Agilent Dako antibodies recommended by EuroFlow / Part 1

An introduction to types of blood cancers

Flow cytometry is a technique that has become ubiquitous in modern day laboratories. Flow cytometry finds use in a plethora of different settings from biobanks to infectious disease laboratories. The technology is important in haematology and immunology laboratories, allowing for the immunophenotyping of cancers such as leukaemias and lymphomas.

Leukaemias and lymphomas, which are also commonly referred to as “blood cancers”, are pathologies of the haemtopoietic and lymphoid tissues ie. cells of the bone marrow, blood circulatory and lymphatic systems. Clinicians will often diagnose these illnesses by analysing biological markers that are associated with these diseases (Cree, 2022).  They will often look for abnormal cell counts, an increase in clonal cell populations, and changes in expression of cell surface and intracellular markers in different cell types (Waldron et al., 2022, Eichhorst et al., 2021, Kappelmayer et al., 2000).

Leukaemias are designated into four general categories, broadly dependent on which cell lineage they originated from (Figure 1) (PDQ Adult Treatment Editorial Board, 2023). There are acute and chronic myeloid leukaemias (AML and CML) which occur in cells of myeloid lineage such as granulocytes (neutrophils, basophils, and eosinophils etc.), and acute and chronic lymphocytic leukaemias (ALL and CLL) which affect cells of lymphoid origin such as B-cells and T-cells. Lymphomas on the other hand can be categorised into two broad categories namely Hodgkin and non-Hodgkin lymphoma. The main difference between the two is the presence of large, abnormal lymphocytes associated with Hodgkin lymphoma known as Reed-Sternberg cells (Sadaf et al., 2023). Myelomas, another important haematological malignancy, are cancers of plasma cells – B-lymphocytes that secrete antibodies. Myelomas are characterised by clonal expansion of plasma cells which can form tumours (plasmacytomas) in bone marrow, and sometimes tumours can form in other soft tissues as well (Rajkumar et al., 2014).

The identification and characterisation of the cell populations play a crucial role in the diagnosis and effective treatment of haematological malignancies. Flow cytometry emerges as a pivotal technique, enabling the simultaneous analysis of multiple cell parameters. Furthermore, this method proves valuable in tracking treatment responses and detecting signs of disease relapse.

Flow cytometry basic principles, intracellular vs extracellular markers

Flow cytometry allows for the sorting and analysis of cells based on various characteristics. Cells are labelled with antibodies conjugated to fluorescent particles which emit light when excited by various lasers within the flow cytometer. Detectors can distinguish cell size and complexity based on light scatter, while other detectors capture information on which markers are expressed by a cell, based on the wavelength of light emitted by fluorescent particles. (Figure 2).

If one considers the basic structure of a mammalian cell, target markers could be found on the surface of the cell embedded in the cellular membrane (extracellular), or within the cell itself in regions such as the cytoplasm or the nucleus (intracellular). Both types of markers would require different approaches for their analysis by flow cytometry. Typically for both preparations some sort of lyse/wash steps will be included which help remove red blood cells and other debris that may interfere with the assay. It is also important to consider other pre-analytical variables such as cell viability, time on ice/freeze thaw cycles, centrifugation and vortexing etc. which can all influence the outcome of the assay (see https://diagnostech.co.za/flow-cytometry-instrument-and-sample-preparation/).

Antibodies targeting extracellular markers are commonly used in immunophenotyping, however, intracellular markers may also add value in disease diagnosis. Extracellular markers identify the cell types, e.g. CD3+ T-cells which co-express either CD4 or CD8 on the cell surface, with intracellular markers in those specific cell phenotypes helping to further characterise those cells e.g. CD3+CD4+FoxP3+ – regulatory T-cells. Staining intracellular markers requires additional preparation, with the cells being fixed with paraformaldehyde and then permeabilised with an alcohol or detergent to allow the conjugated antibodies to enter the cell. Commercial kits are available that can simplify this process, such as IntraStain from Agilent (Agilent cat.no. K2311).

EuroFlow Panels, Agilent Markers

Panel design plays an important role in flow cytometry analysis. The grouping of antibodies labelled with different fluorochromes targeting intracellular or extracellular markers allows the investigator to define and characterise a mixed population of cells. The EuroFlow Consortium has played a significant role in development of flow cytometry panels for leukaemia and lymphoma. The EuroFlow Consortium is a collaborative research initiative focused on the development of standardised flow cytometry protocols and tools for the diagnosis, classification, and monitoring of haematological malignancies. They work on creating standardised antibody panels, data analysis algorithms, and reporting guidelines to ensure consistent and comparable results across different laboratories and clinical settings. This collaboration involves multiple institutions and experts, and it often leads to the publication of guidelines and recommendations for the clinical use of flow cytometry in haematology. The EuroFlow Consortium has put together several standardised screening and testing panels for haematological malignancies. Specific antibodies are recommended as references in these panels, with Agilent Dako antibodies recommended in many of them.

