Contents

4 PCR-Based Diagnostics: Beyond and

Because of COVID
COVID accelerated advances already underway.

8 Tools to Streamline Infectious

Disease Workflows
Novel technology is helping to accelerate workflows and
advance discoveries.

12 Advanced Tools for Influenza Virus

Immunodetection and Vaccine Development
A large number of reagents have an important role in controlling
the influenza virus.

18 Characterization of Cytotoxic Immune Cells

in Human Peripheral Whole Blood
Assessment of phenotypic and functional markers using a 16-color panel
and the BD FACSymphony™ A1 Cell Anaylzer

2
Produced with Support from:

Biocompare Tel: 800-637-1277

395 Oyster Point Blvd., Suite 300 info@biocompare.com
South San Francisco, CA 94080 www.biocompare.com

3
PCR-Based Diagnostics:
Beyond and Because of COVID
COVID accelerated advances already underway.
Nowadays, everyone is familiar with molecular di-
agnostics, or knows what a PCR test is, because of
COVID. However, important advances were taking
place in molecular diagnostics even before COVID.
Most molecular diagnostic assays are PCR-based,
meaning they rely on amplification of specific ge-
netic material where primers, polymerase, and
probes are added. According to a recently pub-
lished report about molecular testing trends on
ResearchAndMarkets.com, the global molecular
diagnostic market is predicted to grow from $19 to
$30 billion in worth from 2021 to 2027.
Advances in infectious disease and cancer diagnos-
tics, the main applications of molecular diagnosis,
can be grouped as: (1) tests that provide more an-
swers (2) whole-genome sequencing/detection (3)
point-of-care molecular testing. Clinicians and re-
searchers interviewed concur that, although many
innovations occurred pre-COVID, they were all ac-
celerated because of COVID.
One test/many answers for
infectious diseases
The use of PCR-based panels or genetic array test-
ing in infectious diseases has drastically increased
4
over the past few years because they offer the con-
venience of providing a lot of information in one
test. Carlos Salama, M.D., Attending Physician in
Global Health at Elmhurst Hospital and Professor at
Mt. Sinai School of Medicine, gives an example of
how, within the past two years, his hospital started
using BioFire Diagnostic’s CSF (cerebrospinal fluid)
panel because one test on a patient’s sample can
reveal the most common infections seen in his city
hospital’s diverse population base.
“Now I don’t have to worry when I am called for
a consult, that the emergency department, espe-
cially when it’s super busy, may have forgotten to
test for certain pathogens.” However, Salama notes
there are still situations where empirical diagnosis
is quicker and cheaper, as well as instances where
panels aren’t designed to detect pathogens com-
mon to a subset of the population.
Array testing was enabled by advances in real time
(RT)-PCR, also known as quantitative (q)PCR, which
uses fluorescent probes that allow for multiplexing
and quantification, explains Heba Mostafa, Ph.D.,
Director of Molecular Virology at The Johns Hop-
kins Hospital. But Mostafa believes COVID enabled
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clinical labs such as hers to more widely adopt pan-
el testing by focusing labs’ and technology provid-
ers’ attention on optimization improvements such
as faster turn-around time and scale-up.
One test/many answers for
cancer diagnostics
In cancer molecular diagnostics, a multiplex equiv-
alent is tumor testing panels. The tumor panels
are based on NGS (next-generation sequencing),
which relies on massively parallel PCR-based se-
quencing and enables the detection of many mu-
tations from one DNA sample. Knowing whether
specific mutations are present in a tumor cell’s DNA
often enables the physician to determine the best
treatment course, says Lyndsay Harris, M.D., Associ-
ate Director of Cancer Diagnostics at the National
Cancer Institute.
Improvements in sample-to-answer speed and
costs have increased adoption of tumor panels
by more physicians. “Some hospitals or practices
have their own pathology labs with PCR-capable
machines and platforms that can couple with the
various NGS tumor testing kits. Practices can also
collect the tumor sample but send it to a reference
lab for analysis and still obtain results within a rea-
sonable time frame now,” adds Harris.
Whole-genome sequencing
Whole-genome sequencing has also vastly im-
proved over the past two years and is another PCR-
based diagnostic application. However, for infec-
tious diseases, the impact of COVID has been more
on “mindset” rather than technological improve-
ments. “Following viral variants such as Omicron
highlighted the importance of access to and ability
6
to share viral genomic sequences and associated
data,” explains Mostafa. Now whole-genome se-
quencing is being applied to influenza and adeno-
viral infections for research-based surveillance.
In cancer, whole-genome sequencing is also a
dream closer to fruition. “Five years ago, people
