Nihms 390146

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Nat Rev Immunol. Author manuscript; available in PMC 2012 August 01.
Published in final edited form as:
NIH-PA Author Manuscript
Nat Rev Immunol. ; 12(3): 191–200. doi:10.1038/nri3158.
Standardizing immunophenotyping for the Human Immunology

Project
Holden T. Maecker1, J. Philip McCoy2,3, and Robert Nussenblatt3,4
1Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine,
Stanford, California 94305, USA.

2National
Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
20892, USA.
3Centerfor Human Immunology, Autoimmunity and Inflammation, National Institutes of Health,
Bethesda, Maryland 20892, USA.
4National Eye Institute, National Institutes of Health, Bethesda, Maryland 20893, USA.
Abstract
The heterogeneity in the healthy human immune system, and the immunological changes that
portend various diseases, have been only partially described. Their comprehensive elucidation has
been termed the ‘Human Immunology Project’. The accurate measurement of variations in the
human immune system requires precise and standardized assays to distinguish true biological
changes from technical artefacts. Thus, to be successful, the Human Immunology Project will
require standardized assays for immunophenotyping humans in health and disease. A major tool in
this effort is flow cytometry, which remains highly variable with regard to sample handling,
reagents, instrument setup and data analysis. In this Review, we outline the current state of
standardization of flow cytometry assays and summarize the steps that are required to enable the
Human Immunology Project.
Flow cytometry has increasingly become a tool of choice for the analysis of cellular
phenotype and function in the immune system (BOX 1). It is arguably the most powerful
technology available for probing human immune phenotypes, because it measures
multiple parameters on many individual cells. Flow cytometry thus allows for the
characterization of many subsets of cells, including rare subsets, in a complex mixture such
as blood. And because of the wide array of antibody reagents and protocols available, flow
cytometry can be used to assess not only the expression of cell-surface proteins, but also that
of intracellular phosphoproteins1 and cytokines2, as well as other functional readouts3–6.
© 2012 Macmillan Publishers Limited. All rights reserved

Correspondence to H.T.M. [email protected].
Competing interests statement
The authors declare competing financial interests: see web version for details.
FURTHER INFORMATION
Holden T. Maecker’s homepage: http://himc.stanford.edu
Clinical Trials in Organ Transplantation: http://www.ctotstudies.org
European Network for Translational Immunology Research and Education: http://entire-net.eu
FlowCAP: http://flowcap.flowsite.org
Spring 2011 FOCIS newsletter: http://www.focisnet.org/FOCIS/newsletter/201104.htm
ALL LINKS ARE ACTIVE IN THE ONLINE PDF
Maecker et al. Page 2
This diversity of reagents and applications has led to a large variety of ways in which flow
cytometry is being used to monitor the immune systems of humans and model organisms
(mostly mice). These uses include the identification of antigen-specific T cells using
tetramers7 or intracellular cytokine staining8,9; the measurement of T cell proliferation using

dyes such as 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)3; and the use of
immunophenotyping assays to identify lymphocyte, monocyte and/or granulocyte subsets.
The information to be gained from such assays is immense and varied. Here, we cite just a
few examples for human disease. First, a high proliferative capacity10 and the production of
multiple cytokines11 by HIV-specific CD8+ T cells have been linked to non-progression in
patients with HIV/AIDS, whereas declining CD4+ T cell counts12, inverted ratios of CD4+
to CD8+ T cells13 and increased numbers of CD38+CD8+ T cells14 have been shown to
signify disease progression. Second, the diagnosis and monitoring of leukaemia and
lymphoma have been aided by the immunophenotyping of blood and bone marrow15. Third,
ageing has been associated with an increase in the number of CD28− late-stage effector T
cells16 and an increase in the number of oligoclonal cytomegalovirus-specific T cells17.
Animal studies tend to be relatively small and self-contained, as the mice used are inbred
(genetically identical) and their environment and treatments are carefully controlled. Human
studies, by contrast, are often larger, to account for genetic and environmental variables.
They may require longitudinal assays over a considerable length of time, owing to the
difficulty of recruiting suitable subjects and/or the need to follow those subjects over time.
There may also be a need to aggregate data from multiple sites, or even across multiple
studies, to achieve sufficient sample sizes for statistical analysis or to compare different
treatments. In these situations, the standardization of reagents and protocols becomes
crucial.
The steps in a typical flow cytometry experiment are shown in FIG. 1, and they present
several variables that need to be controlled for effective standardization. These variables
involve the general areas of reagents, sample handling, instrument setup and data analysis
(TABLE 1). The effects of changes in these variables are largely known18–22. For example,
the stabilization and control of staining reagents through the use of preconfigured
lyophilized-reagent plates, and centralized data analysis, have both been shown to decrease
variability in a multi-site study20. However, the widespread adoption of standards for
controlling such variables has not taken place. This is in contrast to other technologies, such
as gene expression microarrays, which have achieved a reasonable degree of standardization
in recent years23–29. Led by consortia such as the Microarray Quality Control (MAQC)
consortium28,29, the External RNA Controls Consortium (ERCC)24 and the EMERALD
project23, this standardization was facilitated by the move to a few standardized vendor
platforms for microarrays. Of course, microarray data are less complex than flow cytometry
data, which are based on many hierarchical gates. Still, a reasonable degree of
standardization of flow cytometry assays should be possible to achieve.
Beyond facilitating large, longitudinal and multi-site studies, the global standardization of
flow cytometry-based immunophenotyping would enable an even larger goal: the Human
Immunology Project30. By analogy to the Human Genome Project, the Human
Immunology Project would attempt to characterize the detailed immune phenotypes of
healthy individuals, for comparison with various disease states or to elucidate the changes
that occur in response to defined perturbations, such as vaccination. In this context, an
‘immune phenotype’ would encompass not only the proportions of major immune cell
subsets, but also, for example, their activation state, responsiveness to stimuli, and T cell
receptor or B cell receptor repertoires, among other parameters. The Human Immunology
Project would create a repository of immunological data that could be mined for biomarkers
of immunological ageing, of disease diagnosis and prognosis, and of responses to therapy.
But such a comprehensive enterprise can only be successful with informative and well-
standardized assays. Whereas the information content of immunophenotyping assays has
grown exponentially in recent years, their level of standardization has not. Therefore, we
outline here the steps that we feel need to be taken for assay standardization to enable a
successful Human Immunology Project, using flow cytometry of peripheral blood
mononuclear cells (PBMCs) as the prime example from which to start the difficult process
of standardization. Similar principles might then be applied to cells from target organs and/
or lymphoid tissue and to more complex functional assays and rare cell analyses (BOX 2).
Current state of flow cytometry standardization

