EFB Dengue Literature Review

Conventional Flow Cytometry

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Conventional Flow Cytometry

Overview

Conventional flow cytometry uses laser excitation and fluorescence detection to measure multiple surface and intracellular markers simultaneously on single cells. In the context of B cell immunology, it is the primary tool for classifying peripheral blood B cell subsets by surface phenotype. “Conventional” here refers to standard multi-laser instruments (e.g., BD FACSCalibur, FACSCanto, LSRFortessa) running panels of up to ~10–14 colours, as distinct from spectral flow cytometry (which can run 28+ colour panels by full spectral unmixing).

In dengue and related infection studies, conventional flow cytometry has been used to quantify plasmablasts, memory B cell subsets, and atypical/DN B cells during acute and convalescent phases.

Key Points from Literature

  • Wei2007 panel (8–9 color, FACSCalibur): CD19, IgD, CD27, CD38, plus one of: IgG, IgM, IgA, B220, CD10; 9-color protocol adds CD24, CD138, CD3; FcRH4 detected with biotinylated anti-FcRH4 + streptavidin. Standard IgD vs. CD27 dot plot used to define four quadrant populations: naive (IgD⁺CD27⁻), nonswitched memory (IgD⁺CD27⁺), switched memory (IgD⁻CD27⁺), and DN/double-negative (IgD⁻CD27⁻) (see Wei2007 - DN Memory B Cells in SLE).

  • Bm1–Bm5 classification: An alternative IgD/CD38-based gating strategy that identifies B cell subsets from naive (Bm1: IgD⁺CD38⁻) through GC (Bm3–4: IgD⁻CD38⁺) to memory (Bm5: IgD⁻CD38⁻/dull). The Bm5 gate substantially overlaps with classical memory cells but contains both CD27⁺ and CD27⁻ fractions; used as an orthogonal check on IgD/CD27 gating (see Wei2007 - DN Memory B Cells in SLE).

  • Limitation of limited panels: 8-color conventional panels cannot simultaneously resolve all relevant B cell subsets. Classification schemes based on IgD, CD27, and/or CD38 have acknowledged limitations: naive Bm1/Bm2 fractions contain non-switched CD27⁺ memory cells; Bm2ʹ pre-GC cells overlap with transitional B cells (see Wei2007 - DN Memory B Cells in SLE). Newer spectral panels adding T-bet, CD11c, CXCR5, FcRL5 provide finer resolution.

  • Fluorescence-minus-one (FMO) controls used to define positive/negative boundaries; Simply Cellular compensation beads used for spectral compensation (see Wei2007 - DN Memory B Cells in SLE).

  • Anolik2004 panel (≤5 color, FACSCalibur): CD19, CD20, CD27, IgD, CD38 — used in three gating configurations: (1) IgD vs. CD27 four-quadrant plot for naive/memory/DN/plasmablast classification; (2) CD38 vs. CD19 for plasmablast identification (CD38^high CD19^low gate with CD20 overlay); (3) CD38 vs. IgD Bm1–Bm5 scheme for developmental staging. 9G4 antiidiotype added separately for VH4.34 B cell tracking. Cells isolated by Ficoll-Hypaque from heparinized blood; B cells enriched by CD19 magnetic selection for residual cell analysis at maximal depletion timepoints (see Anolik2004 - Rituximab and B Cell Abnormalities in SLE).

  • Combining IgD/CD27 and CD38/IgD axes: Using both gating strategies simultaneously (by adding CD27 to the CD38/IgD plot) resolves the ambiguity between DN memory B cells and Bm5 cells, since DN cells (IgD⁻CD38⁻CD27⁻) and CD27⁺ memory cells (IgD⁻CD38⁻CD27⁺) are indistinguishable on CD38/IgD alone. The recommended practice is to combine both axes; 5-color panels can do this with CD19, IgD, CD27, CD38, and one additional marker (see Bm Classification).

  • Tipton2015 panel (multi-color, FACSAria II): IgD (FITC), IgM (PE-Cy5), CD38 (Pacific Blue), CD23 (PE-Cy7), CD21 (PE-Cy5), CD27 (PE), CD19 (APC-Cy7), CD3/CD14 (Pacific Orange, exclusion dump channel), CD24 (PE-Alexa Fluor 610), IgD (APC, second clone), CD138 (APC), Ki67, and MitoTracker Green (MTG). Ficoll density-gradient PBMC isolation; ~10⁴–3×10⁵ cells sorted per population on FACSAria II (BD Biosciences). Key gating logic: (1) ASC gate: CD19⁺IgD⁻CD27^hiCD38^hi, further subdivided by CD138⁻/CD138⁺; (2) naive compartment: CD19⁺IgD⁺CD27⁻, subdivided by MTG vs. CD24 into resting (MTG⁻CD24⁺), transitional (MTG⁺CD24⁺), and activated naive/acN (MTG⁺CD24⁻) fractions; (3) IgD⁻ memory gate: CD19⁺IgD⁻CD27⁺ (see Tipton2015 - ASC Diversity and Origin in SLE).

