Read, inspect, and write Flow Cytometry Standard 2.0, 3.0

FlowIO

Purpose

Use FlowIO as a lightweight, low-level reader and writer for Flow Cytometry Standard files. Examples in this skill target FlowIO 1.4.0, the current stable release verified on 2026-07-23.

FlowIO is appropriate for:

• Reading FCS 2.0, 3.0, and 3.1 files
• Inspecting HEADER, TEXT, ANALYSIS, and channel metadata
• Retrieving event data as a two-dimensional NumPy array
• Reading legacy files that contain multiple datasets
• Writing list-mode, single-precision FCS 3.1 files
• Preparing data for pandas, machine-learning, or downstream cytometry tools

FlowIO does not perform compensation, logicle/biexponential transforms, gating, clustering, or FlowJo workspace processing. Use FlowKit or another analysis package for those tasks.

Install

Create or activate a Python environment, then install the verified release:

uv pip install "flowio==1.4.0"

Confirm the runtime version:

uv run python -c "import flowio; print(flowio.__version__)"

FlowIO 1.4.0 supports Python 3.9 through 3.13 and depends on NumPy.

Operating Workflow

1. Clarify the operation. Distinguish metadata inventory, event extraction, file repair, conversion, and downstream biological analysis.
2. Inspect before loading events. Use only_text=True for metadata-only work, especially with large or unfamiliar files.
3. Choose event semantics explicitly. Use as_array(preprocess=True) for gain/log/time scaling from FCS metadata, or preprocess=False for values as encoded in the DATA segment. Record the choice.
4. Keep parsing strict by default. Do not automatically suppress offset errors. Relax checks only for a known vendor-format defect, and review the resulting event data.
5. Treat metadata as potentially sensitive. FCS TEXT values can include sample, subject, operator, and instrument identifiers. Export only fields needed for the task.
6. Validate writes by reopening them. Check event/channel counts, labels, metadata, and representative values after any FCS export.

Critical Semantics

TEXT keys are normalized

FlowData.text stores keys in lowercase and strips the leading $ from standard FCS keywords:

from flowio import FlowData

flow = FlowData("sample.fcs", only_text=True)
acquisition_date = flow.text.get("date")
instrument = flow.text.get("cyt")
next_dataset = int(flow.text.get("nextdata", "0"))

Do not look up "$DATE", "$CYT", or other uppercase dollar-prefixed keys. TEXT values remain strings. FlowIO 1.4.0 also removes every $ character from the decoded TEXT segment, including $ characters inside values; preserve the original file when exact metadata fidelity matters.

Events have two representations

• flow.events is the unprocessed, flattened one-dimensional event array.
• flow.as_array() returns shape (event_count, channel_count) as a NumPy float64 array.
• flow.as_array(preprocess=True) applies FCS gain, logarithmic, and time scaling. It does not apply compensation or logicle/biexponential display transforms.
• flow.as_array(preprocess=False) reshapes the encoded event values without those scaling steps.

as_array() creates another in-memory array. FlowIO does not provide chunked or memory-mapped event access.

Channel numbering uses two conventions

• NumPy columns and fluoro_indices, scatter_indices, and time_index use zero-based indices.
• flow.channels uses FCS parameter numbers beginning at 1.
• null_channels contains the PnN label strings supplied through null_channel_list, including supplied labels that were not found.
• pns_labels always matches pnn_labels in length; missing optional PnS labels appear as empty strings.

Writing is intentionally limited

create_fcs() requires:

• An already-open binary file handle
• Flattened one-dimensional event data in row-major event/channel order
• One PnN name per channel
• Optional PnS names and string-valued metadata via metadata_dict

It writes FCS 3.1 list-mode ($MODE=L) single-precision float ($DATATYPE=F) data. Required interpretation keywords are generated by FlowIO and cannot be overridden through metadata.

Quick Start: Read an FCS File

from pathlib import Path

from flowio import FlowData

flow = FlowData(Path("sample.fcs"))
events = flow.as_array(preprocess=True)

print(
    {
        "version": flow.version,
        "events": flow.event_count,
        "channels": flow.channel_count,
        "shape": events.shape,
        "pnn": flow.pnn_labels,
        "pns": flow.pns_labels,
        "date": flow.text.get("date"),
        "instrument": flow.text.get("cyt"),
    }
)

For metadata only:

from flowio import FlowData

flow = FlowData("sample.fcs", only_text=True)
print(flow.version, flow.event_count, flow.pnn_labels)

Do not call as_array() on a metadata-only instance because its event data was not loaded.

Prefer a path or Path over a caller-owned file handle. FlowData closes a provided handle after parsing. In FlowIO 1.4.0, read_multiple_data_sets(handle) can fail after the first dataset because the handle has been closed; pass a filesystem path for multi-dataset files.

Quick Start: Read Multiple Datasets

Use the standalone helper rather than manually interpreting $NEXTDATA offsets:

from flowio import read_multiple_data_sets

datasets = read_multiple_data_sets("legacy-multi-dataset.fcs")
for index, dataset in enumerate(datasets):
    values = dataset.as_array(preprocess=True)
    print(index, dataset.event_count, dataset.pnn_labels, values.shape)

The FCS 3.1 specification deprecated multiple datasets in one file, but FlowIO can read legacy files that use them.

