🏥 Immunophenotyping

Comprehensive guide to identifying and quantifying immune cell populations using surface and intracellular markers in flow cytometry.

Home → Use Cases → Immunophenotyping

Table of Contents

  1. Introduction to Immunophenotyping
  2. Major Immune Cell Populations
  3. The Backbone Panel Concept
  4. CD4 Monitoring in HIV/AIDS
  5. Leukemia & Lymphoma Immunophenotyping
  6. Multi-Color Panel Design
  7. Sample Preparation
  8. Controls for Immunophenotyping
  9. Gating Strategy & Data Analysis
  10. Troubleshooting

1. Introduction to Immunophenotyping

Immunophenotyping is the process of using fluorochrome-conjugated antibodies to identify and quantify cells based on the antigens expressed on their surface or within their cytoplasm and nucleus. In the context of flow cytometry, immunophenotyping enables the rapid, multiparametric analysis of individual cells in suspension, providing both the identity and the relative or absolute count of distinct cell populations within a heterogeneous sample.

What Does Immunophenotyping Measure?

Every immune cell expresses a characteristic set of proteins—receptors, adhesion molecules, enzymes, and structural components—that define its lineage, maturation stage, and functional state. Immunophenotyping leverages monoclonal antibodies that bind these proteins with high specificity. When conjugated to fluorochromes and analyzed on a flow cytometer, these antibodies reveal which antigens are present, at what density, and in what combination on each cell.

The CD Nomenclature System

The Cluster of Differentiation (CD) system provides a standardized nomenclature for cell surface molecules. Originating from the First International Workshop on Human Leukocyte Differentiation Antigens in 1982, the CD system has grown to catalog over 400 recognized molecules (as of CD371). Each CD number designates a specific surface molecule identified by a cluster of monoclonal antibodies that share reactivity with the same antigen.

Key Point — The CD System: CD markers are not exclusive to a single cell type. For example, CD4 is expressed on helper T cells but also on monocytes (at lower density). Immunophenotyping relies on combinations of markers, not individual antigens, to definitively identify cell populations. A mature helper T cell, for instance, is defined as CD45+ CD3+ CD4+ CD8−.

Brief History

Clinical and Research Applications

Immunophenotyping is indispensable in a wide range of settings:

2. Major Immune Cell Populations

Peripheral blood contains a diverse collection of immune cells, each identifiable by a characteristic combination of surface markers and light scatter properties. The table below summarizes the major leukocyte populations encountered in routine immunophenotyping.

Leukocyte Lineage Overview

Cell Type Key Surface Markers FSC/SSC Profile Function
Helper T Cells CD45+ CD3+ CD4+ Low FSC / Low SSC Coordinate adaptive immune responses; secrete cytokines to activate B cells, macrophages, and cytotoxic T cells
Cytotoxic T Cells CD45+ CD3+ CD8+ Low FSC / Low SSC Kill virus-infected and tumor cells via perforin/granzyme and Fas/FasL pathways
B Cells CD45+ CD19+ CD20+ Low FSC / Low SSC Produce antibodies; antigen presentation; immunological memory
NK Cells CD45+ CD3− CD16+ CD56+ Low–Mid FSC / Low SSC Innate cytotoxicity against tumor and virus-infected cells without prior sensitization
NKT Cells CD45+ CD3+ CD16+ CD56+ Low FSC / Low SSC Bridge innate and adaptive immunity; rapid cytokine production
Monocytes CD45+ CD14+ CD33+ HLA-DR+ High FSC / Mid SSC Phagocytosis; antigen presentation; differentiate into macrophages and dendritic cells
Neutrophils CD45+ CD16+ CD66b+ Mid FSC / High SSC First responders to bacterial infection; phagocytosis, degranulation, and NET formation
Eosinophils CD45+ CD193+ (CCR3) Siglec-8+ Mid FSC / Very High SSC Parasite defense; allergic inflammation; granule-mediated cytotoxicity
Basophils CD45+ CD123+ CD203c+ HLA-DR− Low FSC / Mid SSC Allergic responses; histamine and IL-4 release; rare population (<1% of WBCs)
Plasmacytoid DCs CD45+ CD123+ CD303+ HLA-DR+ Low FSC / Low SSC Type I interferon (α/β) production in response to viral nucleic acids

