Flow Cytometry — Hemato-Oncology Cheat Sheet

Universal Gating Hierarchy

FSC-A vs SSC-A viable cells

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FSC-H vs FSC-A singlets

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Viability⁻ 7-AAD / PI / DAPI / Live-Dead

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CD45⁺ leukocytes confirmed

CD45 vs SSC — leukocyte differential

CD45/SSC POPULATIONS

Gate	CD45	SSC	Identity
R1	Bright	Low	Lymphocytes
R2	Bright	Mid	Monocytes
R3	Dim	High	Granulocytes
R4	Dim/Neg	Low-Mid	Blasts
R5	Neg	Low	Erythroid/debris

⚠ BLAST GATE: CD45^dim/SSC-low — always include; critical for AML/ALL

Lymphocyte Subset Panel (T/B/NK)

Gate on: CD45^bright/SSC-low → Lymphs

Population	Markers	Normal %
TT cells	CD3⁺	56–84%
TCD4 Helper	CD3⁺CD4⁺	28–57%
TCD8 Cytotox	CD3⁺CD8⁺	10–39%
CD4:CD8 ratio	1.0–3.5 ↓ HIV, immunodeficiency
BB cells	CD19⁺CD3⁻	6–25%
NKNK cells	CD16⁺CD56⁺CD3⁻	5–27%
NKNKT-like	CD3⁺CD56⁺	variable

GATING CHAIN — T SUBSETS

CD45⁺→ CD3⁺→ CD4⁺ helper

CD45⁺→ CD3⁺→ CD8⁺ cytotoxic

CD45⁺→ CD3⁻→ CD19⁺ B cell

CD45⁺→ CD3⁻→ CD16/56⁺ NK

Leukemia Blast Immunophenotyping

AML Myeloid Blasts

CD45^dim→ CD34⁺ CD117⁺ CD33⁺ CD13⁺

HLA-DR⁺ (except APL M3) · MPO⁺ · CD64 monocytic

B-ALL Precursor B

CD45^dim/neg→ CD34⁺ CD19⁺ CD10⁺ CD20^−/dim

TdT⁺ · CD22⁺ · cIgM variable · surface Ig⁻

T-ALL Precursor T

CD45^dim→ CD7⁺ cCD3⁺ sCD3^−/+ CD2⁺

TdT⁺ · CD1a (cortical) · CD4/CD8 double pos (cortical)

CML Blast Crisis

BCR-ABL⁺ background · blast % defines phase · may express myeloid or lymphoid markers

Mature B-cell Lymphoma / CLL Panel

Gate: CD45^bright → CD19⁺ → assess clonality (κ/λ)

CLL/SLL · CD5⁺ CD23⁺ CD20^dim CD19⁺

sIg^dim, CD200⁺

MCL · CD5⁺ CD23⁻ CD20^bright

Cyclin D1⁺, t(11;14)

Follicular · CD10⁺ CD19⁺ CD5⁻

bcl-2⁺, t(14;18)

DLBCL · CD19⁺ CD20⁺ CD10^±

Large cells, high SSC

Hairy Cell · CD11c⁺ CD25⁺ CD103⁺ CD123⁺

TRAP⁺, "hairy" morphology

Marginal Zone · CD19⁺ CD5⁻ CD10⁻ CD23⁻

Exclusion dx

Burkitt · CD10⁺ CD20⁺ TdT⁻

Ki-67 ~100%, t(8;14)

CLONALITY: κ:λ ratio normal 1:2–3:1 · ratio >6:1 or <1:3 → clonal · assess on CD19⁺ gate

Plasma Cell / MDS / PNH / MRD

PLASMA CELL MYELOMA

CD38^bright CD138⁺ CD19⁻ CD56⁺

Normal PC: CD19⁺CD56⁻ · Myeloma: CD19⁻CD56⁺CD45^−/dim · cIg light chain restriction

MDS DYSPLASIA PANEL

Aberrant myeloid maturation: CD34⁺ blast %, CD13/CD16 maturation plot (neutrophil dysmaturation), CD64 upregulation on monocytes, aberrant CD56 on granulocytes

ALOT: CD45, CD34, CD117, HLA-DR, CD13, CD16, CD11b, CD64, CD56, CD14, CD15

PNH (Paroxysmal Nocturnal Hemoglobinuria)

GPI-anchored protein loss: CD59⁻ on RBCs, CD55⁻/CD59⁻ on granulocytes/monocytes · FLAER — most sensitive for WBCs

Report type I/II/III populations · threshold: >1% clone significant

B-ALL MRD

CD19/CD10/CD34/CD38/CD45/CD20/CD58/CD123/CD81 · 6–8 color minimum · sensitivity 10⁻⁴; NGS 10⁻⁶

AML MRD

CD34/CD117/HLA-DR + lineage aberrancy panel · LAIPs defined at diagnosis · sensitivity 10⁻⁴–10⁻⁵

Mature T-cell / NK Lymphoma

PTCL-NOS · CD3⁺ aberrant loss of CD5/CD7 · CD4 or CD8

ALCL · CD30⁺ CD25⁺ · ALK^+/− · CD3^−/+

Sézary / MF · CD4⁺CD26⁻CD7⁻ · CD4:CD8 >10:1 in blood

LGL Leukemia · CD8⁺CD57⁺CD16⁺ · TCR Vβ clonality

NK/T lymphoma · CD56⁺CD3⁻ · cCD3ε⁺ · EBV⁺

Key aberrancies: loss of CD5, CD7, CD2, CD3 · abnormal CD4:CD8 · CD56 gain on T cells

QC, Controls & Practical Tips

Item	Practice
Daily QC	8-peak bead (or equiv.) · CV <5% target · track MFI shift
FMO Controls	Required for dim populations; viability, κ/λ, CD45^dim blasts
Isotypes	Use sparingly; FMO preferred for gating; isotypes for specificity confirmation
Compensation	Single-stain controls · match fluorochrome to capture channel · use same bead lot
Specimen	PB/BM <24h preferred · BM <4h ideal · EDTA preferred · lyse/wash vs bulk lysis
Event count	Lymphs: ≥2,000 · Blasts/MRD: ≥50,000–100,000 total

PANEL TIP: Brightest fluorochrome on dimmest marker · dump channel (dead + CD14 + CD19 for T cell purity)

CD Antigen Quick Reference

CD	AKA	Lineage/Use
CD2	LFA-2	Pan-T, NK
CD3	TCR complex	Pan-T (surface = mature)
CD4	—	Helper T, monocytes
CD5	—	Pan-T; B: CLL/MCL
CD7	—	Pan-T, NK; T-ALL
CD8	—	Cytotoxic T, NK subset
CD10	CALLA	B-ALL, Follicular, Burkitt
CD16	FcγRIII	NK, neutrophils, monocytes
CD19	—	Pan-B (earlier than CD20)
CD20	MS4A1	Mature B; rituximab target
CD23	FcεRII	CLL (pos); MCL (neg)
CD33	Siglec-3	Myeloid; AML; gemtuzumab
CD34	—	Progenitors/blasts; HSC
CD38	—	Plasma cells (bright); daratumumab
CD45	LCA	Pan-leukocyte; gating anchor
CD56	NCAM	NK; neuroendocrine; aberrant myeloid
CD117	c-KIT	Mast cells, AML blasts, HSC
CD138	Syndecan-1	Plasma cells; myeloma