRBCs 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. 1. Align the instrumentation optics, if necessary.
  2. 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. 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. 4. Set a gate around the lymphocytes and activate the gate. Change the display parameters to FL1 and FL2.
  5. 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. 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. 7. Prepare a fresh dilution of the CRBCs to achieve a flow rate of about 500 - 1000 events/second. Place the tube on the cytometer.
  8. 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. 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. 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 PMTsetting for the CRBCs. This value will be used as the target value for monitoring the SSC stability.
  11. 11. Proceed to the FL1 histogram display. Run the CRBCs using the averaged FL1 PMT setting established by the control donor lymphocytes. Record the mean peak value and C.V.'s of the CRBCs for the FL1 parameter. These values will be used as target values for monitoring the FL1 stability.
  12. 12. Proceed to the FL2 histogram display. Run the CRBCs using the averaged FL2 PMT setting established by the control donor lymphocytes. Record the mean peak value and C.V. of the CRBCs for the FL2 parameter. These values will be used as target values for monitoring the FL2 stability.
  13. 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 CRBCs. 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.

Once target reference values for the CRBCs have been established, monitoring of the parameters is performed on a daily basis. Instrument settings for the target values are recalled and the new daily values compared to the target reference values. Recording of the new daily values onto Levey-Jennings plots provides a laboratory record of instrument stability over time.

Daily Monitoring Procedure

  1. 1. Prepare a fresh dilution of the CRBCs to achieve a flow rate of about 500 - 1000 events/second. Place the tube on the cytometer. Set the instrument parameters as follows: FSC-linear, SSC-linear, FL1-log, FL2-log.
  2. 2. Recall the instrument settings for the FSC parameter. The CRBC high scatter peak and the low scatter peak should both be on scale in the forward scatter histogram display. Calculate the difference in mean channels between the CRBC high scatter peak and the low scatter peak. Both mean channels should be within plus or minus ten channels of the target values. A significant loss (approximately 10%) of the high peak's target mean channel value or delta probably indicates optical system problems. Record the values.
  3. 3. Proceed to the SSC histogram display. Recall the instrument settings for the SSC parameter. Compare the side scatter mean channel number with the mean channel number established for the SSC during the initial target values run. A variation of more than 10% may indicate the need for realignment or instrument servicing.
  4. 4. Proceed to the FL1 histogram display. Recall the averaged FL1 PMT setting established by the control donor lymphocytes. Adjust the PMT voltage in the FL1 parameter (if necessary) to place the CRBCs in the same FL1 channel as determined by the initial target values. Record the adjusted PMT voltage and C.V. A variation of more than 10% may indicate the need for realignment or instrument servicing.
  5. 5. Proceed to the FL2 histrogram display. Recall the averaged FL2 PMT setting established by the control donor lymphocytes. Adjust the PMT voltage in the FL2 parameter (if necessary) to place the CRBCs in the same FL2 channel as determined by the initial target values. Record the adjusted PMT voltage and C.V. A variation of more than 10% may indicate the need for realignment or instrument servicing.
  6. 6. Repeat for FL3 or other parameters, if desired.

  7. Before any realignment or service of the instrument, prepare a fresh dilution of the CRBC standards and repeat the test protocol. If variations persist with the freshly prepared sample of CRBCs, then servicing or realignment may be necessary.

Biological Safety Data

This product is manufactured by BioSure®, Inc. as a biological research product. To the best of our knowledge, this product does not contain concentrations of carcinogenic, teratogenic or chemical agents that exceed 0.1% or greater in accordance with OSHA’s Hazardous Communication Standard 29CFR1910.1200 (d) (5) (i-iv). This product is exempt from the Material Safety Data Sheet requirements. Biological research controls should be handled using proper laboratory safety procedures, such as gloves, eye protection etc. Disposal can be by normal sewage. However, autoclaving or incineration is recommended should this product be mixed with hazardous or infectious materials such as human or animal serum, plasma, whole blood or tissue. If you have further questions, please call us at 800-345-2267 or email us at questions@biosure.com