T Cell Subset Immunophenotyping

Beyond the basic CD4/CD8 distinction, T cells can be further subdivided by differentiation state and functional capacity. The following markers are commonly used to define T cell maturation subsets:

T Cell Subset Phenotype Characteristics
Naïve (TN) CD45RA+ CCR7+ CD27+ CD95− Antigen-inexperienced; high proliferative potential; home to secondary lymphoid organs
Stem Cell Memory (TSCM) CD45RA+ CCR7+ CD27+ CD95+ Self-renewing; superior reconstitution capacity; important in adoptive cell therapy
Central Memory (TCM) CD45RA− CCR7+ CD27+ Home to lymph nodes; high proliferative capacity; produce IL-2 upon restimulation
Effector Memory (TEM) CD45RA− CCR7− CD27− Circulate to peripheral tissues; rapid effector function; produce IFN-γ and TNF-α
Terminally Differentiated (TEMRA) CD45RA+ CCR7− CD27− Re-express CD45RA; high cytotoxic capacity; limited proliferation; senescence-associated
Tip: When phenotyping T cell subsets, always include a viability dye. Dead cells bind antibodies non-specifically and can masquerade as rare memory populations, leading to significant overestimation.

3. The Backbone Panel Concept

A backbone panel is a standardized core set of markers used to identify major leukocyte lineages in every experiment. By keeping these markers constant across studies and timepoints, laboratories achieve consistent gating, reproducible results, and the ability to compare data longitudinally. Additional markers can be added to the backbone to address specific research or clinical questions.

The TBNK Panel

The most widely used backbone panel in clinical immunophenotyping is the TBNK panel, which identifies T cells, B cells, and NK cells in a single tube. The classic 6-color TBNK combination is:

Why CD45 Is Essential

CD45 (leukocyte common antigen) is the cornerstone of the backbone panel. In the CD45 vs. SSC gating strategy, distinct leukocyte populations segregate into recognizable clusters:

Tip: Always include CD45 and a viability dye in your backbone panel. CD45 enables a clean leukocyte gate that excludes debris, unlysed red cells, and platelet aggregates. The viability dye ensures dead cells—which bind antibodies non-specifically—are excluded before any downstream analysis. This combination is the foundation of reliable immunophenotyping data.

Extending the Backbone

The backbone concept is modular. For example, a laboratory studying T regulatory cells would keep the TBNK core and add CD25, CD127, and FoxP3. A study of monocyte subsets might add CD14, CD16, and HLA-DR. The backbone stays the same, ensuring consistent lineage identification regardless of the specific research question.

Clinical TBNK

6 colors: CD3, CD4, CD8, CD19, CD16/56, CD45. Single tube, whole blood. Reports absolute counts and percentages.

Extended T Cell Panel

10–14 colors: TBNK backbone + CD45RA, CCR7, CD27, CD28, CD95, CD127, CD25, PD-1. Deep T cell subset profiling.

Myeloid/DC Panel

10–12 colors: CD45 backbone + CD14, CD16, HLA-DR, CD11c, CD123, CD303, CD1c, CD141. Monocyte and DC subsets.

4. CD4 Monitoring in HIV/AIDS

The CD4+ T lymphocyte count is the single most important laboratory marker for assessing immune function in individuals living with HIV. HIV selectively infects and depletes CD4+ T cells, and the rate of decline predicts progression to AIDS-defining illnesses and death. Flow cytometric CD4 counting is the gold standard method used worldwide.

Clinical Significance

CD4 counts guide critical clinical decisions: when to initiate antiretroviral therapy (ART), when to start prophylaxis against opportunistic infections (e.g., Pneumocystis jirovecii pneumonia at CD4 <200 cells/μL), and how to monitor immune reconstitution after ART initiation. While current WHO guidelines recommend treating all HIV-positive individuals regardless of CD4 count (“treat all” policy), CD4 monitoring remains essential for clinical management.

