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 (www.cdc.gov/mmwr/preview/mmwrhtml/rr5202a1.htm),
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 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%.
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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