ORIGINAL ARTICLE

Standardization of flow cytometric detection of antigen

expression

Linhua Tian 1 | Aaron R. Nelson 2 | Tyler Lowe2 | Linda Weaver2 |

Constance Yuan2 | Hao-Wei Wang2 | Paul DeRose1 |

Maryalice Stetler-Stevenson 2 | Lili Wang1

1

Biosystems and Biomaterials Division,

National Institute of Standards and

Technology (NIST), Gaithersburg,

Maryland, USA

2

Laboratory of Pathology, National Cancer

Institute, National Institutes of Health,

Bethesda, Maryland, USA

Correspondence

Dr. Maryalice Stetler-Stevenson, Laboratory of

Pathology, NCI, NIH, Building 10, Room

3S235, Bethesda, MD, 20892, USA.

Email: mstetstev@comcast.net

Dr. Lili Wang, Biosystems and Biomaterials

Division, NIST, 100 Bureau Drive,

Gaithersburg, MD 20899, USA.

Email: lili.wang@nist.gov

Abstract

Since response to antigen-based immunotherapy relies upon the level of tumor antigen expression we developed an antigen quantification assay using ABC values. Antigen quantification as a clinical assay requires methods for quality control and for

interlaboratory and inter-cytometer platform standardization. A single lot of CytotrolTM Lyophilized Control Cells (Beckman Coulter) used for all studies. The variability

in antigen quantification across 4 different instrument platforms in 2 separate laboratories was evaluated. The effect of the antibody clone utilized, importance of custom

1:1 molar ratio (fluorophore to protein, F/P) verses off-the-shelf antibodies, and

QuantiBrite PE calibration verses linearity calibration combined with a single point

scale transformation with CD4 as reference were determined. Use of single lot control cells allowed validation of reproducibility between flow cytometer platforms and

laboratories and allowed assessment of different antibody lots, cocktail preparation,

and different antibody clones. Off the shelf antibody preparations provide reproducible estimates of antigen density, however custom 1:1 unimolar antibody preparations should be utilized for definitive measurement of antigen expression.Geometric

Mean fluorescent Intensity (GeoMFI) was not comparable across instruments and

inter-laboratory. The use of CD4 as the reference marker can minimize variability in

ABC values. Comparable antigen quantification is vital in managing patients receiving

antigen-based immunotherapy. If this assay is to be utilized in a clinical setting, quality control methods have to be instituted to assure reproducibility and allow validation across laboratories. We have demonstrated that use of a lyophilized cell control

is highly valuable in achieveing these goals.

KEYWORDS

acute leukemia, Flow cytometry, Leukemia, Qunatification, Standardization

1 | INTRODUCTION

Immunotherapies, such as bispecific T-Cell engagers (BiTE) or chimeric

antigen receptor (CAR) T-cell therapy, have radically changed therapy

in cancer patients with relapsed or chemotherapy refractory disease

and their possible incorporation into standard therapy is being investigated (Lee et al., 2015; Topp et al., 2015). Surface expression of the

targeted antigen is required for CAR T-cell binding and killing;

Received: 6 January 2023 Revised: 30 September 2023 Accepted: 29 November 2023

DOI: 10.1002/cyto.b.22155

Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

Cytometry. 2024;1–10. wileyonlinelibrary.com/journal/cytob 1
the level of antigen expression can impact treatment response and

duration. At lower target antigen densities, CAR T-cells are less potent

and produce decreased cytokine levels, such as IFN-gamma or IL-2,

despite augmentation with co-stimulatory molecules or alterations of

CAR T-cell affinity binding (Chmielewski et al., 2011). The finding that

CD22 dim disease is prone for relapse after CD22 CAR therapy (Fry

et al., 2018) and that dim disease limits anti-tumor efficacy

(Ramakrishna et al., 2019) indicates the importance of sufficient antigen density for CAR T-cell efficacy. In a preclinical model of CAR

T-cells targeting anaplastic lymphoma kinase (ALK), lower antigen

expression also limited the anti-tumor efficacy of the CAR (Walker

et al., 2017). In addition, tumor cell evolution resulting in target antigen modulation (the decreased level of expression or antigen loss) is

emerging as a mechanism of resistance (Majzner & Mackall, 2018;

Shalabi et al., 2018; Sotillo et al., 2015; Yu et al., 2017).

Antigen density prior to immunotherapy is receiving attention

and may impact feasibility of future antigen targeted therapies (Libert

et al., 2020; Walker et al., 2017). Furthermore, antigen-dim disease

will likely become a more frequent occurrence with the rise of

sequential immunotherapies. In a study, the majority of patients with

relapsed/refractory disease (B-lymphoblastic leukemia-B-ALL, diffuse

large B-cell lymphoma-DLBCL) post CD19 targeted therapy, had

CD19 negative, CD19 partial, or CD19 dim disease. This impacted the

efficacy of additional immunotherapies (Libert et al., 2020). Some

patients showed dynamic changes in surface CD19 expression, warranting the serial evaluation of surface CD19 expression to monitor

for renewed susceptibility to CD19 targeting (Libert et al., 2020). In

anti-CD22 targeting therapy for B-ALL, prior targeting with antiCD22 Inotuzumab can result in a lower CD22 expression (Bhojwani

et al., 2019; Yates et al., 2018), which leads to decreased subsequent

CD22 CAR T-cell activity (Fry et al., 2018).

