By Andrea Illingworth, Ahmad Al-Attar and Stacy League

Lymphocyte subset enumeration is one of the most common assays performed in clinical flow cytometry laboratories. The assay is used in monitoring the absolute counts of lymphocyte subsets in human immunodeficiency virus (HIV) infected individuals and other immunodeficiency states, and in the follow-up of certain autoimmune diseases, or for assessing the immune response to immunotherapy.

By Andrea Illingworth, Allyson Wong, Katherine Devitt, Weijie Li, Felipe Magalhães Furtado, Jean Oak, Xuehai Wang and Wolfgang Kern

T-cell lymphomas encompass a wide spectrum of hematologic entities. They can manifest in the blood, bone marrow, lymph nodes, intestines, liver, spleen and skin, among others. In the 2022 classification, the WHO increasingly defines hematologic entities based on genetic alterations, however, this does not apply to T-cell lymphomas.

By Teri Oldaker, Katherine Devitt, Kalpesh Shah and Andrea Illingworth

In the clinical laboratory, flow cytometry assays are critical to providing diagnostic and prognostic information to the treating clinicians. A validation or verification provides confidence that the assay will yield reliable results that can be trusted to make critical medical decisions.

By Jolene Cardinali, Bruce Greig, Valerie Miller, Sindhu Cherian and Joseph DiGiuseppe

This Guide has been written by experienced BD FACSLyric^TM users to help laboratories with alternative procedures for spectral compensation using the FACSuite^TM RUO software for laboratory developed multi-color panels. These tips and tricks may be helpful when standard procedures yield results that are not optimal.

By Dietrich Werner, Andrea Illingworth, Ben Hedley, Katherine Devitt and Lorraine Liu

Absolute neoplastic B-cell counts have gained importance in the distinction of monoclonal B-cell lymphocytosis (MBL) from chronic lymphocytic leukemia (CLL) and other chronic B-cell lymphoproliferative disorders as established by the new WHO Classification (Swerdlow WHO IARC 2017 Ref 12). Although the WHO classification establishes a threshold of greater than 5 x 10e9/L neoplastic B-cells to diagnose CLL, there are no guidelines or recommendations about the best laboratory method to accurately obtain this number. The goals of this module are to review the different approaches a flow cytometry laboratory can pursue to report absolute neoplastic B-cell counts and to discuss the variability that each approach may introduce to the final results.

By David Ng, Dietrich Werner, Jean Oak, Katherine Devitt and Teri Oldaker

Flow cytometry is a robust technology that can be used for several applications in oncology, hematology and immunology. The instrumentation for clinical applications has evolved from 2 color to 4 color and now 10 and 12 color. Developing a new 10 color assay or converting an existing 5 color assay into 10 color is appealing on many fronts, however more colors adds more complexity. Therefore, this process needs to be well thought out and designed with input from all affected stakeholders.

By Kalpesh Shah, Amr Rajab, Teri Oldaker, Andrea Illingworth, and Ann Taylor

Multiparametric flow cytometry is one of the leading technologies for cellular analysis because it allows for the simultaneous detection of numerous characteristics of individual cells with relatively high throughput(15). The development of multiparametric flow cytometry assays is a complex task requiring a detailed understanding of flow cytometers, selection of markers for immunophenotype, clone selection, fluorochromes and spectral overlap, surface and/or intracellular staining protocols, and data analysis. Antibody reagents are the key components of multiparametric flow cytometry analysis. The medical director may request additions of new antibodies based on the clinical need to establish a diagnosis and characterize various normal and neoplastic hematolymphoid populations. For reproducible flow cytometry analysis, the quality performance of these antibodies is an absolute requirement. While there are numerous antibody reagents available from various manufacturers, the criteria to select and validate the proper antibody reagents, for flow cytometry analysis, and to interpret the validation results are not well-defined (2).

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.

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).

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.

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.

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.

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.

By Bethany Vallangeon and Ameet R Kini

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.

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.

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.

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.

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.

Click here to download the Excel worksheet for automatic calculation of sensitivity based on your laboratory’s results.

By Maria Arroz

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.

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.

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.

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).

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.

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.

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.

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.

By Melanie O’Donahue, Fernando Ortiz, Ben Hedley, Veronica Sibing Wei, Abigail Kelliher, and Andrea Illingworth

Erythrocyte lysis is commonly performed as part of the processing of bone marrows, biopsies, fluids and peripheral blood specimens for flow cytometric immunophenotyping. Eliminating the red blood cells (RBCs) allows us to primarily focus on the WBCs and not to acquire events which are not needed for analysis. If an acquisition threshold is set to exclude most of the RBCs, it should not be set too high to prevent the loss of lymphocytes.