Flow Cytometry Analysis
Immunophenotype interpretation for suspected leukaemia, lymphoma or plasma cell neoplasm
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Flow Cytometry Immunophenotyping Interpretation
Flow Cytometry Immunophenotyping Interpretation: Flow Cytometry Analysis → Specimen Assessment → If follow-up (MRD) sample: use a validated MRD assay → ...
Pathway Overview
11 steps
Algorithm Steps
11 total
01Start 02Action Specimen Assessment
Check sample quality and treatment history before analysis
- Check viability with a viability dye; poor viability can lose fragile cells (for example large B cells)
- Gate on viable single cells: exclude debris and doublets
- Confirm adequate cell yield for the panel
- Record targeted therapy: anti-CD19 (blinatumomab, CAR-T), anti-CD20 (rituximab), anti-CD38 (daratumumab, isatuximab)
- Note sample type (blood, marrow, node, fluid) and haemodilution of marrow
03Action If follow-up (MRD) sample: use a validated MRD assay
Check first for anti-CD19, CAR-T, anti-CD22 (inotuzumab) or anti-CD38 therapy. Do not use a diagnostic screening panel to call MRD negative
- Compare with the diagnostic immunophenotype and look for difference from normal
- After anti-CD19 therapy or CAR-T: gate B cells on CD22, CD24 and HLA-DR. After inotuzumab, CD22 can be lost: do not rely on CD22. After anti-CD38: use multiepitope CD38 and CD138
- AML (ELN 2021): acquire more than 500,000 CD45+ cells; positive if 0.1% or more of CD45+ cells
- APL, NPM1-mutated or core-binding-factor AML: use molecular MRD (qPCR or dPCR) (ELN 2021)
- Regenerating or stressed marrow (after chemotherapy, transplant or G-CSF) can show transient aberrant myeloid phenotypes: say so in the report; repeat in 2-4 weeks if clinically indicated
- Myeloma: EuroFlow NGF or equivalent, sensitivity at least 10^-5
- Report the lower limit of detection and quantification (LLOD, LLOQ)
04Action Population Identification
Gate all populations and compare each with its normal maturation pattern
- CD45 vs SSC: lymphocytes, monocytes, granulocytes, blasts
- Blasts: usually CD45 dim, low SSC; some B-ALL blasts are CD45 negative
- APL cells and monocytic cells often fall outside the blast gate
- Check mature B, T and NK cells and plasma cells in every sample
- Regenerating marrow (after chemotherapy, transplant or G-CSF): increased normal precursors can mimic leukaemia or MRD
05Warning APL pattern? Emergency: phone the treating team now
Suspected APL: do not wait for genetic confirmation
- Classic APL: high SSC, CD34-, HLA-DR-, CD11b-, CD117+, CD13+, CD33+. Variant APL: lower SSC, may be CD34+ and CD2+
- ELN 2019: ATRA and blood products for coagulopathy start on suspicion; avoid central lines and lumbar puncture
- Confirm PML::RARA urgently (FISH or PCR). NPM1-mutated AML can mimic APL (CD34-, HLA-DR-)
06Action If blasts are increased or abnormal: assign lineage
Children and regenerating marrow: normal B precursors (haematogones) mimic B-ALL but show a normal maturation pattern. After anti-CD19 therapy or CAR-T, B-ALL can lose CD19. New acute leukaemia: tell the treating team the same day
- B-ALL: CD19+, CD22+, cyCD79a+, TdT+; CD10 and CD34 variable
- T-ALL: cyCD3+, CD7+; TdT, CD1a, CD4, CD8 variable. ETP-ALL: CD1a-, CD8-, CD5 weak, stem or myeloid markers
- AML: CD34, CD117, HLA-DR, CD13, CD33, MPO; monocytic: CD64, CD14, CD11b, CD36
- MPAL (WHO-HAEM5): B = CD19 strong with 1 of CD10, CD22, CD79a strong, or CD19 weak with 2
- MPAL T = cyCD3 or sCD3 above 50% of mature T-cell intensity. Myeloid = MPO above 50% of neutrophil intensity, or 2 monocytic markers (NSE, CD11c, CD14, CD64, lysozyme)
- BPDCN: CD123 plus 1 other pDC marker (TCF4, TCL1, CD303, CD304) with CD4 and/or CD56; or 3 pDC markers with CD3, CD14, CD19, CD34, lysozyme and MPO negative
- After CD19-targeted therapy: gate B blasts on CD22, CD24 and HLA-DR. After inotuzumab, do not rely on CD22
07Action If mature B cells are abnormal: characterise
Clonal if kappa or lambda restricted, or no surface light chain. After rituximab, CD20 may be absent: gate on CD19
- CD10+, CD38 bright, BCL2 negative, surface Ig+: consider Burkitt; tell the treating team the same day
- CLL: CD19+, CD5+, CD23+, CD200+, CD43+; CD20, CD79b and light chain weak (ERIC/ESCCA)
- CLL needs 5 x 10^9/L clonal B cells in blood (or nodal disease); fewer, with no other features: MBL
- MCL: usually CD5+ (can be CD5-, for example leukaemic non-nodal MCL), CD23- or weak, CD200- or weak, CD20 and light chain bright. Confirm cyclin D1 (IHC) or t(11;14) (FISH); if cyclin D1 negative: SOX11 IHC and CCND2 or CCND3 studies
- HCL: CD103+, CD25+, CD11c bright, CD123+. CD25- and CD123-: consider splenic B-cell lymphoma with prominent nucleoli
- Follicular and other germinal centre lymphomas: CD10+; BCL2 and BCL6 by IHC on tissue