Additionally, targeting one antigen may impact the level of

expression of another antigen, reducing efficacy of immune-based

therapies. CD19 and CD22 expression levels trend together (Libert

et al., 2020). With CD19 loss post-therapy, there is a corresponding

decrease in CD22 which may impact response to subsequent therapeutic strategies. Furthermore, in immunotherapy-naïve patients, low

CD19 expression had a correlation with low CD22 expression (Libert

et al., 2020).

Due to the inherent variability in qualitative estimates of antigen

density (e.g. 1+, 2+, etc. or dim, moderate, bright), there is an increasing interest in flow cytometric quantification of cell surface expression

of antigens targeted by immunotherapies. Geometric Mean Fluorescent Intensity (GeoMFI) has been utilized in studies in an effort to

provide more accurate quantification, however GeoMFI may not be

reproducible across different instruments and laboratories. Antibodies

bound per cell (ABC) is a unit of measurement for biomarker

expression that is instrument independent (Davis et al., 1998). In the

well-known BDIS QuantiBriteTM method, standard beads with known

quantities of fluorescent phycoerythrin (PE) are run and hence a standard curve of number of PE molecules versus fluorescence intensities

of the beads is constructed. This fluorescent calibration curve allows

measured fluorescence signals given by cells to be converted to the

ABC values as expression levels of cell surface antigens. This technique can be applied to the study of hematolymphoid neoplastic cells

(Salem et al., 2018; Tembhare et al., 2013). This expression analysis

method is straightforward; however, result comparability across different instrument platforms, reagent lots, operators, and laboratories

has not yet been demonstrated. Assessing the assay variability of flow

cytometric quantitation, including interinstrument, intralaboratory,

interlaboratory, and antibody lot-to-lot variability, is therefore critical

in developing assay standardization to enable broad clinical utility and

impact. To study potential sources of assay variability, the levels of

CD4, CD19, CD20, and CD22 expression were investigated using

Beckman-Coulter's CytotrolTM control cells, a known stable and uniform lyophilized PBMC control, across instruments, laboratories, and

antibody lots. Because the CD4 expression was measured in this

study, we compared an alternative quantification scheme based on

the use of CD4 as a reference marker (Degheidy et al., 2016; Wang

et al., 2012, 2014, 2016) to the QuantiBriteTM PE calibration method.

2 | METHODS

Cell processing and data acquisition: To keep the specimen identical

across experiments, a single lot of CytotrolTM Lyophilized Control Cells

(Beckman Coulter) was used for all studies. Cells were reconstituted

at a concentration of 1.0  106 per mL per manufacturers recommendations. A 100 μL aliquot of CytotrolTM cells was incubated with

one of the following single antibodies: CD4PE (PE) clone 13B8.2

Beckman-Coulter (BC); CD4PE clone SK3 Becton Dickinson Biosciences (BD); CD19PE clone J3-119 BC; CD19PE clone SJ25C1 BD;

CD20PE clone B9E9 BC; CD20PE clone L27 BD; CD22PE clone

SJ10.1H11 BC; CD22PE clone S-HCL-1 BD or an antibody cocktail

with CD45v500 clone H130 BD added to the single antibodies per

manufacturers' recommendations and as previously reported (Lee

et al., 2015). Briefly, CytotrolTM cells were incubated at room temperature in the dark for 30 min, washed with PBA (PBS, 0.5% BSA, 0.05%

sodium azide) and resuspended in PBA, and stored at 4C in the dark

before acquisition within 8 h of staining. Flow cytometers were

cleaned prior to acquisition by running 10% bleach solution on the

cytometer sample probe for 5 min followed by 5 min deionized water.

Specimens were acquired on the following flow cytometric instruments: two BD FACSCanto II and one Lyric instruments in Laboratory

1; one Attune NxT and one CytoFlex LX instruments in

Laboratory 2. QuantiBriteTM standard beads (BD Biosciences) were run

the same day on each instrument. Data was analyzed using FSCExpress (De Novo Software) and QuantiCalc (BD Biosciences) software

to generate standard curve with QuantiBriteTM beads and the ABC

values determined. This directly provides accurate antigen quantitation in antibodies with a 1:1 molar ratio of PE to antibody (unimolar).

Due to insufficient availability of unimolar PE-antibody conjugates,

non-unimolar antibodies must be used in some assays. As the fluorescence to protein (F/P) ratio of antibodies may vary in non-unimolar

antibodies, the value of adjusting the ABC value using the known

(F/P) ratio provided by the manufacturers as previously described

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(Tembhare et al., 2013) was investigated by comparing QunatiBrite

generated ABC values with and without adjustment of F/P ratio.

Instrument comparison: Potential variability in antigen quantitation across platforms and laboratories was assessed by simultaneous

evaluation of identical specimens in multiple platforms and in two laboratories. In order to evaluate intralaboratory instrument variation in

ABC values, results were compared across two identical flow cytometer platforms, namely two FACSCanto II instruments, in the same

laboratory by running triplicate identical specimens prepared and

acquired on the same day. To compare cross platform variations in PE

fluorescence channels due to instrument filter differences and to

explore possible interlaboratory variation identical specimens were

run on 2 BD FACSCanto II instruments and 1 BD Lyric instrument in

laboratory 1 as well as an Attune and CytoFlex instrument in laboratory 2 on the same day.

