Instrument and Assay Optimization for BD FACSLyric Instruments: Creating Tube Settings for White Blood Cells using Lyse Wash Methods
By Ahmad Al-Attar, John Andreasen and Abigail Kelliher
Instrument optimization is a critical step in any laboratory, and that holds true for the flow lab as well. A flow cytometer is set up by adjusting various instrument settings to produce optimal resolution of dim populations while ensuring that bright populations are maintained within the dynamic range of each photomultiplier tube (PMT) on the instrument. These settings are crucial to performing flow cytometry testing accurately. In order to perform testing reproducibly, those settings need to be maintained over time in order to draw accurate conclusions when reviewing data collected over a long period of time as well as performing accurate qualitative and quantitative measurements for rare populations.
Current Status of Myeloperoxidase (MPO) Testing by Flow Cytometry - Survey Results
By Ahmad Al-Attar, Silvia T. Bunting, George Deeb, Andrea Illingworth, Wolfgang Kern, and Dietrich Werner
Myeloperoxidase (MPO) is a heme enzyme present in abundant quantity in neutrophils and expressed specifically in cells committed to granulocytic differentiation including the CD34-positive myeloid progenitors in normal marrow (1, 2). MPO protein expression is considered myeloid lineage-specific by the recent WHO classification of myeloid neoplasms and acute leukemia and more precisely the only single marker listed as a myeloid lineage determinant when assessing acute leukemia of ambiguous lineage/ mixed-phenotype acute leukemia (3).
Awareness of Flow Cytometry Software Differences in Default Settings
By Liam Whitby, Charlotte Wynn, Brahmananda R. Chitteti, Jean Oak, Melanie O’Donahue, Maria Arroz, George Deeb, Abigail Kelliher, Ahmad Al-Attar, and Nina Rolf
Post-acquisition analysis of flow cytometry data (FCS files) is a key factor in the generation of robust flow cytometric results. Whilst analysis software is provided by instrument manufacturers, there are also other products available from a variety of independent third-party vendors. When using any software package there will be variation based on the experience of the operator, the instrument configuration and the capabilities of the software program.
Qualification of a 10/12-Colour 3-Laser FACSLyric Flow Cytometer for Clinical Laboratory Testing
By Ahmad Al-Attar and Katherine A. Devitt
Laboratories introducing clinical testing services by flow cytometry are required to meet regulatory and accreditation requirements, verify vendors’ claims and demonstrate the acceptance of their diagnostic method. The scope of this module is to cover the BD FACSLyric flow cytometer qualifica-tion steps. Qualification of instrumentation includes three components: Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ). These will be described in detail in this module. Assay verification and validation scenarios will be described as related to PQ, but will be covered in detail in other modules addressing the assay-specific intent.
Antibody Cocktail Validation for Flow Cytometry
By Melanie O’Donahue, Fernando Ortiz, Ben Hedley, Veronica Sibing Wei and Kim Le
The purpose of this module is to present methodology for the preparation of antibody cocktails for clinical flow cytometry assays, validation of cocktail use, stability/proper preservation and quality control. We will also discuss troubleshooting tips and methods to minimize errors when using antibody cocktails for flow cytometry.
Current Status of TdT Testing by Flow Cytometry - Survey Results
By Andrea Illingworth, Lorraine Liu and Nina Rolf
The goal for this ICCS-initiated TdT survey was to gain a better understanding of the technical and interpretational challenges of flow cytometric testing for TdT. The survey was based on 18 questions developed by the Quality & Standards Committee, which were answered by 81 clinical laboratories in the US, Canada, as well as Europe. The following survey analysis represents a current state of TdT staining across the various clinical flow cytometry laboratories with a summary for each question and some suggestions and guidance for optimization if indicated.
Tissue Disaggregation Methods for Flow Cytometric Immunophenotyping
By Bethany Vallangeon, MD and Ameet R Kini, MD, PhD
Successful flow cytometry analysis requires a single-cell suspension; therefore, peripheral blood, bone marrow, and body fluid samples are all very suitable sample types. In contrast, tissue samples such as lymph node and extranodal tissue require processing into single-cell suspension before flow cytometric analysis can be performed. It is advisable that the selected method of tissue processing preserves the cell viability and antigenicity as much as possible. Our goal is to compare and contrast different tissue disaggregation methods, discuss the factors that impact the results, advantages and disadvantages, and provide a starting point for choosing and validating the optimal method for your laboratory.
Qualification of a 10C 3L Navios Flow Cytometer for Clinical Laboratory Testing
By Amr Rajab, Andrea Illingworth and Teri Oldaker
Laboratories introducing immunophenotyping services by flow cytometry are required to meet regulatory and accreditation requirements, verify vendors’ claims and demonstrate the acceptance of their diagnostic method. The scope of this module is to cover Instrument Qualification (IQ OQ PQ) of the Beckman Coulter Navios flow cytometer. Verification and Validation scenarios will be described, but will be covered in detail in other modules addressing the assay-specific intent.
