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Novel automated blood separations validate whole cell biomarkers.

Burger DE, Wang L, Ban L, Okubo Y, Kühtreiber WM, Leichliter AK, Faustman DL - PLoS ONE (2011)

Bottom Line: To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations.The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure.We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratories, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massaschusetts, United States of America.

ABSTRACT

Background: Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples.

Methods and findings: To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes.

Conclusions: Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.

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Related in: MedlinePlus

Lymphocytes for biomarker assays from Ficoll versus a magnetic method.In order to detect rare lymphocyte populations, it is critical that the separation method yields healthy cells of sufficiently high purity. (A) Schematic depiction of magnetic and gradient methods. The magnetic method is gentle, highly selective and retains the lymphocytes of interest (blue cells), whereas the gradient method has remaining red blood cells (red dots) and low viability (dark cells), while the cells of interest are largely lost due to cell death. (B) Comparison of performance parameters of magnetic and Ficoll gradient separation methods. Autoreactive CD8 T cells were isolated from 56 blood samples from human type I diabetics with both Ficoll and magnetic methods. Using the insensitive WST biomarker assay of rare autoreactive T cells, only magnetically separated cells showed the presence of the cells (Pass); Ficoll isolated cells uniformly lacked autoreactive T cells. The accuracy of the cell separation method was established by determining viability, purity, and yield. The results show that extremely efficient cell isolation procedures with high yield and high viabilities are required to preserve the cells of interest. (C) Flow cytometry with tetramers specific for autoreactive CD8 T cells shows that these cells are present and detectable after separation from patient blood samples using our magnetic method.
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pone-0022430-g003: Lymphocytes for biomarker assays from Ficoll versus a magnetic method.In order to detect rare lymphocyte populations, it is critical that the separation method yields healthy cells of sufficiently high purity. (A) Schematic depiction of magnetic and gradient methods. The magnetic method is gentle, highly selective and retains the lymphocytes of interest (blue cells), whereas the gradient method has remaining red blood cells (red dots) and low viability (dark cells), while the cells of interest are largely lost due to cell death. (B) Comparison of performance parameters of magnetic and Ficoll gradient separation methods. Autoreactive CD8 T cells were isolated from 56 blood samples from human type I diabetics with both Ficoll and magnetic methods. Using the insensitive WST biomarker assay of rare autoreactive T cells, only magnetically separated cells showed the presence of the cells (Pass); Ficoll isolated cells uniformly lacked autoreactive T cells. The accuracy of the cell separation method was established by determining viability, purity, and yield. The results show that extremely efficient cell isolation procedures with high yield and high viabilities are required to preserve the cells of interest. (C) Flow cytometry with tetramers specific for autoreactive CD8 T cells shows that these cells are present and detectable after separation from patient blood samples using our magnetic method.

Mentions: Taken together, the findings from >335 fresh blood samples studied by the magnetic method (Fig. 1C), combined with the ability to automate this blood separation process with robotic platforms (Fig. 2B), were convincing enough to justify the examination of these separated cell preparations in biomarker assays. We were particularly interested in testing the isolated whole blood cells for the reproducible detection of rare CD8 T cell subsets (approximately 0.6–2% of the total cell population) that are the autoreactive T cells in type I diabetes. As Figure 3A depicts, we hypothesized that with whole blood derived cell preparations featuring high viability, purity, yield and only infrequent RBC contamination, it was timely to see if the rare cells of autoimmunity persisted and were detectable. For this initial biomarker testing, autoreactive T cells were first studied by an indirect method of exposing isolated CD8 T cells to Tumor Necrosis Factor (TNF), which selectively kills autoreactive, but not normal CD8 T cells, which continue to proliferate [7]. After TNF challenge, the remaining CD8 T cell isolate was assessed for autoreactive cells by the WST assay for T cell proliferation. The second biomarker method we used to assess the impact of the isolation method on the detection of lymphocyte subpopulations was the more highly sensitive “tetramer” method. This highly sensitive method of autoreactive T cell detection directly evaluates cells for the presence of rare autoreactive CD8 T cells, and involves the incubation of whole blood isolates with self peptide bound with fluorescent HLA class I protein. Only a single clone of autoreactive T cells should recognize the tetramers in flow cytometric studies.