Evaluation of the effect of antibody clone and quality on results:

To evaluate the effect of the antibody clone utilized in antigen quantitation, two different anti-CD4, anti-CD19, anti-CD20, and anti-CD22

antibody clones were evaluated in identical specimens across 4 flow

cytometer platforms (2 FACSCanto II, 1 Attune NxT, and 1 CytoFlex

LX) in two laboratories. To verify the importance of a custom antibody

preparation of a 1:1 molar ratio (fluorophore to protein, F/P), a commercial off-the-shelf 1:1 (fluorophore to protein) CD20 antibody was

compared to the unimolar CD20 antibody conjugate from the same

manufacturer across a FACSCanto II and Lyric instruments in Laboratory 1 and Attune NxT and Cytoflex instruments in Laboratory 2.

Evaluation of potential technical variability due to operator:

Experiments using the identical antibody clone and lot number were

performed on two separate days and acquired on the same 2 intralaboratory instrument platforms, namely two BDIS FACSCanto II in

Laboratory 1.

Comparison of two antigen quantification methods: Intralaboratory variability on CD20 quantification was evaluated using

commercial antibodies CD4 and CD20, two cytometer platforms

(Attune NxT and CytoFlex LX), and two quantification schemes,

namely QuantiBrite PE calibration and a linearity calibration combined

with a single point scale transformation with CD4 as the reference

marker (Degheidy et al., 2016; Wang et al., 2016).

3 | RESULTS

CytotrolTM cells were utilized to evaluate possible variables affecting

ABC values and to determine their usefulness as a possible QC

reagent in developing and monitoring ABC testing. In order to evaluate the use of CytotrolTM cells as a QC reagent for instrument comparison, triplicate CytotrolTM specimens stained with the same lot of CD19

PE and CD22 PE were run on two separate BDIS FACSCanto II instruments on the same day. Use of control cells provided an excellent tool

to evaluate intra- and inter-instrument reproducibility of ABC results

(Table 1). As reproducibility of results over time is critical in a clinical

laboratory, a repeat test was performed on both instruments

3 months later, showing good consistency of the measurements. The

results demonstrated the utility of control cells to evaluate the longitudinal reproducibility of ABC values (Table 2).

Clinical laboratories are required to perform quality control on all

new antibody lots to determine if the quality is the same as previous

lots. This is typically accomplished by comparison of the newly purchased antibody lot to the previous antibody lot. Problems occur

when a laboratory depletes the previous antibody lot before receipt

of the new one or the old lot exceeds the expiration date. To assess

the utility of control cells to perform lot testing, CytotrolTM cells were

stained in triplicate with two different lots of CD19, CD20, and CD22

and run on an Attune NxT (Table 3). The difference in the two

CD20PE lots was larger than that observed with the other antibodies.

This may be due to the greater difference in the F/P ratio for CD20

(difference in the F/P ratio of lots: 0.043 (CD19), 0.213 (CD20), 0.08

(CD22), indicating that this may be an important variable in ABC

determinations. Adjusting the CD20 values for F:P ratio (formula:

ABC/(F/P ratio) = adjusted value) did not correct the difference in

ABC values between the two CD20 lots. This may be due to a number

of causes, including an inherent problem in a specific lot of antibody,

degradation of one antibody lot over time, or that a significant difference in the F/P ratio of an antibody indicates a disparity that cannot

be simply corrected by the F/P ratio adjustment. The results demonstrate the utility of control cells in antibody lot testing.

The utility of control cells in Quality Control of cocktail preparation was also evaluated. Triplicate CytotrolTM specimens stained with

two cocktails containing CD4 PE, clone SK3 and CD45 v500, clone

H130 from identical lots and prepared by two different individuals in

two separate laboratories were acquired on the same Attune NxT cytometer and CD4 PE ABC values determined. The results are CD4PE

ABC mean 26,061 (SD 77) for Cocktail preparation 1 and CD4PE ABC

mean 26,271 (SD 259) for Cocktail preparation 2, respectively, demonstrating result comparability across cocktail preparations can be

easily obtained.

Antibody clone selection is important as antibody clones binding

to different antigen epitopes might result in variation in ABC values.

TABLE 1 Comparison of CD19 and CD22 ABC values determined

on two FACSCanto II instruments in same laboratory.

CD19PE CD22PE

Clone SJ25C1 Clone S-HCL-1

BDIS FACSCanto A

Mean ABC Values 7020 9329

SD 11 108

CV 0.0016 0.0116

BDIS FACSCanto B

Mean ABC Values 7114 9505

SD 58 136

CV 0.0082 0.0143

Note: The mean, standard deviation (SD), and coefficient of variation (CV)

from triplicate CytotrolTM cells stained with CD19PE, Clone SJ25C1 from

Beckman-Coulter and CD22PE, Clone S-HCL-1 from BD Biosciences.