The definition of particular subsets of immune cells using cell-surface markers continues to
evolve, particularly for cell types that are the subject of intense current research. These
include regulatory T (TReg) cells, interleukin-17 (IL-17)-secreting T helper (TH17) cells,
dendritic cells (DCs) and natural killer (NK) cells. However, even well-characterized
subsets, such as naive and memory T cells, are defined differently in various studies. For
example, the classical T cell subsets of naive, central memory, effector memory and effector
T cells were first defined on the basis of their expression of CC-chemokine receptor 7
(CCR7; also known as CD197) and CD45RA31 (where naive T cells are CCR7+CD45RA+,
central memory T cells are CCR7+CD45RA−, effector memory T cells are CCR7−CD45RA−
and effector T cells are CCR7−CD45RA+). Other investigators have since used different
markers, such as CD62L or CD27 in place of CCR7 (REFS 32,33), and CD45RO in place of
CD45RA. Although these different combinations of markers generally define similar cell
subsets, they introduce an unknown amount of heterogeneity that makes comparisons
between studies difficult.
It is reasonable to think that the discovery of new markers and new cell subsets will continue
for some time in the future. However, we propose that there is sufficient maturity in the field
of cellular immunology to reach consensus working definitions for most of the commonly
studied subsets of immune cells. At a high level, these definitions are unlikely to change
very much as new discoveries are made, although there are likely to be new subsets of these
cell types described over time. In other words, it should be possible to define a stable set of
markers that delineate the major classes of B, T and NK cells, monocytes and DCs. Towards
this end, we review here the literature that indicates what the key differentiation markers for
these cell types might be; these markers could then, in our opinion, form the basis of a
standard working definition. We further discuss how consensus is being reached in the
immunological community regarding these markers and definitions, leading to a
standardized immunophenotyping panel for human immune monitoring. Although there may
be disagreements about the choice and utility of certain markers, and the names of the
subsets so defined, we propose that the markers and subsets shown in FIG. 2, and discussed
below, can form a working definition of cell types that will help to drive standardization in
the future.
T cells
The major subsets of T cells can be defined by the expression of CD4 and CD8, together
with, for example, CCR7 and CD45RA31. CCR7 has been a difficult marker for use in flow
cytometry because of its low expression levels; however, a new CCR7-specific antibody
clone (150503; commercially available from multiple vendors) that provides brighter
staining has greatly improved this situation. Furthermore, other substitute markers for CCR7
are more problematic. For example, the expression level of CD62L is highly affected by
density gradient separation34 or cryopreservation35 of PBMCs. As PBMC
cryopreservation is integral to the workflow of many human studies, owing to the need to
batch samples and store cells for future assays, this limits the broad use of CD62L for
immunophenotyping. Another possible CCR7 replacement, CD27, is expressed not only by