talked about the $1K genome. Now the $100 ge-
nome may soon be possible,” says Harris. “Compa-
nies are always looking at different chemistries, dif-
ferent polymerases, and ways to innovate. I have no
doubt that as speed and accuracy increase, costs
will continue to drop.” Harris believes once it is
cost-effective to sequence each person’s genome,
no other tumor diagnostic tests may be needed.
Point-of-care innovations in
infectious disease
For PCR-based assays, point-of-care (POC) diagnos-
tics, which means the test is analyzed onsite and
results are relatively rapid, are not easy. The ther-
mocyclers needed for PCR, especially those with
high throughput, are big and expensive machines
that often only exist in a reference lab, so it can take
a few days before results are available.
Salama explains that BioFire Diagnostics has creat-
ed specialized rapid PCR-based tests where many
institutions can keep the appropriate machines
and cartridges onsite. For example, BioFire has a
rapid PCR detection kit so that he can know within
24 hours if a patient has an MRSA (Methicillin-resis-
tant Staphylococcus aureus) infection or not, which
has enabled him to reduce the number of unneces-
sary antibiotics prescribed.
COVID also spurred innovations in PCR-based
tests that are POC. A few companies (Cephe-
id though it had POC/PCR applications before
COVID, Thermo Fisher Scientific, and Abbott)
make smaller and more affordable machines for
PCR analysis associated with kits/reagents for
SARS-CoV-2 detection that can be performed at
the point of care.
7
Mostafa points out that developing PCR-based di-
agnostics for POC requires a lot of rigorous testing.
Personnel needs to be well-trained, and well-writ-
ten instructions become more critical. However,
she has faith in, and has witnessed, how quickly
industry innovates.
Tools to Streamline Infectious
Disease Workflows
Novel technology is helping to accelerate workflows
and advance discoveries.
The COVID-19 pandemic has highlighted the im-
portance of infectious disease research and its
close relationship with public health. In a global
effort to stem the waves of coronavirus infections
and deaths, the furious storm of pandemic-related
research revealed a silver lining of advanced tech-
nologies now available to streamline workflows in
infectious disease labs worldwide. This article looks
at technologies and tools that are helping to accel-
erate workflows and advance discoveries in infec-
tious disease research.
Automation and multiplexing
As in many fields, reducing bottlenecks using au-
tomated solutions can greatly impact workflow.
“Automation is being used more frequently in to-
day’s laboratories as researchers strive to simplify
their technologies, reduce the chance for opera-
tor error, and also alleviate workforce shortages,”
says Sherry Dunbar, Senior Director of Global Sci-
entific Affairs at Luminex. In addition, automation
coupled with multiplexing is increasing workflow
more than ever. Luminex’s xMAP Technology, for
example, can measure up to 500 analytes in a
single reaction, and Luminex’s high-throughput
8
xMAP analyzers, the FLEXMAP 3D and xMAP IN-
TELLIFLEX, can be connected to plate-handling
systems for automation.
Preconfigured assay panels can also save time in
assay development, screening, and epidemiologi-
cal research. “Most researchers are under pressure
to do more with less,” says Dunbar. “Being able to
test for multiple targets simultaneously in a multi-
plexed panel saves time and cost, as well as reduc-
es the amount of sample required.” Luminex’s assay
panels, such as the xTAG GPP and NxTAG RPP, allow
researchers to test for the most common patho-
gens causing gastrointestinal and respiratory in-
fections, respectively.
Andy Lane, Commercial Director at The Native An-
tigen Company, observes that multiplex testing to
investigate pathogenic targets is becoming more
widespread. Traditionally, a patient’s symptoms
lead to testing for the most likely infection, and
then subsequently the next most likely. However,
this approach can lead to delays and ultimately
worse patient outcomes, with increased costs of
multiple tests. Multiplex testing allows researchers
to look for multiple markers of existing or previous
infections simultaneously. “This not only provides
faster results, but a greater degree of certainty with
regards to the infecting pathogen,” says Lane. “A
common diagnostic example is testing for several
respiratory viruses together—often SARS-CoV-2,
influenza A and B, and respiratory syncytial virus.”
In the lab, more multiplex testing means more mul-
tiplex-enabled methods. “For immunoassays, this is
increasingly taking the form of bead-based tech-
nologies and mass spectrometry,” says Lane. “For
molecular assays, technologies such as microar-
rays, next-generation sequencing, and new forms
of PCR, are proving to be better.”
Antibody neutralization
Among his customers, Lane notes that a major area
of research is assessing the functionality and dy-
