Journal of Immunological Methods 385 (2012) 79–89

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

Research paper

Optimal cellular preservation for high dimensional flow cytometric analysis

of multicentre trials
Amanda A.P. Ng a,⁎, Bernett T.K. Lee a, Timothy S.Y. Teo a, Michael Poidinger a, John E. Connolly a, b
a
Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos Building, Level 3,
Biopolis 138648, Singapore
b
Institute of Biomedical Studies, Baylor University, Waco, TX 76798-7348, USA

a r t i c l e i n f o a b s t r a c t

Article history: High dimensional flow cytometry is best served by centralized facilities. However, the
Received 27 July 2012 difficulties around sample processing, storage and shipment make large scale international
Received in revised form 17 August 2012 studies impractical. We therefore sought to identify optimized fixation procedures which fully
Accepted 17 August 2012 leverage the analytical capability of high dimensional flow cytometry without the need for
Available online 24 August 2012
complex cell processing or a sustained cold chain. Whole blood staining procedure was
employed to investigate the applicability of fixatives including Cyto-Chex® Blood Collection
Keywords: tube (Streck), Transfix® (Cytomark), 1% and 4% paraformaldehyde to centralized analysis of
Cellular preservation field trial samples. Samples were subjected to environmental conditions which mimic field
Immunophenotyping
studies, without refrigerated shipment and analyzed across 10 days, based on cell count and
Whole blood
marker expression. This study showed that Cyto-Chex® demonstrated the least variability in
Cell count
Marker expression absolute cell count relative to samples analyzed directly from donors in the absence of fixation.
Flow cytometry Transfix® was better at preserving the marker expression among all fixatives. However,
Transfix® caused marked increased cell membrane permeabilization and was detrimental to
intracellular marker identification. Paraformaldehyde fixation, at either 1% or 4% concentra-
tions, was unfavorable for cell preservation under the conditions tested and thus not
recommended. Using these data, we have created an online interactive tool which enables
researchers to evaluate the impact of different fixatives on their panel of interest. In this study,
we have identified Cyto-Chex® as the optimal cellular preservative for high dimensional flow
cytometry in large scale studies for shipped whole blood samples, even in the absence of a
sustained cold chain.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction technique permits single cell characterization of various

Flow cytometry is a powerful tool that allows the analysis
of cellular phenotype and function in the immune system
(Maecker et al., 2012; Baumgarth and Roederer, 2000). With
high dimensionality and wide dynamic range capability, this
Abbreviations: BCT, Blood collection tube;PFA, Paraformaldehyde;PBMCs,
peripheral blood mononuclear cells;PBS, phosphate buffered saline;SB, staining
buffer;DC, dendritic cells;mDC, myeloid dendritic cells;pDC, plasmacytoid
dendritic cells;Treg, regulatory T cells;MFI, median fluorescence intensity;NK,
natural killer cells;NMG, NK, monocytes and granulocytes;TB, T cells and B cells
⁎ Corresponding author. Tel.: +65 64070609; fax: +65 64642056.
E-mail address: Amanda_Ng@immunol.a-star.edu.sg (A.A.P. Ng).
immune subsets and detection of intracellular phosphopro-
teins (Wu et al., 2010), transcription factors (Crellin et al.,
2007) and cytokines (Foster et al., 2007). It has become an
important tool widely used in clinical settings such as for the
diagnosis of leukemia (Stetler-Stevenson and Tembhare,
2011), immunological disorders (Puxeddu et al., 2012) and
monitoring of drug therapy (Galligan et al., 2009).
Due to its complexity and sensitivity, high dimensional flow
cytometry is best served by centralized facilities. This allows
batching of samples which minimizes site variability, time and
overall cost. However, the use of centralized facilities repre-
sents a challenge when designing multicentre clinical studies.

0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jim.2012.08.010
80 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89