naive and CCR7+ central memory T cells, but also by a population of effector memory T
cells that lack expression of CCR7 (REF. 36), meaning that CD27 is not a full substitute for
CCR7. So, a panel containing CD3 (to define T cells), CD4, CD8, CD45RA and CCR7
seems most applicable for distinguishing naive, central memory, effector memory and
effector CD4+ and CD8+ T cells. With the addition of activation markers, such as CD38 and
HLA-DR, it is possible to define activated subsets of each of these cell types as well (FIG. 2;
TABLE 2).
B cells
Tonsillar B cells were first classified into five groups, termed Bm1–Bm5, according to their
expression of IgD and CD38 together with that of CD23 and CD77 (REF. 37). Some
versions of all of these cell types can also be found in peripheral blood38. Bm1 and Bm2
cells are CD38−IgD+, and include mostly resting (CD23−) naive B cells and some activated
(CD23+) cells. Bm3 and Bm4 cells are CD38+IgD− germinal centre (or germinal centre
precursor) cells, which can be further subdivided by their expression of CD77, a marker of
germinal centre location (Bm3 cells are CD77−; Bm4 cells are CD77+). Bm5 cells are
CD38−IgD− memory B cells. CD27 can also be used to define naive (CD27−) and memory
(CD27+) B cells39. CD24 has been shown to be useful in defining immature ‘transitional’ B
cells (which are CD24hiCD38hi)40. Recently, circulating plasmablasts were also identified in
peripheral blood, using either CD38 and CD138 (REF. 41) or CD38 and CD27 (REF. 42).
Notably, these plasmablasts are low or negative for CD20 expression and, because of the use
of CD20-specific antibody therapy in various diseases, it is prudent to also include CD19 as
a marker to define the parental B cell population. Although it is not possible to include all of
the potentially relevant B cell markers in a single eight-colour antibody cocktail, a highly
useful panel can be constructed using antibodies specific for CD19 and CD20 (to define B
cells), CD38 (to identify plasmablasts as well as transitional B cells), CD24 (for transitional
B cells), and IgD and CD27 (for naive and memory B cell populations) (FIG. 2).
NK cells
NK cells have been shown in recent years to be much more heterogeneous than previously
thought, with a large variety of activating and inhibitory receptors expressed by overlapping
subsets of cells that vary widely between individuals43. However, at a more basic level, NK
cells can be subdivided into two major categories based only on the markers CD16 and
CD56. The vast majority of peripheral blood NK cells are CD16+CD56low, whereas a
smaller subset is CD16+CD56hi. These populations differ in terms of function and tissue
localization, and the latter subset has been shown to be an intermediate phenotype that can
give rise to the former subset44.
Dendritic cells
Circulating blood DCs have long been identified as HLA-DRhi cells that are negative for
various lineage-specific markers, including CD3 (which defines T cells), CD14 (which is
monocyte specific), and CD19 and CD20 (which define B cells)45. DCs can be divided into
cells of the myeloid lineage (CD11c+ cells) and the plasmacytoid lineage (CD123+ cells).
Myeloid DCs can be further subdivided by their expression of CD16, CD1c and CD141
(REFS 46,47). Although these myeloid DC subsets may have functional significance, their
enumeration in cryopreserved PBMC populations is too problematic to warrant the inclusion
of these markers in a general phenotyping panel. Thus, one can construct a basic DC panel
based on a lineage cocktail, and antibodies specific for HLA-DR, CD11c and CD123.
Monocytes
With further study, monocytes might also reveal substantial heterogeneity, but currently two
major categories are widely recognized: classical monocytes (which are CD14hiCD16−) and
non-classical monocytes (which are CD14lowCD16hi)48. Because monocyte, NK cell and

DC phenotyping can all include CD16, it is possible to imagine a phenotyping cocktail for
all three cell types that includes a lineage cocktail, together with antibodies specific for
HLA-DR, CD11c, CD14, CD16, CD56 and CD123.
Activation markers
Activation markers for these cell types are of course of equal interest to the differentiation
markers discussed above, as the activated subsets can provide clues to an individual’s
response to infection, vaccination, cancer or autoimmune disease. For this purpose, one can
assess intracellular markers of recent cell division (such as Ki67) or surface markers of
activation that have varying kinetics of expression (for example, CD69 is a transient marker,
whereas HLA-DR, CD38 and CD71 are expressed for longer periods). The choice of
marker(s) will certainly vary depending on the experimental question, and the expression of
these markers may not be perfectly correlated with proliferation in vivo49. However, the
combination of CD38 and HLA-DR has proven to be useful for identifying activated T cells
in various contexts, including HIV infection14. It can be hypothesized that the frequencies of
activated T cells of various phenotypes could become biomarkers of several conditions or
outcomes, including: vaccine-induced protection (at defined time point(s) post-vaccination);

disease activity (for T cell-mediated autoimmune or infectious diseases); and chronic
inflammation (as has been shown in HIV infection). The similar use of HLA-DR to track
activated NK cells is a possibility that has been less well explored, but that might be equally
fruitful. Plasmablasts — the activated (CD38+) subset of antibody-secreting B cells — have
been shown to increase in number post-vaccination50 and might also be used as biomarkers
of disease status and/or vaccine efficacy.
From the above discussion, it should be clear that it is possible, using current reagents and
instrumentation, to carry out immunophenotyping in a far greater level of detail than is
currently achieved clinically. Furthermore, there are some clear choices that we think could
form the basis of standard working definitions for different immune cell subsets using eight
colours of fluorescence or less (FIG. 2; TABLE 2). However, no current clinical test
includes all, or even most, of the subsets and cell types mentioned above. As a result,
clinical immunophenotyping is carried out only at the level of lymphocytes, monocytes and
granulocytes, or using limited four- to six-colour staining for major cell types in diseases
such as HIV/AIDS and leukaemia or lymphoma. All other immunophenotyping is carried
out in the research laboratory, with essentially no standardization. A clinician who wishes to
know the status of a patient’s immune system cannot yet order a ‘complete
immunophenotype’ in the way that we currently order a ‘complete blood count’.
How can this situation be improved?