- Marginal zone: CD5-, CD10-; diagnosis of exclusion, needs tissue and clinical correlation
- Large B cells can be lost in processing: a negative result does not exclude DLBCL
08Action If T or NK cells are abnormal: characterise
Reactive T cells can alter the CD4:CD8 ratio or lose CD7; confirm clonality
- Aberrant: loss or abnormal intensity of CD2, CD3, CD5 or CD7; abnormal CD4:CD8 pattern
- Clonality: TRBC1 or TCR V-beta repertoire by flow, or TCR gene rearrangement
- T-LGL: CD3+, CD8+, CD57+, CD16 variable
- Sezary cells: CD4+ with loss of CD7 and/or CD26
- ATLL: CD4+, CD25 bright, CD7-; confirm HTLV-1 serology
- Nodal TFH lymphoma (AITL): CD4+, CD10+, PD1+, surface CD3 dim or lost; needs node biopsy
- NK cells: CD3-, CD16 and/or CD56+; KIR restriction suggests a clonal NK disorder
09Action If plasma cells are abnormal: assess
Gate on CD38 bright, CD138+. After anti-CD38 therapy (daratumumab, isatuximab), CD38 is masked: use multiepitope CD38 and CD138. Haemodilute marrow: flow underestimates the plasma cell %
- Clonal: cytoplasmic kappa or lambda restriction
- Aberrant: CD19-, CD45- or dim, CD56+, CD27 low, CD81-, CD117+
- Normal plasma cells are polyclonal; about 30% are CD19- and a small subset is CD56+. Call a clone only with light chain restriction
- Use aspirate morphology or trephine, not flow, for the plasma cell %
10Action Clinical and Morphologic Correlation
Integrate all findings before a diagnosis
- Review blood film, marrow aspirate and trephine, or tissue biopsy
- Blast % for diagnosis comes from the morphology count; flow % can differ
- Cytogenetics, FISH and molecular studies as indicated
- Clinical context: blood count, nodes, organomegaly, prior treatment
11Outcome Integrated Report and Diagnosis
Classify by WHO-HAEM5 or ICC 2022. If no abnormal population is found, say so: this does not exclude lymphoma or focal disease
Guideline Source
WHO Classification of Haematolymphoid Tumours, 5th edition (WHO-HAEM5): lymphoid (Alaggio 2022) and myeloid (Khoury 2022) summaries
Clinical Safety Information
Clinical Decision Support — Not a Substitute for Clinical Judgment
Individual patient factors may require deviation from these recommendations.
Known Limitations
- Flow cytometry alone does not give the diagnosis: integrate morphology, genetics and clinical findings (WHO-HAEM5 or ICC 2022)
- A normal result does not exclude lymphoma (large B cells, Hodgkin lymphoma, focal marrow disease): biopsy if suspected
- Panels, gating and MRD sensitivity vary by laboratory: use locally validated assays
- Cyclin D1, BCL2, BCL6 and other tissue markers need immunohistochemistry
- Targeted antibody and CAR-T therapy can remove or mask CD19, CD20 and CD38
Contraindicated Populations
Non-neoplastic flow cytometry (lymphocyte subsets for immunodeficiency, PNH testing, CD34 stem cell counts): use the specific protocol
Applicable Regions
USAUUKEU
AU: Classify by WHO-HAEM5 or ICC 2022. Report suspected APL to the treating team at once.
EU: EuroFlow standardised panels; ERIC/ESCCA CLL marker set (Cytometry B 2018).
UK: BSH (formerly BCSH) guideline on multicolour flow cytometry in haematological neoplasms, Br J Haematol 2014;165:455-488.
US: 2006 Bethesda International Consensus (ICCS) recommendations on immunophenotypic analysis of haematolymphoid neoplasia.
Version 2Next review: 2027-09-30
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Related Resources
Frequently Asked Questions
What is the Flow Cytometry Immunophenotyping Interpretation?
The Flow Cytometry Immunophenotyping Interpretation is a diagnostic clinical algorithm for Pathology. It provides a structured decision tree to guide clinical decision-making, based on WHO Classification of Haematolymphoid Tumours, 5th edition (WHO-HAEM5): lymphoid (Alaggio 2022) and myeloid (Khoury 2022) summaries.
What guideline is the Flow Cytometry Immunophenotyping Interpretation based on?
This algorithm is based on WHO Classification of Haematolymphoid Tumours, 5th edition (WHO-HAEM5): lymphoid (Alaggio 2022) and myeloid (Khoury 2022) summaries (DOI: 10.1038/s41375-022-01620-2).
What are the limitations of the Flow Cytometry Immunophenotyping Interpretation?
Known limitations include: Flow cytometry alone does not give the diagnosis: integrate morphology, genetics and clinical findings (WHO-HAEM5 or ICC 2022); A normal result does not exclude lymphoma (large B cells, Hodgkin lymphoma, focal marrow disease): biopsy if suspected; Panels, gating and MRD sensitivity vary by laboratory: use locally validated assays; Cyclin D1, BCL2, BCL6 and other tissue markers need immunohistochemistry; Targeted antibody and CAR-T therapy can remove or mask CD19, CD20 and CD38. Individual patient factors may require deviation from these recommendations.
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