2014 duke cfar flow cytometry workshop
Download
1 / 23

2014 Duke CFAR Flow Cytometry Workshop - PowerPoint PPT Presentation


  • 76 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' 2014 Duke CFAR Flow Cytometry Workshop' - madonna-finley


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
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



Example of completed miflowcyt checklist for cytometry publication
Example of Completed MIFlowCyt Checklist for Cytometry Publication





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



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




ad