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Updated: Mar 18, 2026
Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis
Published on: March 20, 2021
Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective
Peixuan Zhu1, Melissa L Stanton2, Erik P Castle3
1Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac, MD, 20854, USA.
Background:
Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples.
Methods:
Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration.
Results:
MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples.
Conclusions:
To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.
Insights
This study demonstrates that microfiltration effectively isolates circulating tumor cells (CTCs) and cancer-associated macrophage-like cells (CAMLs) from cryopreserved blood samples. This method preserves cell integrity, enabling retrospective analysis of patient outcomes.
Area of Science:
- Oncology
- Cell Biology
- Biobanking
Background:
- Cryopreserved peripheral blood mononuclear cells (PBMCs) are common in biobanks, but data on tumor-associated cell preservation are limited.
- Assessing circulating tumor cells (CTCs) and cancer-associated macrophage-like cells (CAMLs) in cryopreserved samples is crucial for retrospective studies.
Purpose of the Study:
- To evaluate the feasibility of using the CellSieve™ microfiltration assay for isolating CTCs and CAMLs from cryopreserved PBMC samples.
- To determine the analytical accuracy, efficiency, and reproducibility of this method after cryopreservation.
Main Methods:
- Blood samples were spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines.
- Samples underwent Ficoll separation, cryopreservation, thawing, and microfiltration using the CellSieve™ assay.
- CAMLs were quantified in blood samples from renal cell carcinoma (RCC) patients.
Main Results:
- High recovery efficiencies for MCF-7 (87.8-89.0%), PC-3 (84.7-85.3%), and 786-O (81.3-82.7%) cells were achieved after cryopreservation.
- Recovered cells maintained their morphology and immunohistochemical markers post-thaw.
- No statistically significant difference in recovery rates of CTCs and CAMLs was observed between cryopreserved and fresh samples.
Conclusions:
- This is the first report demonstrating successful cryopreservation and microfiltration analysis of CAMLs.
- Microfiltration of cryopreserved samples facilitates extensive retrospective studies on cancer patients' long-term outcomes.
- The CellSieve™ assay is a viable method for analyzing CTCs and CAMLs in cryopreserved biobanked samples.