namics of antibodies in disease states, particularly
neutralizing antibodies. Normally, the neutralizing
ability of an antibody is evaluated by testing the ef-
fect of patient sera on viral infectivity in cell culture
assays. But such experiments require specialized
training and can take up to a week.
9
Instead, alternative immunoassay formats have
been developed to measure indicators of neutral-
ization, using methods that are both faster and
cell-free. “For example, in vitro assays can be de-
veloped to measure the competitive interactions
between viral antigens, their target receptor,
and neutralizing antibodies,” says Lane. “Wheth-
er patient-derived or monoclonal, different anti-
bodies at different titers can be evaluated in this
way to assess neutralizing ability as a means of
understanding host protection from natural or
vaccine-induced immunity, as well as identify-
ing therapeutic candidates.” The Native Antigen
Company offers a range of such immunoassay re-
agents, particularly for SARS-CoV-2, as well as oth-
er viral and bacterial pathogens.
NGS and nucleic acid synthesis
NGS has become a critical tool for infectious disease
workflows, allowing faster viral sequencing and
vaccine development. Sylvain Gariel, Co-founder
and COO at DNA Script, believes that NGS will be
critical to detecting and combating the next pan-
demic in the earliest days when it’s easier to stop.
“The wide adoption of NGS will be critical for viral
detection and surveillance, for monitoring its evo-
lution and mutations,” he says, also predicting that
the combination of NGS and single-cell techniques
will be powerful for treating patients with severe
disease. “Single-cell sequencing can help us un-
derstand the immune system and how a virus [like
SARS-CoV-2] is interacting with a patient’s lungs,”
he notes. “This will be a critical tool in understand-
ing severe reactions such as cytokine storms in
some patients.”
DNA Script offers the benchtop SYNTAX System,
which uses an enzyme-based process to synthe-
size oligonucleotides within several hours, accel-
erating infectious disease workflows by reducing
the time required to obtain nucleic acids. Most labs
outsource this step, but waiting for the arrival of
these important reagents can cause delays of days
or weeks (even when not in the middle of a pan-
demic). “We want to enable our clients to be au-
tonomous and get access to the nucleic acids they
need in a few hours instead of a few days or weeks,”
says Gariel.
mRNA-based vaccines
The technology behind the successful develop-
ment of mRNA vaccines is also likely to accel-
erate infectious disease research. For example,
BioNTech is developing a vaccine for malaria
with the same mRNA technology used in its
successful Pfizer-BioNTech coronavirus vaccine,
with clinical tests planned by the end of 2022.
The University of Oxford’s Jenner Institute is also
working on a non-mRNA-based malaria vaccine.
The only malaria vaccine currently available to-
day, Mosquirix from GlaxoSmithKline, does not
10
reliably reach the 75% efficacy goal set by the
World Health Organization.
Besides being an important public health tool,
vaccines also comprise a valuable asset for nation-
al defense—especially when they can be created
rapidly. DNA Script is working with multiple part-
ners to develop tools for detecting and responding
to emerging biothreats. The SYNTAX System will
allow the French Department of Defense to manu-
facture their own primers and probes without out-
sourcing. In addition, Moderna and GE Research
have partnered with DNA Script to develop mobile,
rapid production of mRNA-based vaccines and
therapeutics as part of the U.S.’s Defense Advanced
Research Projects Agency’s (DARPA) Nucleic Acids
On-Demand World-Wide (NOW) Program. “The first
step is to make short pieces of DNA, and then as-
semble them into larger pieces that might become
a plasmid for a DNA vaccine, or could go through
an individual transcription step to become an
mRNA molecule for an mRNA vaccine,” says Gariel.
“We’re also developing multiple different biochem-
istries that we hope will enable customers to make
long RNA molecules more efficiently than the exist-
ing chemistry.”
In contrast to the agents of infection, host cells
may also shed new light on diagnosing and treat-
ing infectious diseases. “Another exciting area of
infectious diseases research is understanding the
role of host proteins and host protein networks in
the pathogenicity of microorganisms,” says Dunbar.
“This research could identify host protein biomark-
ers as early indicators of infection and the class of
microorganism responsible, as well as detect new
targets for therapeutics.” Approaching infectious
diseases from multiple angles will give us our best
chance at defeating infectious agents.
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Advanced Tools for Influenza
Virus Immunodetection and
Vaccine Development
A large number of reagents have an important role in controlling
the influenza virus.