Acute Leukaemia Orientation Tube (ALOT)

The EuroFlow ALOT panel was developed to assess the characteristics of immature blast cell populations in acute leukaemia samples, allowing the investigator to distinguish between B-cell, T-cell, non-lymphoid, or mixed cell phenotypes (Van Dongen et al., 2012). It aids in directing towards the requisite complementary antibody panels such as BCP-ALL, T-ALL, or AML/MDS which further refine the analysis. Using the ALOT panel in combination with other protocols, it allows for the detection or exclusion of most types of haematological malignancies. As shown in Table 1., two markers from Agilent Dako are recommended as reference antibodies, the FITC-labelled anti-MPO clone ‘MPO-7’ (Agilent cat.no. F0714) and the PE-labelled anti-CD79α clone ‘HM57’ (Agilent cat.no. R7159 ). EuroFlow also recommends the PacB-labelled anti-CD3 clone ‘UCTH1’ from Agilent as an alternative to the BD reference (Agilent cat.no. PB982).

Table 1. ALOT

Lymphoid Screening Tube (LST)

The purpose of this panel is for initial diagnostic screening in suspected haematological maligancies. This panel efficiently detects phenotypically aberrant mature B-, T-, and NK-cells in various body tissues and fluid, which can then direct further analysis with other antibody panels for accurate diagnosis and classification of lymphoproliferative disorders (Van Dongen et al., 2012). The panel was developed to evaluate various medical conditions such as lymphocytosis in peripheral blood, lymphoid infiltrates in bone marrow, monoclonal components in serum, or enlargement of various tissues like lymph nodes and spleen. The FITC-labelled anti-CD8 ‘DK25’ clone from Agilent can be used as an alternative to the clone from Cytognos (Table 2.) (Agilent cat.no. PB982).

Table 2. LST

Antibody panel for B-cell precursor ALL (BCP-ALL)

The BCP-ALL panel serves the purpose of recognising and classifying all immature B-cell lineage malignancies (Van Dongen et al., 2012). This includes classically defined BCP-ALL such as pro-B-ALL, common-ALL and pre-B-ALL. The information obtained from the BCP-ALL panel needs to be combined with the ALOT panel data. This integration is crucial for a comprehensive evaluation and accurate classification of BCP-ALL. The ALOT panel offers a broader orientation to acute leukaemias, while the BCP-ALL panel focuses specifically on the detailed characterisation of BCP-ALL, allowing for a more precise diagnosis. As shown in Table 3., two markers from Agilent Dako are recommended as reference antibodies, FITC-labelled rabbit anti-human IgM polyclonal sera (Agilent cat.no. F0058) and the FITC-labelled anti-TdT clone ‘HT-6 ‘(Agilent cat.no. F7139). The APC-labelled anti-CD117 ‘104D2’ clone from Agilent can be used as an alternative to the clone from BD Biosciences (Table 3.) (Agilent cat.no. C7244).

Table 3. BCP-ALL

T-cell acute lymphoblastic leukaemia (T-ALL)

The T-ALL panel is used when the ALOT panel indicates T-lineage precursor expansion (Van Dongen et al., 2012). T-ALL is an aggressive haematological malignancy characterised by the clonal proliferation of immature T-lymphoblasts. These cells are arrested at various stages of T-cell development, resulting in their accumulation in the bone marrow, peripheral blood, and potentially other tissues. As shown in Table 4., one marker from Agilent Dako is recommended as a reference antibody, the FITC-labelled anti-TdT clone ‘HT-6 ‘(Agilent cat.no. F7139).

Table 4. T-ALL

Acute myeloid leukaemia/myelodysplastic syndrome (AML/MDS)

Several panels act as compliments to the ALOT panel, this includes the AML/MDS antibody panel. This panel is used for patients suspected of having AML or MDS and can characterise myeloid lineages and abnormal phenotypes (Van Dongen et al., 2012). Unlike other leukaemias such as T-ALL and BCP-ALL, the cell populations involved can be highly heterogenous and affect various lineages at different maturation stages. As shown in Table 5., one marker from Agilent Dako is recommended as a reference antibody, the FITC-labelled anti-TdT clone ‘HT-6 ‘(Agilent cat.no. F7139).