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Triplicate CytotrolTM specimens stained with CD19 PE (clone SJ25C1),

CD19 PE (clone J3-119), CD20 PE (clone L27), CD20 PE (clone B9E9),

CD22 PE (clone S-HCL-1), CD22 PE (clone SJ10.1H11), CD4 PE

(clone SK3), and CD4 PE (clone 13B8.2) from BD Biosciences and

Beckman Coulter, respectively, were run a BDIS FACSCanto

instrument on the same day. As shown in Table 4, the use of control

cells allowed the assessment of comparability of antibody clones targeting the same antigen. In all cases there were differences in ABC

values with the highest difference observed between the CD20

clones. Again, adjusting the ABC values for the F/P ratio did not correct the difference in ABC values. Final testing should be validated on

live cells as observed differences may be secondary to the effect of

lyophilization on the epitopes targeted.

Flow cytometer platforms differ across laboratories introducing a

possible source of variability in ABC values. Four different flow cytometer platforms were used, including BD FACSCanto II, BD FACS

Lyric, Thermo Fisher's Attune NxT, and Beckman-Coulter's CytoFlex

LX cytometers, with which optical configurations of the PE channel

are different, that is, under different laser excitations, 488 or 561 nm

and with different bandpass filters such as 585 ± 21 nm and

585 ± 8 nm. In order to utilize the QuantiBrite PE calibration scheme

across different instrument platforms, the shapes of PE fluorescence

spectra from PBMC stained with PE-labeled antibodies and QuantiBrite PE beads have to be closely matched at the wavelength range

defined by the bandpass filters. Figure 1 shows four reasonably

matched PE fluorescence spectra obtained from PBMC stained with

PE-labeled antibodies and QuantiBrite PE beads within the wavelength range of 585 ± 21 nm under either 488 nm or 561 nm laser

excitation. Higher fluorescence background signals for QuantiBrite PE

beads with regard to those for PE-labeled PBMC were observed

under 488 nm laser excitation due to unavailability of unlabeled beads

(QuantiBrite beads without PE labels) for proper background subtraction. The two pairs of fluorescence spectra under the two laser excitations match reasonably well within the wavelength range defined by

bandpass filter of 585 ± 21 nm. These results indicate that the ABC

values determined based on QuantiBrite PE calibration can be independent of the cytometer platforms used.

We next compared ABC values obtained using CytotrolTM control

cells stained with cocktails prepared separately in the two individual

laboratories containing the same lots of CD4 PE, CD19 PE, and CD22,

and run on the four cytometers in the two laboratories. As shown in

Table 5, control cells allow comparison of quantification across different flow cytometer platforms and laboratories. ABC values obtained

are consistent across instrument platforms as expected and with cocktail preparation by two different laboratorians using the same

reagents. This indicates the use of control cells can evaluate and

TABLE 2 Evaluation of ABC values

at two different time points testing

longitudinal reproducibility.

CD19PE CD19PE CD22PE CD22PE

Clone S125C1 Clone S125C1 Clone S-HCL-1 Clone S-HCL-1

Time zero Time point 2 Time zero Time point 2

BDIS FACSCanto A 7020 7714 9329 8945

BDIS FACSCanto B 7114 7504 9505 9244

Note: Mean ABC values from CytotrolTM cells stained with CD19PE, Clone SJ25C1 from BeckmanCoulter and CD22PE, Clone S-HCL-1 from BD Biosciences. Time Zero is the mean value from triplicate

determinations (Table 1) and is used for future testing evaluation. Time Point 2 is a single determination

3 months later indicating longitudinal reproducibility.

TABLE 3 Comparison of ABC values obtained with two different

antibody lots.

Antibody (lot) ABC value (values adjusted for F/P)

CD19PE (9151856): F/P 1.018

Mean 3854 (3785)

SD 163

CV 0.0423

CD19PE (9066849): F/P 0.975

Mean 4073 (4177)

SD 83

CV 0.0204

CD20 PE (9176021): F/P 0.897

Mean 14,597 (16,273)

SD 282

CV 0.0193

CD20 PE (9044581): F/P 1.11

Mean 13,001 (11,712)

SD 749

CV 0.0576

CD22 PE (9207363): F/P 1.03

Mean 7114 (6906)

SD 64

CV 0.0090

CD22 PE (9038654) F/P 1.11

Mean 6908 (6224)

SD 61

CV 0.0088

Note: The mean, standard deviation (SD), and coefficient of variation (CV)

from triplicate CytotrolTM cells stained with 2 different lots of CD19PE

(clone SJ25C1), CD20PE (clone L27), and CD22PE (clone S-HCL-1) from

BD Biosciences. Specimens run on the same day using the same Attune

NxT cytometer.

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validate interlaboratory agreement in determining ABC values across

multiple platforms.

To minimize the variabilities due to antibody lot difference, cocktail preparation, and sample staining on the determination of ABC

values and focus soley on instrument variability, CytotrolTM cells were

stained in triplicate with a cocktail containing CD4 PE, clone SK3 and

CD45 v500, clone H130 (BDIS) prepared by a single individual using

the same antibody lots on all specimens. The stained cells were

acquired on a FACSCanto II and a Lyric in Laboratory 1 and an Attune

and a CytoFlex in Laboratory 2. CD45 staining was utilized to gate

out debris. The data shown in Table 6 demonstrate a large variation in

GeoMFI across instruments making the comparison of GeoMFI across

cytometers not useful, however, the ABC values based on QuantiBrite

PE calibration were consistent across instruments.

For definitive quantification of the actual number of antigens

expressed on cell surface, antibodies with a 1:1 molar ratio of PE to

antibody (unimolar) should be used. Unfortunately, very few unimolar

antibodies are available and nonunimolar antibodies must be used.