Influenza (also known as flu) is an acute respiratory

illness caused by the influenza viruses A, B, C, and
D. Among them, influenza A, B, and C can affect hu-
mans (Table 1). Influenza A and B viruses can cause
epidemic (seasonal or interpandemic) influenza,
and influenza A viruses can also cause pandemics.
Figure 1. Diagram of Influenza Virus
Inflfluenza Virus Structure
Influenza A viruses contain eight negative-sense, nucleoprotein (NP). Polymerase complexes, which
single-stranded viral RNA (vRNA) gene segments consist of three polymerase proteins, PB1, PB2, and
encoding ten viral proteins (Figure 1). The eight PA, are located at the ends of nucleocapsids. These
genome segments are loosely encapsulated by the helical capsids are encircled by the matrix protein

Table 1. Classification of Influenza Virus

Influenza A Influenza B Influenza C
Hosts Humans, waterfowl, poultry, pigs, Humans, seals Humans, pigs, dogs
horses, sea mammals, bats
Gene Segments 8 8 7
Proteins 11 11 9
Clinical Features Moderate to severe illness Milder disease than Influenza A Largely subclinical
Epidemiological Features Causes pandemics Less severe epidemics than Does not cause epidemics
Influenza A; no pandemics or pandemics

12
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Human
Coronaviruses
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SARS-CoV-2
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HCoV-OC43

HCoV-HKU1

Influenza ●
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Virus
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HCoV-229E
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SARS-CoV

MERS-CoV
Influenza B

Influenza A Many Other Hard-to-find Viral Proteins from

CMV, Ebola, RSV, Zika, etc.

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1 (M1) and a host-derived lipid bilayer envelope
in which the viral surface glycoprotein hemagglu-
tinin (HA) and neuraminidase (NA) and the matrix
protein 2 (M2) are embedded.
HA, NA, and NP are three major antigens of the
influenza virus, which play essential roles in the
lifecycle of a virus (Table 2). Their recombinant pro-
teins are widely used in basic research, therapeu-
tics, vaccine development, and diagnosis.
Table 2. Major Antigens of Influenza Virus
Antigens Functions in Lifecycle
HA Binding to sialic acid receptors on the surface of the
host cell membrane, helping the interfusion of the viral antibody development; Detection reagents development
envelope with the host cell membrane.
NA Mediating the release of nascent viral particles
assembled in the host cells by cleaving sialic acid
groups from glycoproteins.
NP A major component of the ribonucleoprotein complex,
engaging in viral gene replication, transcription,
and translation.
Inflfluenza Pandemics and Strains
Historically, influenza pandemics were caused by
influenza A viruses, which can be further divided
into different subtypes depending on HA and NA
differences. Four influenza pandemics occurred in
the past 100 years: the H1N1 Spanish flu pandemic
(1918–1920), the H2N2 Asian influenza pandemic
(1957–1958), the H3N2 Hong Kong flu pandemic
(1968–1969), and the H1N1/09 swine flu pandemic
(2009–2010) (Table 3).
Applications
Basic research; Vaccine development; Anti-hemagglutinin
Antiviral drug development; Basic Research;
Vaccine development; Antibody development;
Detection reagents development
Antiviral drug development; Basic Research; Antibody
development; Detection reagents development