Novel automated blood separations validate whole cell biomarkers.

Burger DE, Wang L, Ban L, Okubo Y, Kühtreiber WM, Leichliter AK, Faustman DL - PLoS ONE (2011)

Lymphocytes for biomarker assays from Ficoll versus a magnetic method.In order to detect rare lymphocyte populations, it is critical that the separation method yields healthy cells of sufficiently high purity. (A) Schematic depiction of magnetic and gradient methods. The magnetic method is gentle, highly selective and retains the lymphocytes of interest (blue cells), whereas the gradient method has remaining red blood cells (red dots) and low viability (dark cells), while the cells of interest are largely lost due to cell death. (B) Comparison of performance parameters of magnetic and Ficoll gradient separation methods. Autoreactive CD8 T cells were isolated from 56 blood samples from human type I diabetics with both Ficoll and magnetic methods. Using the insensitive WST biomarker assay of rare autoreactive T cells, only magnetically separated cells showed the presence of the cells (Pass); Ficoll isolated cells uniformly lacked autoreactive T cells. The accuracy of the cell separation method was established by determining viability, purity, and yield. The results show that extremely efficient cell isolation procedures with high yield and high viabilities are required to preserve the cells of interest. (C) Flow cytometry with tetramers specific for autoreactive CD8 T cells shows that these cells are present and detectable after separation from patient blood samples using our magnetic method.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3142167&req=5

pone-0022430-g003: Lymphocytes for biomarker assays from Ficoll versus a magnetic method.In order to detect rare lymphocyte populations, it is critical that the separation method yields healthy cells of sufficiently high purity. (A) Schematic depiction of magnetic and gradient methods. The magnetic method is gentle, highly selective and retains the lymphocytes of interest (blue cells), whereas the gradient method has remaining red blood cells (red dots) and low viability (dark cells), while the cells of interest are largely lost due to cell death. (B) Comparison of performance parameters of magnetic and Ficoll gradient separation methods. Autoreactive CD8 T cells were isolated from 56 blood samples from human type I diabetics with both Ficoll and magnetic methods. Using the insensitive WST biomarker assay of rare autoreactive T cells, only magnetically separated cells showed the presence of the cells (Pass); Ficoll isolated cells uniformly lacked autoreactive T cells. The accuracy of the cell separation method was established by determining viability, purity, and yield. The results show that extremely efficient cell isolation procedures with high yield and high viabilities are required to preserve the cells of interest. (C) Flow cytometry with tetramers specific for autoreactive CD8 T cells shows that these cells are present and detectable after separation from patient blood samples using our magnetic method.
Mentions: Taken together, the findings from >335 fresh blood samples studied by the magnetic method (Fig. 1C), combined with the ability to automate this blood separation process with robotic platforms (Fig. 2B), were convincing enough to justify the examination of these separated cell preparations in biomarker assays. We were particularly interested in testing the isolated whole blood cells for the reproducible detection of rare CD8 T cell subsets (approximately 0.6–2% of the total cell population) that are the autoreactive T cells in type I diabetes. As Figure 3A depicts, we hypothesized that with whole blood derived cell preparations featuring high viability, purity, yield and only infrequent RBC contamination, it was timely to see if the rare cells of autoimmunity persisted and were detectable. For this initial biomarker testing, autoreactive T cells were first studied by an indirect method of exposing isolated CD8 T cells to Tumor Necrosis Factor (TNF), which selectively kills autoreactive, but not normal CD8 T cells, which continue to proliferate [7]. After TNF challenge, the remaining CD8 T cell isolate was assessed for autoreactive cells by the WST assay for T cell proliferation. The second biomarker method we used to assess the impact of the isolation method on the detection of lymphocyte subpopulations was the more highly sensitive “tetramer” method. This highly sensitive method of autoreactive T cell detection directly evaluates cells for the presence of rare autoreactive CD8 T cells, and involves the incubation of whole blood isolates with self peptide bound with fluorescent HLA class I protein. Only a single clone of autoreactive T cells should recognize the tetramers in flow cytometric studies.

Bottom Line: To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations.The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure.We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratories, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massaschusetts, United States of America.

ABSTRACT

Background: Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples.

Methods and findings: To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes.

Conclusions: Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.

Show MeSH
Related in: MedlinePlus