The difficulties around sample processing, storage and ship-
ment often result in surface marker degradation making large
scale multicentre studies impractical. This is particularly true in
international pivotal trials involving developing countries,
where the infrastructure for cell processing and logistics of a
sustainable cold chain are largely non-existent.
Various methods have been developed to address
compromised sample integrity upon shipment (Pinto et al.,
2005). The most commonly used reagent is paraformaldehyde
(PFA), which cross links cellular proteins (Lanier and Warner,
1981). Stabilization reagents were initially used to develop
biological standards to evaluate the performance of cytometers
(Barnett et al., 1996). Companies such as Streck and Cytomark
have developed stabilizing reagents called Cyto-Chex® (Streck
Laboratories, La Vista, NE) and Transfix® (United Kingdom
National External Quality Assessment Service [UK NEQAS]) for
cellular preservation. These stabilizing reagents are available in
vacutainer form which allows direct blood draw and cellular
preservation up to a week at room temperature (Canonico et al.,
2004; Davis et al., 2011; Warrino et al., 2005).
In laboratory immunological studies, the most commonly
acquired human samples for flow cytometry are peripheral
blood mononuclear cells (PBMCs) which require gradient
separation of cells from whole blood (Fuss et al., 2009). This
manipulation has an impact on marker expression (Nieto et al.,
2012; Zhou et al., 2012) which results in the loss of specific
cellular populations (Renzi and Ginns, 1987; Romeu et al.,
1992; Tamul et al., 1995). Furthermore, clinical sites may have
varying levels of infrastructure to support proper cellular
separation and storage. In many clinical studies, whole blood
isolation methods have replaced density gradient separation of
PBMCs as a preparative technique for immunophenotyping
(Romeu et al., 1992; De Paoli et al., 1984). Whole blood analysis
results in decreased sample manipulation and reduced blood
volume. Consequently, there are lower risks associated with
sample handling and an increase in information from volume
limited samples (De Paoli et al., 1984).
With the clear advantages of whole blood collection, there
is a need to identify optimized fixation procedures. An ideal
procedure should permit us to fully utilize high dimensional
flow analytical capability without the need for complex cell
processing or sustained refrigeration.
In this study, we investigated the impact of cellular
preservation by different fixatives at various time points with
and without shipment. The data demonstrated that some
methods of fixation were better at preserving cell count while
others were better at preserving marker expression. An online
interactive tool has been created to visualize the impact of
different fixatives on individual markers on different cellular
populations. This data will be useful for identifying an optimal
method of preservation depending on the analytical require-
ment needed by multicentre clinical studies.
2. Materials and methods
2.1. Subjects
Informed consent was obtained from the donors according
to NUS-IRB 09‐256. Blood was drawn into 10 ml EDTA (K2)
vacutainers (Becton Dickinson) and Cyto-Chex® blood collec-
tion tubes (BCT) (Streck) from 4 individuals, aged between 24
and 36 years old, equally distributed between both sexes and
all of Asian ancestry. No subjects had taken any medication 6 h
prior to blood collection.
2.2. Reagents
Transfix® reagent (United Kingdom National External
Quality Assessment Service [UK NEQAS]), Cyto-Chex® Blood
Collection tubes (Streck Laboratories, La Vista, NE) and PFA
(Electron Microscopy services) diluted in phosphate buffered
saline (PBS) (Gibco) were used as fixatives. ACK lysis buffer
(Lonza) was used for red blood cells lysis. The monoclonal
antibodies used are stated in Table 1. AccuCount Blank
counting beads (Spherotech) were used for enumerating
cell count.
2.3. Storage conditions
Unfixed samples, Transfix® and Cyto-Chex® treated
samples were shipped at room temperature through courier
service to a destination within Singapore and were sent
back to the lab within 4 days. These samples were kept at
room temperature till Day 10. For non-shipped samples,
unfixed and PFA treated samples were stored at 4 °C while
Cyto-Chex® and Transfix® treated samples were kept at
room temperature for 10 days.
2.4. Cellular fixation
Cyto-Chex® treated samples were directly fixed in the
vacutainer by inverting the vacutainer 10 times. For Trans-
fix® treated samples, 2 ml of Transfix® was added to 10 ml
of blood as recommended by the manufacturer. Samples
were fixed for 2 h at room temperature before staining at Day
0. In order to fix samples with PFA, red blood cells were lysed
at room temperature for 8 min with rotation (17 rpm) using
ACK lysis buffer (Lonza) in a 1/1 volume ratio. PBS was then
added and cells were centrifuged for 2 min, 1000 g, at room
temperature. Cells were fixed with 2 ml of 1% PFA/PBS or 4%
PFA/PBS for 20 min on ice. Fetal bovine serum (8 ml)
(HyClone Laboratories Inc., South Logan, Utah) was added
to neutralize the cross linking effect of PFA.
2.5. Sample preparation
1.5 ml of unfixed and Cyto-Chex® treated samples and
1.8 ml of Transfix® treated samples (to account for the
dilution factor of Transfix®) were aliquoted. Red blood cells
in these samples were then lysed based on the protocol
mentioned above. 1.5 ml of PFA treated samples was
aliquoted into a new tube and PBS was added. All samples
were centrifuged and resuspended with 1.5 ml staining
buffer (SB) (2% FCS, 2 mM EDTA [Gibco], 1.5% HEPES
[Gibco], PBS [pH7.4]). 200 μl of cell suspension was seeded
into a 96 well v-bottom plate (Corning) for staining with
panels for T and B cells (TB), natural killer cells (NK),
monocytes and granulocytes (NMG) and dendritic cells
(DC) (shown in Table 1). The remaining cell suspension
was used for staining regulatory T cells (Treg).
 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89 81

Table 1
Antibodies staining panel.

Flurophore TB panel Treg panel DC panel NMG panel

FITC CD45RA (HI100, Biolegend) CD4 (RPA-T4, Biolegend) BDCA3 (AD5-14H12, Macs Miltenyi) CD66 (B1.1, BD Pharmingen)
PE CD45RO (UCHL1, Biolegend) CD127 (HIL-7R-M21, BD BDCA2 (AC144, Macs Miltenyi) CD14 (M5E2, Biolegend)
Pharmingen)
ECD CD19 (J3.119, Beckman Coulter) CD45RA (2H4DH11LDB9, CD62L (DREG56, Beckman Coulter)
Beckman Coulter)
PECy5 7AAD viability solution (Ebioscience)
PECy7 CD4 (RPA-T4, Biolegend) CD25 (M-A251, BD Pharmingen) CD86 (Fun1(2331)BD Pharmingen) CD11b (ICRF44, BD Pharmingen)
APC CD8 (RPA-T8, BD Pharmingen) CD49D (9F10, BD Pharmingen) CD11c (S-HCL-3, BD Pharmingen) CD16 (3G8, Invitrogen)
APC-Cy7 HLADR (L243, BD Bioscience) HLADR (L243, BD Bioscience) HLADR (L243, BD Bioscience)
Alexa Fluro 700 CD3 (UCHT1, BD Pharmingen) CD3 (UCHT1, BD Pharmingen) CD80 (L307.4, BD Pharmingen) CD56 (B159, BD Pharmingen)
Pacific Blue CD86 (FUN1(2331), BD Horizon) FOXP3 (PCH101, Ebioscience) BDCA1 (L161, Biolegend) NKp46 (900, Biolegend)
Pacific Orange CD45 (HI30, Invitrogen) CD45 (HI30, Invitrogen) CD45 (HI30, Invitrogen)
Qdot655 CD20 (2H7, Ebioscience) CD123 (6H6, Ebioscience)