Methods for Absolute CD4 Counting

CD4 Count Interpretation

CD4 Count (cells/μL) Immune Status Clinical Implications
>500 Normal / Mild Suppression Low risk of opportunistic infections; initiate ART per current guidelines; monitor every 6–12 months
350–500 Moderate Suppression Increased risk of bacterial infections; ART strongly recommended; monitor every 3–6 months
200–349 Advanced Suppression High risk of opportunistic infections; begin prophylaxis for Pneumocystis; ART urgent; monitor every 3 months
<200 Severe Immunodeficiency (AIDS) AIDS-defining; high risk of Pneumocystis, toxoplasmosis, cryptococcal meningitis; start cotrimoxazole prophylaxis; urgent ART
<50 Critical Immunodeficiency Very high mortality risk; MAC prophylaxis; CMV retinitis screening; immediate ART with close monitoring for IRIS
Caution — CD4% vs. Absolute Count: The CD4 percentage is more stable than the absolute count because it is less affected by diurnal variation, exercise, stress, and intercurrent illness. In pediatric patients (especially children under 5 years), the CD4 percentage is preferred because normal absolute CD4 counts are much higher in young children and decline with age. A CD4% <25% in a child corresponds approximately to the adult threshold of <350 cells/μL. Always report both values.

WHO Staging and CD4 Correlation

The WHO clinical staging system (Stages 1–4) broadly correlates with CD4 decline. Stage 1 (asymptomatic) typically corresponds to CD4 >500; Stage 2 (mild symptoms) to 350–500; Stage 3 (advanced symptoms) to 200–350; and Stage 4 (AIDS-defining illness) to <200 cells/μL. However, clinical staging alone is insufficient, and CD4 counting provides objective, quantitative assessment of immunosuppression.

5. Leukemia & Lymphoma Immunophenotyping

Immunophenotyping by flow cytometry is integral to the diagnosis, classification, and monitoring of hematological malignancies. The WHO classification of hematolymphoid neoplasms relies heavily on immunophenotypic data alongside morphology, cytogenetics, and molecular studies to define disease entities.

Acute Lymphoblastic Leukemia (ALL)

B-cell ALL (B-ALL): The most common malignancy of childhood. Blasts express CD19, CD10 (CALLA), CD34, TdT, and cytoplasmic CD79a. They are negative for surface immunoglobulin in precursor stages. CD20 expression is variable and may be weak or absent. The presence of CD10 positivity generally confers a more favorable prognosis.

T-cell ALL (T-ALL): Blasts express cytoplasmic CD3 (the earliest T lineage marker), CD7, CD2, CD5, and TdT. Surface CD3 is often negative in immature forms. CD1a expression marks cortical T-ALL, which has a relatively better prognosis. CD4 and CD8 may be co-expressed (double-positive) in cortical thymocyte stages.

Acute Myeloid Leukemia (AML)

AML blasts characteristically express CD13, CD33, CD117 (c-kit), and myeloperoxidase (MPO). HLA-DR is positive in most subtypes but notably absent in acute promyelocytic leukemia (APL, formerly M3). Additional markers help define subtypes: CD14 and CD64 for monocytic differentiation, CD41/CD61 for megakaryoblastic leukemia, and CD235a (glycophorin A) for erythroleukemia.

Chronic Lymphocytic Leukemia (CLL) vs. Mantle Cell Lymphoma (MCL)

Both CLL and MCL can present as CD5+ B cell neoplasms, making immunophenotypic distinction crucial. The following markers differentiate these entities:

Marker CLL MCL
CD5 Positive Positive
CD19 Positive Positive
CD20 Dim / Weak Bright / Strong
CD23 Positive Negative (usually)
CD200 Positive Negative
FMC7 Negative / Dim Positive
Surface Ig (sIg) Dim Moderate–Bright
Cyclin D1 Negative Positive (diagnostic)
SOX11 Negative Positive (nuclear, by immunohistochemistry)
Key Point: The Matutes scoring system assigns one point each for weak sIg, CD5+, CD23+, FMC7−, and CD79b dim/negative. A score of 4–5 strongly supports CLL. Scores of 0–2 suggest a non-CLL B cell lymphoproliferative disorder and warrant further investigation including cyclin D1 and SOX11 assessment.

Minimal Residual Disease (MRD)

After treatment, flow cytometry can detect residual leukemia cells at sensitivities of 10−4 to 10−5 (1 leukemic cell among 10,000–100,000 normal cells). MRD assessment uses ≥8-color panels that exploit the aberrant antigen expression patterns of neoplastic cells—combinations not found on normal counterparts. MRD negativity is a powerful prognostic indicator and increasingly drives treatment decisions in both ALL and CLL.