Recognizing the need for the standardization of immunophenotyping, a consortium of
laboratories was formed in 2009, led by the National Institutes of Health (NIH) in the United
States and the Federation of Clinical Immunology Societies (FOCIS). In 2010, the
consortium convened an international group of laboratories, representing FOCIS Centers of
Excellence (FCEs), recipients of NIH grants in the Human Immunophenotyping Consortium
(HIPC), and key academic and industrial leaders in flow cytometry. About 50 participants
from these groups met at a workshop in January 2011 called the Flow Immunophenotyping
Technical Meeting at NIH (FITMaN; see the spring 2011 FOCIS newsletter). The main
outcome of the meeting was the proposal of a set of desired standard markers for the
phenotyping of B, T and NK cells, monocytes and DCs. These evolved into a set of defined
eight-colour antibody cocktails, which could be expanded by the addition of more marker-
specific antibodies but which, by themselves, could enumerate the basic subsets of the above
cell types and could determine their activation status (TABLE 2).
To see where this effort may lead, and to define the remaining obstacles to the
standardization of flow immunophenotyping, let us examine in more detail the major areas
of flow cytometry variability and how these can be addressed through this and similar
community efforts.
Reagents
How important is it to define the actual antibody cocktails used for immunophenotyping? As
described above, the choice of markers is clearly important, but different antibody clones
and fluorochromes can also greatly influence results. As shown in FIG. 3, two clones of
CD38-specific antibody give widely disparate staining results for PBMCs from the same
healthy donor. Moreover, the fluorescence separation of positive and negative populations
for a particular marker can vary by about 20-fold between the brightest commercially
available fluorochromes (such as phycoerythrin and allophycocyanin) and the dimmest ones
(such as AmCyan and Pacific Blue)51. This difference affects whether a population that is
positive for a particular marker can be reliably separated from the negative population and
may even influence the apparent percentage of positive cells (as dim positive cells become
mixed with negative cells when the separation is poor). Needless to say, the antibody titre
also has an important role in this variability.
The use of preformatted lyophilized-reagent plates (Lyoplates, BD Biosciences) can help, by

‘locking in’ the above reagent variables, once optimal antibody clones, fluorochromes and
titres have been established. The use of such lyophilized-reagent plates has been shown to
decrease staining variability compared with using individual liquid reagents in multiple
studies20,52. The decrease in variability can be attributed to the stabilization of reagents
against degradation, as well as to the elimination of pipetting variation and the avoidance of
errors in reagent addition. Lyophilized-reagent plates have also been used as a means of
standardizing stimulation reagents for functional assays53,54.
Of course, standardization does not end with defining the reagents (that is, the antibody
specificities, clones and titres, and the fluorochromes) in a staining cocktail. Sample
handling, instrument setup and data analysis are crucial variables as well.
Sample handling
The presumed ideal sample-handling protocol (staining fresh whole blood or PBMCs
immediately after blood draw) is not always practical in large clinical trials, and it may not
even be desirable with regard to comparing samples longitudinally. The cryopreservation of
PBMCs is an attractive alternative for the purpose of batching samples over time, and for
banking samples for future use. But it has the disadvantage of depleting certain interesting
cell types (such as plasmablasts and DCs), and generally decreasing cell yield and function.
Cryopreservation is also technically demanding, such that not all clinical trial sites are
equipped and competent to carry it out. This leads to the compromise of shipping whole
blood or PBMCs to a central laboratory for cryopreservation, which can further decrease cell
viability and function and can introduce additional variability owing to shipping time and
conditions.
A case has been made for empowering individual laboratories to carry out their own
processing, including cryopreservation and possibly even flow cytometry, in large
multicentre studies18. Done correctly, this could significantly decrease the compromises
associated with shipping and/or cryopreservation of samples. However, it requires an

infrastructure commitment in terms of equipment, training and the maintenance of
standardization between a set of disparate laboratories to minimize variability. In many
cases though, such empowerment is becoming a requirement for collaboration with