Table 3. Four Influenza Pandemics in the Past 100 Years

Name Date Subtype Infected (est.) Deaths Case Fatality Rate
Spanish Flu 1918-1920 H1N1 500 million or >1 billion 17–100million 2–3%, or ~4%, or ~10%
Asian Flu 1957-1958 H2N2 >500 million 1–4 million <0.2%
Hong Kong Flu 1968-1969 H3N2 >500 million 1–4 million <0.2%
Swine Flu 2009-2010 H1N1/09 0.7–1.4 billion 151,700–575,400 0.01%
Source: https://en.wikipedia.org/wiki/Influenza_pandemic

Figure 2. Emerging Potential Pandemic Strains in the Last 20 Years

14
 Table 4. Influenza Pandemic Strains
Subtype Strain Year Antigen Proteins Antigen Antibodies
H1N1 A/Brevig Mission/1/1918 1918 HA, HA1, M1, NP HA
H1N1 A/California/04/2009 2009 HA, HA0, HA1, NA HA, NA
H2N2 A/Guiyang/1/1957 1957 HA, HA1
H3N2 A/Hong Kong/1/1968 1968 HA, HA1,NP NP
H5N1 A/Hong Kong/483/97 1997 HA,HA1 HA
H7N7 A/Netherlands/219/2003 2003 HA, HA1, NA HA, NA
H7N9 A/Shanghai/1/2013 2013 HA, HA1, HA1+HA2, NA, NP HA
H9N2 A/Hong Kong/35820/2009 2009 HA, HA1

The strains responsible for the four pandemics,
H1N1 (Spanish flu and swine flu), H2N2 (Asian flu),
and H3N2 (Hong Kong flu), have attracted wide-
spread attention. In the last 20 years, multiple po-
tential pandemic strains have emerged with sev-
eral zoonotic influenza events (Figure 2).
Sino Biological has developed a panel of antigen
proteins and antibodies for influenza pandemic
strains (Table 4) designated to be used for var-
ious biochemical assays, such as the antibody
titer, antigen detection, and antigenic character-
ization assays.
Flu Prevention and Control
Early diagnosis and vaccination are important
ways to prevent and control flu. Early diagnosis
and treatment can effectively shorten the illness
duration, relieve symptoms, and control the vi-
rus spread. The World Health Organization (WHO)
recommends annual vaccination to prevent sea-
sonal influenza.
Immunodetection
Immunodetection is a common method to di-
agnose flu. Antibodies targeting NP proteins are
commonly used for immunodetection in different
assays, including enzyme-linked immunosorbent
assay, lateral flow assay, and direct fluorescent
antibody tests. Due to the high frequency of an-
tigenic drift or shift in various influenza strains,
broad-spectrum influenza antibodies are particu-
larly desired to diagnose flu.
Sino Biological has identified six pan-antibody
pairs each against the NP protein of influenza A
and B (Table 5). These matched pairs can detect
broad-spectrum strains within the target subtype
without any cross-reactivity with other subtypes
(Figure 3).

Table 5. Pan Antibody Pairs for NP Detection

Pair No. Capture Ab Detection Ab Pair No. Capture Ab Detection Ab
PanA-1 40205-MM16 40208-R014 PanB-1 40438-R004 40438-MM10
PanA-2 40205-R063 40205-MM18 PanB-2 40438-R036 40438-MM10
PanA-3 40208-R117 40205-MM18 PanB-3 40438-MM05 40438-R036

