Syndecan-1 (SDC1), also known as CD138, is a well-known and specific plasma cell marker that is highly expressed on the surface of multiple myeloma plasma cells. As it can be a challenge detecting the small number of malignant plasma cells within the large proportion of cells obtained from a bone marrow (BM) aspirate, CD138 plasma cell enrichment is key to isolating plasma cells from bone marrow aspirates. We recently sat down with Janelle Salkowitz-Bokal, PhD, Maria Coronesi, and Lucas Rifkin, MD, to explore how CD138 plasma enrichment enhances the sensitivity of downstream analysis for multiple myeloma testing.
Targeted cell isolation (TCI)
Due to the fact that higher multiple myeloma plasma cell abnormality rates can be seen on enriched CD138 cells, in comparison to routinely cultured BM cells, CD138 enrichment has become a mainstay in gaining reliable data from fluorescence in situ hybridization (FISH) and flow cytometry, among other downstream techniques. The Covance Targeted Cell Isolation (TCI) team uses immunomagnetic cell isolation technology to complement downstream analysis. (See Box 1 for Covance TCI capabilities.) This way, CD138+ plasma cells from primary bone marrow mononuclear cell (BMMC) and peripheral blood mononuclear cell (PBMC) samples can be targeted using antibody complexes with affinity for CD138 on the cell surface to enable cross-linking of the labeled cells to magnetic particles for positive selection immunomagnetic cell separation. Covance achieves this by using Miltenyi Biotec Microbeads for CD138 magnetic cell separation of BM analytes. When the sample is placed into a magnetic field, magnetically labeled cells are retained, while unlabeled cells are removed.
TCI techniques: then and now
Changes to TCI techniques at Covance have greatly reduced the loss of plasma cells during the cell isolation process. Previously, plasma cells were isolated from BM aspirates using a 2-step process; with the first step involving the isolation of BMMCs followed by isolation of plasma cells. The shift to utilizing StraightFrom® MicroBeads has enabled a much more rapid and efficient process of plasma cell isolation directly from BM aspirate, resulting in little loss of plasma cells. Studies at Covance have shown that up to 40% of CD138+ plasma cell enrichment can be generated from as little as <1% BM aspirate through use of StraightFrom® MicroBeads.
The technicians at Covance use a column-based technology with nano-sized superparamagnetic beads and an amplified magnetic force. With minimal labeling, there is no alteration to cell characteristics and there is no nonspecific labeling of the target cell fraction.
Flow cytometry available in all five central lab locations
Following the cell enrichment process, the purity of both the positive and negative fractions can be confirmed with flow cytometry. This allows for the exclusive tracking of plasma cells prior to their further downstream analysis, which may be a requirement in some clinical studies. In many cases, flow cytometry is seen as a crucial accompaniment to the enrichment process and overall workflow.
Extensive flow cytometry technologies are available across the global network of Covance central labs, where a dedicated team of highly trained operational staff, data analysts and validation scientists play a pivotal role in controlling technical variability and ensuring high quality results. Instrumentation at Covance laboratories includes that of FACSCanto™ II, FACSLyric™ and Bio-Rad ZE5. Furthermore, board certified, specifically trained hematopathologists interpret flow cytometry results, including in the assessment of minimal residual disease for plasma cell neoplasms. All questionable data is reviewed in-house by the experienced TCI Scientific team to ensure expert interpretation is applied.
Flow cytometric quantitative analysis as standard practice
In conducting clinical studies, it is important to ensure that the first BM aspirate is used as the source of plasma cells or, failing this, that the needle is repositioned in order for a new draw to be obtained. This is necessary to minimize the risk of hemodilution. These recommendations form part of the consensus guidelines on performing interface FISH testing in multiple myeloma. At Covance, flow cytometric quantitative analysis of plasma cells is performed in every case. Consequently, sponsors can be assured as to the validity of samples used in the studies. We understand how vital it is for techniques to be used that limit the peripheral blood content and consequently support plasma cell collection for your study.
Improvements in accuracy and throughput
Validation of the Beckman Coulter CytoFLEX system
Flow cytometry has been shown to offer greater accuracy when assessing PBMC count and viability using CD45 and 7-AAD (DURAClone IM Count Tube) versus Trypan Blue exclusion methods. An absolute count is generated by a known number of fluorescent beads per tube being counted and specific gated events using a mathematical formula. Covance now has CytoFLEX flow cytometer instruments available at each global location – there are two instruments for Singapore, Shanghai and Tokyo, and three instruments for Geneva and Indianapolis.
Covance is currently in the process of validating the count, viability and purity of CD138 by flow cytometry globally. Completion of the validation process for the CytoFLEX panel for all TCI processes is expected in January 2021. This offers an exciting development for sponsors as results will be more accurate and available immediately in real-time.
Automated PBMC isolation
Covance is also validating instrumentation to support automated PBMC isolation to increase efficiency and save valuable hands-on time. Automated PBMC isolation is a key innovation in this field and is currently unique to Covance. It is designed to increase throughput and help maintain the optimal viability and recovery of cryopreserved PBMCs. The Covance team is expecting to offer this service in Q1, 2021 from the Indianapolis site.
Worldwide shipment of samples within the stability timeframe
BM samples can only remain stable for up to 48 hours following collection and, since BM samples cannot be frozen, it is vital that BM aspirates are transferred rapidly to a lab for processing. This is achieved at Covance through the globally located team of more than 50 logistics experts to assure on-time and in-stability shipments are achieved.
Following aliquot, plasma samples are obtained and rapidly frozen on dry ice where they can then be sent to one of the central labs for subsequent FISH analysis and interpretation. Importantly, these samples may also be stabilized and stored for up to three months, under the auspices of our highly trained Anatomic Pathology and Histology (APH) team.
Continued operations despite COVID-19
COVID-19 continues to affect communities and healthcare systems across the globe. However, due to the robust business continuity plans and highly skilled logistics professionals, Covance is in a unique position to offer sample shipment to multiple global locations, ensuring that sample stability timeframes are met. This is especially important when it comes to the short stability of BM aspirates.
Efficiency, reliability and robust clinical interpretation
By engaging with Covance to support you in CD138 plasma cell enrichment, you can be assured of the high quality of plasma cell samples on which the results of your key clinical trials rely. We bring efficiency, reliability and robust clinical interpretation to your data and studies by employing the latest technologies backed by expert insights and guidance from our technical staff. You can be confident in our rigorous, well-documented practices from initial sample collection all the way through to provision of study findings.
Other Blog Posts in the Advancing Multiple Myeloma Clinical Trials series: