Plasma Cell Enrichment

Plasma Cell Myeloma (Multiple Myeloma) presents a unique challenge for cytogeneticists. The neoplastic plasma cells (PCs) often have a very low proliferation rate (low mitotic index), making standard metaphase karyotyping difficult; conventional cytogenetics yields normal results in 60-70% of active myeloma cases because only the normal myeloid cells divide. Furthermore, in many patients, the plasma cells comprise a small fraction of the total bone marrow cellularity (\(<5\%\)). Performing Interphase FISH on a standard marrow smear is risky because the analyst might inadvertently score hundreds of normal lymphocytes and neutrophils, missing the rare abnormal plasma cells (false negative). To solve this, Plasma Cell Enrichment (Selection) is mandatory for accurate prognosis

The CD138 Target

The enrichment process relies on the specific immunophenotype of plasma cells

• CD138 (Syndecan-1): This proteoglycan adhesion molecule is strongly and uniformly expressed on the surface of both normal and malignant plasma cells. It is generally absent from other bone marrow lineages (myeloid, erythroid, lymphoid)
• The Strategy: We use magnetic beads coated with anti-CD138 antibodies to physically pull the plasma cells out of the complex marrow mixture

Magnetic Activated Cell Sorting (MACS) Protocol

This is the industry standard for positive selection

1. Preparation: The bone marrow aspirate is washed to remove plasma and fat
2. Labeling: The cell suspension is incubated with Anti-CD138 MicroBeads. These are microscopic iron-containing beads attached to specific antibodies. They bind exclusively to the CD138+ plasma cells
3. Separation (The Magnet)
   • The sample is passed through a column placed inside a strong magnetic field
   • Capture: The magnetically labeled plasma cells stick to the wall of the column
   • Flow-Through: The unlabeled cells (neutrophils, T-cells, B-cells) flow straight through the column and are collected in a “Negative Fraction” tube
4. Elution: The column is removed from the magnet. A plunger is used to flush buffer through the column, releasing the trapped plasma cells into a “Positive Fraction” tube
5. Processing: This positive fraction (now nearly 100% plasma cells) is then harvested and used for FISH slide preparation

Alternative: Cytoplasmic Immunofluorescence (cIg-FISH)

If magnetic sorting is not available or the sample is too small, a simultaneous staining method is used

• Mechanism: Plasma cells are factories for Immunoglobulin (Ig). Their cytoplasm is packed with light chains (Kappa or Lambda)
• Protocol: The FISH slide is stained with a fluorescent dye (e.g., AMCA-blue) targeted against cytoplasmic Immunoglobulin
• Analysis: The laboratory scientist looks for the probe signals (Red/Green) only inside the cells that glow blue (Ig-positive plasma cells). This is a “visual enrichment” rather than a physical one

Clinical Impact

• Sensitivity: Enrichment increases the detection rate of abnormalities (like IGH translocations or del(17p)) from ~30% in unenriched samples to >90% in CD138-enriched samples
• Prognosis: Since myeloma is defined by specific high-risk markers (e.g., t(4;14), del(17p)), failing to enrich leads to missed high-risk diagnoses and inappropriate therapy
• Limitations
  • CD138-Negative Myeloma: In rare cases, the tumor cells downregulate or “shed” CD138 (especially in relapsed/aggressive disease). Magnetic sorting will discard these tumor cells into the negative waste fraction
  • Clumping: Plasma cells are sticky. If the marrow clots, the magnetic column will clog

Processing the Enriched Pellet

Once enriched, the CD138+ pellet is often tiny (barely visible)

• Handling: The laboratory scientist must use extreme care not to aspirate the pellet during the hypotonic/fixation steps
• Slide Making: Because the volume is small, the entire pellet is often used to make just 1 or 2 hybridization areas (“spots”) on a slide