In part 1 of this series on EuroFlow panels we delved into the basics of flow cytometry and blood cancers, and looked at the various EuroFlow panels used for immunophenotyping haematological malignancies. Here we will expand on the biology of some of the key reference markers targeted by these various panels.
Myeloperoxidase (MPO)
MPO is a peroxidase enzyme found in certain white blood cells, particularly granulocytes such as neutrophils. MPO plays a crucial role in the immune system by aiding in the destruction of pathogens, such as bacteria and fungi. It does this by catalysing the formation of hypochlorous acid and other reactive oxygen species (Figure 1.) (Aratani, 2018).
MPO is often used as a diagnostic marker in flow cytometry for the classification of AML and can help distinguish AML from other leukaemias, as it is one of the defining characteristics of cells of the myeloid lineage (Döhner et al., 2022). EuroFlow recommends the FITC-labelled anti-MPO clone ‘MPO-7’ from Agilent (Agilent cat.no. F0714) for use in their ALOT (Acute Leukaemia Orientation Tube) panel, which is used for the assessment of the nature of immature blast cell populations in acute leukaemia samples (Van Dongen et al., 2012).
Kappa/Lambda
B-cells are the antibody factories of the immune system (Alberts et al., 2002). Antibodies or immunoglobulins are glycosylated proteins that are vital components of the adaptive immune system, able to neutralise pathogens and direct immune responses against them. The basic structure of a typical IgG antibody is composed of two identical heavy chains and two identical light chains (Figure 2.). These light chains are either kappa (κ) or lambda (λ) chains, and they are held together by disulfide bonds, forming a Y-like configuration. The genes for the kappa and lambda light chains of antibodies are located on two different chromosomes (Mcbride et al., 1982). Each light chain, whether kappa or lambda, consists of two main regions: the variable (V) region and the constant (C) region. The variable region is responsible for antigen recognition. It contains the antigen-binding site, also known as the paratope, which exhibits a high degree of specificity for a particular epitope on an antigen. The variable regions of the light chains, together with the variable regions of the heavy chains, provide the antibody with its antigen-binding specificity. Importantly, a normal B-cell population should express an expected ratio of kappa vs. lambda chains (~2:1) (Marti et al., 2005). If this ratio deviates greatly from the expected range, it can be indicative of a B-cell malignancy.
Analysis by flow cytometry can be used to assess kappa and lambda light chain ratios, with the light chain proteins able to be stained either extracellularly or intracellularly dependent on the investigator’s requirements. Kappa and lambda light chains are particularly important in the immunophenotyping of B-cell malignancies (Paiva et al., 2018). By analysing the expression of these light chains, pathologists can determine the clonality of B-cells in a patient’s sample (Figure 3.). Aberrant ratios or the presence of one light chain type over the other can be an indicator of a B-cell related malignancy. These ratios can also be monitored to assess disease progression and responses to treatment. EuroFlow recommends APC-labelled anti-kappa polyclonal antibodies from Agilent (Agilent cat.no. C0222) for use in their MM-MRD (multiple myeloma-minimal residual disease) and PCD (plasma cell dyscrasias) panels which aim to detect, discriminate between, and monitor B-cell lymphoproliferative disorders.
IgM
B-cells go through a gamut of stages during their development in the bone marrow and secondary lymphoid organs (Pieper et al., 2013). Various extracellular and intracellular markers are expressed, that can inform at which stage the B-cells are in their developmental cycle. One of the markers often used to detect and classify different B-cell populations by flow cytometry is IgM (Sevilla et al., 2009). IgM antibodies are one of the five major classes of antibodies, having a distinct pentameric structure when secreted, which consists of five basic antibody units (Figure 4.) (Keyt et al., 2020). IgM is rapidly produced upon initial exposure to an antigen, providing immediate protection, with B-cells then later isotype switching to production of other antibody types. IgM will be detectable on the surface or in the cytoplasm of a B-cell dependent at which stage of development the cell is in. Mu-chains (μ-chains) are the heavy chains of IgM antibodies. Pre-B-cells express cytoplasmic μ-chains but no light chains, whereas early B-cells express membrane IgM.
Detection of IgM (via the μ-chains) is important for characterising and diagnosing acute lymphoblastic leukaemias (ALL) (Sevilla et al., 2009). Detection of μ-chains, in conjunction with other markers, can help differentiate between various subtypes of lymphoblastic leukaemias, such as precursor B-cell ALL (pre-B ALL) which affects immature B-cell precursors (Theunissen et al., 2017). The information provided can aid in guiding treatment decisions. EuroFlow recommends FITC-labelled anti-IgM (μ-chains) polyclonal antibodies from Agilent (Agilent cat.no. F0058 ) for use in their BCP-ALL panel (Van Dongen et al., 2012).
Terminal Deoxynucleotidyl Transferase (TdT)
TdT is a DNA polymerase that plays an important role in V(D)J Recombination, the process whereby segments of genes encoding antigen receptors of developing B-cells and T-cells are rearranged to generate a diverse array of receptors (Fowler and Suo, 2006). This diversity allows the immune system to recognise and respond to various pathogens. More specifically, TdT is a polymerase that can add nucleotides to a DNA chain without requiring a template strand. During recombination of the V, D and J gene segments of B-cell and T-cell receptors (BCR, TCR), TdT will introduce nucleotides between the recombining gene sections, resulting in junctional diversity (Figure 5.) (Roth, 2014).
