Imsol - Image Solutions

The Duet is an automated microscope for scanning and sorting cells in bright- light and in fluorescent illumination (FISH). The cells are identified according to their features. In the bright field mode the system automatically classifies the cells into 6 groups (PMN, lymphocytes, normoblasts, myelocytes, blasts and plasma cells) based on the cell morphology after MGG staining. After Immunohistochemical staining the system automatically classified the cells into 2 groups, positive stained and negative stained. In the fluorescent mode it accomplishes fast and accurate spot counting and identifying other chromosomal aberrations such as translocations, inversions etc. All of this is done at a very high rate (~10,000 cells per minute in bright field mode and thousands of cells per hour in fluorescent mode), in true color and high resolution.

Moreover, BioView has developed the software and the biological protocols enabling scanning a slide stained with Giemsa or labeled for the presence of a specific antigen by IHC, subsequently wash the initial stain, apply FISH probes of interest and rescan the slide on the Duet. The two scans are fully synchronized and for each and every cell in the sample the system can present two aspects of the same cell – morphology/IHC result, and the FISH signals of the probes that were applied to it.

The combination of the two scans provides a full picture of a suspicious cell, and therefore increases the accuracy and specificity of the diagnosis (Ref. 2, 5).

The power of the Duet system is in several aspects: 

• Combination between two tests (genetic and morphology or genetic and IHC).
• Automating the now-manual tedious work.
• Enumeration of the cells of interest in an accurate manner.
• Increase the number of cells examined per case - hence reducing the false rates
• Increase sensitivity - in our system we were able to detect in automated fluorescent scanning cells at a frequency of 1:40,000. This is very important in follow up of Minimal residual disease cases (Ref. 2).

Below, several applications in Hematology malignancies where this approach may prove beneficial are described: Bone Marrow Transplantation (BMT) Multiple Myeloma (MM) and other types of blood diseases (CLL, SCID, CML).

Bone marrow Transplantation (BMT)

Following BMT, patients are routinely followed up in order to identify relapses.

Currently, the method of follow up is to extract a blood or a bone marrow sample, divide it into two, and perform two tests: morphological analysis and FISH count. The morphological part looks for immature cells (blast cells), and existence of more than 5% of these cells in the bone marrow indicates a high risk for relapse. The FISH protocol is performed on the other part of the sample. Identification of more than 5% of the cells as host cells is indicative of a high risk for relapse.

BioView suggests to combine both tests into one tests: in the first step, a sample will be prepared and stained with a stain that is suitable for the identification of blast cells.

The images and coordinates of these cells will be recorded. The same slide will next be stained by the FISH markers suitable for the identification of the host cells (typically XY/XX in sex-mismatched transplants, or the disease typical chromosomal rearrangements where the donor and host are of the same gender).

The system now looks on the FISH pattern only in the blast cells. A host blast is indicative of relapse. For example, a case of a patient where only 4 blast cells were identified in 5000 cells, but all of them had the disease markers, indicate a clear upcoming relapse. Studies done in Sheba medical center (Tel Aviv, Israel) indicate that relapses can be identified up to two months prior to being diagnosed by conventional methods, without loss of specificity (Ref. 1, 4, 6).

Figure 1: The DuetTM main classification screen showing pairs of morphology and FISH images. The left image in each pair is of a myeloblast, and the right image shows the same cell with the XY genotype of the host cells (X in green and Y in red).

Multiple Myeloma (MM)

Being a disease of plasma cells, this may well be the most striking example of the usefulness of the method. In normal patients, or for patients in remission, the percentage of plasma cells in the bone marrow is expected to be 0.5%-1%. The MM FISH probes have a cutoff of 5-10%. The background level is especially problematic in MM as in this group of patients it is common to find a low percentage of PC in BM samples regardless of the disease severity. This phenomenon is referred as a "sample error" and is due to the patchy pattern of PC distribution in BM. Difficulty in BM aspiration and dilution of the BM sample with PB may add to this variation. This limitation is overcome by identifying the plasma cells by their morphology or by their immuno-histochemical staining, and then counting FISH signals within the plasma cells population only.

In one sample, we found 1.2% of plasma cells, and 7% cells with deletion 13q. Both results are within normal. However, within the plasma cells, 40% of the cells had a 13q deletion. This is, of course, a clear indication of a relapse. More results are described in the references (Ref. 9).

Figure 2: The right hand image shows a cell with del-13q (one red signal). The left hand image shows the morphology of the same cell within the red rectangle) – a plasma cell.

Other types of blood diseases

The efficiency of the system has also been demonstrated for other diseases, most notably for: 

• CLL and ALL – where only lymphocytes, to their various stages of maturity should be considered (Ref. 3).
• SCID (Severe Combined Immuno-Deficiency), where the B and T cell should be distinguished in order to follow up the remission process (Ref. 10).

Figure 3: The right hand image shows a host cell with XY genotype (one green and one red signal). The left hand image shows the - a positive CD19 stained B – lymphocyte (a brown cell – within the red rectangle). The CD3 pink stained cells seen on the left image are T- lymphocytes. The left image shows that these cells are donor cells with XX genotype. 

• CML research – follow up of accelerated disease progression in CML patients can be connected to secondary aberrations. This may have a significant impact on the efficiency of disease medications such as Glivec (Ref. 11).

Figure 4: The right hand image shows a cell with Double Ph+ (2 yellow, 2 red and 1 green signals). The left hand image shows the morphology of the same cell within the red rectangle) – a blast. 

• Other myeloproliferative disorders (Ref. 8).

Metaphase finder (G-banding and DAPI)

From cultured peripheral blood and bone marrow.

G-banding: The system automatically identifies the metaphases and images them. For karyo typing purposes the Duet metaphase finder outputs images of metaphases and coordinate list of their location on the slide. The Duet enables to choose the best metaphases using thumbnail images display. A schematic display of the metaphases on the slide enables to choose metaphases from different colonies. Using the coordinate AutoCovert feature the Duet enables to locate the metaphases on each and every microscope in the lab for karyotyping analysis.