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CD4 Flow Cytometry Questions & Answers

IQA guidelines

Are there guidelines for a lab that wants to switch from 3 color flow cytometry to 4 color flow cytometry?

If your lab wants to switch from 3 color flow cytometry to 4 color flow cytometry, click here for guidelines. Please let your PNL and the IQA know that you will be performing this study prior to starting. Send the results to the IQA statisticians listed in the document. The data will then be discussed with the cross network IQA QA working group and you will be notified about the outcome of the data. Do not switch until you hear from the committee.

On the Facscount report, do the sites need to keep track and plot any of the following Slope intercept R values?

The acceptability of these statistical values is embedded in the FACSCount software and the software will trigger a failure if these values are outside of the defined acceptable limits.  Since the software is monitoring these parameters for acceptability, it seems redundant to manually plot these values for tracking purposes.

What is the recommended allowable difference in absolute count between 2 FACScounts, 2 similar Flow cytometers, and /or between FACScount and another flow cytometer?

 We recommend that each lab define the acceptable limits of variablility for each analyte within the guidelines outlined by the manufacturer. Our testing shows variations within 10% for the CD3, CD4, and CD8 parameters when testing normal samples between different FACScount instruments, between flow cytometers, and between Facscount and other instrument/methodology.  Out testing shows variations within 15% when testing samples with CD4 counts below 200.

What do you do if you do not have a normal donor each day that you need to run your instruments?

If a normal donor is not available each day the instrument is run, a previously tested patient sample that displays marker values within normal ranges may be substituted.  This specimen must be used within the pre-defined limits of sample stability for the testing system.  This alternative testing method may also be applied to satisfy the requirement for a daily CD4 low control.  A patient sample with low CD4 level (<200) may be selected as the CD4 low control.

If you need to refer a sample to a referral lab that uses the same analyzer, would you recommend that the primary sample be sent or would it be better to prepare and fix the sample?

Samples should be sent as an unprocessed specimen to control for variables between testing sites unless a study was conducted between the sites to show correlation of testing prepared and fixed samples sent from a primary lab.  It is important that should the need arise to send a sample to a referral lab that the sample is received at the referral site within the defined stability limits of the specimen.

Can you provide a list of all websites/companies to order the CD4 controls from?

Note that some control materials may not be compatible with the testing system.

  • Beckman Coulter Cytometry www.beckman.com Immunotrol Cells, CD4 Normal Immunotrol Low Cells, CD4 Low
  • Becton Dickinson ImmunoCytometry Systems www.bdbiosciences.com
    Multi-Check Control, CD4 Normal Control
    Multi-Check Control, CD4 Low Control
    These controls may not be compatible with the Beckman Coulter Q-Prep Cytometry System
  • Streck Labs www.streck.com
    CD Chex Plus, CD4 Normal
    CD Chex Plus, CD4 Low
    CD Chex Plus BC, CD4 Normal designed for Beckman Coulter Q-prep
    Cytometry system
    CD Chex Plus BC, CD4 Low designed for Coulter Q-prep Cytometry system.


Can you explain the difference between single and dual platforms?

In the CDC MMWR Guidelines for Performing Single Platform Absolute CD4+ T-Cell Determinations, the definition of single-platform echnology (SPT) is a process in which absolute counts of lymphocyte subsets are measured from a single tube by a single instrument. SPT incorporates internal calibrator beads of known quantity in the analysis of specimens by three- or four-color flow cytometry.  Compare this with the definition of a dual platform; dual-platform technology is a method in which absolute counts are derived from measurements obtained from two instruments---a flow cytometer and hematology analyzer.

The FACSCount flow cytometer is a single platform system.  The absolute marker values tested in this system may be reported as a percentage of the total lymphocytes with the use of a hematology count for the specimen.  For example, a CD4 count of 1218 cells/┬Ál obtained from the FACSCount instrument divided by the total lymphocyte count of 2230 obtained from the hematology analyzer equals a CD4% value of 54.62.

Can you provide instructions on how to gate the UK NEQAS samples?

UKNEQAS samples are stabilized whole blood samples with light scatter and fluorescence staining properties that are different from fresh blood specimens and as such may not satisfy the automatic gating algorithm defined by the instrument software.  The instrument software may flag the analysis with an error identifying questionable sample integrity. In this instance, a manual gating should be applied to the analysis. The gating of the populations should be reviewed to ensure that the population clusters are gated to include the entire cluster without any interfering background and debris events.

Example of software-defined automatic gating of specimen of questionable sample integrity.



Example of manual gating to correct for overlapping interfering events in the analysis.




Can you provide details on what needs to be done to validate a new flow cytometer?