  • MitoTracker Green (MTG) as activation discriminator: MTG is a mitochondrial membrane potential dye retained by activated and transitional B cells but not by resting naive B cells. Combined with CD24 staining, MTG distinguishes three populations within the IgD⁺CD27⁻ naive compartment: MTG⁻CD24⁺ (resting naive), MTG⁺CD24⁺ (transitional), and MTG⁺CD24⁻ (acN/activated naive). This is a non-standard reagent not captured by antibody-based panels; it was validated by concordance with activated B cell surface markers (CD19^hi, CD21⁻, CD23⁻) and with disease activity (see Tipton2015 - ASC Diversity and Origin in SLE).

  • Jenks2018 DN1/DN2 resolution panel (multi-color, LSRFortessa + FACSAria II): Panel includes: CD19, IgD, CD27, CD38, CXCR5, CD21, CD11c, CD24, MTG, T-bet (intracellular), BLIMP-1 (intracellular), Ki67 (intracellular), with additional markers in extended panels: FCRL4, FCRL5, CD62L, CD32b, CD22, CD69, HLA-DR, CD86. The critical gating logic for DN1/DN2 subdivision: within IgD⁻CD27⁻CD19⁺ (DN gate), DN1 = CXCR5⁺CD21⁺ and DN2 = CXCR5⁻CD21⁻CD11c⁺CD19^hi. This panel also resolves aNAV cells within IgD⁺CD27⁻ as CXCR5⁻CD19^hiCD21⁻MTG⁺CD24⁻. T-bet intracellular staining confirms highest expression in DN2 and aNAV. Phospho-flow (pERK, pMAPKp38) after R848 stimulation used separately for TLR7 responsiveness (see Jenks2018 - DN2 B Cells and EF Pathway in SLE).

  • Minimum markers for DN2 identification: CXCR5 is the single most discriminating marker between DN1 and DN2 within the DN gate; adding CD11c and CD21 provides redundancy. A practical 8-color panel for DN2 screening would require: CD19, IgD, CD27, CXCR5, CD21, CD11c + two additional channels (e.g., CD38 for plasmablast exclusion, viability dye). This is achievable on conventional instruments (see Jenks2018 - DN2 B Cells and EF Pathway in SLE).

  • Sanz2025 recommended classification scheme (Table 1, Figure 2): The definitive gating strategy for human B cell subsets uses IgD vs. CD27 for the four parental populations (naive, USM, SM+PB, DN), then CD21 vs. CD11c (or equivalently CXCR5 vs. CD11c, or CXCR5 vs. T-bet) within each parental gate to resolve subsets: resting naive (rN: CD21⁺CD11c⁻), activated naive (aNAV: CD21loCD11c⁺); resting memory (rMem: CD21⁺CD11c⁻), ABC memory (CD21⁺CD11c⁺ resting; CD21loCD11c⁺ activated); DN1 (CD21⁺CD11c⁻), DN2 (CD21loCD11c⁺⁺), DN3 (CD21loCD11c⁻). CXCR5 can substitute for CD21, and T-bet or FcRL5 for CD11c, with each alternative identifying similar populations (see Sanz2025 - Human Atypical B Cells Overview, review, Table 1 + Figure 2).

  • Classification inconsistency is the root problem in cross-study comparison: Different studies define AtB/ABC using different subsets of CD27⁻, CD21lo, CD11c⁺, T-bet⁺, FcRL5⁺ — often without IgD measurement. This makes it impossible to compare AtB/ABC frequencies across diseases or to assign function from phenotype alone. Sanz (2025) recommends comprehensive phenotyping with at least IgD, CD27, and one of {CD21/CXCR5} × {CD11c/T-bet/FcRL5} to resolve subsets properly (see Sanz2025 - Human Atypical B Cells Overview, review).

  • Ansari2025 panel — first dengue EF B cell panel with CD21/CD11c resolution: Two multi-color panels used on acute dengue PBMCs: (1) T cell panel: CXCR5, PD-1, CD38, HLA-DR, CD4, CD8, CD45RA — resolves Tph (CXCR5⁻PD-1⁺) vs. cTfh (CXCR5⁺PD-1⁺) within CD38⁺HLA-DR⁺ activated CD4⁺ T cells. (2) B cell panel: IgD, CD27, CD21, CD11c, CD38, CD20, Ki67, CD71, CXCR3 — resolves DN gate (IgD⁻CD27⁻) with CD21 vs. CD11c for EF phenotype (CD21⁻CD11c⁺ ≈ DN2) and defines plasmablasts as CD20⁻CD38⁺⁺CD27⁺Ki67⁺CD71⁺CXCR3⁺. Includes IgD (passes Sanz2025 audit). Lacks CXCR5 in the B cell panel — cannot formally subdivide DN1/DN2 by CXCR5; uses CD21/CD11c as equivalent (per Woodruff2020). Lacks intracellular T-bet — cannot confirm T-bet expression on EF B cells (see Ansari2025 - Peripheral T Helper Subset Drives B Cell Response in Dengue, n=170 acute dengue).

