Analyzing Flow Cytometry

1. Specimen type — Peripheral blood, bone marrow aspirate, lymph node, body fluid, or tissue disaggregate? Default: peripheral blood.
2. Clinical indication — New diagnosis, relapse assessment, MRD monitoring, staging, or T-cell subset enumeration? Default: new diagnosis workup.
3. Panel requested — Acute leukemia panel, mature B-cell panel, T-cell panel, plasma cell panel, or MRD panel? Default: acute leukemia screening panel.
4. CBC/differential — Current CBC with automated differential available? Default: not provided; flag [VERIFY].
5. Morphology correlation — Is a concurrent peripheral smear or bone marrow aspirate smear reviewed? Default: pending.
6. Prior flow cytometry — Are there prior immunophenotyping results for comparison? Default: none on file.
7. Specimen viability — Time from collection, anticoagulant used (EDTA vs. heparin), specimen temperature? Default: EDTA, < 24 hours.

Documents to Request

• CBC with automated differential
• Peripheral blood smear or bone marrow aspirate smear review
• Prior flow cytometry reports
• Prior pathology/cytogenetics/molecular reports
• Clinical notes including suspected diagnosis and treatment history
• Panel configuration and antibody-fluorochrome conjugate assignments
• Instrument QC records (daily bead calibration, compensation matrix)

Step 1: Pre-Analytical Assessment and Quality Check

Validate specimen and instrument quality before interpretation:

• Specimen viability: > 90% viability by 7-AAD or propidium iodide exclusion for diagnostic interpretation; < 70% viability compromises antigen detection.
• Cell count: Minimum 100,000 events acquired for screening panels; 500,000-1,000,000 events for MRD assays (sensitivity target: 10^-4 to 10^-5).
• Compensation verification: Confirm single-stain compensation controls were run and compensation matrix applied correctly. Flag any spillover artifacts.
• Instrument QC: Verify daily calibration beads are within acceptable CV (< 3% for fluorescence channels per manufacturer specifications).

Step 2: Gating Strategy and Population Identification

Apply systematic gating following ICCS/Euroflow consensus recommendations:

1. Singlet discrimination: FSC-H vs. FSC-A to exclude doublets and aggregates.
2. Viability gating: Exclude dead cells (7-AAD or viability dye negative).
3. CD45 vs. SSC backbone: Identify major populations:
   • Lymphocytes: CD45 bright, SSC low
   • Monocytes: CD45 moderate-bright, SSC intermediate
   • Granulocytes: CD45 moderate, SSC high
   • Blasts: CD45 dim, SSC low
   • Erythroid precursors: CD45 negative-dim, SSC low
4. Lineage assignment for abnormal populations:
   • B-cell lineage: CD19, CD20, CD22, CD79a, cytoplasmic CD79a
   • T-cell lineage: CD2, CD3 (surface and cytoplasmic), CD5, CD7
   • Myeloid lineage: CD13, CD33, CD117, MPO
   • Monocytic: CD14, CD64, CD36
   • Megakaryocytic: CD41, CD61
   • Erythroid: CD71, CD235a (glycophorin A)

Step 3: Immunophenotype Interpretation by Disease Category

Acute Leukemia Classification

Mature B-Cell Neoplasms — Key Discriminators

MRD Detection

• Apply leukemia-associated immunophenotype (LAIP) approach or different-from-normal (DfN) approach.
• Report MRD as percentage of evaluable cells and absolute events counted.
• Sensitivity: state achieved sensitivity level (e.g., 0.01% = 10^-4).

Step 4: Integration with Morphology and Molecular Data

• Correlate immunophenotype with peripheral smear or bone marrow morphology (blast percentage, Auer rods, dysplasia).
• Integrate cytogenetic results: t(9;22) BCR::ABL1, t(8;21) RUNX1::RUNX1T1, inv(16) CBFB::MYH11, t(15;17) PML::RARA.
• Cross-reference molecular findings: FLT3-ITD, NPM1, CEBPA, TP53, IKZF1 for risk stratification.
• For mature B-cell neoplasms, integrate FISH for t(11;14) cyclin D1 (MCL), t(14;18) BCL2 (FL), and MYC rearrangement.

Step 5: Report Construction

Structure the flow cytometry report per ICCS recommendations:

1. Specimen information: Type, collection time, anticoagulant, viability.
2. Clinical indication: Stated reason for testing.
3. Panel summary: List antibodies tested with fluorochrome assignments.
4. Findings: Describe each abnormal population with percentage, antigen expression profile, and light chain status.
5. Interpretation: Provide a conclusive statement linking immunophenotype to WHO diagnostic category.
6. Correlation statement: Note agreement or discordance with morphology/molecular data.
7. Comment: Recommendations for additional testing (cytogenetics, molecular, biopsy).

Checkpoint B: Post-Draft Alignment (Mandatory)

1. Were all populations identified using standardized CD45/SSC gating with singlet and viability discrimination?
2. Does the immunophenotype assignment match a recognized WHO 5th edition entity?
3. Is light chain restriction (kappa:lambda ratio) quantified for B-cell populations?
4. Are MRD results reported with sensitivity level and event count?
5. Is there documented correlation with concurrent morphology and any available molecular data?

Quality Audit

• Specimen viability assessed and documented
• Minimum event acquisition thresholds met for assay type
• Compensation matrix validated with single-stain controls
• CD45/SSC backbone gating strategy applied per ICCS guidelines
• Singlet and viability discrimination gates documented
• Lineage assignment supported by multiple markers (not single-antigen calls)
• Light chain restriction quantified with kappa:lambda ratio
• Immunophenotype matched to WHO 5th edition diagnostic entity
• MRD sensitivity level explicitly stated
• Morphology correlation documented
• Molecular/cytogenetic integration addressed
• Instrument QC records reviewed and within specification
• Report signed by qualified hematopathologist

Guidelines

• Never assign lineage based on a single marker; require at least two lineage-defining antigens per WHO/ICCS criteria
• Report light chain restriction quantitatively (kappa:lambda ratio) rather than qualitatively; a ratio > 3:1 or < 0.3:1 supports clonality
• For MRD, always state the achieved sensitivity level and whether the result is below, at, or above the detection threshold
• Use WHO 5th edition terminology for all diagnostic classifications; retire outdated FAB nomenclature in final reports
• Flag specimens with < 70% viability as compromised and note potential for false-negative antigen loss
• Document the gating strategy in sufficient detail that another hematopathologist could reproduce the analysis
• Correlate flow cytometry with morphology and molecular results in every case; never issue a standalone flow report without context
• Maintain daily instrument QC logs and review compensation matrices at least weekly per CAP FLO checklist requirements