The ICCS Quality and Standards committee is dedicated to the optimization of fundamental flow cytometric testing components. Its purpose is to identify major areas of variability, determine critical components needing standardization, develop and define acceptability standards and criteria, and provide guidance and measures for practical implementation in the laboratory. This group will work closely with the ICCS education committee and other entities as necessary.
The Q&S committee is comprised of 4 groups (instrument optimization, reagents and panels, specimen preparation and reporting) which will address the most common areas of variability in flow cytometry. The information will be presented in peer-reviewed “modules” with the goal to provide the laboratory staff with a practical reference guide in optimizing their procedures.
Lysing Methods and Reagents for Flow cytometric Immunophenotyping
By Melanie O’Donahue, MT, ASCP, CCy and Laura Johnson, MT, ASCP, SM, SH
There is no consensus in the flow cytometry industry on which method of lysing erythrocytes is optimal. Different protocols might be more appropriate in different situations and differences in specimen preparation are a potential source of variability regarding the final results. In this first ICCS Quality and Standards module two experienced flow cytometry technologists present their findings about how the most commonly used lysing reagents may impact the quality of the results.
Instrument optimization - Adjusting PMT voltages and compensation on a Beckman Coulter System
By Andrea Illingworth
Multicolor flow cytometry has evolved over the past years and has become more complex due to the number of PMT's and the associated potential for incorrect voltage and compensation settings. Instrument optimization is a much underestimated source of variability and it is important to optimize the voltages for each PMT in order to place the antigen-negative and antigen-positive population visibly "on-scale" and to maximize the potential resolution (signal/noise ratio). This is important to produce good resolution for dimly expressed antigens as well as visualization of antigen negative populations (e.g. PNH). This process should be followed at initial assay setup and this protocol can be used for QC purposes to document and track MFI and signal/noise ratio.
Instrument Optimization for BD FACSCanto Instruments - Creating Application
Settings for White Blood Cells using Lyse/Wash, or Lyse/No Wash methods
By Marsha L. Griffin, Joan Batchelder, Bob Hoffman, Lili Wang, Marybeth
Sharky; Virginia Litwin
Instrument optimization is an often underestimated source of low resolution and high
variability. It is important to optimize voltages for each PMT to determine and maximize
the dynamic range available for positivity. An optimal dynamic range provides the best
resolution for dim staining, while maintaining maximum range for very bright staining.
The process described below, using objective values obtained from CS&T, should be
followed to create an objective, optimized setup prior to assay validation. Once
determined, CS&T Application Settings can be used to maintain optimized settings,
while target particles can be used to standardize multiple instruments and reset
optimization after a service visit.
Identifying appropriate reagents to assess CD5 expression
By Andy C. Rawstron
The relative signal (CD5 median fluorescence intensity on T-cells vs. polyclonal B-cells) was
used as a basic measure of CD5 reagent quality. The relative normal T:B-cell CD5 signal was
calculated in 25 cases with optimal vs. sub-optimal discrimination of CLL cells from normal Bcells.
A target relative signal on normal T-cells vs. normal B-cells of ≥30 was identified as a
threshold to achieve optimal separation of CLL cells from normal B-cells. This target was
subsequently evaluated by ten centers using a series of 100 control cases and was met in 61%
of cases. There are several commercial reagents available which routinely achieve a median
fluorescence intensity on T-cells of ≥30 relative to polyclonal B-cells in the same sample.
Suboptimal signals may reflect laboratory processes and/or reagent quality. CD5 reagents that
do not achieve this target need to be independently validated for the specific diagnostic assay.
Analysis-reporting – CD5-positive B cells – Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma versus Mantle Cell Lymphoma
By Michael A. Linden
CD5-positive chronic lymphoproliferative disorders/lymphomas are characterized by their morphologic, immunophenotypic, and cytogenetic characteristics. In clinical flow cytometry labs, panels are designed to distinguish between the different immunophenotypic subtypes.