6. Multi-Color Panel Design

Designing an effective multi-color immunophenotyping panel requires balancing biological questions with the physics of fluorescence detection. The goal is to maximize signal resolution while minimizing spectral spillover between fluorochromes. Thoughtful fluorochrome-to-marker assignment is the single most important step in panel design.

Fluorochrome Brightness and Assignment Rules

The cardinal rule of panel design is: assign the brightest fluorochromes to the dimmest antigens, and the dimmest fluorochromes to the brightest antigens. This compensates for the limited number of antigen molecules on the cell surface by maximizing the signal from each bound antibody.

  1. Rank your antigens by expression density — from lowest (e.g., cytokines, checkpoint receptors like PD-1) to highest (e.g., CD45, CD3, CD4).
  2. Rank your fluorochromes by brightness — PE and APC are among the brightest conventional fluorochromes; FITC and PerCP are moderate; BV421 is bright but UV-excitable alternatives exist.
  3. Minimize spectral overlap between co-expressed markers — If two markers are always on the same cell (e.g., CD3 and CD4 on helper T cells), place them on fluorochromes with minimal spillover into each other.
  4. Consider spread from compensation — Even after mathematical compensation, spreading error increases the variance of dimly stained populations. Avoid placing dim antigens in channels that receive heavy compensation from bright neighbors.

Example 10-Color T Cell Panel

Fluorochrome Laser Marker Expression Level Rationale
BV421 Violet (405 nm) CCR7 Dim–Moderate Bright fluorochrome for moderately expressed chemokine receptor
BV510 Violet (405 nm) CD45 Bright Backbone marker; bright expression tolerates a dimmer fluorochrome
FITC Blue (488 nm) CD3 Bright Bright antigen allows use of a moderate fluorochrome
PerCP-Cy5.5 Blue (488 nm) CD8 Bright Good brightness, minimal spread into PE channel
PE Blue/Yellow-Green (488/561 nm) PD-1 Dim Brightest conventional fluorochrome assigned to dimmest antigen
PE-Cy7 Blue/Yellow-Green (488/561 nm) CD127 Dim–Moderate Good brightness for low-density IL-7Rα
APC Red (633 nm) CD25 Dim Bright fluorochrome needed for dim IL-2Rα on Tregs
APC-Cy7 Red (633 nm) CD4 Bright Tolerates tandem dye; bright expression compensates for higher spread
BV785 Violet (405 nm) CD45RA Bright Bright antigen works with higher-wavelength violet polymer dye
Zombie Aqua Violet (405 nm) Viability N/A Amine-reactive viability dye; excludes dead cells from analysis
Tip — The Spread Matrix: Before finalizing your panel, generate a spread matrix (also called a spillover spreading matrix) using single-stained controls. The spread matrix quantifies the additional variance introduced into each detector by compensation of spectral overlap from every other fluorochrome. Tools like FluoroFinder, the BD panel design tool, or Cytek’s SpectroFlo software can model spread computationally before you run your experiment, saving time and reagents.

7. Sample Preparation

Proper sample preparation is essential for high-quality immunophenotyping data. The choice of method depends on the clinical or research application, the target cell population, and the downstream analysis requirements.

Whole Blood Staining (Lyse/No-Wash)

For clinical immunophenotyping, whole blood staining with subsequent red cell lysis is the preferred method because it preserves the natural distribution of leukocytes and avoids cell loss during washing steps. The lyse/no-wash approach is especially important for absolute counting.

  1. Aliquot 50–100 μL of EDTA-anticoagulated whole blood into a test tube.
  2. Add the fluorochrome-conjugated antibody cocktail at pre-titrated volumes.
  3. Incubate for 15–20 minutes at room temperature in the dark.
  4. Add 500 μL of 1× lysing solution (e.g., BD FACS™ Lysing Solution).
  5. Incubate for 10 minutes at room temperature to lyse red blood cells.
  6. Acquire on the flow cytometer within 1 hour (or fix with 1% paraformaldehyde for delayed acquisition up to 24 hours).

PBMC Isolation

For research applications requiring purified mononuclear cells—particularly when intracellular staining or functional assays are planned—peripheral blood mononuclear cells (PBMCs) are isolated by density gradient centrifugation using Ficoll-Paque (density 1.077 g/mL). PBMCs are then washed, counted, assessed for viability, and stained according to the surface staining protocol.