  • Woodruff2020 Table 1 — complete standardised B cell population definitions: The most comprehensive published lookup table for B cell gating, defining 5 primary and 14 secondary populations with precise marker criteria. Primary: Tr (CD19⁺CD27⁻CD38^intCD24⁺), N (CD19⁺CD27⁻CD38⁻CD24⁻IgD⁺), DN (CD19⁺CD27⁻CD38⁻CD24⁻IgD⁻), M (CD19⁺CD27⁺CD38⁻/lo), ASC (CD19⁺CD27⁺CD38^hi). Secondary: Tr→CD21lo/CD21hi; N→aN(CD11c⁺)/rN(CD11c⁻); DN→DN1(CD11c⁻CD21⁺)/DN2(CD11c⁺CD21⁻)/DN3(CD11c⁻CD21⁻); M→sM/usM/mM/dM by IgM/IgD; ASC→CD138⁻/CD138⁺. This table is the reference standard for designing dengue EF pathway panels (see Woodruff2020 - EF B Cell Responses in COVID-19, Table 1; see also Spectral Flow Cytometry for the 24-marker spectral implementation).

  • Intracellular T-bet staining integrated with surface phenotyping: True-Nuclear Transcription Factor Buffer Set (BioLegend) used for intracellular T-bet staining combined with the spectral surface panel. This validated the T-bet hierarchy (aN > DN2 > DN1 > rN) in an infection context (see Woodruff2020 - EF B Cell Responses in COVID-19).

  • Wrammert2012 panel (5-color, whole blood) — simplest dengue plasmablast panel: CD19-FITC (555412, Pharmingen), CD38-PE (555460, Pharmingen), CD3-PerCP (340663, Pharmingen), CD20-PerCP (347674, Pharmingen), CD27-APC (17-0279-73, eBiosciences). Whole blood staining with erythrocyte lysis (BD FACS lysing solution) and 2% phosphonoformic acid fixation. Gating: CD19⁺CD3⁻ → CD20⁻/low → CD27^high CD38^high on extended lymphocyte gate (to include blasting cells). Absolute counts by BD Trucount bead system. This is the minimum viable dengue plasmablast panel — 5 markers identify PBs but cannot resolve memory subsets (no IgD), EF populations (no CD21, CD11c, CXCR5), or proliferation/apoptosis (no Ki-67, caspase-3). Every subsequent dengue panel in this wiki adds markers beyond this foundation (see Wrammert2012 - Plasmablast Responses in Acute Dengue, n=46 dengue + controls).

  • GarciaBates2013 panel (multi-color, LSRII) — earliest dengue B cell subset panel with severity stratification: Markers: CD19 (HIB19), CD20 (2H7), CD10 (HI10a), CD27 (M-T271), CD69 (L78), CD95 (DX-2), CD3 (SP34-2), CD21 (B-ly4), CD38 (AT-1, Stem Cell Technologies), Ki-67 (B56, intracellular), active caspase-3 (C92-605, intracellular), LIVE/DEAD (Invitrogen). Gating: (1) Live cells → CD19⁺CD3⁻ → CD10⁻ (mature B cells); (2) CD27 vs. CD21 defines naive (CD27⁻CD21⁺), resting memory (CD27⁺CD21⁺), atypical memory (CD27⁻CD21⁻); (3) CD27⁺CD21⁻ fraction further resolved by CD20 vs. CD38 into plasmablasts (CD20⁻CD38⁺) and activated memory (CD20⁺CD38⁻/lo). Lacks IgD (fails Sanz2025 IgD audit — cannot confirm naive vs. unswitched memory within CD27⁻ gate). Lacks CD11c, CXCR5, T-bet (cannot resolve DN1/DN2). Includes Ki-67 and active caspase-3 for proliferation/apoptosis — unique among dengue panels in this wiki (see GarciaBates2013 - Plasmablast Response and Dengue Severity, LSRII, n=84 dengue + controls).