scientists in countries such as India and Japan, as the primary samples are not usually
allowed to leave these countries.
Another solution is to use some type of automation to carry out either the staining or, for
functional assays, the activation of whole blood, followed by fixation and freezing, at
individual laboratories where samples are drawn. The fixed and frozen samples can then be
batch-shipped to a central laboratory for further processing and analysis, or this could be
carried out at the local site in a batch manner. A commercial instrument to automate the
activation and fixation of whole blood is available (from Smart Tube, Palo Alto, California),
as are instruments to stain and fix whole blood samples (such as Sample Prep Assistant, BD
Biosciences; and Biomek, Beckman Coulter).
Instrument setup
In addition to optimizing certain settings, such as laser time delays, instrument setup is
mostly concerned with the setting of the voltage gains applied to each fluorescence detector,
which influences the sensitivity of the detector to dim versus bright signals. This used to be
entirely a matter of user preference, but some guiding principles have now emerged55 based
on measuring the variance of a population of dim particles over a series of voltage gains.
Equally important, software to automatically achieve a predetermined setup has become
available (such as BD Cytometer Setup and Tracking, BD Biosciences; and Beckman
Coulter’s MXP software). There are still, however, marked differences in performance
associated with instruments of different configurations and from different manufacturers. In
fact, a high proportion of multicolour flow cytometers are customized by the manufacturer
and/or by individual laboratories with a variety of laser and filter options. This makes
standardization between sites (or even between two different instruments at the same site)
difficult. The only method currently available to overcome these variations is to establish
target values across the cytometers using standardized control particles. By setting voltage
gains to place the control particles at a particular target channel, a relatively similar
instrument setup is achieved. The best particles in this regard are ones that contain the actual
fluorochromes to be used experimentally, such as stained antibody capture beads56,
so that the performance using actual samples is most closely mimicked.
Data analysis
Data analysis is one of the largest variables in flow cytometry, as shown by studies
analysing data at multiple sites versus centrally20. It is also one of the easiest variables to
address, as re-analysis of existing data is always possible, whereas choices made about
sample handling and instrument setup are irrevocable. Central analysis is simple in concept,
but it can be overwhelming in terms of the demands placed on one or a few coordinated
personnel who must review all of the data to achieve consistent gating. Fortunately,
automated gating algorithms have proliferated and improved, such that they now compete
favourably with expert manual gating57. A group of interested experts (FlowCAP), with
support from the NIH, has completed two workshops aimed at comparing and improving the
use of automated gating algorithms for flow cytometry analyses. Their continued work in
this area should lead to standard software packages that can carry out easy and unbiased
analyses of flow cytometry files.
From the above, it is clear that individual solutions are available in each of the areas that
lead to flow cytometry variability. What is missing is: guidance for those designing clinical
trials on the integrated use of these solutions; resources to enable the implementation of all
appropriate solutions; and some standardized approaches to the most common flow
cytometry immunophenotyping challenges. With regard to such standardized approaches,
the preformatted antibody cocktails being produced by the HIPC might provide a good start.
By predetermining the reagent choices and simplifying sample handling, they should
alleviate much of the variability in these areas. Because they represent a single set of reagent
cocktails, they are also amenable to standardized instrument setup and data analysis
routines, which should further decrease variability. Their use by the HIPC and by other
interested laboratories should produce an example body of data that could demonstrate to the
field the benefits of a standardized approach to flow cytometry, and to immunological
assays in general.
What needs to happen next?

The efforts of the HIPC with regard to eight-colour immunophenotyping of the major
PBMC subsets are of course only a start to the process of standardization. For a glimpse at
where this process could lead, one needs only to consider another immunophenotyping assay
that has already been standardized — namely, CD4+ T cell counting for the staging of HIV/
AIDS. As HIV directly infects and depletes CD4+ T cells, the enumeration of these cells is
crucial for determining disease progression and for tracking the efficacy of therapeutic
intervention. Because this assay requires multiple markers for accurate quantitation
(generally CD3 and CD4 at a minimum), it is well-suited to a flow cytometry platform. And
because of the large global burden of HIV infection, manufacturers have responded with
multiple systems-level solutions. As a result, the counting of CD4+ T cells has been
standardized to achieve an inter-laboratory coefficient of variation of <10%58. It can be
carried out around the world on one of several standardized platforms, at least one of which
takes advantage of lyophilized reagents (BD FACSCount, BD Biosciences), and several of
which use automated analysis software (TetraCXP, Beckman Coulter; BD Multiset, BD
Biosciences; Guava EasyCD4, Millipore). Sample-handling integrity is ensured by simple
protocols and the mandatory proficiency testing that is prescribed by government agencies
overseeing clinical laboratories.
Why not apply these principles to a broader set of immunophenotyping assays (BOX 2)?
With the availability of standard antibody panels (such as those described above), specific
instrument-setup and staining protocols, and centralized and/or automated data analysis, the
potential exists to create highly comparable data. But will such systems be widely adopted?
And how will the participating laboratories be monitored? As long as the assays are strictly
research based, there is little or no enforcement power, other than that of the funding
agencies of grant recipients. Beyond that, we believe that it is only in the power of example
that the adoption of standardized panels and practices can be encouraged. Still, this can be a
powerful force in itself; after all, the microarray community quickly adopted standard
commercial platforms once they became available and their quality became apparent.
Eventually, when the clinical utility of immunophenotyping has been demonstrated for
specific disease indications, quality assurance can be achieved through the same regulatory
means as for other clinical assays.
Extending to other assays

