Advertisement

Real-Time Compensation in Flow Cytometry: A Real Need of Time

  • Aroonima Misra
  • Sunanda Chauhan
  • Pranay Tanwar
  • Saroj Singh
  • Saurabh Sharma
  • Sandeep Rai
Correspondence

Dear Sir,

Compensation is done to prevent spectral overlap and spill-over during data acquisition in fluorescence activated cell sorting (FACS). Spectral overlap occurs because the fluorophores used in flow cytometry emit photons of multiple energies and wavelengths, where emission spectra overlap, fluorescence from more than one fluorochrome may be detected in a channel [ 1, 2, 3]. With newer advanced machines with more lasers and many more detectors or “channels”, the importance of compensation cannot be undermined. The conventional single Antibody (Ab) stained cell tube run before standardization of a protocol experiment, and getting a desired compensation settings is not sufficient in this age of multicolor and multi-tube experiments, real time compensation takes care of other problems apart from compensation like steric hinderance and tube specific “spill overs”. In our experience, even if an experimental protocol is standardized beforehand with single tube run experiments, a...

Notes

Author Contribution

AM and SS wrote the paper, AM, SC and PT made the concept and experimentation setup, SR and SS did the experiment and instrumentation, PT, SR and SS reviewed the paper.

Compliance with Ethical Standards

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

References

  1. 1.
    Baumgarth N, Roederer M (2000) A practical approach to multicolour flow cytometry for immunophenotyping. J Immunol Methods 243:77–97CrossRefPubMedGoogle Scholar
  2. 2.
    Gratama JW, D’Hautcourt JL, Mandy F, Rothe G, Barnett D, Janossy G, Papa S, Schmitz G, Lenkei R (1998) Flow cytometric quantitation of immunofluorescence intensity: problems and perspectives. Cytometry 33:166–178CrossRefPubMedGoogle Scholar
  3. 3.
    Roederer M (2001) Spectral compensation for flow cytometry: visualization artefacts, limitations, and caveats. Cytometry 45:194–205CrossRefPubMedGoogle Scholar
  4. 4.
    Alberti S, Parks DR, Herzenberg LA (1987) A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry 8:114–119CrossRefPubMedGoogle Scholar
  5. 5.
    Bagwell CB, Adams EG (1993) Fluorescence spectral overlap compensation for any number of flow cytometry parameters. Ann N Y Acad Sci 677:167–184CrossRefPubMedGoogle Scholar

Copyright information

© Indian Society of Hematology and Blood Transfusion 2018

Authors and Affiliations

  • Aroonima Misra
    • 1
  • Sunanda Chauhan
    • 2
  • Pranay Tanwar
    • 3
  • Saroj Singh
    • 3
  • Saurabh Sharma
    • 4
  • Sandeep Rai
    • 3
  1. 1.Indian Council of Medical Research- NIOPNew DelhiIndia
  2. 2.Ananta Institute of Medical Sciences and Research CentreRajasamandIndia
  3. 3.Department of Laboratory Oncology, DR B R A IRCHAll India Institute of Medical SciencesNew DelhiIndia
  4. 4.Indian Council of Medical Research- NIMSNew DelhiIndia

Personalised recommendations