2.6. Surface staining
Samples were centrifuged and supernatant were aspirat-
ed with a multichannel pipette. Antibody cocktails were
prepared at an optimized concentration and diluted with SB.
50 μl of antibody cocktail (excluding FOXP3 for Treg panel)
was added to each respective well for each panel. Samples
were incubated on ice in the dark for 30 min. 100 μl of SB was
added and samples were centrifuged. Supernatant was aspirat-
ed and cells were washed with 150 μl of SB. Samples were
resuspended with 100 μl of SB.
2.7. Intracellular staining for Treg population
After surface staining, cells were centrifuged and re-
suspended with 100 μl of diluted fixation/permeabilization
concentrate (Ebioscience) according to manufacturer's proto-
col. Cells were fixed on ice in the dark for 30 min and washed
with 100 μl of SB. Supernatant was aspirated and 100 μl of
permeabilization buffer (Ebioscience) was added. Cells were
incubated for 10 min on ice in the dark and supernatant was
aspirated after centrifugation. 3 μl of FOXP3 antibodies was
added directly. Cells were incubated on ice in the dark for
30 min followed by washing with 100 μl of permeabilization
buffer. Washing step was repeated with 200 μl of perme-
abilization buffer. Samples were then centrifuged and re-
suspended with 100 μl of SB.
2.8. Absolute cell counting
AccuCount Blank counting beads (Spherotech) were used
for absolute cell enumeration according to the manufacturer's
instructions. Beads were sonicated for 3 min, after which 50 μl
was added into 1.2 ml propylene tubes (Corning). 100 μl of
samples was then transferred into these tubes.
2.9. Sample acquisition
Acquisition by flow cytometry was performed with the
LSRFortessa™ cell analyzer (Becton Dickinson) using BD
FACSDiva Version 6.1.3. The machine was calibrated with
Cytometer Setup and Tracking beads (BD Biosciences) to
check for laser delay and voltage settings so as to provide
reproducible day-to-day cytometer performance for increased
data consistency over time. In addition, SPHERO™ Rainbow
Calibration Particles (Spherotech) were used before sample
acquisition to check for laser and machine performance. For
each sample, the whole volume was acquired.
2.10. Data and statistical analysis
Samples were analyzed with FlowJo 7.6.1. Hierarchical
clustering was used to cluster the unfixed population cell counts.
To identify cell count with similar profiles, Pearson correlation
was used for distance measurement and Unweighted Pair Group
Method with Arithmetic Mean (UPGMA) was used for clustering.
The cell counts for unfixed samples were then presented as a
percentage scale where the maximum cell count is denoted as
100% and lowest as 0%.
To determine if treatments for each measurement
(cell count and marker median fluorescence intensity
[MFI]) differed significantly, one-way repeated measures
ANOVA were used and P values b0.05 were considered
statistically significant. Results with P values >0.05 meant that
the measurement was not significantly affected by time or
fixation. Repeated measures ANOVA were carried out using
groups defined by treatment and days. Multiple testing correc-
tions were then done using the Benjamini–Hochberg method.
To better understand the effects of time on marker MFI of the
unfixed, standardized Euclidean distances [(original value−
mean)/standard deviation] were computed for the marker MFI
values using day 0 of the unfixed treatment as the reference
value. It measures the sum of absolute difference of the distance
between all possible datasets and Day 0 unfixed. Given that this
is an absolute value of difference, it does not indicate if
measurements increased or decreased. The longer the distance,
the bigger the difference compared to Day 0 unfixed. A graphical
illustration for Euclidean distance calculation is stated in Fig. 1.
Hence, standardized Euclidean distances allow comparisons
across cell populations and measurement types (cell counts or
marker MFI).
For comparing various treatments against the unfixed the
cell count or marker MFI of a particular treatment and cell
population was compared to the same cell population cell
count or marker MFI of the unfixed or data obtained from
days 4, 7, and 10. The comparison was then scored a 1 if the
summation of the standardized Euclidean distances was less
than the unfixed and 0 otherwise (Supplementary Table 1).
The scores were then counted and expressed as a percentage
of the times the treatment for the particular cell count or
82 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89
Fig. 1. Graphical illustration on Euclidean distance analysis. A, B, C and D
refer to the difference in distance between the data points of a particular
treatment with reference to unfixed Day 0 on a particular day for each
respective donor. The Euclidean distance is the sum of these differences in
distance between the two data points in each donor. The bigger the
Euclidean distance, the more different the data (marker MFI or cell count) is
with respect to Day 0 unfixed.
marker MFI in the cell population was better than the unfixed
over all comparisons made. This summarization allowed for a
higher level interpretation of the results. Day 0 data was
omitted since day 0 unfixed was used as the reference for the
Euclidean distance computations.
The data was analyzed using both Accelrys Pipeline Pilot
(www.accelrys.com) and R statistical language. The comput-
ed results were imported into TIBCO Spotfire and visualiza-
tions were generated.
3. Results
3.1. Impact of 4 °C storage in the absence of fixation on absolute
cell count
Any form of chemical modification may interfere with
antibody antigen interaction. Therefore, we first set out to
access the impact of simple 4 °C storage on our ability to
detect cellular subsets over a period of 10 days. Flow cytometry
analysis was performed at Days 0, 4, 7 and 10. Following
identification of immune cellular subsets with their lineage
markers (Supplementary Fig. 1), absolute cell count was
enumerated with counting beads. The data was analyzed by
clustering cell types with similar kinetic profile of cell count,
resulting in four distinct clusters which range from the least to
the most stable cell profile (Fig. 2a).
The least stable cell types for cell count preservation were
BDCA3 + mDC, granulocytes and pDC (Fig. 2a[I]). This is not
surprising as BDCA3+ mDC and pDC are among the most
rare cell types in our analysis. Additionally, granulocytes are
known to be particularly sensitive to manipulations.
Populations that were slightly more stable in cell count
include the T and B lymphocytes (Fig. 2a[II]). The B cells
population displayed a slightly different trend where the
population stabilized at Day 4–7 and then dropped at Day 10.
Populations including BDCA1+ mDC, CD14 +CD16 + mono-
cytes, CD14 loCD16 +monocytes, CD56 +NKp46 + NK cells and
Treg showed a more gradual decrease over time (Fig. 2a[III]).
The most stable populations were CD14 + monocytes,
CD14+CD16− monocytes, CD56+ NK cells and CD56+CD16+
NK cells (Fig. 2a [IV]). In general, there was a decrease in cell
count in all populations and their subsets (Fig. 2a).
3.2. Impact of 4 °C storage in the absence of fixation on
marker expression
Given the observations that our ability to recover cellular
population was subset dependent, we set out to investigate
the impact of 4 °C storage on the marker MFI expressed on
specific cellular populations. Based on repeated ANOVA analysis
with multiple testing corrections, the results showed that of the
117 measurements, only 4 were not significant, meaning that the
majority of the measurements were affected by time and fixation
(Supplementary Fig. 2).
Fig. 2b demonstrates the impact of time on individual
markers for each cell population. Standardized Euclidean
distances were used to quantify the absolute difference within
a population at different time points in relation to Day 0. CD45
and 7AAD were expressed in all populations. Generally, based
on the Euclidean distance of CD45 expression analyzed across
the TB, DC and NMG panels, we determined that this marker
was less stable than 7AAD with time of storage at 4 °C,
particularly in B cells, BDCA1 and BDCA3 mDCs, CD14 loCD16+
monocytes and CD4 T cells. A higher 7AAD expression was
observed in Treg and mDC populations among the others due
to an increase in cell death.
All markers were different compared to Day 0; the most
dramatic difference was observed in CD62L especially in the
monocytes, granulocytes and CD56 +NKp46 + populations.
This may be due to the ability of CD62L to be rapidly shed
from the surface of the cell (Feuerecker et al., 2012). Two
other activation markers were also studied; CD86 was most
affected in BDCA3 + population while CD80 levels were
different on Day 7 in mDC and pDC. CD11b and CD16 markers
were impacted specifically in the granulocyte population. As
expected, long term storage at 4 °C had an effect on marker
expression. Unlike the impact on cell count, however, overall
individual marker expression was largely well maintained.
3.3. Impact of fixation on absolute cell count
We next evaluated the impact of fixation on cellular marker
expression. Cells were fixed in Cyto-Chex®, Transfix® or PFA
for 10 days and compared to unfixed 4 °C samples at Days 0, 4,
7, and 10. Given that Transfix® causes cell membrane
permeabilization (Canonico et al., 2004), our data demonstrat-
ed that all cellular populations under Transfix® treatment
became 7AAD+ from Day 4 onwards. Thus, the gating for
Transfix® treated samples on cell count was determined by
expanding the analysis to CD45+ population regardless of
7AAD fluorescence.
To determine if the variation was greater in fixed than
unfixed, the comparison between fixatives and unfixed samples
were made based on the summation of Euclidean distance
between Days 4, 7 and 10. An example of the scoring method is
indicated in Supplementary Table 1.
As seen in Fig. 3a, the best preservative was Cyto-Chex®
as it maintained cell count in most populations better than
unfixed. Transfix® was not as good in preserving cell counts
 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89 83