Table 5. AML/MDS

Plasma cell disorders (PCD)

Plasma cell disorders are a group of diseases characterised by abnormal growth and proliferation of plasma cells. They typically exhibit an overproduction of a single monoclonal antibody that can be detected in blood or urine. Multiple myelomas (MM) and monoclonal gammopathy of undetermined significance (MGUS) are typical plasma cell disorders. The PCD panel has been developed to identify and count plasma cells as well as to discriminate between normal and pathological monoclonal cells (Van Dongen et al., 2012). As shown in Table 6., two markers from Agilent Dako are recommended as reference antibodies, the PacB-labelled anti-CD45 clone ‘T29/33’(Agilent cat.no. PB986) and APC-labelled rabbit anti-human cyIgκ polyclonal sera ‘(Agilent cat.no. C0222). The APC-labelled anti-CD117 ‘104D2’ clone from Agilent can be used as an alternative to the clone from BD Biosciences (Table 6.) (Agilent cat.no. C7244).

Table 6. PCD

Antibody panel for B-cell chronic lymphoproliferative diseases (B-CLPD)

B-CLPD are a group of disorders characterised by the abnormal proliferation of mature B-cells. Types can include Chronic Lymphocytic Leukaemias (CLL), Mantle Cell Lymphoma (MCL) and Hairy Cell Leukaemias (HCL) (Van Dongen et al., 2012). As shown in Table 7., one marker from Agilent Dako is recommended as a reference antibody, the FITC-labelled anti-CD23 clone ‘MHM6‘(Agilent cat.no. F7062).

Table 7. B-CLPD

Minimal (or Measurable) Residual Disease in Multiple Myeloma (MM-MRD)

Minimal measurable disease refers to the small number of cancerous cells that remain in a patient following treatment, even when the patient is in remission and no symptoms of the disease are present (Kumar et al., 2016). These residual cells can eventually cause a relapse, making MRD analyses an important factor in monitoring effectiveness of therapy and the risk of disease recurrence. Many MM patients in remission will experience relapse therefore it is vital to monitor MRD using a sensitive and reliable method. As shown in Table 8., one marker from Agilent Dako is recommended as a reference antibody in the EuroFlow MM-MRD panel (Flores-Montero et al., 2017), the APC-labelled polyclonal anti-cyIgκ (Agilent cat.no. C0222). The APC-labelled anti-CD117 ‘104D2’ clone from Agilent can be used as an alternative to the clone from BD Biosciences (Table 8.) (Agilent cat.no. C7244).

Table 8. MM-MRD

In conclusion

Flow cytometry remains a vital tool in the modern clinical laboratory. The ability to assess the landscape of immune cells, identifying and distinguishing them with precision, is an invaluable asset for clinicians and researchers alike. The clinical significance of flow cytometry extends from initial diagnosis to assessing treatment responses and monitoring MRD. EuroFlow panels require precise and standardised reagents for diagnosing haematological diseases. Agilent antibodies are valued for their consistent and reliable performance, contributing to the robustness and comparability of flow cytometric data globally.

References:

Board, P. a. T. E. 2023. PDQ Acute Myeloid Leukemia Treatment: Health Professional Version [Online]. Bethesda, MD: National Cancer Institute. Available: https://www.cancer.gov/types/leukemia/hp/adult-aml-treatment-pdq [Accessed 13/09/2023].

Cree, I. A. 2022. The WHO Classification of Haematolymphoid Tumours. Leukemia, 36, 1701-1702.

Eichhorst, B., Robak, T., Montserrat, E., et al. 2021. Chronic lymphocytic leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology, 32, 23-33.

Flores-Montero, J., Sanoja-Flores, L., Paiva, B., et al. 2017. Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Leukemia, 31, 2094-2103.

Häggström, M. 2014. Medical gallery of Mikael Häggström 2014. WikiJournal of Medicine.

Kappelmayer, J., Gratama, J. W., Karászi, É., et al. 2000. Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a: Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols. Journal of Immunological Methods, 242, 53-65.

Kumar, S., Paiva, B., Anderson, K. C., et al. 2016. International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma. The Lancet Oncology, 17, e328-e346.

O’neill, K., Aghaeepour, N., Špidlen, J., et al. 2013. Flow Cytometry Bioinformatics. PLOS Computational Biology, 9, e1003365.

Rajkumar, S. V., Dimopoulos, M. A., Palumbo, A., et al. 2014. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. The Lancet Oncology, 15, e538-e548.

Sadaf, H., Ambroziak, M., Binkowski, R., et al. 2023. New molecular targets in Hodgkin and Reed-Sternberg cells. Frontiers in Immunology, 14.

Van Dongen, J. J. M., Lhermitte, L., Böttcher, S., et al. 2012. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia, 26, 1908-1975.

Waldron, D., O’brien, D., Smyth, L., et al. 2022. Reliable Detection of T-Cell Clonality by Flow Cytometry in Mature T-Cell Neoplasms Using TRBC1: Implementation as a Reflex Test and Comparison with PCR-Based Clonality Testing. Laboratory Medicine, 53, 417-425.