Studies of ABC values using nonunimolar antibodies have, however,

resulted in clinically useful prognostic data across serial patient specimens collected over time periods ranging from months to years and

using different antibody lots with F/P ratios that were close to 1.0

(Bhojwani et al., 2019; Libert et al., 2020). We undertook a comparison of unimolar antibody to commercial, off-the-shelf antibody with

an F/P ratio close to 1. CytotrolTM cells were stained in triplicate

with either a cocktail containing off-the-shelf CD20 PE, clone L27 and

CD45 v500, clone H130 or a second cocktail with unimolar CD20 PE,

clone L27 and CD45 v500, clone H130 (BD Bioscences), prepared by

the same individual using the same antibody lots on all specimens.

The stained cells were acquired on a FACSCanto II, a Lyric (Laboratory

1), an Attune, and a CytoFlex (Laboratory 2). The mean GeoMFI

values and associated SDs of CD20 obtained are given for the offthe-shelf CD20 PE and the unimolar CD20 PE in Table 7. Large variation in GeoMFI values were observed across instrument platforms;

however, the ABC values based on QuantiBrite PE calibration were

very consistent for the use of unimolar CD20 PE and reasonably comparable for the off-the-shelf CD20 PE across instruments (Table 7).

Additionally, CD20 ABC values were higher with unimolar verses offthe-shelf CD20 PE antibody, despite the off-the-shelf CD20 PE antibody having a F/P ratio close to 1. Adjusting the ABC value using the

known (F/P) ratio provided by the manufacturers did not result in

agreement between the unimolar and off the shelf antibodies. This is

interpreted as intrinsic differences in the antibody preparation with the

unimolar antibody undergoing further purification. An antibody with a

F/P ratio of close to 1 still contains antibodies without fluorophore

label, labeled with a single PE, or labeled with ≥2PE molecules, and that

unlabeled antibody has a much higher binding affinity to antigen, resulting in lower GeoMFI and ABC values in the off-the shelf antibody.

We further explore the use of CD4 as a reference marker for

quantifying CD20 expression levels (Degheidy et al., 2016; Wang

et al., 2016). Corrected CD20 ABC values can be calculated by using

the following equation:

ABCCD20 1⁄4 ABCCD20

ABCCD4

40,000, ð1Þ

where the value of 40,000 is a known value of CD4 ABC for fixed

whole blood and Cytotrol control cells using the off-the-shelf CD4 PE

clone SK3 reagent (Davis et al., 1998; Wang et al., 2012, 2014). The

resulted CD20 ABC values with CD4 measured on the same cytometer serving as the reference marker are provided in the last column of Table 7 for the use of the off-the-shelf CD20 PE and unimolar

CD20 PE. Both ABC values of CD20 and CD4 on the right side of the

TABLE 4 Comparison of different antibody clones in ABC value

determination.

Conjugate/Mfr. ABC value (F/P adjusted)

CD19 BC clone J3-119: F/P 1.19

Mean 9941 (8354)

SD 95

CV 0.0096

CD19 BDIS clone SJ25C1: F/P 1.018

Mean 6789 (6669)

SD 185

CV 0.0272

CD20 BC clone B9E9: F/P 1.0

Mean 12,398 (12,398)

SD 430

CV 0.0346

CD20 BDIS clone L27: F/P 0.897

Mean 19,480 (21,717)

SD 397

CV 0.0204

CD22 BC clone SJ10.1H11: F/P 0.8

Mean 5066 (6333)

SD 69

CV 0.0136

CD22 BDIS clone S-HCL-1: F/P 1.03

Mean 9710 (9427)

SD 14

CV 0.0014

CD4 BC clone 13B8.2: F/P 1.2

Mean 33,715 (28,096)

SD 340

CV 0.0101

CD4 BDIS clone SK3: F/P 1.094

Mean 29,721 (27,167)

SD 160

CV 0.0054

Note: The mean, standard deviation (SD), and coefficient of variation (CV)

from triplicate CytotrolTM cells stained with different antibody clones from

BC-Beckman Coulter and BDIS-BD Bioscences and run on a BDIS

FACSCanto.

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Equation 1 are without F/P ratio adjustment. Again, the ABC values

based on CD4 reference are very consistent for the unimolar CD20

PE and fairly comparable for the off-the-shelf CD20 PE across instruments (Table 7) but the values are decreased for the off-the shelf antibody in comparison to the unimolar preparation.

The two quantification approaches, QuantiBrite PE calibration

and CD4 as a known reference marker enable quantitative measurement of CD20 expression levels in cytometer platform independent

manner. In addition the value of correcting ABC values using the F/P

ratios was examined, however, this correction requires the F/P ratios

from the manufacturer and is based upon the assumption that the F/P

ratios are accurate and explainable. These F/P ratios are obtained by

absorbance measurements and are averaged results from bulk solutions. The same F/P ratio of two antibody solutions does not mean

equivalence of the molecular constituents of the two solutions, for

example, unlabeled, singlely labeled, and doublely labeled antibodies.

Correction of ABC values using the F/P ratio did not improve the

comparability and it can be concluded from the study that this additional calculation is not useful and hence not needed.