Fig. 2. Impact of time on cell count and median fluorescence intensity (MFI) of individual marker on each population under unfixed 4 °C storage condition.
a) Hierarchical clustering based on Pearson correlation distance matrix was used to profile the cell count for unfixed population. Data was presented
according to kinetic cell profile, where the Y axis shows the cell counts expressed in a percentage scale where the lowest cell count for the particular
treatment is denoted as 0% and the highest as 100%. Each cell population is depicted as a line in the figure with time at 4 °C storage on the X axis. (I) The
least stable cell types were defined by a sharp drop of cell count from Day 0 to Day 4 and the levels continued to decrease gradually. (II) The slightly more
stable cell type would be populations that showed a less drastic drop from Day 0 to Day 4 and a gradual decrease in cell numbers over time. (III) The more
stable populations were those that decrease in a more gradual manner over time. (IV) The most stable populations belonged to populations that do not
have a decrease in cell count within the first 4 days and decrease gradually over the next week. b) Data presented indicates the distance of the particular
marker MFI compared to the marker MFI at Day 0. It was calculated based on Euclidean distance score between unfixed Day 0 and the respective day as
described in Materials and methods. The gradient in color represents the distance of the data from Day 0; the bigger the difference, the stronger the color.
All markers that were expressed in different population are shown here.
84 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89

as compared to Cyto-Chex® but it still performed better
compared to unfixed samples in DC and CD8 T cell
populations. Transfix® was the best overall in preserving
the BDCA1 population. PFA treatments were the most
detrimental to cell count as they did not preserve any
population better than unfixed. In summary, these data
demonstrated that Cyto-Chex® outperformed both Trans-
fix® and PFA fixations with regard to preserving cell
count.
3.4. Impact of fixation on marker expressions
We next set out to investigate the impact of fixations on
marker expression. As shown in the gating strategy (Supple-
mentary Fig. 1), the identity of a population is defined by a
set of markers. The data represented in Fig. 3b indicate the
percentage of time when the fixative was better in preserving
the identity of the population than unfixed (> 50%). It can be
seen that Transfix® was better at preserving the identity of

Fig. 3. Impact of fixatives on cell count, identity of population and individual marker MFI of all population. a) Summation of Euclidean distance in each treatment
was used to compare against unfixed treatment. The scoring method was illustrated in Supplementary Table 1. For treatments which performed better than
unfixed, the score is denoted as 1 (indicated as green), otherwise it is given 0 (indicated as red). Day 0 was not used for comparison as unfixed Day 0 was used as
the reference point for calculating Euclidean distance. b) Using the same scoring method as Fig. 3a, the sum of scores was computed from the set of markers that
defines the population and normalized by the number of markers. This would indicate the percentage of time a particular fixative is better in preserving the
identity of the population. c) In order to study the effects of fixation on individual marker expression, the same scoring method employed in Fig. 3a was used. The
sum of counts was computed from all populations expressing the particular marker and normalized by the number of populations. This would indicate the
percentage of time a particular fixative is better in preserving the specific marker in all populations expressing it. d) 7AAD MFI values (Y-axis) plotted against
different time points (Day 0, 4, 7, 10) (X-axis) for all treatments. Standard error was plotted with the mean value.
 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89
the following populations based on their respective set of
markers: CD4 T cells, BDCA1 mDC, BDCA3 mDC, pDC, CD56
NK and granulocytes, whereas Cyto-Chex® was better at
preserving these populations: BDCA1 DC, BDCA3 DC, pDC and
CD56 NK cells. None of the PFA preparations was better in
preserving the overall MFI of any specific population.
Fig. 3c shows the ability to detect a given marker in all of
the populations which expresses it. When we compared all
fixatives to unfixed samples as described in materials and
methods, clear performance patterns emerged. Overall, Trans-
fix® was the best fixative among all fixatives in terms of MFI
preservation. This was reflected particularly in BDCA3, CD11b,
CD123, CD19, CD4, CD45, CD45RA, CD56, CD66, CD80 and CD86
staining. Cyto-Chex® performance in marker expressions was
relatively robust, with preservation of BDCA2, BDCA3, CD123,
CD127, CD25, CD3, CD45, CD45RA, CD56 and CD80. However,
although Transfix® was the better fixative for preserving
marker expression, it displayed detrimental effects on cell
membrane integrity. Fig. 3d illustrates that Transfix® treated
samples displayed elevated 7AAD levels in all cell populations,
which is a marker of increased cell membrane permeabilization.
Among all the different fixatives, PFA demonstrated the worst
performance in preserving marker expression. 1% PFA was
favorable in preserving CD123, CD80 and FOXP3 while 4% PFA
was better in preserving BDCA3, CD127 and CD80. In the whole
list of markers that were studied, CD8 was the most sensitive
towards any fixation (Supplementary Fig. 3). Transfix® was the
most detrimental to this marker compared to all fixation
procedures employed. This highlights the fact that optimal
performance is dependent on the markers of interest although
overall Transfix was the best in MFI preservation.
3.5. Impact of fixation on cell count in shipped samples
We observed that certain fixatives were superior at preserv-
ing cell count while others were better at marker preservation.
However, all these procedures were carried out in a laboratory
setting. To analyze samples from field studies, the preparation
procedure must minimize any effects of shipment. In addition, in
many large scale international studies, a reliable cold chain is
difficult to establish, so the procedure must also preserve
samples in the absence of refrigeration. Thus, we studied the
impact of ambient temperature shipment on the two best
performing fixatives, Cyto-Chex® and Transfix®. We added a
condition where unfixed sample was shipped at room temper-
ature to examine if room temperature storage without fixative is
detrimental. All samples were shipped under field condition at
ambient temperature (~31 °C) on Day 0 to a destination within
Singapore, shipped back to the laboratory at Day 3 (~23 °C) and
stored at room temperature (~23 °C) for the remaining study. It
can be seen from Fig. 4a that shipment did not have a major
impact on cell count with either fixation procedures. Both
fixation procedures out performed shipped unfixed samples in
cell count preservation.
3.6. Impact of fixation on marker preservation in shipped samples
Since shipment did not have a significant impact on cell
count, we went on to compare the impact of shipment on cell
population identity to non-shipped samples. The percentage
of time where the specific treatment was better than unfixed
85
in each cell population is depicted in Fig. 4b. Samples which
were fixed with Transfix® and then shipped reduced the
ability to identify CD4 T cells and CD14 monocytes, with the
effect on the latter more pronounced. Cyto-Chex® treated
samples impaired our ability to identify BDCA3 DC, pDC and
granulocytes. The effect on granulocytes was more pro-
nounced as compared to the other populations where
changes were less than 15%. Shipment of samples without
prior fixation was detrimental to the ability to detect specific
cellular populations.
Fig. 4c depicts the effects on individual markers in all
populations expressing it. When compared to non-shipped
samples, shipping after Cyto-Chex® fixation had an effect on
BDCA2 and BDCA3 markers, and shipping after Transfix®
fixation affected CD4. Shipment of unfixed samples had an
adverse effect on most markers, though there were some that
were not affected (BDCA1, BDCA3, CD8 and CD80). In summary,
shipment did not have a major impact on marker expression in
most cell populations for Transfix® and Cyto-Chex® preserved
samples as compared to similarly preserved non-shipped
samples.
As a general conclusion, we have shown that Cyto-Chex® is
better at preserving cell count and Transfix® is better at
preserving marker MFI (Fig. 5a) among all fixatives. However,
marker expression is best preserved in the absence of both
shipment and fixation (Fig. 5b). As the selection of the optimal
fixation method is dependent on the condition of the study, the
time point and markers of interest, we created an online tool
for users to best interrogate the dataset according to their
particular study design (link: http://public.tableausoftware.
com/views/fixation_treatment/MainPage?:embed=y).
4. Discussions
Flow cytometry has long been the tool of choice for the
analysis of cells in the immune system. The diversity of reagents,
applications and the ability to dissect cellular phenotype and
function in ways that is impossible with other assays made it
arguably the most powerful technology available for probing
immune phenotype.
It is important to limit those study parameters which
introduce data variability and error due to compromised sample
integrity. A study done by Bergeron et al. (2002) demonstrated
that storing whole blood at room temperature for 2 days has an
impact on the light scatter properties and an alternative gating
strategy that based exclusively on cell-lineage specific marker
was required. Another study (Schumacher and Burkhead, 2000)
suggested that peripheral blood and umbilical cord blood held
up to 2 days displayed decreased B cell numbers and a reduction
in marker intensity. Given that not all laboratories have the
capacity to receive and analyzed blood samples within a
day, the problem of sample degradation raises the need
for a fixative that preserves sample integrity, even when
shipped under suboptimal conditions.
There had been several studies investigating the effects of
fixatives on cellular analysis. A study conducted by Canonico
and co workers had shown that Transfix® was supreme in
preserving scatter characteristics, immunophenotyping and
absolute cell count in the lymphocyte population. Other studies
(Warrino et al., 2005; Davis et al., 2011) demonstrated that
Cyto-Chex® was ideal in maintaining the immunological
86 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89

Fig. 4. Effects of shipment on cell count, identity of population and individual marker MFI of all populations. Cyto-Chex-shipped refers to shipped samples fixed
with Cyto-Chex. Transfix-shipped refers to shipped samples fixed with Transfix. Unfix-shipped refers to shipped unfixed samples. a) To determine if the variation
of dataset was bigger in fixed than unfixed samples, the comparison between fixatives and unfixed were made based on the summation of Euclidean distance
between Days 4 and 10. The same scoring method was used as Fig. 3a. The score is denoted as 1 (indicated as green) for treatments which performed better than
unfixed. Otherwise, it is given 0 (indicated as red). b) The same scoring method employed in Fig. 4b was used. The sum of counts was computed and normalized
by the number of markers defining a given population. This would indicate the percentage of time a particular fixative is better in preserving the identity of
specific cell population. c) In order to study the effects of fixation on individual marker expression, the same scoring method employed in Fig. 4c was used. The
sum of counts was computed and normalized by the number of populations expressing the marker. This would indicate the percentage of time a particular
fixative is better in preserving specific marker in all populations expressing it.
 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89 87

Fig. 5. Summary on the impact of fixatives on cell count and shipment. Cyto-Chex‐shipped refers to shipped samples fixed with Cyto-Chex. Transfix-shipped
refers to shipped samples fixed with Transfix. Unfixed-shipped refers to shipped unfixed samples. a) Based on all markers or all cell counts in all populations, the
sum of counts was computed from all conditions and normalized by the number of comparisons made. This would indicate the percentage of time a particular
fixative is better in preservation compared to unfixed condition. b) The sum of counts for unfixed was computed from marker MFI in all populations and
normalized by the number of comparisons made. Unfixed treatment was compared to the respective fixatives indicated on the X-axis and the percentage of time
unfixed outperform in preservation compared to respective fixatives was indicated.

markers for the white blood cells in whole blood sample. These
studies were in contrast to work done by Plate et al. (2009),
which illustrated that neither Transfix® nor Cyto-Chex® BCT
was useful in preserving CD4+ quantification. In all of these
studies, less than 5-color fluorophore staining was employed.
Although useful, such a panel cannot be said to leverage the
analytical capability of modern flow cytometers which utilize
FRET paired and Qdot nanocrystals reagents. Furthermore, given
that cellular populations are defined by coordinated expression
of several markers and the effect on any one marker could mean
a loss of the population in the analysis. Therefore, we set out to
analyze fixation procedures which utilize whole blood with high
dimensional flow in an environment that mimics field studies.
We examined the effects of commercially available fixatives
such as Transfix® (Cytomark) and Cyto-Chex® (Streck) on cell
count and MFI. Sample processing is simple as these reagents
come in either vacutainer which allows direct draw or liquid
form which is added directly into the whole blood. In addition,
these fixatives permit storage at room temperature according
to the manufacturer's protocol. From the data we concluded
that Cyto-Chex® was generally the best in preserving cell
numbers. This result was supported by study from Warrino et
al. which showed that Cyto-Chex® displayed good correlation
between 6 h and 7 days on absolute cell count of some
leukocytes in normal and HIV infected patients. However, due
to the constraints of the 4-color staining panel, the number of
88 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89
cellular subsets identified was restricted. Transfix®, displayed
better cell count preservation in specific populations (BDCA1+,
BDCA3+, CD8 T cells, pDC) and this result corresponded to the
study conducted by Canonico et al. which showed that absolute
cell count of lymphocytes was better preserved compared to
monocytes and granulocytes. However, the gating strategy of
this group was solely based on side scatter and CD45 marker
which do not allow the dissection of cellular subsets. Next, we
studied the impact of these fixatives on marker MFI and our data
shows that Transfix® was the better preservative compared to
Cyto-Chex® for all extracellular and intracellular markers
studied. However, Transfix® caused increased membrane
permeabilization as reflected by increased 7AAD staining. This
result was also shown in the study done by Canonico et al.
where they illustrated the effects through propidium iodide
staining. Surprisingly, unfixed samples stored at 4 °C was the
best in retaining marker expression hence indicating that any
form of fixation could potentially have an impact on conforma-
tional shape of the epitopes resulting in decreased antibody
binding.
Besides commercial fixatives, the commonly used fixative
(PFA) was employed in this study. In this study, we performed
red blood cell lysis prior to PFA fixation and excess amount of
FBS was added to neutralize the cross linking effects of PFA
after incubation. PFA treatments consistently yielded poor
results in terms of cell count and marker MFI, thus this
preservation method was not optimal. PFA fixation before
staining was chosen due to the ease of transporting items as
stained samples might degrade faster if the delay between
sample staining and acquisition is too long. In addition, PFA
was not directly added into the whole blood as it resulted in
fixation of red blood cells which was not possible to lyze at later
time points (data not shown). Although these factors may have
contributed to the poor performance of PFA in this study,
multicolor staining or red blood cells lysis at clinical sites prior
to fixation and shipment is in most cases impractical.
The numbers of studies analyzing the impact of shipment
on fluctuating temperatures on flow cytometry analysis is
limited and were mainly done on controlled temperature to
mimic shipment conditions (Antonenas et al., 2006; Ekong et
al., 1993). Studies conducted by Ahmann et al. (2008) showed
that tissue sample shipment on dry ice within USA did not have
a drastic impact on plasma cell isolation, viability and RNA
integrity. Olson et al. (2011) had investigated the effects of
overnight ambient temperature shipment at different seasons
and samples subjected to 40 °C for more than 8 h showed
reduced viability. In Olson et al. study, PBMCs were isolated for
analysis and 4 color flow staining panel was employed.
Furthermore, the duration of shipment was overnight with
sample analysis was done the following day. Over the last
5 years, multi-plex (>12 color) flow cytometry has become
the mainstay, with international shipping becoming essential
in multicentre studies. In this situation, samples will require
3 days for shipment excluding custom clearance delays. Thus,
we went on to explore the effects of shipment in our study to
assess if shipment could have an impact on cell count and the
marker MFI; this shipment effect would mimic field sites
located within the tropical region. The results demonstrated
that both Transfix® and Cyto-Chex® fixatives were stable in
the presence of shipment and displayed the same trend as the
non-shipped samples across a period of 10 days.
Since populations of interest depend on the study design
and the scientific question asked we have developed an
online interactive tool to allow scientists to explore and
identify the preservative of choice that is most applicable to
their study.
5. Conclusions
To our knowledge, this is the first study of its kind analyzing
whole blood fixation procedures for high dimensional flow
cytometry under condition amenable to multicentre trials in
the absence of a sustained cold chain. Our data demonstrated
that the appropriate choice of fixative depends on the markers
of interest. However, the study reveals general conclusions on
multi-dimensional flow analysis. We have determined that if
an investigator intends to immunophenotype prominent
populations based on lineage and activation markers
where cell numbers are not a critical issue, cold storage
in the absence of fixative is recommended. In the absence
of available refrigeration, Transfix® demonstrates supe-
rior performance for the analysis of surface markers
expression but not intracellular markers. Overall, based
on the studies presented here, we would recommend
Cyto-Chex®, as the preservative proved effective at retaining
cell numbers, extracellular and intracellular surface markers
following ambient temperature shipment.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jim.2012.08.010.
Acknowledgments
The study was supported by BMRC/EDB grant (IAF 311006,
Clinical Immunomonitoring; JC) and A*STAR core funds. The
authors would like to thank Dr. Anna-Marie Fairhurst for her
guidance; Nivashini Kaliaperumal, Wu Xue Lin, Lin Yufang and
Dr. Richard Hopkins for their valuable contributions on the