Surface Staining Protocol (PBMCs or Washed Cells)

  1. Resuspend cells at 1–5 × 106 cells per tube in 100 μL of staining buffer (PBS + 2% FBS + 0.1% NaN3).
  2. Add Fc receptor blocking reagent (e.g., Human TruStain FcX) and incubate for 10 minutes at 4°C.
  3. Add viability dye (amine-reactive, e.g., Zombie Aqua or LIVE/DEAD Fixable dye) and incubate for 15 minutes at 4°C in the dark. Viability dyes must be added before any protein-containing buffer.
  4. Without washing, add the surface antibody cocktail and incubate for 20–30 minutes at 4°C in the dark.
  5. Wash twice with 2 mL of staining buffer (300 × g, 5 minutes).
  6. Resuspend in 200–400 μL of staining buffer and acquire on the cytometer (or fix with 1–2% paraformaldehyde).
Caution — Viability Dye Compatibility: Amine-reactive viability dyes (e.g., Zombie, LIVE/DEAD Fixable) must be added in protein-free buffer (PBS only). If added in buffer containing FBS or BSA, the dye will react with free amines in the serum proteins rather than the cells, resulting in dramatically reduced staining of dead cells and false-negative viability results. Always stain viability first, then add protein-containing buffer with antibodies.

Sample Timing and Stability

8. Controls for Immunophenotyping

Proper controls are the foundation of reliable immunophenotyping. Without appropriate controls, it is impossible to distinguish true antigen expression from non-specific binding, autofluorescence, or spectral spillover. The two principal types of gating controls are isotype controls and Fluorescence Minus One (FMO) controls.

Isotype Controls

Isotype controls use antibodies of the same immunoglobulin class, subclass, and conjugated fluorochrome as the test antibody, but directed against an irrelevant antigen not present on the cells. In theory, they account for non-specific Fc receptor binding and background fluorescence. However, isotype controls have significant limitations:

Fluorescence Minus One (FMO) Controls

An FMO control contains all fluorochromes in the panel except the one being assessed. This reveals the maximum fluorescence a cell can exhibit in that channel due to spectral spillover from all other fluorochromes, defining the precise boundary between negative and positive populations for the missing marker.

Key Point — FMO Controls Are the Gold Standard: FMO controls are universally recommended for multi-color immunophenotyping because they account for the actual spectral environment of the experiment. They are especially critical for markers with continuous (dim-to-bright) or poorly resolved expression patterns such as CD25, CD127, CCR7, PD-1, and cytokine staining. For clearly bimodal markers with distinct positive and negative populations (e.g., CD3, CD19), FMO controls may be less essential but are still best practice. When resources are limited, prioritize FMO controls for your most problematic channels.

Other Essential Controls

9. Gating Strategy & Data Analysis

A systematic gating strategy is the backbone of reproducible immunophenotyping. The standard hierarchical approach removes artifacts sequentially, narrowing focus to the cell populations of interest with high purity.

Standard Gating Hierarchy

The following sequence represents a consensus gating hierarchy for peripheral blood immunophenotyping. Each gate builds on the previous one:

  1. Time Gate: Plot fluorescence (any channel) vs. time to exclude fluidic events (air bubbles, clogs, pressure fluctuations). Remove any periods where events are absent, shifted, or show sudden spikes.
  2. Scatter Gate (Debris Exclusion): FSC-A vs. SSC-A plot to exclude small debris and cellular fragments near the origin. Set a generous gate that includes all leukocyte populations.
  3. Singlet Gate (Doublet Discrimination): FSC-H vs. FSC-A (or FSC-W vs. FSC-A) to exclude cell doublets and aggregates. Doublets appear off the diagonal with disproportionately high area relative to height. This step is critical—doublets can appear as false double-positive events.
  4. Viability Gate: Viability dye vs. any other parameter; gate on the viability dye-negative (live) population. Dead cells should form a clearly separate positive population.
  5. CD45 Leukocyte Gate: CD45 vs. SSC-A to identify leukocyte clusters and exclude residual debris, unlysed red cells, and platelets (CD45negative).
  6. Lineage Gates: Apply lineage markers (CD3, CD19, CD14, etc.) to segregate populations from the clean CD45+ live singlet parent gate.
  7. Subset Gates: Within each lineage, apply subset markers (e.g., CD4/CD8 within CD3+ T cells; CD45RA/CCR7 within CD4+ or CD8+ subsets).

