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• 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

MIFlowCyt

Example of Completed MIFlowCyt Checklist for Cytometry Publication

MIATA- Minimal Information

MIATA-Minimal Information about T-cell Assay

Data Standards

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?

• 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

Why is Annotation important?

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 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!

Study IDMethodAssay NameBatch #OperatorSample IDVisit IDAccession #% Viable (Flow)% Viable (Guava)RecoveryCD4 countCD8 countGate Name (Parameter Names)Tube NameFile Name

Checking:X1 - for electronic dataX3 - 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)

Folder Investigator StudyExperiment Investigator & Researcher Init Study & Date [Batch]

& Date JS_GA 29Sept08 ATTACK 29Sept08Specimen Investigator & Researcher Init Donor ID & Visit

JS_GA Attack 9 PreTube Sample ID Stim & Stain

1_Ms 21 FS CMV FSParameters mAb & Fluor mAb & Fluor

Green E CD4 PE Green E CD4 PE[per detector/sample] [per detector/sample]

Research Clinical

JS_GA 1_Ms 21 FS Attack 9 Pre CMV FSFile Name

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

Characters & Dates

• Characters• Avoid : / > & % # . + -

• Examples: CD4 FITC 1:50; Tube#1; CD14+CD19• Use

• Space• _

• Clarify dates• ddmonyyyy (12Nov2009)• ddmonyy (121109)• monddyy (111209)

Tube List

Folder: MurdochExperiment: DMM Lyo V450 AV 09Jul09

ParametersSpecimen Tube name Tube # Blue F Blue E Blue B Blue A Red C Red B Red A Violet A Violet B

SS_CD57 FITC 1 CD57 FITCSS_CD38 PE 2 PESS_CD3 PE 3 PE

SS_CD4 PerCPCy55 4 CD4 PerCPCy55SS_CD27 APC 5 CD27 APCSS_CD8 A700 6 CD8 A700

SS_DR APCCy7 7 APCCy7SS_CD8 APCCy7 8 APCCy7SS_CD3 AmCy 9 CD3 AmCy

Unstain 1 10Unstain 2 11

SS45_20uL 12 CD45 V450SS45_10uL 13 CD45 V450SS45_5uL 14 CD45 V450

SS45_2 5uL 15 CD45 V450SS45_1 25uL 16 CD45 V450

SS45_0 625uL 17 CD45 V450SSro_20uL 18 CD45RO PECy7SSro_10uL 19 CD45RO PECy7SSro_5uL 20 CD45RO PECy7

SSro_2 5uL 21 CD45RO PECy7SSro_1 25uL 22 CD45RO PECy7SSro_0 625uL 23 CD45RO PECy7

FS 24 CD57 FITC PE CD4 PerCPCy55 CD27 APC CD8 A700 APCCy7 CD3 AmCy -FS45 25 CD57 FITC PE CD4 PerCPCy55 CD27 APC CD8 A700 APCCy7 CD3 AmCy CD45 V450Fsro 26 CD57 FITC PE CD4 PerCPCy55 CD45RO PECy7 CD27 APC CD8 A700 APCCy7 CD3 AmCy -

FS_45ro 27 CD57 FITC PE CD4 PerCPCy55 CD45RO PECy7 CD27 APC CD8 A700 APCCy7 CD3 AmCy CD45 V450FS_45ro_iso 28 CD57 FITC PE CD4 PerCPCy55 CD45RO PECy7 CD27 APC CD8 A700 APCCy7 CD3 AmCy -

Ms Comp Beads CD57 FITC 19 CD57 FITC - - - - - - - -Ms Comp Beads CD3 PE 20 - PE - - - - - - -Ms Comp Beads CD4 PerCPCy55 22 - - CD4 PerCPCy55 - - - - - -Ms Comp Beads CD45RO PECy7 23 - - - CD45RO PECy7 - - - - -Ms Comp Beads CD27 APC 24 - - - - CD27 APC - - - -Ms Comp Beads CD8 A700 25 - - - - - CD8 A700 - - -Ms Comp Beads CD8 APCCy7 26 - - - - - - APCCy7 - -Ms Comp Beads CD3 AmCy 27 - - - - - - - CD3 AmCy -Ms Comp Beads CD14 V450 28 - - - - - - - - CD45 V450Ms Comp Beads Unstained 29 - - - - - - - - -

fAMJn

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

Keywords within Experiment Layout

Green Laser

(532nm; 150mW)

Duke LSRII

PE

Cy7

PE

Cy5

PE

TR

PE

Cy5.5

PE-

Green780/40

610/20

710/50

660/40

575/25

740LP640LP

600LP

690LP

Violet Laser

(405nm; 25mW)

Duke LSR II

QDot

705

QDot

605

QDot

545

QDot

585

QDot

655

QDot

565705/70

585/42

660/40

605/40

560/40

670LP595LP

570LP

630LP

557LP

560/40

535LP

Am

Cyan

515/20

505LP

Cas

Blue

450/

50

FITC

Red Laser

(642nm; 25mW solid state)

Duke LSRII

APC

Alexa

680

710/50

685LP

780/60

740LP

APC

Cy7

660/20

vAminePacBlue

APC-HL750

Blue Laser

(488nm; 20mW)

Duke LSRII

SSC

Blue

515/20

505LP

710/50

685LP

PerCP-Cy5.5

488LP

CFSE

17Apr08, jhe

CD57 FITC

CD4 PerCP-Cy5.5

TNF Alexa700

CD8 APC-Cy7

Alexa700

CD27 APC

IL-2 PE

CD45RO ECD

INF PE-Cy7CD107 PE-Cy5

CD3 AmCyan

vAmine + CD14 PacBlue + CD19 PacBlue

Duke University Medical Center

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

Softwares

FlowRepository.org