  • Scharer2019 intracellular TF staining — PD-1 and ATF3 validated by flow: Beyond subset sorting, Scharer2019 used conventional flow cytometry for protein-level validation of two novel EF markers: (1) PD-1 surface staining on sorted subsets (mean ~60% PD-1⁺ on DN2 vs. ~10% rN, ~20% aN, ~15% SM; n=4 SLE); (2) ATF3 intracellular staining (significantly elevated MFI in rN, aN, SM, and DN2 in SLE vs. HC; P=0.034, 0.034, 0.033, 0.048 by Wilcoxon rank-sum). These represent practical flow cytometry readouts for EF pathway activation that could be added to dengue panels (see Scharer2019 - Epigenetic Programming in SLE B Cells).

  • Appanna2016 panel (5-marker sort panel + antigen-specific probes, FACSAria) — first dengue panel with CD138 and live virus antigen probes: Markers: CD19, CD20, CD27, CD38, CD138 for B cell subset sorting; Alexa Fluor-labelled live DENV-1, -2, -3 virions for antigen-specific MBC identification. Gating: (1) Plasmablasts: CD19⁺CD20⁻CD27^hiCD38^hi (acute phase, days 3–7); (2) DENV-specific MBCs: CD19⁺CD20⁺CD27⁺, gated by binding to fluorescent live DENV particles (convalescence, days 16–166). Includes CD138 — first dengue study to incorporate this marker in sorting, though used only to refine subset boundaries rather than CD138⁺/CD138⁻ ASC subdivision (cf. Tipton2015, Woodruff2020). Lacks IgD (fails Sanz2025 audit — cannot distinguish naive from unswitched memory within CD27⁻). Lacks CD21, CD11c, CXCR5, T-bet (cannot resolve DN subsets or EF populations). The live virus antigen probe approach enables direct identification of DENV-binding B cells without recombinant protein; however, it selects for surface-accessible epitopes on intact virions and cannot resolve specificity for individual viral proteins (E vs. prM vs. NS1) by flow cytometry alone (see Appanna2016 - Plasmablasts as Subset of Memory B Cell Pool, FACSAria, n=12 dengue).

  • Singh2026 panel (12-color, BD LSRFortessa) — first dengue MBC subset panel with antigen-specific detection: Dump: CD3-V500, CD14-V500, CD16-V500. Viability: Aqua L/D. B cell: CD19-APC-Cy7. Subset: CD20-PerCP-Cy5.5, IgD-V450, IgM-BV605, IgG-BV786, CD21-PE-CF594, CD27-PE-Cy7, CD38-PE. Antigen probes: AF488-DENV (1+2+3), AF647-DENV (1+2+3) — 6-antigen cocktail using whole virions grown on Vero-furin cells, dual-labelled for double-positive gating. DENV-specific B cells defined as AF488+/AF647+ double-positive. Defines 9 B cell subsets: naive (CD20+/IgD+), IgD+/IgM+ naive, class-switched MBC (CD20+/IgD⁻), activated MBC (CD20+/IgD⁻/CD27+/CD21⁻), resting MBC (CD20+/IgD⁻/CD27+/CD21+), atypical MBC (CD20+/IgD⁻/CD27⁻/CD21⁻), IgG+ MBC (CD20+/IgD⁻/IgG+), IgM+ MBC (CD20+/IgD⁻/IgM+), IgD⁻/IgM⁻/IgG⁻ MBC. FMO for CD21 and CD27; no-antigen control for DENV-specific threshold. Includes IgD (passes Sanz2025 audit); lacks CXCR5 and CD11c (cannot resolve DN1/DN2/DN3) (see Singh2026 - DENV-Specific Memory B Cell Subsets).

Contradictions & Debates

  • Conventional panels with limited colour capacity may undercount DN B cells or conflate them with transitional B cells if CD10 or CD24 are not included. The Wei2007 data (Fig. 2B) show that DN cells are CD10⁻, which resolves this ambiguity — but earlier studies using 4- or 5-color panels may have misclassified these cells.
  • The DN1/DN2 distinction requires CXCR5 staining, which was not included in any of the prior Sanz lab panels (Wei2007, Anolik2004, Tipton2015). Studies using IgD/CD27 gating alone capture both DN1 and DN2 without discrimination — this affects interpretation of all prior DN frequency data.
  • Lack of IgD is a common omission: Many studies defining AtB/ABC do not include IgD in the panel, which means they cannot distinguish naïve-derived aNAV (IgD⁺) from DN2 (IgD⁻) among CD11c⁺ T-bet⁺ cells. This conflation is a major source of confusion in the literature (see Sanz2025 - Human Atypical B Cells Overview).

Bm Classification, FACS Sorting, Double-Negative B Cell, DN2 B Cell, Activated Naive B Cell, Memory B Cell, Plasmablast, CD27, IgD, CD38, CD19, CD20, CXCR5, CD11c, T-bet, PD-1, ATF3

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