Commercial reality for Australian-developed leukaemia diagnostic test

University of Sydney spin-out company Medsaic has developed a proprietary technology, DotScan, which can be used to diagnose haematological malignancies, solid tissue tumours and autoimmune diseases. This year, the company was awarded the NSW BioFirst Commercialisation Award for outstanding achievement in technology for its leukaemia and lymphoma diagnostic. The award is given to the biotechnology company deemed most likely to achieve international success with its proof of concept technology.

The first product based on Medsaic's DotScan technology is for diagnosing leukaemia and lymphoma. It has been assigned a Medicare rebate item number and Mayne Health is Medsaic's first customer. Mayne is providing the test in four states in Australia including QML Pathology in Queensland, Dorevitch Pathology in Victoria, Western Diagnostic Pathology in Western Australia and Mayne Health Laverty Pathology in NSW.

"Mayne Pathology is pleased to have played a role in the development of this exciting new technology. Improving patient care and clinical outcomes are important to our business and the implementation of the Medsaic array represents a real step forward for our patients and the medical community," said Mr Paul Cosban, Mayne's general manager Pathology.

The test results will assist clinicians to achieve a better understanding of their patient's condition and to provide better treatment as a consequence.

The technology first mooted in a casual conversation at a tennis match, received seed funding from an angel investor and major funding from Symbion Ltd. Later grants from the COMET and BIF schemes of the Commonwealth Government and more recently from R&D Start, together with BioFirst and BioBusiness programs of the NSW Department of State and Regional Development enabled the development to continue.

The technology behind DotScan

The technology is based on cell capture by microarrays of antibodies bound to nitrocellulose-coated microscope slides. It is suitable for in-vitro diagnostic assessments of mononuclear leukocytes in peripheral blood or marrow by characterisation of surface antigens in patients with known or suspected leukaemias/lymphomas where the malignant population is dominant.

Leukaemias and lymphomas express on their plasma membranes particular sub-sets of the defined cluster of differentiation (CD) antigens, also found on cells along the lineages of differentiation to mature myeloid and lymphoid leukocytes. The use of CD antigen expression (immunophenotyping) for characterisation of leukaemias and lymphomas is well established.

DotScan enables screening for expression of more than 80 CD antigens on leukocytes in a single assay using a microarray of monoclonal antibodies.

A suspension of leukocytes is applied to the microarray, and cells only bind to antibody dots for which they express the corresponding CD antigen. The cells captured on the microarray form a dot pattern (mosaic) that is stored as a digital image; the pattern defines the repertoire of surface molecules present. Distinctive and reproducible dot patterns have been obtained for normal leukocytes and various leukaemias and lymphomas from myeloid, B-cell and T-cell lineages. Where a clonal leukaemic cell population is the dominant cell type present, the array results typically represent an accurate immunophenotype of the leukaemia.

The test is appropriate for in-vitro diagnostic applications where the leukaemic cell count is -10 x 109 cells/L or higher in peripheral blood or the dominant population in bone marrow. A complementary method with DotScan should be considered for samples where the leukaemic population is below 10 x 109 cells/L in peripheral blood or less than 50% of the cells in bone marrow samples.

Test methodology

The antibody array is prepared on a nitrocellulose-coated region of a microscope slide. The advantage of nitrocellulose is that the monoclonal antibodies (Mabs) applied to the surface as drops dry as symmetrical circles, giving an unambiguous pattern to the array.

Robotic arrayers apply 10 nL samples in duplicate, one set on the left side of the nitrocellulose patch, the other on the right. This effectively provides two tests in one, and internal quality control.

For the leukaemia and lymphoma applications, 82 discriminatory, diagnostic dots are applied in duplicate. The slides are stable for 12 months when refrigerated with desiccant. Control antibodies are also included.

A suspension of the patient's cells, from either blood or bone marrow, is applied to the array and incubated for 30 minutes to allow cell capture to take place.

The slide is then washed to remove unbound cells by placing them vertically in a series of wells containing wash buffer, followed by immersion in fixative to fix bound cells to the surface of the slide.

While still moist, the slide is placed in the reader for scanning and the image is captured and interrogated using Medsaic's proprietary DotScan software.

DotScan finds the corner dots on each slide as a reference point, then places a grid across the dotted area and adds labels for each antibody. The image of the test slide appearing on the computer screen is viewed as a mosaic of dots, where the intensity of each dot reflects the density of cell binding. After normalisation, each dot is quantified above background using a scale from 0 to 255.

The cell binding density data is presented on screen in a number of different numerical, pictorial and graphical formats which are collated with patient information to produce a written report.

The report includes a table summarising the binding density data, grouped in accordance with recognised lineages of differentiation. A colour-coded histogram based on these lineages shows at a glance which cell types are present in the sample. A comparison of the binding pattern with the disease-specific signatures leads to diagnosis.

Clinical data

The DotScan has been clinically evaluated using Peripheral Blood Leukocytes (PBL) and Bone Marrow Aspirate (BMA) samples in diseases including:

• Acute Myeloid Leukaemia (AML)
• B-Cell Chronic Lymphocytic Leukaemia (B-CLL)
• Hairy Cell Leukaemia (HCL)
• Non-Hodgkin's Lymphoma (NHL)
• Precursor B-Cell Acute Lymphoblastic Leukaemia (Precursor B-ALL)
• T-Cell Lymphoproliferative Disease (T-LPD)

The components of the technology

The DotScan technology has three components, each of which is patent protected. These are:

• A single use diagnostic kit containing a nitrocellulose coated glass slide carrying an array of dots corresponding to a range of discriminatory antibodies, and the reagents needed to perform the test;
• A peripheral slide reader, DotReader, for capturing an optical image of the test slide; and
• A software package to handle the information generated by the slide reader and to produce a comprehensive report of the result.

Future developments

Other products at various stages of development employ DotScan's platform technology for the diagnosis of solid tissue tumours, autoimmune diseases such as lupus, viral infections and cardiovascular disorders.

The platform potentially allows the diagnosis of any disease where antibodies are employed to determine the antigenic profile of abnormal cells. Theoretically it can be used to monitor disease progression and to determine the efficacy of antibody treatment and/or chemotherapy for an individual patient.