Quick Start: Create an FCS 3.1 File

from pathlib import Path

import numpy as np
from flowio import FlowData, create_fcs

values = np.asarray(
    [[100.0, 200.0, 50.0], [150.0, 180.0, 60.0]],
    dtype=np.float32,
)
pnn_labels = ["FSC-A", "SSC-A", "FITC-A"]
pns_labels = ["Forward scatter", "Side scatter", "CD3"]

output = Path("output.fcs")
with output.open("xb") as handle:
    create_fcs(
        handle,
        values.ravel(order="C"),
        pnn_labels,
        opt_channel_names=pns_labels,
        metadata_dict={
            "date": "23-JUL-2026",
            "cyt": "Example instrument",
            "src": "Validated NumPy array",
        },
    )

roundtrip = FlowData(output)
assert roundtrip.event_count == values.shape[0]
assert roundtrip.pnn_labels == pnn_labels
np.testing.assert_allclose(
    roundtrip.as_array(preprocess=False),
    values,
    rtol=1e-6,
    atol=1e-6,
)

Metadata keys may be supplied in mixed case or with $, but lowercase keys without $ match FlowIO's normalized representation and are less error-prone. Metadata values must be strings.

Copy or Rewrite an Existing File

Use write_fcs() when the event data does not need to change:

from flowio import FlowData

flow = FlowData("source.fcs")

# Preserve selected source metadata (cyt, date, and spill/spillover when present).
flow.write_fcs("copy.fcs")

# Write only required metadata plus the custom fields supplied here.
flow.write_fcs("deidentified.fcs", metadata={"src": "Deidentified export"})

Passing metadata=None preserves FlowIO's selected defaults. Passing any dictionary, including {}, replaces those defaults rather than merging with them. write_fcs() always produces FCS 3.1 floating-point output; non-float source events are preprocessed before writing. It opens the destination for overwrite, so reject an existing output path before calling it unless replacement is intentional. For floating-point sources it can preserve encoded events while dropping PnG or timestep, changing later as_array(preprocess=True) results. Validate both raw and preprocessed round-trips.

Use create_fcs() instead when event values, event count, or channel layout changes.

Bundled Inspector

scripts/inspect_fcs.py inventories one or more datasets without network access. By default it reads metadata only, emits structural fields and channel labels without full TEXT/ANALYSIS values, and refuses files above a configurable size limit.

Set FLOWIO_SKILL_DIR to the installed skill directory. From this repository's root, use skills/flowio:

FLOWIO_SKILL_DIR="skills/flowio"

# Metadata and channel inventory
uv run --no-project --with "flowio==1.4.0" \
  python "$FLOWIO_SKILL_DIR/scripts/inspect_fcs.py" sample.fcs

# Include all normalized TEXT metadata; review output for identifiers
uv run --no-project --with "flowio==1.4.0" \
  python "$FLOWIO_SKILL_DIR/scripts/inspect_fcs.py" sample.fcs --include-text

# Load events and compute finite-value statistics using FlowIO preprocessing
uv run --no-project --with "flowio==1.4.0" \
  python "$FLOWIO_SKILL_DIR/scripts/inspect_fcs.py" sample.fcs --stats

# Compute statistics from encoded values instead
uv run --no-project --with "flowio==1.4.0" \
  python "$FLOWIO_SKILL_DIR/scripts/inspect_fcs.py" sample.fcs --stats --raw

Use --help for output files, input/array memory limits, null-channel labels, and controlled offset-recovery options.

References

Read only the reference needed for the current task:

• references/api_reference.md — exact FlowIO 1.4.0 public API and signatures
• references/workflows.md — inventory, DataFrame/CSV, batch, write, and round-trip patterns
• references/fcs_semantics.md — FCS structure, metadata normalization, preprocessing equations, indexing, and writer behavior
• references/troubleshooting.md — offset failures, multi-dataset files, memory limits, validation, security, and privacy
• references/sources.md — authoritative upstream docs, release notes, source, and FCS 3.1 publications used for this refresh

Non-Negotiable Checks

• Never claim FlowIO applies compensation or gating.
• Never treat as_array(preprocess=True) as raw acquisition values.
• Never pass a two-dimensional array or a path directly to create_fcs().
• Never assume TEXT keys retain $ or uppercase spelling.
• Never silence offset errors without documenting why and validating the data.
• Never describe FlowIO event loading as streaming or chunked.

Citing Scientific Agent Skills

This skill is part of Scientific Agent Skills by K-Dense. If it materially contributed to a manuscript, report, presentation, or code release, add the paper to the references or software section and tell the user you did so:

Kassis, T., Agarwal, V., He, Y., Patel, D., & Brueckner, A. M. (2026). Scientific Agent Skills: A Library of Procedural Knowledge for Research Agents. arXiv:2609.00065. https://doi.org/10.48550/arXiv.2609.00065

Always cite the current version. The DOI and https://arxiv.org/abs/2609.00065 resolve to the latest arXiv version, so never append a version suffix such as v1. When network access is available, fetch https://arxiv.org/abs/2609.00065 (or http://export.arxiv.org/api/query?id_list=2609.00065) before writing the reference and take the author list, year, and version from that record. If the record lists a journal reference or publisher DOI, cite the published version instead.