manuscript and their support rendered in the experimental
work.
References
Ahmann, G.J., Chng, W.J., Henderson, K.J., Price-Troska, T.L., DeGoey, R.W.,
Timm, M.M., Dispenzieri, A., Greipp, P.R., Sable-Hunt, A., Bergsagel, L.,
Fonseca, R., 2008. Effect of tissue shipping on plasma cell isolation,
viability, and RNA integrity in the context of a centralized good
laboratory practice-certified tissue banking facility. Cancer Epidemiol.
Biomarkers Prev. 17 (3), 666.
Antonenas, V., Garvin, F., Webb, M., Sartor, M., Bradstock, K.F., Gottlieb, D.,
2006. Fresh PBSC harvests, but not BM, show temperature-related loss of
CD34 viability during storage and transport. Cytotherapy 8 (2), 158.
Barnett, D., Granger, V.P., Storie, I., Wilson, G.A., Reilly, J.T., 1996. Evaluation
of a novel stable whole blood quality control material for lymphocyte
subset analysis: results from the UK NEQAS immune monitoring
scheme. Cytometry Commun. Clin. Cytom. 26, 216.
Baumgarth, N., Roederer, M., 2000. A practical approach to multicolor flow
cytometry for immunophenotyping. J. Immunol. Methods 243 (1–2), 77.
Bergeron, M., Nicholson, J.K.A., Phaneuf, S., Ding, T., Soucy, N., Badley, A.D.,
Hawley Foss, N.C., Mandy, F., 2002. Selection of lymphocyte gating
protocol has an impact on the level of reliability of T cell subsets in aging
specimens. Cytometry Clin. Cytom. 50, 53.
Canonico, B., Zamai, L., Burattini, S., Grangerd, V., Mannelloc, F., Gobbi, P.,
Felici, C., Falcieri, E., Reilly, J.T., Barnett, D., Papa, S., 2004. Evaluation of
leukocyte stabilisation in Transfix®-treated blood samples by flow
cytometry and transmission electron microscopy. J. Immunol. Methods
295, 67.
 A.A.P. Ng et al. / Journal of Immunological Methods 385 (2012) 79–89
Crellin, N.k, Garcia, R.V., Levings, M.K., 2007. Flow cytometry-based methods
for studying signaling in human CD4+CD25+FOXP3 + T regulatory
cells. J. Immunol. Methods 324 (1–2), 92.
Davis, C., Wu, X., Li, W., Fan, H., Reddy, M., 2011. Stability of immunophenotypic
markers in fixed peripheral blood for extended analysis using flow
cytometry. J. Immunol. Methods 363, 158.
De Paoli, P., Reitano, M., Battistin, S., Castiglia, C., Santini, G., 1984. Enumeration of
human lymphocyte subsets by monoclonal antibodies and flow cytometry: a
comparative study using whole blood or mononuclear cells separated by
density gradient centrifugation. J. Immunol. Methods 72, 349.
Ekong, T., Kupek, E., Hill, A., Clark, C., Davies, A., Pinching, A., 1993. Technical
influences on immunophenotyping by flow cytometry. The effect of time
and temperature of storage on the viability of lymphocyte subsets.
J. Immunol. Methods 164 (2), 263 (15).
Feuerecker, M., Kaufmann, I., Salam, A.P., Choukèr, 2012. Effects of cryopreser-
vation with polyethylene glycol on the expression of CD11B and CD62L on
the surface of polymorphonuclear leukocytes. CryoLetters 33 (2), 150 (10).
Foster, B., Prussin, C., Liu, F., Whitmire, J.K., Whitton, J.L., 2007. Detection of
intracellular cytokines by flow cytometry. Curr. Protoc. Immunol. (Chapter
6: Unit 6.24).
Fuss, I.J., Kanof, M.E., Smith, P.D., Zola, H., 2009. Isolation of whole
mononuclear cells from peripheral blood and cord blood. Curr. Protoc.
Immunol. (Chapter 7: Unit7.1).
Galligan, C.L., Siebert, J.C., Siminovitch, K.A., Keystone, E.C., Bykerk, V., Perez,
O.D., Fish, E.N., 2009. Multiparameter phospho-flow analysis of lympho-
cytes in early rheumatoid arthritis: implications for diagnosis and
monitoring drug therapy. PLoS One 4 (8), e6703.
Lanier, L.L., Warner, N.L., 1981. Paraformaldehyde fixation of hematopoietic
cells for quantitative flow cytometry (FACS) analysis. J. Immunol. Methods
47, 25.
Maecker, H.T., McCoy, J.P., Nussenblatt, R., 2012. Standardizing immuno-
phenotyping for the Human Immunology Project. Nat. Rev. Immunol. 12
(3), 191.
Nieto, J.C., Canto, E., Zamora, C., Ortiz, M.A., Juarez, C., Vidal, S., 2012.
Selective loss of chemokine receptor expression on leukocytes after cell
isolation. PLoS One 7 (3), e31297.
Olson, W.C., Smolkin, M.E., Farris, E.M., Fink, R.J., Czarkowski, A.R., Fink, J.H.,
Chianese-Bullock, K.A., Slingluff Jr., C.L., 2011. Shipping blood to a central
laboratory in multicenter clinical trials: effect of ambient temperature on
specimen temperature, and effects of temperature on mononuclear cell yield,
viability and immunologic function. J. Transl. Med. 9, 26 (2011 Mar 8).
89
Pinto, L.A., Trivett, M.T., Wallace, D., Higgins, J., Baseler, M., Terabe, M.,
Belvakov, I.M., Berzofsky, J.A., Hildesheim, A., 2005. Fixation and
cryopreservation of whole blood and isolated mononuclear cells:
influence of different procedures on lymphocyte subset analysis by
flow cytometry. Cytometry B Clin. Cytom. 63B, 47.
Plate, M.M., Louzao, R., Steele, P.M., Greengrass, V., Morris, L.M., Lewis, J.,
Barnett, D., Warrino, D., Hearps, A.C., Denny, T., Crowe, S.M., 2009.
Evaluation of the blood stabilizers TransFix and Cyto-Chex BCT for low-
cost CD4 T-cell methodologies. Viral Immunol. 22 (5), 329.
Puxeddu, I., Bongiorni, F., Chimenti, D., Bombardieri, S., Moretta, A., Bottino,
C., Migliorini, P., 2012. Cell surface expression of activating receptors and
co-receptors on peripheral blood NK cells in systemic autoimmune
diseases. Scand. J. Rheumatol. 41 (4), 298–304.
Renzi, P., Ginns, L.C., 1987. Analysis of T cell subsets in normal adults.
Comparison of whole blood lysis technique to Ficoll–Hypaque separa-
tion by flow cytometry. J. Immmunol. Methods 98 (1), 53.
Romeu, M.A., Master, M., Gonzalez, L., Valls, A., Verdaguer, J., Corominas, M.,
Bas, J., Massip, E., Buendia, E., 1992. Lymphocyte immunophenotyping
by flow cytometry in normal adults. Comparison of fresh whole blood
lysis technique, Ficoll-Paque separation and cryopreservation. J. Immunol.
Methods 154, 7.
Schumacher, M.J., Burkhead, T., 2000. Stability of fresh and preserved fetal
and adult lymphocyte cell surface markers. J. Clin. Lab. Anal. 14, 320.
Stetler-Stevenson, M., Tembhare, P.R., 2011. Diagnosis of hairy cell leukemia
by flow cytometry. Leuk. Lymphoma 52 (Suppl. 2), 11.
Tamul, K.R., Schmitz, J.L., Kane, K., Folds, J.D., 1995. Comparison of the effects
of Ficoll–Hypaque separation and whole blood lysis on results of immuno-
phenotypic analysis of blood and bone marrow samples from patients with
hematologic malignancies. Clin. Diagn. Lab. Immunol. 2 (3), 337.
Warrino, D.E., DeGennaro, L.J., Hanson, M., Swindells, S., Pirruccello, S.J.,
Ryan, W.L., 2005. Stabilization of white blood cells and immunologic
markers for extended analysis using flow cytometry. J. Immunol.
Methods 305, 107.
Wu, S., Jin, L., Vence, L., Radvanvi, L.G., 2010. Development and application of
‘phosphoflow’ as a tool for immunomonitoring. Expert Rev. Vaccines 9
(6), 631.
Zhou, L., Somasundaram, R., Nederhof, R.F., Dijkstra, G., Faber, K.N.,
Peppelenbosch, M.P., Fuhler, G.M., 2012. Human granulocyte and
monocyte isolation procedures: impact on functional studies. Clin.
Vaccine Immunol. (Epub ahead of print).