Since the three antibodies, CD4 PE, CD20 PE, and CD20 PE 1:1 are

all produced by BD Biosciences, the PE conjugation processes are very

similar except for narrower sized band collection for purifying CD20 PE

1:1 relative to CD20 PE and their F/P ratios provided by the vendor are

shown to be similar. The Equation 1 above can be simplified without application of QuantiBrite PE calibration to both CD4 and CD20,

ABCCD20 1⁄4 GeoMFICD20

GeoMFICD4

40,000: ð2Þ

The CD20 ABC values obtained by using Equation 2 are provided

in the 4th column and 7th columns of Table 8 for the use of the offFIGURE 1 Normalized PE fluorescence spectra of PBMC stained with CD4 PE (clone SK3) and QuantiBrite PE beads in PBS, pH 7.4 under

either 488 or 561 nm laser excitation using a calibrated CCD-based fluorimeter (DeRose et al., 2020). The fluorescence spectra of CD4 PEstained PBMC were corrected by subtracting the background signal from unstained PBMC; however, for QuantBrite PE beads, background

subtraction was made using PBS only. The wavelength range enclosed by the two black vertical lines is defined by the bandwidth filter of

585 ± 21 nm. [Color figure can be viewed at wileyonlinelibrary.com]

TABLE 5 Comparison of different flow cytometer platforms in

ABC determination.

Platform CD4 ABC CD19 ABC CD22 ABC

Canto A

Mean 33,715 9942 5066

SD 408 113 55

CV 0.0121 0.0114 0.0109

Canto B

Mean 34,353 11,163 5133

SD 481 159 12

CV 0.0140 0.0142 0.0023

Attune NxT

Mean 32,297 9712 3932

SD 271 162 14

CV 0.0084 0.0167 0.0036

CytoFlex LX

Mean 36,422 9116 4843

SD 215 147 94

CV 0.0059 0.0161 0.0194

All Platforms

Mean 34,197 9983 4757

SD 1581 788 504

CV 0.0462 0.0789 0.1059

Note: The mean, standard deviation (SD), and coefficient of variation (CV)

from triplicate CytotrolTM cells stained with the same clone and lot of

antibody (CD4 PE clone 13B8.2, CD19 PE clone J3-119, and CD22 clone

SJ10.1H11) from Beckman Coulter using cocktails prepared in two

different laboratories and acquired on four different flow cytometry

platforms (2 FACSCANTO instruments in Laboratory 1, Attune NxT and

CytoFlex LX cytometers in Laboratory 2).

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the-shelf CD20 PE and unimolar CD20 PE conjugate, respectively. By

comparing the %CV associated with the mean ABC value across

instruments, the simplified quantification approach using Equation 2 is

as good as the approach using Equation 1. The use of CD4 as the reference marker does minimize the variability in the determination of

the CD20 ABC values compared to the QuantiBrite PE calibration

method.

4 | DISCUSSION

If ABC values are to be clinically relevant, they need to be independent of cytometers used and reproducible across flow cytometry

instruments, laboratories, and time. Furthermore, minimizing variability in antigen quantification is crucial in large clinical studies of antigen

directed therapies that examine the utility of expression levels of target antigens in predicting outcome. The same results must be generated by all participating laboratories. The ability to detect variability in

results is especially important when monitoring changes in antigen

expression overtime because decreased antigen expression serves as

a potential indicator of loss of therapeutic efficacy. Methods of assessing interlaboratory and interinstrument variability are therefore

needed. In addition, laboratories quantifying antigen expression need

quality control reagents to evaluate new antibody lots, cocktail preparation, comparability of multiple intralaboratory flow cytometers,

instrument performance over time, and determine the optimal antibody clone when setting up an assay. We studied the use of

Beckman-Coulter CytotrolTM control cells, a known stable and uniform

lyophilized PBMC control, for quality control processes and evaluation

of possible interlaboratory and cross platform variability. We found

that a stable control cell product such as CytotrolTM is highly useful in

validating synchronization of multiple intralaboratory instruments to

produce consistent results (Table 1). In addition, shelf stable control

cells provided an excellent identical specimen for evaluation of instrument function over time (Table 2). Shelf stable control cells provide an

excellent quality control product for evaluating new antibody lots,

which may be superior since it does not rely on comparison to an old

antibody lot that may have degraded over time (Table 3). Lastly, CytotrolTM cells served an excellent Quality Control reagent for double

checking cocktail proparation by different individuals.

When designing a flow cytometry assay the choice of antibody

clone is important. Our studies indicate it is especially critical in assays

that measure levels of antigen expression. While the two CD4 clones

were similar, notable differences were observed between the CD19,

CD20, and CD22 clones (Table 4). It should be noted that the

observed differences in these antibody clones may be secondary to

different antigen epitopes being targeted by the clones and possible

differential preservation of epitope sites during lyophilization. Confirmation in biological samples without lyophilization is therefore recommended. When validating an assay run in multiple institutions, cocktail

preparation and flow cytometer platform are possible sources of variability in the data collected. Mimicing these conditions, CytotrolTM cells

were stained with cocktails prepared separately by the two participating laboratories using the same lots of reagents, CD4 PE, CD19 PE,

and CD22 PE and run on four different flow cytometer platforms.

Consistent ABC values were obtained across instrument platforms, as

expected (Table 5). Use of an identical control cell product allowed

confident assessment of potential variability between the two laboratories and different platforms.