A correlation study using both normal and patient samples representing the expected range of reportable result values is recommended to validate a new instrument or method.  A minimum of thirty samples should be analyzed on both instruments and correlation statistics applied to determine acceptability of data.  In addition, a normal level and low CD4 level should be assayed in replicates (10 times each) on the new instrument to evaluate intra-instrument variations.

What are the IQA's recommendations for the running of calibration beads for clinical labs running CD4 counts?

Clinical labs performing CD4 enumerations should follow manufacturer's recommendations for running daily calibration beads.

 For the BD FACSCount System, they are the low, medium, and high control beads.  For the BD FACSCalibur or FACScan systems, it is the FACSComp Calibrite beads.  For the Beckman Coulter Cytometry System, they are the FlowCheck and FlowSet Fluorospheres.  In addition, the BD FACSCalibur and Beckman Coulter Cytometer Systems should be monitored monthly for fluorescence linearity.  Spherotech Rainbow Beads and ImmunoBrite beads are available to evaluate the linearity of the Becton Dickinson and Beckman Coulter systems respectively.

What are the IQA's recommendations for commercial controls?

Commercial controls are available to monitor the precision and accurately of the testing system.  The CD4 normal and CD4 low controls are characterized with expected target range values.  The manufacturer defined ranges have variations greater than 25%.  Labs should determine their own target ranges for these control material.

The advantage of using a commercial control is that commercial controls are stable over a period of several months.  In addition, the results from the testing of commercial controls can be plotted onto a Levey Jennings chart for monthly monitoring of the system.  At the start, a lab should use the manufacturers provided ranges to monitor the daily QC of the lab.  As the lab gains more confidence and experience, the ranges provided by the manufacturer should be adjusted.  Running commercial controls are not meant to be used to establish the normal ranges.

Does the IQA recommend running replicate samples to check day to day instrument performance? If the answer to this question is yes, do you recommend monitoring % CD4 counts or absolute CD4 counts or both?

It is not necessary to run replicate samples routinely to check day to day instrument performance.  The daily control runs would serve as the monitor for instrument and system performance.

Is it necessary to run 2 other controls (? patient bloods) where one has a ratio of <1.0 and the other has a ratio >1.0?

The use of the control beads; low, med, and high beads, serve to monitor the testing system to assess the pipetting accuracy and system linearity.  The use of the normal and low level controls serves to monitor the precision and accuracy of the testing protocol.  Labs should demonstrate their proficiency to test for values at the levels of routine patient testing.  To achieve this, normal level and low CD4 level controls with known target value are recommended.  Commercially available controls with normal and low CD4 levels are ready sources for the daily control runs.  In addition, a fresh specimen from a normal donor should be included to monitor any variations in testing fresh daily patient specimens.

Patient specimens may be used as a substitute to the commercial controls but are limited to evaluating the target values based on the 1-2 day stability of the sample.  A patient specimen which is selected as the daily control has been tested with a reported CD4 value and will be used to monitor the instrument ability to replicate this target value. The tolerance limit should be determine based on previous replicate studies performed on the instrument as part of  the instrument validation and set up.  The advantage of a commercial control over a patient sample is the stability of the sample over several months.  A monthly Levey Jennings chart can be used to monitor the performance of the system in the testing of the commercial controls.  This monitoring system can not be applied when using a different patient control daily as the target control.

For reagent lot parallel testing, is it necessary to run a patient with a ratio >1.0 and a patient with a ratio <1.0.  If so, how reproducible should the results be?

For lot-to-lot reagent comparisons, it is not necessary to run two different levels of samples.  A normal sample should suffice and the range of acceptability is the same as that defined in the replicated study performed as part of the instrument validation.  Generally, the range of the marker values defined in intra-instrument variation studies are less than 10%.

Are the CD4 + CD8 values supposed to equal the CD3 values and if so +/- what percentage is acceptable?  Would you think a difference of 1500 between the CD3 value and the total of the CD4 + CD8 is problematic?

The CD4 plus CD8 absolute values (T sum) should equal the CD3 value +/-  15% in most cases.  A difference of 1500 between CD3 and the T sum values does seem rather high and may warrant further investigation.  When considering percentage values, the acceptable range between the CD3 and T sum values is +/- 5%.  Samples may test outside of these ranges but should be identified for additional testing.

Other QC measures that you suggest by the IQA

For labs with more than one flow cytometer, a replicate sample should be run daily on each additional instrument in use to show instrument correlation.  The tolerance limits of the monitoring parameters should be previously defined by instrument correlation studies.

Full service cytometers should be monitored monthly for instrument linearity.  Calibration beads are available to monitor system fluorescence linearity.  Beckman Coulter provides ImmunoBrite Beads for this purpose for their cytometry systems.  Spherotech Rainbow Beads are available for use in BD cytometry systems.