Yan Lu1, Chuixin Liao1, Ming Lei1,Xiaohuan Wang1, Peifang Ye1, GarretGuenther2, Nan Li2

Capability of spectral flow cytometry using fluorochromes withhighly overlapping spectra in conjunction

1Agilent BIO (Hangzhou) CO., Ltd.,

Hangzhou, China; 2Agilent Biosciences,
Inc., San Diego, CA, United States

Introduction

Results and Discussions

Full spectrum flow cytometers use a large number ofdetectors with narrow bandpath filters to measure thefull spectrum emission of every fluorochrome acrossmultiple lasers to create a more detailed signature foreach fluorochrome. Compared to conventional flow
cytometry which uses compensation to correct for
fluorescence spillover, spectral flow cytometry uses a
process called spectral unmixing to resolve eachfluorochrome.
Spectralunmixingusesamathematical algorithm to decompose the spectral
signature of a multicolor sample into a set offluorochromes and their corresponding abundances,
based on the unique spectral signature of eachfluorochrome. Through this approach, fluorochromeswith near-identical peak emissions but different offpeak emission characteristics can be distinguishedand used in conjunction in one panel.

Examples of spectrum similar fluorochrome combinations

CYTO 2024
POSTER#58

Several panels containing fluorochromes with similar spectra were designed and compared in parallel with panels
containing the same markers but conjugated to fluorochromes with different spectra. The spectrum-similar
fluorochrome combinations include BV421/Pacific Blue, Qdot 705/BV711, BB515/FITC, APC/Alexa Fluor 647,
PerCP-Cy5.5/PerCP-eFluor 710, and PerCP-Cy5.5/PE-Cy5.5. Fluorochromes in these combinations can be resolved
when used in conjunction with each other and the subpopulation results are not affected. However, the spillover
spreading of spectrum-similar fluorochromes cannot be eliminated.
EDTA anticoagulated human peripheral blood was stained. Red blood cells were lysed. Afterward, the samples
were washed and analyzed on a five-laser NovoCyte Opteon flow cytometer.
Spectral density plots generated by the NovoCyte Opteon flow cytometer and theoretical spectral plots are shownin the figures.
(A) Brilliant Violet 421 and Pacific Blue
Brilliant Violet 421

Herein, the capability of distinguishing spectrum
similar fluorochromes were demonstrated using theAgilent NovoCyte Opteon spectral flow cytometer.

(D) APC and Alexa Fluor 647
Pacific Blue

BV421

Control

Test

Pacific Blue

Results and Discussions

APC

APC

Alexa Fluor 647

Control

Test

Alexa Fluor 647(E) PerCP-Cy5.5 and PerCP-eFluor 710(B) Qdot 705 and Brilliant Violet 711
BV711

FITC combined with fluorochromes ofdegrees of similarity

FITC used in conjunction with fluorochromes ofvarying degrees of similarity are compared using theNovoCyte Opteon spectral flow cytometer. As thesimilarity decreases, the fluorescent spreading error
becomes smaller and the resolution increases. When
FITC was used in conjunction with BB515, theSimilarity Index is 0.99, indicating the spectra of thesetwo dyes are almost the same. Even with such highsimilarity, their signals still can be resolved.

FITC

11FITC

1111

AF488

1111KB520

11BB515

FITC

FITC

KB520

FITC

FITC

AF488

FITC

FITC

FITC

Similarity Index

FITC

10.99

BB515

0.99

1PerCP-Cy5.5

Control

Test

Brilliant Violet 711

Control

Test(F) PerCP-Cy5.5 and PE-Cy5.5
PerCP-Cy5.5

BB515
FITC

Brilliant Blue 515

Control

FITC

PerCP-eF710

PerCP-eFluor 710(C) Brilliant Blue 515 and FITC

CD4 VS CD4

PerCP-Cy5.5

PE-Cy5.5

PerCP-Cy5.5

Test

ControlTest

PE-Cy5.5

Figure 2. The detection of spectrum-similar fluorochrome combinations. The combinations include BV421/Pacific Blue(A), Qdot
705/BV711(B), BB515/FITC(C), APC/Alexa Fluor 647(D), PerCP-Cy5.5/PerCP-eFluor 710(E), and PerCP-Cy5.5/PE-Cy5.5(F). Control: nospectrum-similar fluorochromes included; Test: spectrum similar fluorochromes included.

Multiple fluorochrome combinations excited by the 405-nm, 561-nm, or 637-nm lasers
Compensation beads (Cat# CMIgP-30-5H, Spherotech) were stained with different fluorescent antibodies. Thoselabeled with fluorochromes primarily excited by one laser (violet, yellow, or red laser) were mixed together tosimulate multi-color panels and acquired on the Agilent NovoCyte OpteonTM spectral flow cytometer. Not mixed
single-stained compensation bead samples were used to generate spectrum signatures for each fluorochrome.
The unmixed results are presented here. All populations can be well distinguished.
(A) Violet excited fluorochromes

AF532

FITC10.56

AF532

0.56

1APC

FITC

(B) Yellow excited fluorochromes

FITC

FITC & APC

FITC & AF532

FITC & BB515

FITC & KB520

FITC & AF488

FITC & FITCSpectrum

Qdot 705

Qdot 705

varying

FITC

10.08

APC

0.08

1Figure 1. FITC combined with fluorochromes of varying degrees
of similarity. PBMC were single stained with CD3 PE-Cy7 andFITC, Alexa Fluor 488, KIRAVIA Blue 520, Brilliant Blue 515,Alexa Fluor 532, or APC-conjugated CD4 antibody. CD3 PECy7/CD4 FITC-stained PBMC samples were mixed withsamples labeled with CD3 PE-Cy7 and different fluorochromeconjugated CD4 antibodies correspondingly and analyzed on a
five-laser NovoCyte Opteon spectral flow cytometer.

(C) Red excited fluorochromes

Figure 3. Unmixing of multiple fluorochromes with overlapping emission spectra excited by the 405-nm, 561-nm, or 637-nm lasers. (A)Combination of violet laser excited fluorochromes, BV421, eFluor 450, BV480, BV510, eFluor 506, and Pacific Orange. (B) Combinationof yellow laser excited fluorochromes, PE, CF568, PE-Dazzle 594, PE-Alexa Fluor 610, PE-Fire 640, PE-Cy5, PE-Cy5.5, and PE-Alexa
Fluor 700. (C) Combination of red laser excited fluorochromes APC, Alexa Fluor 647, Spark NIR 685, APC-R700, Alexa Fluor 700, andZombie NIR.

Conclusions•Fluorochromes with highly overlapping spectra can be used in conjunction using spectral flow cytometry,
increasing the flexibility in fluorochrome selection.

•Spillover spreading between these fluorochromes cannot be eliminated and should be considered carefully inpanel design .
For Research Use Only. Not for use in diagnostic procedures. DE24594451.