Questions and Answers for CD4 flow cytometry

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					                        Questions and Answers for CD4 flow cytometry

                                Non-SMILE created resources
Author: John Wong-IQA                          Document Number:             Pro63-A-05
                                            Effective (or Post) Date:      13 March 2009
Review History                                 Date of last review:         7 May 2010
                                                  Reviewed by:              Heidi Hanes
SMILE Comments: This document is provided as an example only. It must be revised to
accurately reflect your lab’s specific processes and/or specific protocol requirements.
Users are directed to countercheck facts when considering their use in other applications.
If you have any questions contact SMILE.

                                          IQA guidelines

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 is not necessary to manually plot
these values for tracking purposes.

What is the recommended allowable difference in absolute count between 2 FACSCounts
and/or between FACSCount and another flow cytometer?

We recommend that each lab define the acceptable limits of variability for each analyte within the
guidelines outlined by the manufacturer. Our testing show variations within 10% for the CD3, CD4
and CD8 parameters when testing between different FACSCount instruments and between a
FACSCount and other instrument/methodology.

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

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

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 all testing systems.

Beckman Coulter Cytometry Immunotrol Cells, CD4 Normal Immunotrol Low
Cells, CD4 Low

Becton Dickinson ImmunoCytometry Systems
Multi-Check Control, CD4 Normal Control
Multi-Check Control, CD4 Low Control
BD Multi-Check controls may not be compatible with the Beckman Coulter Q-Prep Cytometry

Streck Labs
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.
Streck CD Chex Plus controls may not be compatible with the BD FACSCount 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 technology (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/µl 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.

BD has determined the use of a conversion factor to calculate the CD4% values from the
CD4/CD3 ratio value obtained in the FACSCount system. To convert the FACSCount data,
multiply the CD4/CD3 ratio by 100% and the conversion factor of 0.813. For example; a
FACSCount CD4/CD3 ratio of 0.220 is first changed to a percentage by the calculation, 0.220 x
100% = 22.0%, next the % of CD4 of CD3 is multiplied by the conversion factor, 22.0% x 0.813 =
17.9%, which results in the CD4 percentage value of the total lymphocytes. This conversion
calculation formula is limited for use in fresh whole blood specimens from adults and is not
applicable for pediatric samples or stabilized whole blood samples such as commercial controls
or some proficiency test material.
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 (non HIV) and patient samples (HIV 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

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 that
has been tested with a reported CD4 value may be selected as the daily control to monitor the
ability of the testing system to replicate this target value. The tolerance limit should be
determined based on previous replicate studies performed as part of the instrument validation
and set up. The advantage of using a commercial control over a patient sample is that the
commercial control is stable over 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. This monitoring approach 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%.

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.

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