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2014 Duke CFAR Flow Cytometry Workshop. Data Standards and Annotation. What standards currently exist?. International Society for the Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF ): Data file standards Flow Cytometry Standard (FCS) – version FCS 3.1 Gating ML

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2014 duke cfar flow cytometry workshop

2014 Duke CFAR Flow Cytometry Workshop

Data Standards and Annotation

what standards currently exist
What standards currently exist?
  • International Society for the Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF):
    • Data file standards
      • Flow Cytometry Standard (FCS) – version FCS 3.1
    • Gating ML
    • Calibration and Measurement Standards
      • Instrument calibration
      • Fluorescence intensity calibrations
        • Molecules of Equivalent Soluble Fluorescence (MESF)
        • Antibody Binding Capacity (ABC)
        • Equivalent Reference Fluorophore (ERF)
      • Counting Standards
      • Immunophenotyping
      • Clinical cytometry standards
    • MIFlowCyt: the minimum information about a Flow Cytometry Experiment
      • Requirement for Cytometry publication
    • Training and Education
      • International Cytometry Certification Examination
  • Flow Cytometry:
    • International Cytometry Certification Examination
    • Optimized ImmunoPhenotyping Panel (OMIP)
    • Bioinformatics Standards for Flow Cytometry – working group
  • Cellular Assays:
    • Biosafety
      • Biosafety in Microbiological Laboratories
      • OSHA Bloodborne pathogens
      • Risk Group and Biosafety Level Definitions
      • Pathogen Safety Database
      • Biological Risk Assessment Generic Form (CDCs)
      • World Health Organization Laboratory Safety Manual
      • ISAC Biosafety Standard for Sorting Unfixed Cells
    • “Miata” Minimal Information about T Cell Assays
what is annotation
What is Annotation?
  • Webster’s dictionary:
    • Annotation:
      • a note added by way of comment or explanation
  • Wikipedia:
    • An annotation is a note that is made while reading any form of text.
    • These notes.. establishes a summary for and expresses the relevance of each source..
why is it significant
Why is it significant?
  • A statistian can quickly batch analyze a large amount of information
  • Data management: Metadata
    • Data about data. Metadata describes how and when and by whom a particular set of data was collected, and how the data is formatted. Metadata is essential for understanding information stored in data warehouses and has become increasingly important in XML-based Web applications.
  • Tracking
    • Easily track which experiments/data are relevant
  • Repository within a particular institution
    • Institutions are interested in archiving data
goal of data annotation
Goal of Data Annotation
  • John Altman: “…to include enough information within the FCS file to allow someone else to make sense of the data without requiring cross-references to your notebook.”

- Another operator

- Researcher

- Investigator

- Study Monitor

- Auditor

example of h ow not to annotate data
Example of how not to annotate data

No annotation of experiment:

What’s in the experiment?

No Annotation of data files:

Which file is which?

Where are the controls?

data annotation for all 143 280 data points
Data Annotation - for all 143,280 data points!

Study ID

Method

Assay Name

Batch #

Operator

Sample ID

Visit ID

Accession #

% Viable (Flow)

% Viable (Guava)

Recovery

CD4 count

CD8 count

Gate Name (Parameter Names)

Tube Name

File Name

  • Checking:
  • X1 - for electronic data
  • X3 - for manual entry
  • Requires STRONG statistical support:
    • Quickly exceeds limits of excel
    • Format data for statistical analysis
      • FJ: column (gates) vs row (file)
      • CSV: column (identifiers) vs row (single value)
    • Check data
      • Manual check: 8sec/value x 143280 = 49 days!!!
data annotation define in requisition protocol billing tracked by investigator study date
Data Annotation:Define in Requisition & Protocol(Billing tracked by Investigator/Study & Date)

Research

Clinical

Folder Investigator Study

Experiment Investigator & Researcher Init Study & Date [Batch]

& Date JS_GA 29Sept08ATTACK 29Sept08

Specimen Investigator & Researcher Init Donor ID & Visit

JS_GAAttack 9 Pre

Tube Sample ID Stim & Stain

1_Ms 21 FSCMV FS

Parameters mAb & Fluor mAb & Fluor

Green E CD4 PE Green E CD4 PE

[per detector/sample][per detector/sample]

JS_GA 1_Ms 21 FS

Attack 9 Pre CMV FS

File Name

Abbreviations: FS, SS, FMO, Iso, RR, RP

characters dates
Characters & Dates
  • Characters
    • Avoid : / > & % # . + -
      • Examples: CD4 FITC 1:50; Tube#1; CD14+CD19
    • Use
      • Space
      • _
  • Clarify dates
    • ddmonyyyy (12Nov2009)
    • ddmonyy (121109)
    • monddyy (111209)
keywords
Keywords
  • a word that serves as a key, as to the meaning of another word, a sentence, passage, or the like: Search the database for the keyword “Ireland.”
  • Keywords can be entered into the data of a data file for easy searching within a database
slide19

Green Laser

(532nm; 150mW)

Duke LSRII

Red Laser

(642nm; 25mW solid state)

Duke LSRII

Blue Laser

(488nm; 20mW)

Violet Laser

(405nm; 25mW)

Duke LSRII

Duke LSR II

CFSE

Alexa

680

515/20

QDot

655

505LP

QDot

585

488LP

710/50

685LP

660/40

710/50

PE

Cy5.5

SSC

Blue

685LP

630LP

585/42

PerCP-Cy5.5

PE

TR

Am

Cyan

710/50

570LP

QDot

545

690LP

610/20

515/20

505LP

600LP

560/40

535LP

557LP

575/25

640LP

PE-

Green

740LP

560/40

660/40

780/40

740LP

450/50

595LP

QDot

565

670LP

660/20

PE

Cy5

PE

Cy7

Cas

Blue

780/60

605/40

705/70

APC

QDot

605

APC

Cy7

QDot

705

Duke University Medical Center

Alexa700

FITC

CD57 FITC

TNF Alexa700

CD8 APC-Cy7

CD4 PerCP-Cy5.5

CD27 APC

APC-HL750

CD45RO ECD

CD3 AmCyan

vAmine

PacBlue

IL-2 PE

vAmine + CD14 PacBlue + CD19 PacBlue

INF PE-Cy7

CD107 PE-Cy5

17Apr08, jhe

instrument configurations
Instrument configurations
  • Lasers and parameters of your instrument
    • May be unique to your instrument
  • Name style: which give more information?
    • Fluorochorme and marker
      • CD4 FITC, CD8 APC Cy-7
    • Laser/filter/ fluorochrome/marker
      • 488nm 530/30 CD4 FITC
      • 647nm 660/20 CD8 APC Cy-7
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