Tips to enhance FISH workflows for CD138 cells

Bone marrow aspirates are a challenging sample type from which to obtain accurate and conclusive FISH results due to the low abundance of plasma cells in which the cytogenetic abnormalities would be present.

To overcome this, labs commonly incorporate the plasma cell marker CD138+ into their selection methods to enrich plasma cells before performing FISH to enhance the detection of high-risk cytogenetic abnormalities. While effective in isolating the cells of interest, these extraction methods can have knock-on effects that impede probe penetration and alter cell morphology, leading to weak signals and inconclusive results. The two most frequently used methods labs use to enrich plasma cells are flow cytometry cell sorting and Magnetic Activated Cell Sorting (MACS). Each of these methods presents challenges that, if not addressed, can lead to weak signals and inconclusive results.

In this blog, we will explore common obstacles encountered in CD138+ cell selection workflows and offer practical tips to maximise success in FISH analysis, ensuring clearer signals and better probe penetration.

Loss of surface markers

The MACS process for CD138+ cells can lead to a significant loss of important cell surface markers (e.g., CD71, CD11b, CD11a, CD49e, and CD69) on plasma cells.¹ Similarly, CD138+ sorting by flow cytometry can also lead to a marked loss of these important cell surface markers.²

The loss of surface markers impacts downstream FISH analysis as they play key roles in the structural integrity and phenotypic characteristics of the plasma cells, which may affect probe penetration and hybridization. In some cases, plasma cell subsets can be lost entirely during sorting2. This can skew the results of downstream FISH by limiting the diversity of the plasma cell population being analysed. When plasma cells are already scarce, overlooking plasma sub-populations could mean missing important genetic abnormalities.

Residual antibodies/magnetic beads

Both flow-based and magnetic-based cell separation methods can leave behind residual artifacts, such as antibodies or magnetic beads, which may skew FISH results in several ways. Plasma cells are naturally protein-rich, and as such, naturally pose a barrier to probe penetration. Residual artifacts exacerbate this issue by creating a physical barrier on the cell surface, further preventing FISH probes from accessing the DNA within the cells. This often results in weak or incomplete hybridization, leading to lower signal intensity and reduced sensitivity of the assay.

Residual antibodies and magnetic beads can also increase the likelihood of non-specific probe binding. The FISH probes may bind to these foreign materials instead of the target sequences, increasing background fluorescence and making it difficult to distinguish between true signals and non-specific noise.

Optimizing CD138+ cell selection for FISH

Challenges with plasma cell enrichment are commonplace, but there are a number of strategies and workflow adjustments being discussed in the field which seek to overcome these issues. Here are some of the inside tips used by FISH experts to enhance CD138+ cell selection and FISH results:

Strategies to address surface marker loss:

• Optimize enrichment protocols: Flow cytometry or magnetic bead separation protocols can be fine-tuned to minimize the disruption of cell surface markers. Reducing the number of sorting cycles and using more selective antibodies or beads can help retain more surface antigens, preserving the plasma cells' phenotypic integrity​.
• Pre-fixation treatment: Using mild hypotonic treatments before fixing CD138+ cells can help the cells retain phenotype and surface markers.

Minimizing residual antibodies or magnetic beads:

• Post-separation washing: After cell sorting or magnetic separation, employing multiple wash steps with gentle centrifugation can help reduce the number of residual antibodies or beads left on the cell surface which impede probe hybridization. Take care to balance effective washing with cell retention to avoid losing too many plasma cells in the process.
• Prolonged pre-assay fixation: Storing plasma cells in methanol-acetic acid (Carnoy’s fixative) for 4–7 days at –20°C post-enrichment can improve probe penetration. The prolonged fixation helps stabilize the cell membrane and reduces the impact of residual materials on hybridization​. Use fresh fixative for the FISH assay to ensure optimal conditions for probe hybridization. Alternatively, prior to applying FISH probes, prepared slides of sorted CD138 cells can be stored at -20°C for 24 hours to help increase signal intensity.
• Pre-treatment digestion: Enzyme digestion, a common step in pre-treating plasma cells for FISH, can also help eliminate residual antibodies or beads from the cell surface. By fine-tuning enzyme concentration and incubation time, it’s possible to break down these surface obstructions without damaging the cells.

Improving probe penetration:

• Extended denaturation times: For CD138+ plasma cells, slightly extending the denaturation time during FISH setup can help the probes better penetrate the highly proteinaceous cell membranes. However, care must be taken not to over-denature. Always monitor signal quality and adjust denaturation times based on signal clarity to avoid over-denaturation, which can increase non-specific binding and background noise.
• Increased enzyme digestion: For plasma cells with thick proteinaceous membranes, increasing enzyme digestion times during pre-treatment may help expose the target DNA more effectively, allowing for better probe binding​. Take care to avoid over-digestion, which can damage your sample and compromise FISH results.

Hook onto high quality plasma cells for better FISH results

Optimizing the enrichment and preparation of CD138+ plasma cells can boost your chances of obtaining accurate and conclusive FISH results. Experts in the field are addressing common enrichment issues posed by separation techniques, such as the loss of surface markers, residual materials and poor probe penetration, through strategic workflow adjustments.

By carefully refining CD138+ cell selection protocols, adjusting washing and fixation techniques, and optimizing probe penetration and denaturation conditions, it’s possible to improve the clarity and consistency of FISH results. Addressing these workflow challenges will help ensure that even with scarce plasma cells, your FISH analyses will yield strong, reliable signals.

Top tips recap

• Thoroughly wash cells post-selection to remove antibody/magnetic bead residue.
• Consider using a mild hypotonic treatment before fixation to maintain cell morphology
• Extend storage at –20°C in methanol-acetic acid fixative for 4–7 days before performing FISH can improve probe penetration.
• Optimize enzyme digestion and denaturation time to improve probe penetration.

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References

1. Bansal R et al. Clin Lymphoma Myeloma Leuk 2021;21:e48-e51. doi: 10.1016/j.clml.2020.08.003.
2. Sanz I et al. Front Immunol 2019;10:2458. doi: 10.3389/fimmu.2019.02458.