There is no reason to believe that similar standardized panels cannot be developed for
functional assays, such as intracellular cytokine staining and phosphoepitope flow
cytometry. Efforts are already underway in Europe to standardize functional assays that
are used in clinical diagnostics, through the
European Network for Translational Immunology Research and Education (ENTIRE). And
an NIH-supported cooperative research programme, Clinical Trials in Organ Transplantation
(CTOT), is engaged in cross-validating cellular function assays as well as

immunophenotyping panels. Their findings should help to inform reagent choices for
standardized functional assay panels, including those used in non-flow cytometric assays
such as enzyme-linked immunosorbent spot (ELISPOT) assays and immunohistochemistry.
Adopting new technologies

To some degree, the need for standardization and the effort required to achieve it will be
reduced by new technologies. One area of technology growth is in systems that can both
process samples and analyse them by flow cytometry, all in a highly automated manner
(such as the Load & Go systems from Blue Ocean Biomedical). This type of system works
from a blood draw tube, processing and staining cells, acquiring data and carrying out
routine analysis, all in a pre-programmed manner. The proliferation and continued
development of such systems could eliminate, to a large degree, the need to ship blood to
central laboratories for flow cytometry assays. In the process, the quality and reproducibility
of data should also be improved.
Another new technology, mass cytometry (CyTOF, DVS Sciences), is poised to

revolutionize flow cytometry by allowing many more antibody probes to be combined in a
single assay, while simultaneously eliminating the issue of optical spillover59–61. If
reagents for this platform become standardized, the design of antibody panels would be
much easier, because the inclusion of certain reagents would not influence the performance
of other reagents (as it does for conventional flow cytometry). This would allow for
standardized immunophenotyping panels to be extended as desired for particular
investigations, without compromising the core reagents or the quality of data.
Data centralization and mining

To realize the full benefit of standardized assays, data need to be centrally collected and
mined. In this way, normal ranges can be determined across the variables of age, gender and
ethnicity, for cell subsets and functions about which little is currently known. Disease
cohorts can be compared with matched healthy controls. Patients treated in various ways can
be compared with each other and with controls. Data can be used and reused, beyond the
intention of the original research, if the same standardized assays have been applied, and if
the data are centrally aggregated in a way that allows this type of exploration.
To this end, we have recently collaborated with CytoAnalytics to develop an online system
for data integration and exploration. By modifying existing open-source tools in the business
intelligence field (such as online analytical processing (OLAP)), we were able to create a
data-mining environment in which laboratory results are integrated with clinical and
demographic data (‘metadata’), filtered using any desired variables, and displayed with
automated aggregate statistics (such as the mean, median and sample number). Similar
systems could be used for the aggregation of flow cytometry phenotyping (and functional)
data in general, and they should be made publicly available as quickly as possible to allow
other groups to integrate their own data sets.
All of the above elements should help to facilitate the pursuit of the Human Immunology
Project, the value of which is directly dependent on the ability to compare the results of
immunological assays across studies and across laboratories.
Conclusions
With the major exception of CD4+ T cell counting for immune monitoring in patients with
HIV and the immunophenotyping of leukaemias and lymphomas, flow cytometry is mostly
carried out in a research setting. Particular assays may be internally validated for a given
clinical study, but comprehensive standardization is rare to non-existent.
However, there is a clear and growing interest in the standardization of human

immunophenotyping. This is evidenced by standardization efforts, such as those of the
European ENTIRE and United States CTOC networks. The NIH have also invested in a set
of awards focused on human immunophenotyping, and both FOCIS and the NIH are
working to create standard immunophenotyping panels.
The application of attention and resources to this area should produce tangible results.
Previous studies have identified crucial variables in flow cytometry in the areas of sample
handling, reagents, instrument setup and data analysis. Control of these variables can
markedly decrease variability in clinical studies. What remains to be achieved is the
widespread adoption of standard reagents and protocols for the collection of human
immunophenotyping data. In addition, an infrastructure for aggregating and mining results
will be needed to achieve the vision of the Human Immunology Project. With such
standardization and accompanying infrastructure, it should be possible to rapidly mine the
data for metrics of immunological health and disease, much as we can currently mine
genetic data using common databases and tools. The result will be the more rapid discovery
of biomarkers that can aid in the diagnosis, prognosis and therapeutic monitoring of
immunologically related diseases.
Acknowledgments
The authors thank all the contributors to the HIPC/FITMaN immunophenotyping panel, in particular S. Heck, F.
Nestle, A. Biancotto, S. Gupta, M. Malipatlolla, L. Devine, R. Montgomery and D. Hafler. The mention of any
commercial products in this manuscript does not imply endorsement by the US Government or the US National
Institutes of Health.
Glossary
Immune phenotypes Measurable aspects of the immune system, such as the