15
 Figure 3. Six Pan antibody pairs each against the NP protein can detect different NP proteins of influenza A or B virus by ELISA assay
Vaccine Development
Flu, especially seasonal flu, is a global threat to
human health. Vaccination is the most effective
way to prevent the spread of infection. Each year,
several different flu strains are selected as vaccine
strains based on the surveillance data of the recent
isolates and their performance in the previous sea-
son. In recent decades, most vaccines are trivalent
or quadrivalent, including one H1N1, one H3N2,
and Yamagata and/or Victoria-type flu B.
Sino Biological has released a panel of recombinant
antigen products covering all WHO-recommend-
ed vaccine strains in recent years (Table 6). These
16
products include HA, NA, and NP and are designed
for vaccine development.
Summary
Despite the ongoing coronavirus disease 2019
pandemic, influenza continues to occur each year,
resulting in substantial morbidity and mortality.
Many relevant studies are underway to help con-
trol the influenza virus.
The advanced tools developed by Sino Biological
contain a large collection of recombinant virus
proteins and a library of antiviral antibodies. These
reagents can be widely used for immunodetection
and vaccine development for the influenza virus.
 Table 6. Antigens for 2015-2022 Vaccine Strains
Strains HA NA NP Year
A/Brisbane/02/2018 (H1N1) 40719-V08H 40767-V08B 40776-V08B 2019-2020
A/California/7/2009 (H1N1) 11085-V08B 40205-V08B 2016-2017,
2015-2016
A/Cambodia/e0826360/2020 (H3N2) 40789-V08H 40784-V08B 40778-V08B 2021-2022
40789-V08H1
A/Guangdong-Maonan/SWL1536/2019 (H1N1) 40717-V08H 40723-V08B 2020-2021
A/Hawaii/70/2019 (H1N1) 40717-V08H 40724-V08B 2020-2021
A/Hong Kong/2671/2019 (H3N2) 40721-V08H 40753-V08B 2020-2021
A/Hong Kong/45/2019 (H3N2) 40765-V08H 40754-V08B 2020-2021
A/Hong Kong/4801/2014 (H3N2) 40555-V08B 40569-V08B 40781-V08B 2017-2018, 2016-2017
A/Kansas/14/2017 (H3N2) 40720-V08H 40766-V08B 40779-V08B 2019-2020
A/Michigan/45/2015 (H1N1) 40567-V08H1 40568-V08B 40777-V08B 2018-2019, 2017-2018
A/Singapore/INFIMH-16-0019/2016 (H3N2) 40580-V08H 40779-V08B 2018-2019
A/Switzerland/9715293/2013 (H3N2) 40497-V08B 40499-V08B 2015-2016
A/Victoria/2570/2019 (H1N1) 40787-V08H 40785-V08B 40774-V08B 2021-2022
40787-V08H1
A/Wisconsin/588/2019 (H1N1) 40787-V08H 40785-V08B 40785-V08B 2021-2022
40787-V08H1
B/Brisbane/60/2008 40016-V08B 40203-VNAHC 40783-V08B 2017-2018, 2016-2017,
2015-2016
B/Colorado/06/2017 40581-V08H 40782-V08B 2019-2020, 2018-2019
B/Phuket/3073/2013 40498-V08B 40502-V07B 40500-V08B 2021-2022, 2020-2021,
2019-2020,2018-2019,
2017-2018, 2016-2017,
2015-2016
B/Washington/02/2019 40722-V08H 40790-V08B 40755-V08B 2021-2022, 2020-2021

Additional Resources
Viral Antigen Bank

17
Characterization of Cytotoxic
Immune Cells in Human
Peripheral Whole Blood
Assessment of phenotypic and functional markers using a 16-color
panel and the BD FACSymphony™ A1 Cell Analyzer
Features
• Attain premium flow cytometry performance
with the BD FACSymphony™ A1 Cell
Analyzer that leverages BD FACSymphony™
instrument technology in a compact size
• Analyze up to 16 colors and 19 parameters
(including optional small particle side
scatter channel) to detect various subsets
of cytotoxic cells in blood using four high-
power lasers
• Get clear resolution of dim signals when
optimal panel design is combined with high
instrument sensitivity
Cytotoxic T lymphocytes (CTLs) and natural killer
(NK) cells have become attractive therapeutic tar-
gets for cancer immunotherapy because of their ef-
ficient mechanisms of killing cancerous cells. Both
effector CTLs and mature NK cells present a com-
plex expression pattern of cytolytic proteins that
dictates their ability to kill target cells. Using an op-
18
timized buffer system that allows simultaneous red
blood cell lysis and cell fixation, we could examine
the intracellular levels of cytolytic proteins directly
in peripheral blood cells. The cells were analyzed in
a BD FACSymphony™ A1 Cell Analyzer, which en-
abled the use of several bright or very bright fluo-
rochrome-conjugated antibodies for optimal reso-
lution of the cytolytic proteins and other markers
that are rather low expressed in cytotoxic cells.
Altogether, a coordinated analysis of a total of 16
intracellular or surface markers helped to detect
various subsets of cytotoxic cells in blood.
Protocol
Human peripheral blood was collected in sterile
BD Vacutainer® Blood Collection Tubes containing
sodium heparin. Predetermined optimal concen-
trations of fluorochrome-conjugated antibodies
against surface markers (Table 1) were mixed in the
presence of BD Horizon™ Brilliant Stain Buffer Plus
and dispensed in 5 mL Corning™ Round-Bottom
Polysterene Test Tubes. Next, 200 µL of heparinized
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premium BD FACSymphony™ technology with low-noise electronics to lower background noise and increase sensitivity
in up to 16 colors for a broad spectrum of research applications from analyzing small particles to immunophenotyping.