Backgating

Backgating is a verification technique where a downstream population (e.g., CD3+ T cells) is highlighted on an upstream plot (e.g., FSC vs. SSC) to confirm that the gating strategy is not inadvertently excluding events. For example, if you backgate CD14+ monocytes onto the FSC/SSC scatter plot and find many monocyte events falling outside your initial leukocyte gate, the gate needs to be adjusted. Backgating should be performed routinely when establishing a new panel or analyzing unfamiliar sample types.

All Events ↓ Time Gate (exclude fluidic anomalies) ↓ FSC-A vs. SSC-A (exclude debris) ↓ FSC-H vs. FSC-A (exclude doublets) ↓ Viability Dye− (exclude dead cells) ↓ CD45+ vs. SSC (leukocyte gate) ├─ Lymphocytes (CD45bright SSClow) │ ├─ CD3+ T cells → CD4+ / CD8+ → subsets │ ├─ CD19+ B cells │ └─ CD3−CD56+ NK cells ├─ Monocytes (CD45bright SSCmid) └─ Granulocytes (CD45dim SSChigh)

Figure: Standard immunophenotyping gating hierarchy from all events to defined lineage subsets.

Reporting Results

Immunophenotyping results should be reported with both relative (percentage of parent) and absolute values where applicable. Standard reports include:

10. Troubleshooting

Even with a well-designed panel and careful preparation, immunophenotyping experiments can encounter issues. The table below summarizes common problems, their likely causes, and recommended solutions.

Problem Likely Cause Solution
High background / poor resolution of positive and negative Insufficient Fc receptor blocking; antibody concentration too high; inadequate washing Add Fc block before antibody staining; titrate antibodies to optimal concentration; add or increase wash steps
Viability dye not working (dead cells not staining) Viability dye added in protein-containing buffer; dye expired or improperly stored Stain viability in protein-free PBS; use fresh aliquot of dye; include heat-killed positive control
Excessive doublets in singlet gate Cell aggregation; sample clumping; delayed processing Filter sample through 40 μm mesh; add DNase I for thawed PBMCs; vortex gently before acquisition
Unexpected population in wrong scatter region Activated or apoptotic cells; sample aging; wrong tissue type Backgate to verify; process samples promptly; adjust gates based on CD45/SSC profile
Loss of tandem dye signal (PE-Cy7, APC-Cy7) Tandem dye degradation from light exposure, fixation, or age Protect from light; minimize fixation time; use fresh reagents; match compensation controls to experimental conditions
Compensation errors (over- or under-compensated) Single-stained controls not matching panel antibodies; beads vs. cell mismatch Use same antibody lots for compensation and experiment; ensure compensation controls are brighter than experimental samples; verify with FMO controls
Low event count for rare population Insufficient cells acquired; aggressive upstream gating; low cell input Acquire more events (aim for ≥100 events in target gate); relax scatter and viability gates slightly; increase starting cell number
CD45 dim population overlapping with debris Immature cells (blasts) or nucleated RBCs present; insufficient lysis Use a second lysis step; extend lysis time; verify with backgating from lineage markers
Inconsistent results between runs Instrument drift; variable sample handling; lot-to-lot reagent variation Run QC beads daily (CS&T or equivalent); standardize sample processing SOPs; track reagent lots and MFI trends over time
High autofluorescence Monocytes/macrophages; fixation-induced fluorescence; dead cells Gate out autofluorescent cells; minimize fixation; ensure viability exclusion; use red-shifted fluorochromes (less affected by autofluorescence)
Tip — Troubleshooting Dim Staining: If a marker that should be clearly positive appears dim or poorly resolved, systematically check the following: (1) Confirm the antibody has been titrated—using too much antibody increases background without improving signal. (2) Verify the correct clone is being used for your application and species. (3) Check that the antigen is not being lost during preparation (e.g., enzymatic digestion can cleave certain surface markers like CD62L). (4) Ensure the fluorochrome assignment is appropriate—dim antigens require the brightest fluorochromes. (5) Examine FMO controls to confirm the spread from neighboring channels is not obscuring the positive population.

When to Repeat an Experiment

Consider repeating the experiment if any of the following occur: