• Document: A previous ICCS Module entitled Instrument optimization - Adjusting PMT voltages and compensation 1 should be read as a prerequisite to this module.
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FLOW-373905.18 Sponsored and reviewed by ICCS Quality and Standards Committee Title: Compensation Tips for Beckman Coulter 10-Color Navios Platform Written by: Salima Janmohamed-Anastasakis Ph.D., Applications Scientist, Beckman Coulter Life Sciences, Division of Beckman Coulter, Inc. Amr Rajab BSc, MLT, SCYM (ASCP)CM, Flow Cytometry Technical Specialist, LifeLabs, Toronto, Canada Andrea Illingworth MS, H(ASCP)SCYM, Manager of Flow Cytometry, Clinical Trials and HPV Testing, Dahl-Chase Diagnostic Services Date: June 18 2018 ______________________________________________________________________ OUTLINE Compensation is an important component of assay-specific optimization of a flow cytometer. Incorrect compensation has the potential to lead to false-positive or false-negative interpretation of antigen expression. This module will help the reader understand the technical background of compensation, provide guidance in optimizing instrument settings and some basic troubleshooting tips specific to the Navios. A previous ICCS Module entitled “Instrument optimization - Adjusting PMT voltages and compensation”1 should be read as a prerequisite to this module. Applicable fluorochromes for the 10-color Navios flow cytometer: Blue laser line Navios Detector FL1 FL2 FL3 FL4 FL5 ECD PC5.5 PC7 Fluorochrome FITC PE (PE-Texas Red) (PE-Cy5.5) (PE-Cy7) Red laser line Violet laser line Navios Detector FL6 FL7 FL8 FL9 FL10 Fluorochrome APC APC-A700 APC-A750 Pacific Blue Krome Orange The recommendations in this document are based on the spectral properties of these 10 fluorochromes. Other fluorochromes are commercially available that can be used in place of some of these fluorochromes, however their spectral properties may be different. 1 FLOW-373905.18 Spectral emission properties of applicable Navios fluorochromes: Spectral emission graph obtained from: https://www.thermofisher.com/us/e n/home/life-science/cell- analysis/labeling- chemistry/fluorescence- spectraviewer.html Spectral spillover occurs most significantly from a fluorochrome into an adjacent detector on the same laser line. For example, light from the FITC fluorochrome will also be detected by FL2, which is used to detect PE. This is illustrated in this uncompensated plot of a FITC single-stained sample. The positive signal observed in FL2 is due to spillover from FITC. Spectral spillover also occurs between laser lines. For example, this is an uncompensated plot of a PC5.5 single-stained sample. Light emitted by PC5.5 spills over into FL7, which is the detector used for APC-A700. 2 FLOW-373905.18 Typical compensation matrix from a 10-color application on the Navios To correct for spectral spillover, interfering light is subtracted from the total light entering each detector. The relative amount of light subtracted is represented as a percentage in a compensation matrix. In the Navios software, the columns of the compensation matrix represent each fluorochrome, and the rows represent each detector (schematically shown below). Therefore, when reading down a column, these are the compensation values required to correct spillover from the fluorochrome listed (at the top of the column) into other detectors (listed as FL1 through FL10). The size of each bubble is directly proportional to the amount of spillover from the fluorochrome (column) into the detector (row). (This schematic matrix was based on a compensation matrix obtained on a Navios flow cytometer following voltage setup as per the previous module.1 Spillover values will vary depending on voltage setup.) The percentage obtained for each position in the matrix is influenced by the voltages set for each detector. Therefore, voltages can be adjusted to minimize compensation values (refer to the previous module for guidance on setting voltages).1 The larger the value in the matrix, the more distorted or spread-out the population may appear. Antibody conjugate selection and panel design will also influence how distortion/spreading affects se

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