SubscribeThe Duet™ is an automated microscope for scanning and sorting cells in bright-light and fluorescent illumination (FISH).
The cells are identified according to their features. The Duet ™ is developed and manufactured by Bioview but supplied and supported in the UK by Preston based Image Solutions (UK) Ltd.
In the bright field mode the system automatically classifies cells into six groups - PMN, lymphocytes, normoblasts, myelocytes, blasts and plasma cells - based on cell morphology after MGG staining. After immunohistochemical (IHC) staining the system automatically classified the cells into two groups, positive stained and negative stained. In the fluorescent mode it accomplishes fast and accurate spot counting plus identification of other chromosomal aberrations such as translocations and inversions.
All of this is done at a very high rate, about 10,000 cells per minute in bright field mode and thousands of cells per hour in fluorescent mode, in true colour and with high resolution.
Duet’s advanced software technology rapidly links morphological analysis with Fluorescent in-situ hybridisation (FISH) technology allowing the pathologist to analyse the same cell by both methods. This ability to carry out combined analysis is unique and has resulted in several leading clinical and hospital laboratories increasing the accuracy of their leukemia diagnosis.
Laboratory staff can easily use Bioview’s advanced technology and biological protocols to scan slides stained with Giemsa or labelled for the presence of a specific antigen by IHC. The initial stain is then simply washed off and the specific FISH probes are applied and the slide is rescanned on the Duet ™. The two scans are fully synchronised and for each and every cell in the sample the system can present two aspects – morphology/IHC results and FISH signals of the applied probes. The combination of these two scans provides a full picture of a suspicious cell, increasing both the accuracy and specificity of diagnosis (Ref. 2, 5) and aiding the clinician.
The power of the Duet ™ system is based on a number of important features:
- The combination of two tests (genetic and morphology or genetic and IHC).
- Automation of tedious manual work.
- Accurate enumeration of the cells of interest.
- Increased number of cells examined per case, which reduces false rates.
- Increased sensitivity. The ability to detect cells at a frequency of 1:40,000 in automated fluorescent scanning is very important in the follow up of minimal residual disease cases (Ref. 2).
Several applications in hematology malignancies where this approach may prove beneficial are described below. They include bone marrow transplantation (BMT) multiple myeloma (MM) and other types of blood diseases (CLL, SCID, CML).
Bone Marrow Transplant (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 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) whose existence at more than 5% in the bone marrow indicates a high risk of 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 too.
BioView combines both tests into one. To begin with, a sample is prepared and stained with a stain that is suitable for the identification of blast cells. The images and coordinates of these cells are recorded. The same slide is then stained with FISH markers suitable for the identification of the host cells. Typically these are XY/XX in sex-mismatched transplants, or the disease typical chromosomal rearrangements where the donor and host are of the same gender.
The system next looks at the FISH pattern in the blast cells. A host blast is indicative of relapse. For example, a patient with just four blast cells having disease markers is indicating an upcoming relapse. Studies done in the Tel Aviv-based Sheba medical center in 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, the morphology was analysed to be 1.2% of plasma cells and using FISH analysis, 7% of the white blood cells in the sample were shown to have the deletion 13q. Both results are within normal ranges. However, within the plasma cells, 40% of the cells had a 13q deletion. This is, again, 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:
- 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 and images the metaphases. For karyotyping purposes the Duet ™ metaphase finder outputs images of metaphases and lists the coordinates of their location on the slide. A thumbnail display image enables users to select the best overall metaphases, while a schematic display of them on the slide allows selection of the best examples from individual colonies. The AutoCovert coordinate feature simplifies karyotyping analysis by allowing metaphase location to be achieved on each and every microscope in the laboratory.
Figure 5: The report of the DuetTM G-banding metaphase finder. The colonies are marked in green, and the red dot identifies the location of the specific metaphase in the colonies. The coordinates of each metaphases on microscope karyotyper in the lab appear under the slide.
Fluorescent-DAPI: The system automatically identifies the metaphases and images them with high resolution (x63). All commercially available probes can be analysed.
This application minimises technician time and increases the test capacity of the lab.
Conclusions
Combining information from two stains on the same cell enables the user to focus diagnostic effort on those cells that are the target of the assay. This leads to an enhanced sensitivity of the assay.
The fact that these two indications are completely independent leads to enhanced specificity. Also, in some applications, rare cells (down to 1:40000) still give significant and meaningful results. So the method also leads to more accurate diagnosis, which reduces overall medical costs and minimises the loss of quality of life among patients.
The ability to analyse metaphases of the same sample may also contribute to diagnosis accuracy.
For further information, please contact:
Mr. Tim Fletcher, Image Solutions (UK) Ltd., IMSOL House, Cable Court, Pittman Way, Fulwood, Preston PR2 9YW. Tel: 01772 663 140 Fax: 01772 663 150
Email: tim@imsol.co.ukwww.imsol.co.uk
The Duet ™ is developed and manufactured by Bioview Ltd. but supplied and supported by Image Solutions (UK) Ltd.
Authored by Dr. Malka Reichart of BioView Ltd. and Mr. Ian Corless MD of Image Solutions (UK) Ltd.