In the context of immunophenotyping, antibodies to TdT help distinguish between immature (TdT present) and mature (TdT absent) lymphocytes. TdT is significant in diagnosing ALL, in which there are large numbers of immature or progenitor lymphoid cells. The presence of TdT in leukemic lymphoblasts is a distinguishing feature of ALL, and it helps differentiate it from acute myeloid leukaemia (AML), in which lymphocytes typically lack TdT expression (Sędek et al., 2014). TdT is also used in the monitoring of minimal residual disease in ALL patients (MRD refers to the small number of cancer cells remaining in a patient following treatment) (Van Dongen et al., 2015). Detecting MRD is crucial for evaluating the effectiveness of treatment and predicting disease recurrence. EuroFlow recommends the FITC-labelled anti-TdT monoclonal antibody, clone ‘HT-6’, from Agilent (Agilent cat.no. F7139) for use in their BCP-ALL (B-cell precursor-acute lymphoblastic leukaemia) and T-ALL (T-cell acute lymphoblastic leukaemia) panels, which are used for the recognition and classification of all classically defined BCP-ALL and classically defined T-ALL, respectively (Theunissen et al., 2017, Van Dongen et al., 2012).
Cluster of differentiation 79 alpha (CD79α)
CD79α is a component of the BCR complex found on the surface of B-cells. It forms a heterodimer with CD79β and associates with membrane-bound immunoglobulin (Ig), forming the BCR complex, and is vital for BCR signalling when the Ig component binds to an antigen (Figure 6.) (Tanaka and Baba, 2020). This binding to an antigen and subsequent signal transduction leads to B-cell activation. This activation includes the proliferation of the B-cell and its differentiation into an antibody-secreting plasma cell.
CD79α is an important marker for the diagnosis and classification of malignancies, especially B-cell malignancies. Malignant B-cells often retain the expression of CD79α, and its presence on the cell surface is a distinguishing feature of these cells (Chu and Arber, 2001). Therefore, in flow cytometry analysis, CD79α is used to identify and confirm the presence of B-cell lymphomas and leukaemias. EuroFlow recommends the PE-labelled anti-CD79α monoclonal antibody, clone ‘HM57’, from Agilent (Agilent cat.no. R7159 ) for use in their ALOT panels and it is a key marker for diagnosis of B-ALL.
Cluster of differentiation 23 (CD23)
CD23 is a cell-surface protein involved in a gamut of immunological activities. Besides its role in allergy, acting as a receptor for IgE antibodies, it also plays a useful role in diagnosing and classifying lymphomas (Engeroff and Vogel, 2021, Van Dongen et al., 2012). CD23 is typically expressed in chronic lymphocytic leukaemia (CLL), and its presence helps distinguish CLL from other B-cell lymphoproliferative disorders (Iova et al., 2012). For instance, mantle cell lymphoma (MCL), which can present similarly to CLL, usually lacks CD23 expression. EuroFlow recommends the FITC-labelled anti-CD23 monoclonal antibody, clone ‘MHM6’, from Agilent (Agilent cat.no. F7062) for use in their B-CLPD (B-cell chronic lymphoproliferative diseases) panel.
Cluster of differentiation 45 (CD45)
CD45, also known as leukocyte common antigen (LCA), is a type I transmembrane protein found on the surface of all nucleated haematopoietic cells, such as white blood cells (Rheinländer et al., 2018). It constitutes a significant percentage of the total glycoprotein complement located on the surface of B- and T-cells. It plays a critical role in regulating immune cell function by acting as a phosphatase, which is essential for activating signalling pathways within these cells. CD45 modulates the activity of protein tyrosine kinases involved in T-cell and B-cell receptor signalling, making it crucial for immune responses (Al Barashdi et al., 2021). Additionally, CD45 exhibits multiple isoforms due to alternative splicing, with each form playing a role in fine-tuning immune cell activation and differentiation (Dawes et al., 2006).
CD45 is an important marker used in flow cytometry, particularly in diagnosing and classifying haematological malignancies. Its broad expression on all nucleated haematopoietic cells makes it a fundamental marker for distinguishing leukocytes from non-haematopoietic cells (Gorczyca et al., 2011). EuroFlow recommends the PacB-labelled anti-CD45 clone ‘T29/33’(Agilent cat.no. PB986) for use in their PCD panel, to aid in identifying plasma cells and distinguishing between normal/reactive and clonal plasma cell populations, based on their most frequent abnormal phenotypes.
Conclusion
The EuroFlow consortium took great consideration into the markers they would select to target in their panels for diagnosing and classifying haematological malignancies. Each marker was carefully selected based on its specificity, sensitivity, and relevance to different stages of cell development and disease states. By incorporating a combination of lineage-specific markers, differentiation antigens, and aberrant markers, the EuroFlow panels offer a comprehensive approach to accurately identify and classify a wide range of haematological disorders. Agilent Dako antibodies selected as references in these panels ensure high-quality and reliable detection, further enhancing diagnostic precision.
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