From the CD4 GeoMFI values measured using the same sample

preparation on different cytometers shown in Table 6, it is clear that

instrument-dependent GeoMFI cannot be used to report the expression levels across instruments and laboratories. To quantify antigen

expression levels in a cytometer independent manner, a well-adapted

antigen quantification scheme utilized in clinical laboratories relies on

the use of Quantibrite PE beads and PE labeled antibodies (Davis

et al., 1998; Salem et al., 2018; Tembhare et al., 2013). The method

enables antigen expression analysis in the PE channel with an assumption that the absorption and fluorescence properties of QuantiBrite PE

beads and PBMC stained with PE labeled antibodies are the same.

Because cytometer platforms used in this study have different optical

configurations, including laser wavelength and bandpass filters, PE fluorescence spectra of QauntiBrite PE beads and PE labeled PBMC were

measured to verify this assumption. The closely matched PE fluorescence spectra of the two samples with different bandpass filters and

under either 488 or 561 nm laser excitation (Figure 1) support that the

ABC values based on the QuantiBrite PE calibration method should be

comparable across different cytometer platforms used.

Although the QuantiBrite PE calibration assay can be easily utilized in a clinical laboratory for reproducible results allowing

TABLE 6 Comparison of CD4 Raw GeoMFI and ABC value

variability across instruments.

Flow cytometer CD4 raw GeoMFI CD4 ABC value

CytoFLEX LX

Mean 75,294 24,472

SD 1204 385

CV 0.0160 0.0157

Attune NxT

Mean 47,602 23,129

SD 363 181

CV 0.0076 0.0078

CANTO

Mean 18,717 25,166

SD 29 39

CV 0.0015 0.0015

Lyric

Mean 27,134 26,089

SD 509 488

CV 0.0188 0.0187

Note: The mean, standard deviation (SD), and coefficient of variation (CV)

from triplicate CytotrolTM cells stained with CD4PE, clone SK3 and CD45

v500, clone H130 (BDIS). No F/P ratio adjustment was made to the ABC

values.

TIAN ET AL. 7

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TABLE 8 Comparison of CD20 ABC variability using three different quantification approaches across instruments.

CD20

OTS ABC

(ABCCD20OTS/

ABCCD4OTS)  40,000

(GeoMFICD20OTS/

GeoMFICD4OTS)  40,000

CD20

1:1 ABC

(ABCCD201:1/

ABCCD4OTS)  40,000

(GeoMFICD201:1/

GeoMFICD4OTS)  40,000

CytoFlex

LX

11,247 18,384 18,128 16,843 27,530 26,978

Attune

NxT

8063 13,945 14,308 15,373 26,587 26,856

CANTO 8358 13,285 13,160 16,735 26,600 26,505

Lyric 11,469 17,584 17,872 17,282 27,469 26,482

Mean 9784 15,800 15,867 16,558 26,956 26,705

SD 1824 2558 2509 825 436 250

CV 0.186 0.162 0.158 0.050 0.016 0.009

Note: CD20 OTS: Non-unimolar off the shelf CD20. CD20 1:1: Unimolar CD20. ABC values are from Table 7 and are not corrected by the F/P ratio.

TABLE 7 Comparison of CD20 ABC variability using the off-the-shelf (OTS) and unimolar (U) CD20 PE conjugates across instruments.

Conjugate/Mfr.

Raw

GeoMFI

ABC

valuea

ABC value with F/P

corrected

(ABCCD20/ABCCD4)  40,000 (CD4 ABC from

Table 8 as a reference marker)a

CytoFLEX LX

CD20 OTS, [F/P = 0.946]: Mean

(SD) [CV]

34,123 11,247 11,889 18,384

(1014) (328) (347) (1074)

[0.0297] [0.292] [0.0292] [0.0584]

CD20 U, [F/P = 1.094]: Mean

(SD) [CV]

50,782 16,843 17,550 27,530

(692) (222) (235) (363)

[0.0136] [0.0132] [0.0134] [0.0132]

Attune NxT

CD20 OTS: Mean (SD) [CV] 17,028 8063 8524 13,945

(3649) (1767) (1869) (3058)

[0.2143] [0.2191] [0.2193] [0.2193]

CD20 U, [F/P = 1.094): Mean (SD)

[CV]

31,961 15,373 16,234 26,587

(2129) (1049) (1108) (1815)

[0.0666] [0.682] [0.0682] [0.0683]

Canto II

CD20 OTS, [F/P = 0.946]: Mean

(SD) [CV]

6158 8358 8835 13,285

(1711) (2301) (2433) (3658)

[0.2778] [0.2753] [2754] [0.2753]

CD20 U, [F/P = 1.094): Mean (SD)

[CV]

12,402 16,735 17,672 26,600

(803) (1075) (1135) (1709)

[0.0647] [0.0642] [0.0642] [0.0642]

Lyric

CD20 OTS, [F/P = 0.946]: Mean

(SD) [CV]

11,916 11,469 12,124 17,584

(224) (216) (224) (343)

[0.0188] [0.0188] [0.0185] [0.0195]

CD20 U, [F/P = 1.094): Mean (SD)

[CV]

17,964 17,282 18,249 26,497

(1178) (1132) (1196) (1799)

[0.0656] [0.0655] [0.0655] [0.0679]

a

Indicates no F/P ratio adjustment. The mean, standard deviation (SD), and coefficient of variation [CV] from triplicate CytotrolTM cells stained with offthe-shelf CD20 PE, clone L27 and CD45 v500, clone H130 or a second cocktail with unimolar CD20 PE, clone L27 and CD45 v500, clone H130, both

from BD Bioscences.

