1. 2014 Duke CFAR Flow Cytometry Workshop Data Standards and Annotation

2. 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

3. MIFlowCyt

4. Example of Completed MIFlowCyt Checklist for Cytometry Publication

5. MIATA- Minimal Information

6. MIATA-Minimal Information about T-cell Assay

7. Data Standards

8. 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..

9. 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.Datadata warehousesXML Tracking Easily track which experiments/data are relevant Repository within a particular institution Institutions are interested in archiving data

10. Why is Annotation important?

11. 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

12. 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?

13. 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!!!

14. Data Annotation: Define in Requisition & Protocol (Billing tracked by Investigator/Study & Date) FolderInvestigatorStudy ExperimentInvestigator & Researcher InitStudy & Date [Batch] & Date JS_GA 29Sept08ATTACK 29Sept08 SpecimenInvestigator & Researcher InitDonor ID & Visit JS_GAAttack 9 Pre TubeSample IDStim & Stain 1_Ms 21 FSCMV FS ParametersmAb & FluormAb & Fluor Green E CD4 PEGreen E CD4 PE[per detector/sample] ResearchClinical JS_GA 1_Ms 21 FSAttack 9 Pre CMV FS File Name Abbreviations: FS, SS, FMO, Iso, RR, RP

15. Characters & Dates Characters Avoid : / > & % #. + - Examples: CD4 FITC 1:50; Tube#1; CD14+CD19 Use Space _ Clarify dates ddmonyyyy (12Nov2009) ddmonyy (121109) monddyy (111209)

16. Tube List

17. 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.”wordkey Keywords can be entered into the data of a data file for easy searching within a database

18. Keywords within Experiment Layout

19. Green Laser (532nm; 150mW) Duke LSRII PE Cy7 PE Cy5 PE TR PE Cy5.5 PE- Green 780/40 610/20 710/50 660/40 575/25 740LP 640LP 600LP 690LP Violet Laser (405nm; 25mW) Duke LSR II QDot 705 QDot 605 QDot 545 QDot 585 QDot 655 QDot 565 705/70 585/42 660/40 605/40 560/40 670LP 595LP 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 vAmine PacBlue 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-Cy7 CD107 PE-Cy5 CD3 AmCyan vAmine + CD14 PacBlue + CD19 PacBlue Duke University Medical Center

20. 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

21. Softwares

23. FlowRepository.org