proportions of various cell subsets or measures of cellular
immune function.
Gates Sequential filters that are applied to a set of flow cytometry data
to focus the analysis on particular cell subsets of interest.
Human Immunology The comprehensive mapping of immune phenotypes in healthy
Project and diseased human populations.
Density gradient The isolation of mononuclear cells from blood or other sources
separation by centrifugation over a density gradient, which usually consists
of a carbohydrate polymer solution (Ficoll).
Cryopreservation The processing and storage of cells at sub-zero temperatures
under conditions that preserve their viability for later assays.
Lineage cocktail A mixture of antibodies specific for various lineage-specific
markers, such as CD3 (for T cells), CD14 (for monocytes) and
CD19 and CD20 (for B cells).
Antibody capture Microparticles conjugated with immunoglobulin-specific
beads antibodies. These beads can be stained with fluorescent
antibodies to create single-colour controls for flow cytometry
instrument setup and compensation.
Phosphoepitope flow (Also known as ‘phospho-flow’). A technique that uses

cytometry antibodies specific for phosphorylated versions of proteins to
analyse cell signalling by flow cytometry.

Mass cytometry Flow cytometry using antibodies tagged with heavy metal ions,
which are detected by mass spectrometry (as opposed to
classical flow cytometry, which uses antibodies tagged with
fluorophores and optical detection).
Optical spillover The presence of signals from fluorescent antibody staining in
multiple detectors of a cytometer, resulting in a loss of
resolution sensitivity.
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Box 1 | Flow cytometry

The study of cells moving in suspension through an image plane — flow cytometry — is
a potent tool for immunology research and immune monitoring. Its main advantages are
that it makes multiparameter measurements and that it does so on a single-cell basis. The
result is that this technique can dissect the phenotypes and functions of cell subsets in
ways that are not possible using bulk assays, such as Western blots, microarrays or
enzyme-linked immunosorbent assays (ELISAs). Nowhere has this proven more useful
than in a mixed suspension of immune cells, such as the blood. Newer instrumentation
allows for the analysis of eight or more parameters at flow rates of thousands of cells per
second; the resulting rich data sets are unparalleled for the knowledge of immune
function that they have contributed. Flow cytometry will thus be used in this Review as
the prime example of an immune-monitoring technology that would benefit from
standardization.
Box 2 | Extending standardization to other assays

We recognize that eight-colour phenotyping of peripheral blood mononuclear cells
(PBMCs), although useful, is unlikely in the long term to be the most important assay in
our planned immunophenotyping armamentarium. Instead, it is a relatively simple place
to start, paving the way for the standardization of more complex assays, such as:
• Functional flow cytometry assays, including intracellular cytokine staining,
proliferation and phosphoepitope flow cytometry;
• Whole blood analysis of rare cells, such as circulating tumour cells, stem cells
or dendritic cell subsets;
• Analysis of other tissue types, such as tissue biopsies, or cerebrospinal or
synovial fluid.
These types of assay represent new frontiers for standardization that are more challenging
than the surface-marker phenotyping of PBMCs because they involve the enumeration of
rare cells, in vitro activation or tissue processing. However, the rewards for global
standardization of such assays are likely to be high, in terms of our understanding of
immune mechanisms and eventual clinical utility.
Figure 1. A typical flow cytometry experiment

Sample preparation from blood often involves Ficoll gradient separation of mononuclear
cells, and sometimes cryopreservation, before staining with fluorescent antibody conjugates.
Each of these steps can introduce variability in the assay results. Instrument setup involves
setting voltage gains for the photomultiplier tubes (PMTs) so as to achieve optimal
sensitivity. To the extent that this is not standardized, it becomes a source of variability as
well. Data acquisition involves passing the stained cells through a laser beam and recording
the fluorescence emission from all of the bound antibody conjugates. Here, the main
variable is the type of instrument, including the lasers and optical filters used. This is
followed by data analysis, in which cell populations of interest are defined and reported on,
which is another significant source of variation. Ref., reference; SD, standard deviation.
Figure 2. Identification of immune cell subsets by eight-colour antibody staining

The figure shows the cell populations that can be identified using the markers targeted by
each of the five antibody cocktails of the Human Immunophenotyping Consortium (HIPC)
phenotyping panel shown in TABLE 2. CCR, CC-chemokine receptor; CXCR3, CXC-
chemokine receptor 3; DC, dendritic cell; NK, natural killer; PBMC, peripheral blood
mononuclear cell; TH, T helper; TReg, regulatory T.
Figure 3. The importance of antibody choice

The staining patterns of two commercially available clones of human CD38-specific
antibody are very different, despite the fact that both antibodies were conjugated to
allophycocyanin (APC) by the same vendor, and were used to stain peripheral blood
mononuclear cells (PBMCs) from the same healthy subject under identical conditions.
V450, violet 450. Data courtesy of Angelique Biancotto, National Heart, Lung and Blood
Institute, USA.
Table 1
Technical variables in immunophenotyping and approaches to minimize them
Variable Approaches to minimize effects