Expression of CD5 on Normal Hematolymphoid Cells
By Weina Chen and “Buddy” Frank Fuda
CD5 was one of the first surface markers used to identify T-cells. It is a transmembrane glycoprotein expressed on both immature and mature T-cells. Functionally, it is a co-receptor that can either inhibit or promote T-cell activation by modulating the T-cell receptor (TCR)/peptide major histocompatibility (pMHC) signaling pathway. Co-inhibitory or co-stimulatory effects depend upon the maturation state and the location of the T-lineage cell being activated1. Clinical flow cytometric analysis identifies subsets of T-lineage cells that show different intensities of CD5 expression. While CD5 is generally considered a T-lineage associated marker, its expression extends to other lineage cells, such as subsets of B-cells, NK-cells, and dendritic cells. We will briefly describe normal CD5 expression patterns on various hematolymphoid cells and briefly comment on its diagnostic implications.
Instrument Installation, Operational, and Performance Qualification for BD FACS Canto II
By Brahmananda R. Chitteti and Virginia Litwin
BD FACSCanto II is a flow cytometer intended for the qualitative and quantitative measurement of biological and physical properties of cells and other particles to generate multiparametric results for in vitro diagnostic use. FACSCanto II system is majorly comprised of a flow cytometer, a fluidics cart, and computer workstation. Flow cytometer utilizes fluidics, optics, and electronics sub-systems to acquire and analyze cells in suspension. Fluidics cart contains operational fluids – FACSFlow cubitainer, FACSClean solution, FACS shutdown solution, and waste container. Computer workstation runs two software packages - FACSCanto clinical software for automated immunophenotyping and BD FACSDiva software for manual immunophenotyping of Laboratory Developed Tests (LDT). The instrument can simultaneously measure forward scatter, side scatter, and up to eight fluorescent parameters using spatially separated 405 nm solid state, 488 nm solid state, and 633 nm HeNe lasers.
Laboratories testing patient specimens by flow cytometry are required by accreditation agencies to document Installation (IQ), Operational (OQ) and Performance Qualification (PQ) of the instrument before bringing the instrument into use. In this document, we briefly outline the steps involved in IQ, OQ, and PQ processes according to the laboratory and manufacturer specifications for BD FACSCanto II analyzers and their critical components.
Verification of PNH assay sensitivity through spiking experiment
By A Illingworth, T Oldaker, R Sutherland, S Kotanchiyev, and G Deeb
The PNH Assay is considered a “quasi-quantitative” assay where numeric results are reported, but the results are considered only an estimate due to the lack of reference standards and a calibration curve. Certain parameters, such as accuracy and recovery cannot be demonstrated in these assays. The 2018 ICCS/ESCCA PNH Consensus Guidelines provide detailed information for establishing performance specifications for this assay, which is required prior to testing and reporting patient results. Since this high-sensitivity assay is a rare event analysis, it is mandatory to validate the ability of the assay to distinguish true signals (PNH cells) from background (LOB) and to precisely measure a very small amount of PNH cells. This particular document will focus on the practical aspects of establishing an acceptable Limit of Blank (LOB), as well as verifying the Limit of Detection (LOD) and Lower Limit of Quantification (LLOQ) using a “spiking experiment” as noted on in the recently published ICCS/ESCCA PNH Consensus Guidelines.
Clonality of a B cell expansion is usually the basis for the diagnosis of B-cell chronic lymphoproliferative disorders (B-CLPDs). Nevertheless only considering light chain ratios can be misguiding, a bi-clonal pattern of immunoglobulin light chain expression or polyphenotypic pattern is rare but does exist, the reported incidence of bi-clonal CLL among all CLL cases varies from 3.4% (1) to 1.4% in a larger study (2). It is known that normal and malignant B-cells could show double productive IGVH rearrangements; however, only one rearrangement will be translated to protein and expressed on the cell surface due to allelic exclusion. Therefore, bi-clonal CLL may reflect lack of allelic exclusion (3). Thus, the absence of two different B-cell receptor rearrangements might be found in bi-clonal CLL.
Compensation Tips for Beckman Coulter 10-Color Navios Platform
By Salima Janmohamed-Anastasakis, Amr Rajab and Andrea Illingworth
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” should be read as a prerequisite to this module.
Quality of Reagents – Monoclonal Antibodies
By Ruud Hulspas, Mike Keeney, Ben Hedley and Andrea Illingworth
In clinical flow cytometry, monoclonal antibodies should be validated in the context of the assay as part of the assay validation procedure. It is highly recommended to use well described monoclonal antibodies derived from clones described by the Human Leukocyte Differentiation Antigen (HLDA) Workshops. Reference material is used to validate the reactivity, specificity, selectivity and sensitivity of an antibody. The type of reference material is determined by the assay and may be comprised of (in order of preference if the material is available) ‘normal’ cells from ‘healthy’ donors, a known positive cell line, or other quality control material including commercially available QC material. A titration assay is used to verify antibody reactivity and specificity, and also to determine the antibody amount and concentration resulting in the lowest level of non-specific binding and the highest amount of specific binding.
Flow Cytometric Testing for Kappa and Lambda light chains
By Melanie O’Donahue, Laura Johnson, Ben Hedley and Erin Vaughan
Assessment of immunoglobulin light chain (i.e., kappa or lambda) expression by flow cytometry is a key component in the diagnosis and monitoring of B cell lymphoid neoplasms. Normal and reactive B cell lymphocyte populations typically exhibit expression of both kappa and lambda light chains at an expected ratio, while neoplastic cells exhibit monotypia (over expression of either kappa or lambda).
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.
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.
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.
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.
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.