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blood and 2 µL of BD Horizon™ Fixable Viability
Stain 575V at 0.6 µg/µL were sequentially added to
the tubes and briefly vortexed. After 20 min incu-
bation at room temperature in the dark, 4 mL of BD
Phosflow™ Lyse/Fix Buffer (1X) was added to the
tubes for red blood cells lysis and leucocytes fixa-
tion. Following 15 min incubation, the cells were
spun down and washed twice with BD Pharmin-
gen™ Stain Buffer (FBS). Then, the fixed cells were
permeabilized in 100 µL of BD Phosflow™ Perm/
Wash I Buffer for 20 min at 4 °C and incubated with
antibodies against intracellular markers for an ad-
ditional 60 min protected from light. After two
washes, the cells were acquired in the BD FACSym-
phony™ A1 Cell Analyzer.
Cell Analysis Results
The BD FACSymphony™ A1 Cell Analyzer is equipped
with four lasers (Violet, Blue, Yellow-Green and Red)
and allowed the detection of cells stained with 13
bright or very bright fluorochrome-conjugated
antibodies. The high sensitivity of the instrument
combined with the bright fluorochromes resulted
in highly efficient resolution of markers that are ex-
pressed at variable or low levels among cytotoxic

Table 1. Instrument configuration and reagents in the cytotoxic immune cells panel
Laser Filter Fluorochrome Specificity Clone Catalog Purpose §
Antibody Volume
Number (mL)/ mL Blood or
Perm Buffer
450/50 BV421 Perforin dG9 563393 Cytolytic abilities 5
525/50 BV480 CD159a (NKG2A) 131411 747923 Inhibitory receptor 5
CD19 HIB19 740394 Exclusion/B cells 1.25
CD14 M5E2 564054 Exclusion/monocytes 5
BV605 Exclusion/plasmacytoid dendritic cells
610/20 CD123 7G3 564197 5
and eosinophils
CD141 1A4 740421 Exclusion/myeloid cells and platelets 5
Violet 405 FVS575V - - 565694 Viability 1
nm 670/30 BV650 CD3 UCHT-1 563851 T cells 5
710/50 BV711 CD314 (NKG2D) 1D11 563688 Activating receptor 5
780/60 BV786 HLA-DR G46-6 564041 Activation marker 2.5
530/30 FITC CD57 NK-1 555619 Maturation and differentiation marker 10
Blue 488 nm
710/50 PerCP-Cy5.5 CD8 RPA-T8 560662 Cytotoxic T cells 5
586/15 PE CD158 (KIRs) HP-MA4 567158 Maturation and differentiation marker 5
610/20 PE-CF594 CD56 R19-760 564963 NK cells/activation marker 5
Yellow-
670/30 PE-Cy5 CD95 (Fas) DX2 559773 Differentiation marker 20
Green 561
Differentiation marker/innate
nm 710/50 PE-Cy5.5 CD127 (IL7R-a) eBioRDR5 35-1278-42# 1.25
lymphoid cells
780/60 PE-Cy7 CD38 HIT2 560677 Differentiation marker 2.5
670/30 AF647 Granzyme K G3H69 566655 Cytolytic abilities 5
Red 637 nm 710/50 R718 Granzyme B GB-11 566964 Cytolytic abilities 1.25
780/60 APC-H7 CD16 (FcgRIII) 3G8 560195 NK cells/cytolytic abilities 5
BV, BD Horizon Brilliant Violet™; FVS, BD Horizon™ Fixable Viability Stain; AF, Alexa Fluor™; #Thermo Fisher Scientific; §See Certificate of Analysis for antibody
concentration; Perm: Permeabilization.