Reagents • Definition of standard antibody panels for immunophenotyping
• Use of preconfigured lyophilized reagents
Sample handling • Point-of-collection automation of sample processing (as is being developed by Smart Tube and Blue Ocean
Biomedical, for example)
• Training of sites to cryopreserve PBMCs and/or to do staining on-site
Instrument setup • Use of software (such as BD Biosciences Cytometer Setup and Tracking or Beckman Coulter MXP) for
automated setting of voltage gains
• Setting the fluorescence of standard beads to defined target channels, for reproducible setup across instruments
Data analysis • Central analysis by one or a few coordinated experts

• Use of automated gating algorithms
PBMC, peripheral blood mononuclear cell.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Table 2
Eight-colour antibody panels proposed by the Human Immunophenotyping Consortium
Fluorochrome Marker
T cells TReg cells TH1, TH2 and B cells DCs, monocytes and
Maecker et al.
TH17 cells NK cells
FITC Live or dead Live or dead Live or dead Live or dead Live or dead
PE CCR7 CD25 CXCR3 CD24 CD56
PerCP-Cy5.5 CD4 CD4 CD4 CD19 CD123
PE-Cy7 CD45RA CCR4 CCR6 CD27 CD11c
APC CD38 CD127 CD38 CD38 CD16
APC-H7 CD8 CD45RO CD8 CD20 CD3, CD19 and CD20

V450 CD3 CD3 CD3 CD3 CD14
V500 HLA-DR HLA-DR HLA-DR IgD HLA-DR
APC, allophycocyanin; APC-H7, allophycocyanin–cyanine H7 tandem; CCR, CC-chemokine receptor; CXCR3, CXC-chemokine receptor 3; DC, dendritic cell; FITC, fluorescein isothiocyanate; NK,
natural killer; PE, phycoerythrin; PE-Cy7, phycoerythrin–cyanine 7 tandem; PerCP-Cy5.5, peridinin chlorophyll protein–cyanine 5.5 tandem; TH, T helper; TReg, regulatory T; V450, violet 450; V500,
violet 500.
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Nat Rev Immunol. ; 12(3): 191–200. doi:10.1038/nri3158.

Standardizing immunophenotyping for the Human Immunology

Stanford, California 94305, USA.

© 2012 Macmillan Publishers Limited. All rights reserved

tetramers7 or intracellular cytokine staining8,9; the measurement of T cell proliferation using

Current state of flow cytometry standardization

immunophenotyping. Another possible CCR7 replacement, CD27, is expressed not only by

non-classical monocytes (which are CD14lowCD16hi)48. Because monocyte, NK cell and

outcomes, including: vaccine-induced protection (at defined time point(s) post-vaccination);

How can this situation be improved?

The use of preformatted lyophilized-reagent plates (Lyoplates, BD Biosciences) can help, by

associated with shipping and/or cryopreservation of samples. However, it requires an

cases though, such empowerment is becoming a requirement for collaboration with

What needs to happen next?

Extending to other assays

(CTOT), is engaged in cross-validating cellular function assays as well as

such as enzyme-linked immunosorbent spot (ELISPOT) assays and immunohistochemistry.

Adopting new technologies

Another new technology, mass cytometry (CyTOF, DVS Sciences), is poised to

Data centralization and mining

However, there is a clear and growing interest in the standardization of human

Immune phenotypes Measurable aspects of the immune system, such as the

Phosphoepitope flow (Also known as ‘phospho-flow’). A technique that uses

analyse cell signalling by flow cytometry.

BMC Immunol. 2003; 4:9. [PubMed: 12952557]

MicroArray Quality Control (MAQC) project. Nature Biotech. 2006; 24:1140–1150.

separation. Clin. Lab. Haematol. 2002; 24:353–359. [PubMed: 12452816]

blood. Pathobiology. 1991; 59:127–130. [PubMed: 1715711]

rounds of proficiency testing. J. Immunol. Methods. 2010; 363:143–157. [PubMed: 20727897]

Box 1 | Flow cytometry

Box 2 | Extending standardization to other assays

Figure 1. A typical flow cytometry experiment

Figure 2. Identification of immune cell subsets by eight-colour antibody staining

Figure 3. The importance of antibody choice

Variable Approaches to minimize effects

Data analysis • Central analysis by one or a few coordinated experts

PBMC, peripheral blood mononuclear cell.

TH17 cells NK cells

PE CCR7 CD25 CXCR3 CD24 CD56

PerCP-Cy5.5 CD4 CD4 CD4 CD19 CD123

PE-Cy7 CD45RA CCR4 CCR6 CD27 CD11c

APC CD38 CD127 CD38 CD38 CD16

APC-H7 CD8 CD45RO CD8 CD20 CD3, CD19 and CD20

V500 HLA-DR HLA-DR HLA-DR IgD HLA-DR

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