8 TIAN ET AL.

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comparison of antigen expression among patients and detection of

changes in levels of antigen expression in response to therapy, it is

not without limitations. The method only allows expression analysis in

the PE channel and has issues associated with lot-to-lot variability on

antibody PE conjugates (Table 3) (Wang et al., 2011) as well as limited

availability of high quality unimolar antibody-PE conjugates such as

CD20, CD38 and HLA-DR. It is evidentally clear that the use of offthe-shelf antibody PE conjugates (CD20 OTS) will result in lower ABC

values of antigens than the use of unimolar antibody conjugated with

PE (CD20 U) as shown in this study. An alternative antigen quantification scheme reported in the literature (Degheidy et al., 2016; Wang

et al., 2016) is to use a biological cell reference, which is known to

possess a fixed number of known biomarkers such as CD4 on CytotrolTM control cells characterized by using three orthogonal methods,

quantitative flow cytometry, mass cytometry, and mass spectrometry.

This approach enables antigen expression analysis in different fluorescence channels (Degheidy et al., 2016). Because the quantification

method involves both antibodies for reference marker and antigen of

interest, it is recommended to utilize two antibodies produced by the

same manufacturer to ensure a consistent antibody-fluorophore conjugation process for the two labeled antibodies. The source of variances on the CD4 based quantification scheme was investigated and

reported in the previous study that showed relatively small uncertainty in CD19 quantification using CD4 as a reference marker (Wang

et al., 2021). Because no uncertainty was provided for the number of

PE molecules associated with each Quantibrite PE bead population,

the variance with the use of Quantibrite PE calibration could not be

assessed in that study.

Our results on the CD20 expression analysis on CytotrolTM cells

using Equation 1 shown in Table 7 and Equation 2 in Table 8 demonstrate that the antigen quantification method based on CD4 as the

reference marker performs well with no larger uncertainty compared

to the Quantibrite PE calibration approach. The accurate level of

CD20 expression, however, is likely higher than the values obtained

on the basis of QuantiBrite PE calibration. An antibody with a F/P

ratio of close to 1 still contains antibodies without fluorophore label,

labeled with a single PE, or labeled with ≥2 PE molecules, and that

unlabeled antibody has a much higher binding affinity to antigen. This

likely contributes to the variable results obtained with different antibody lots. Having a stable control to compare antibody lots is vital for

longitudinal studies of antigen expression. Importantly, this study

focuses on the comparability and reproducibility of antigen expression

analysis across different cytometer platforms and laboratories rather

than the accuracy/trueness of the biomarker expression levels. If the

goal of an assay is to accurately determine the number of antigen molecules residing on the cell surface an unimolar antibody should be

used. As antigen directed therapy expands it is hoped that manufacturers provide more high quality unimolar antibody preparations for

antigen quantification but until such products become available, differences in antigen expression between patients or over time in the

same patient can be determined using ABC values and off-the-shelf

antibodies with an F/P ratio close to one with minimal variability demonstrated by accurate lot testing. It is worth mentioning that CD20

quantification was demonstrated previously using CD4 on CytotrolTM

as the reference marker in a single-tube assay format where both

stained specimen and CytotrolTM were added in the same tube (Wang

et al., 2016). Spiking a single PE calibration bead, either a part of

QuantiBrite PE bead kit or a different PE calibration bead material

such as PE calibration beads made by Slingshot Biosciences and BD's

FC beads PE, into the specimen could help to evaluate the precision

of antigen expression analysis.

In conclusion, as response to antigen based immunotherapy

can rely upon the level of antigen expression by tumor cells and

as decreasing levels of antigen expression can be an early indicator of developing resistance to therapy, a reliable antigen quantification assay is needed. Due to the ease of use and reliability

of the QuantiBrite assay as well as its general utility in quality

control, determination of ABC values can meet this need. As in

all flow cytometric assays, the quality of data is dependent upon

the quality of the antibodies used and unimolar antibodies, if

available, are preferable. On the other hand, if antigen quantification is needed in non-PE channel, CD4 reference marker

approach with the use of Equation 2 serves the quantification

purpose. If this assay is to be utilized in a clinical setting quality

control methods have to be instituted to assure reproducibility

and allow validation across laboratories. We have demonstrated

in this study that use of a lyophilized cell control is highly valuable in achieving these goals.

ACKNOWLEDGMENTS

Research reported in this publication was supported in part by the

intramural program, COCR, the National Cancer Institute, the National

Institutes of Health and the Biosystems and Biomaterials Division,

National Institute of Standards and Technology.

DISCLAIMER

To specify an experimental procedure as completely as possible, certain commercial materials, instruments, and equipment are identified

in this manuscript. In no case does identification of the manufacturer

of particular equipment or materials imply a recommendation or

endorsement by the National Institute of Standards and Technology

or the National Institutes of Health, nor does it imply that the materials, instruments, and equipment identified are necessarily the best

available for the purpose.

ORCID

Constance Yuan https://orcid.org/0000-0002-2601-3665

Hao-Wei Wang https://orcid.org/0000-0002-5408-9253

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