20
cell populations, such as CD3, CD56, perforin, CD314
(NKG2D) and CD159 (NKG2A) (Figures 1 and 2).
Detection of rare populations was achieved by re-
cording a minimum of 150,000 lymphocytes and
then excluding doublets, dead cells and non-rele-
vant cell lineages during cell analysis using Flow-
Jo™ v10.7.2 Software. A total of eight healthy do-
nors were pre-evaluated and the results shown
in this panel sheet are from one donor whose pe-
ripheral blood presented high frequencies of five
major cell subsets of interest: (pink) CD56brightCD3–
cytokine-producing NK cells, (blue) CD56dimCD3–
cytotoxic NK cells, (brown) CD56dimCD3+ including
NKT cells, (green) CD56dimCD3bright including γδ T
cells and (purple) conventional CD8 cytotoxic T
(Figure 1).
NK and cytotoxic T cell subsets utilize common
mechanisms to kill infected or transformed cells
via the granule exocytosis pathway. The key com-
ponents of the cytolytic granules are perforin and

Figure 1. Clear separation of cytotoxic immune cell populations in healthy human peripheral blood. Based on forward and side scatter
cell features, an initial gate around lymphocytes excluded debris and most monocytes/granulocytes. Subsequent gates eliminated cell
doublets. Then, FVS575V-labeled dead cells and other lineage cells (CD19+/CD14+/CD123+/CD141+) were also excluded enriching the
samples with T cells and NK cells. Within live and lineage-negative cells, analysis of CD56 versus CD3 revealed various cell populations
that were color coded as cytokine-producing NK cells (pink), cytotoxic NK cells (blue), CD56+ T cells containing NKT cells (brown), CD56+ T
cells containing γδ T cells (green) and cytotoxic CD8+ T cells (purple).

21
several serine esterases, termed granzymes. Herein,
we simultaneously assessed the intracellular levels
of granzyme K (GrzmK), granzyme B (GrzmB) and a
series of differentiation markers for a comprehen-
sive analysis of the functional status of these cells
(Figures 2 and 3).
Conclusion
In this study, we described an assay that provid-
ed a means to compare different types of circu-
lating cytotoxic cells based on the expression of
effector cytolytic proteins. These proteins as well
as other differentiation markers were expressed
in a range from low to high levels across the dif-
ferent cell populations. Thus, the use of high-per-
formance reagents and instrumentation were
critical to examine even the lowest expressed
proteins for a thorough assessment of circulat-
ing cytotoxic cell populations.
Additional Resource
FACSymphony™ A1 Cell Analyzer

Figure 2. Comparative analysis of circulating cytotoxic cells. Each colored histogram represents a cell population gated as depicted in Fig-
ure 1. A. The top row reveals the expression of key granzymes (GrzmK and GrzmB) and perforin. B. The bottom row shows the expression
of two major activating (CD314, NKG2D) and inhibitory (CD159a, NKG2A) receptors and the death receptor CD95 (Fas). The fluorescence
minus one (FMO) histograms were generated after gating on the total live and lineage negative cells.

22
Figure 3. Phenotyping of circulating cytotoxic cells using a 16-color panel. The plots represent the analysis of cytolytic proteins in
combination with various cell differentiation markers, enabling a deeper characterization of the cell populations gated in Figure 1. A.
Overlay of NK cell subsets. B. Analysis of CD56dimCD3+ T cell subsets, highlighting cell subsets that express either GrzmB or GrzmK. C.
Analysis of CD56dimCD3bright T cell subsets also showing GrzmB and GrzmK in the different subsets. D. Identification of activated CD8 T
cells based on the expression of CD38 and HLA-DR. The HLA-DR FMO staining helped to determine the gating boundaries for proper
detection of the double positive cells. The figure also depicts different CD8 T cell subsets based on the expression of GrzmB versus
GrzmK or CD95 versus CD57.

23