Postscript

DUMMY

MECHANICAL
Sign-Off

PRINT PROOF

NEW PDF

REVISED PDF
MLO
PG.24

CIRCLE/RS#

LIT#

SHOWLINE

I/O CHECK

PROD MGR
Nelson Publishing
2500 Tamiami Tr N
Nokomis, FL 34275
1-800-226-6113
Tandem dyes for ow cytometry:
Can we overcome quality concerns?
By Meryl A. Forman and Ravinder K. Gupta, PhD
M
ulticolor analysis is an important tool used in the iden-
tication and investigation of cellular subpopulations.
Many of the most common immunophenotyping tests
involve analyzing multiple parameters. The additional parameters
needed to perform these tests require the use of an increasing
number of uorochromes. Tandem dyes have been developed
to expand the dye portfolio to meet these needs and are widely
available in the marketplace. The quality of these reagents,
however, has suffered from several issues that impact the ease
of use and consistency of performance in the laboratory setting.
As clinical laboratories continually strive to improve the quality
of patient test results, they must consider the variability and quality
of the tandem dyes used in their testing protocols, especially when
combining specic reagents from multiple vendors. Building qual-
ity into tandem dye products to enhance their utility is a process
that begins at the point of design and manufacture, and continues
through performance of the product in the customer hands. Sev-
eral patented and proprietary processes have been developed by
manufacturers to address these problems and are incorporated
into products currently on the market. With a little knowledge,
it is possible to sort through the various offerings and select tan-
dem dyes that produce more consistent, higher quality results.

What is the source of the problems?
Tandem dyes may suffer from one or more of the following
problems:
1. inadequate/variable energy-transfer efciency resulting in
changing compensation requirements;
2. low uorescence intensity;
3. lot-to-lot variation;
4. non-specic binding; and
5. dye degradation.
Tandem dyes exploit the principle of uorescence resonance
energy transfer, or FRET. A donor chromophore (e.g., phycoery-
thrin [PE], allophycocyanin [APC]) is excited by a suitable light
source and transfers its absorbed energy to an acceptor uoro-
phore, which then emits this energy as uorescence at the acceptor-
dye wavelength. Because the energy-transfer efciency depends
on the distance between the donor and acceptor chromophores, it
is important to conjugate them in close proximity.
1,2
One patented
approach to this process
3
uses a method of intramolecular unfold-
ing of the donor phycobiliprotein molecules (e.g., PE or APC).
This makes it possible for the acceptor-reactive-dye molecules to
react preferentially with the newly exposed hydrophobic regions
containing the bilin chromophores (see Figure 1).
The quality of a tandem dye can be measured by the intensity
of its acceptor uorescence and crossover value (ratio of peak
emissions for the donor and acceptor dyes). In the aforemen-
tioned process, after the coupling step the heterogeneous mixture
is separated using hydrophobic-interaction chromatography
(HIC), further improving the quality of the tandem dye. Figure
2 shows the uorescence scans of PE-Cy7 dye before and after
HIC purication as well as the scan of the discarded portion in a
typical preparation. This process has been validated for lot-to-lot
reproducibility within tight target specications, a key require-
ment for use of antibody conjugates of these dyes in longitudinal
or multisite studies.
A comparison of CD3-tandem dye conjugates (PE-Texas Red,
PE-Cy5, PE-Cy7) from different manufacturers demonstrates the dif-
ferences in both uorescence intensity and energy-transfer efcien-
cy. Figure 3 shows the comparative uorescence spectral emission
scans (3A) and the performance (3B) of these conjugate products.

Dealing with non-specic binding
A unique and undesirable characteristic of most tandem dye
products is the low level non-specic binding to monocytes
of the acceptor dyes used (e.g., Cy5, Cy7, Texas Red). This
binding can produce false results that are not always obvious to
inexperienced users. The level of this non-specic staining varies
considerably from dye to dye and vendor to vendor. When analyz-
ing lymphocyte subpopulations, gating approaches can be used to
minimize the impact to data results; however, use of reagents with
C L I N I C A L I S S U E S
Postscript

DUMMY

MECHANICAL
Sign-Off

PRINT PROOF

NEW PDF

REVISED PDF
MLO
PG.25

CIRCLE/RS#

LIT#

SHOWLINE

I/O CHECK

PROD MGR
Nelson Publishing
2500 Tamiami Tr N
Nokomis, FL 34275
1-800-226-6113
The impact of packaging
Another factor affecting quality is the degradation that occurs
over time with exposure to light, which may cause erroneous data
due to changing spectral characteristics. It is, therefore, important
to consider the protective packaging used for the reagent. Most
commercially available tandem-dye reagents are bottled in stan-
dard amber-glass vials that protect against UV radiation but not
from visible and, in particular, the red radiation that corresponds
to the absorption of both donor and acceptor molecules of most
tandem dyes (see Figure 5A). The use of patented black-coated
vials has been shown to protect dyes against photo degradation.
Tandem-dye reagents (PE-Cy5 data shown in Figure 5B) bottled
in these black-coated vials and exposed to controlled lighting
3

conditions showed no degradation compared to the amber-vi-
aled product measured by both uorimetry and ow cytometry.

Conclusion
The advancements in the quality of reagents used to perform
clinical ow-cytometry analyses ultimately improve the utility
of patient test results. The pitfalls associated with tandem-dye
technology have been addressed by patented and proprietary
manufacturing processes, formulation chemistries, and packag-
ing approaches, making delivery of high-quality dye conjugates
available to the market for use in routine testing. These tandems
are offered in in vitro diagnotics- (IVD-) cleared CD4 enumeration
kits and a variety IVD and assay specic reagent conjugates. l
high background staining of monocytes poses a major problem
when evaluation of these populations is critical. An example ap-
plication is the immunophenotyping of bone-marrow specimens
where the targeted populations of interest are both myeloid and
lymphoid lineages. Incorporating proprietary chemistries in the
formulation of the products eliminates this background staining.
Flow-cytometry labs should be aware that there are commer-
cially available tandems that minimize or eliminate background
staining. As seen in the CD3-PECy5 histograms presented (see
Figure 4), the background staining is completely eliminated
after formulation using one of these proprietary chemistries.

Meryl A. Forman and Ravinder K. Gupta, PhD, are both employed with Beckman Coulters Cel-
lular Analysis R&D Center in Brea, CA. Dr. Gupta holds several tandem dye process patents,
while another Beckman Coulter associate, J.P. Daziano, holds black-coated vial patents.
References
1. Glazer AN, Stryer L. Fluorescent Tandem Phycobiliprotein Conjugates, Biophys J.
1983;43:383.
2. Waggoner AS, Ernst LA, Chen C, Rechtenwald DJ. A New Fluorescent Antibody
Label For Three-Color Flow Cytometry With A Single Laser, Anals N.Y. Acad. of
Sciences. 1993;677:185.
3. ICH Expert Working Group. ICH Harmonized Tripartite Guideline. Stability Testing:
Photostability Testing of New Drug Substances and Products. Step 4. Geneva,
Switzerland; November 1996.
C L I N I C A L I S S U E S
Reprinted from Medical Laboratory Observer, October 2007
Copyright 2007 by Nelson Publishing Inc. www.mlo-online.com