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Flow Cytometry Immunophenotyping: Neuron-Glial Antigen 2 Positivity In Mixed Lineage Leukaemia

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Immunophenotyping is used to analyse heterogeneous populations of cells to identify the presence and quantities of the various populations of interest. This is performed by adding antibodies with fluorochromes to the cells which expresses specific antigens expressed by these cells, these are referred to as markers. This prepared sample is connected to the flow cytometer where by the cells are oriented in sheet fluid and a laser is directed onto the fluid. There are a number of detectors aimed at the same point, these detectors detect forward scatter and side scatter; forward scatter detects the size of the cell and side scatter detects the inner complexity of the cells. There are also fluorescence detectors which allows for the detection of what markers the cells express or lack. This allows for deriving various types of information about the physical and chemical structure of each individual particle.

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The marker of interest in our study was a monoclonal antibody neuron glial antigen 2 (NG2) otherwise known as chondroitin sulphate proteoglycan 4 (CSPG4). The protein belongs to the transmembrane chondroitin sulphate proteoglycans family. NG2 is a 250kDa glycoprotein, and has a glycosaminoglycan (GAG) chain. It is expressed on human glial cells. This antigen regulates signalling events that are important for both cell proliferation and cell migration through at least two distinct mechanisms: the focal adhesion kinase (FAK) and the extracellular signal-regulated kinases (ERK). Most leukemic cells carrying KMT2A rearrangements in both B-ALL and AML cases can express a NG2. The expression of NG2 has a high sensitivity and specificity for KMT2A rearrangement. Specificity approaches 100% in B-ALL and in childhood AML cases and the sensitivity ranges between 50-80% in AML and exceeds 80% in B-ALL. Physiologically, NG2 homologue is expressed primarily in glial, muscle and cartilage progenitor cells but not in normal hematopoietic cells. NG2 homologue binds to matrix molecules including type VI collagen, NG2 oligodendrocyte. The progenitors are activated my demyelination rather than by inflammation. This may be related to the frequent CNS involvement in KMT2A rearrangements. In malignant disease, NG2 has been proven to promote metastatic potential of melanoma. The monoclonal antibody NG2 was developed to identify immature haematopoietic and stromal cell antigens. This antibody detects a 220-240 kDa cell surface chondroitin surface homolog. It has already been seen that NG2 does not react with normal haematopoietic cells, but reacts with cells from patients with AML or lymphoid leukaemia with 11q23/KMT2A gene rearrangement aberrations.

Little is known about the mechanisms regulating its expression. The expression of NG2 on flow cytometry is strongly correlated to the chromosomal abnormality involving the KMT2A gene. Cases expressing NG2 without the chromosomal aberration have also been described as well as cases with the KMT2A gene that have not expressed NG2. It has been discovered that NG2 is a marker of over-all bad prognosis in acute leukaemia and is an excellent marker of minimal residual disease.

18 May 2020

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