Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials
© Letzkus et al.; licensee Springer. 2014
Received: 16 June 2014
Accepted: 7 October 2014
Published: 13 November 2014
Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood.
Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based).
Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS.
The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.
KeywordsCell sorting Transcriptomics Clinical
There is an ever-increasing demand for the discovery, validation, and application of clinically useful molecular biomarkers that enable patient stratification, diagnosis, monitoring of disease progression, or a better understanding of drug response (safety and efficacy) -. Gene expression biomarkers have been investigated in various diseases such as autoimmune diseases ,, cancer -, neurological diseases ,, infections , and in transplantation ,. Whole blood or peripheral mononuclear cells (PBMCs) have long been the preferred surrogate tissues for clinical transcriptomics in biomarker research for two major reasons: Firstly, blood is an easily accessible human tissue, and secondly, robust sampling methods of whole blood (e.g. PAXgene Blood RNA Tubes) ,,- and PBMCs , are well established. As is the case with any tissue sample with mixed cell population, whole blood or PBMC transcriptome analysis approaches are sensitive to not only to variation in cell-type composition of the sample, but also to the physiological state of the donor, and technical factors such as sample storage ,. It is therefore important to understand these factors and consider them prior to the study.
Gene expression analysis of isolated cell populations instead of whole blood or PBMC circumvents the need to adjust for potential cell composition variation. Cell populations need to be sorted from the mixture of blood cells by means of a complex cell sorting technology, which would need to be translated to clinical trial settings. The decoupling process needs to guarantee the quality of the source of the cells from limited access to sophisticated cell sorting equipment. Variation in the duration of transport to central laboratories needs to be considered by ensuring sample stability and proper storage conditions. Only a high level of reproducibility assures comparability and reliability of analysis results within a set of samples from a patient and within patient groups.
We assessed a cell separation technology using antibody-coated paramagnetic beads for sorting cells directly from human whole blood under conditions representing a clinical trial. Positive cell selection protocols based on Miltenyi Biotec’s whole blood microbeads were considered for the following reasons: 1) Miltenyi Biotec’s autoMACS Pro Separator is capable of sorting approximately 106 cells per second in a rapid, semi-automated fashion . Rapid throughput minimizes the potential perturbation of the transcriptome. The time required for preparative sorting for transcriptomics analyses is sample dependent. 2) As Lyons et al. reported, the cell purity after positive selection is higher than that after negative selection . 3) Magnetic cell sorting can be performed in a multiplex format, thus further decreasing sorting time when a number of cell types are being purified.
Here we present the results of a feasibility study and discuss the possibilities and limitations of gene expression analysis of sorted cells in future clinical studies.
Study subjects and blood samples
Blood samples were obtained prospectively from 45 adult, consented Caucasians (18 females and 27 males) from a donor pool of healthy volunteers. Venous blood samples were drawn from the cubital region using evacuated tubes containing K2-EDTA as anticoagulant (BD™ Vacutainer, Becton Dickinson, Franklin Lakes, New Jersey, USA) or as control samples into PAXgene® Blood RNA Tubes (Qiagen, Hilden, Germany).
Samples from a subset of 23 donors (10 females and 13 males) were used for hematology and flow cytometry evaluation. Those were the only samples used for cell sorting.
Blood samples were stored at 4°C for up to 6 hours after venipuncture until analysis on a Siemens ADVIA 120 analyzer (Siemens Healthcare Diagnostics, Erlangen, Germany). The analyzer was calibrated and controlled according to the manufacturer’s user manual. Only original reagents produced by the manufacturer were used and maintenance had been performed at the recommended intervals.
Magnetic MicroBead-based cell sorting
Magnetic cell separation by MACS® Technology is based on small superparamagnetic microbeads that are bound to a highly specific antibody against a particular cell marker, thus allowing for magnetic labeling of individual cell types. Separation occurs in a MACS Column which induces a high-gradient magnetic field (~0.6 Tesla) when placed in a MACS Separator.
Cell separation directly from whole blood was performed using the respective Whole Blood MicroBeads (Miltenyi Biotec) as recommended by the manufacturer. A more detailed description of the experimental procedure can be found in the Additional file 1: Supplementary material.
Whole blood samples and corresponding sorted cells were stored at 4°C until analysis on a MACSQuant® Flow Cytometer (Miltenyi Biotec GmbH).
Anti-human monoclonal antibodies against CD3+ T-lymphocytes, CD4+ helper T-cells, CD8+ cytotoxic T-cells, CD14+ monocytes, CD15+ granulocytes, CD19+ B-lymphocytes, CD45+ leukocytes and CD56+ NK cells were obtained from Miltenyi Biotec GmbH. For flow cytometry 250 μL suspensions of isolated cells were incubated with 10 μL of corresponding antibodies for 10 minutes at 2-8°C in the dark. The cells were washed and re-suspended in 500 μL analysis buffer and afterwards immediately analyzed on a MACSQuant Flow Cytometer using MACSQuantify™ Software. Data were further processed with FlowJo® software (http://www.flowjo.com).
Total RNA extraction
The total RNA from the sorted and lysed cells was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and from whole blood with the PAXgene Blood RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. A more detailed description can be found in the in the Additional file 1: Supplementary material.
Whole transcriptome analysis
Whole transcriptome analysis was conducted essentially as described by Lockhart et al.  on expression microarrays. The human genome U133 plus 2.0 array (Affymetrix, Inc.) was used. GeneChip arrays were scanned using a GS 3000 scanner (Affymetrix, Inc.).
Experiments were conducted as recommended by the manufacturer (GeneChip® Expression Analysis Technical Manual) using the cRNA labeling, GeneChip® hybridization, wash, and stain protocols (Affymetrix, Inc., Santa Clara, CA, U.S.A.). Quality control was performed by visual inspection and expert judgment. The primary raw data images, the .dat-files, were processed to .cel-files and to numerical signal values.
Data analysis/Bioinformatics analysis
All gene expression analyses were performed in Partek® Genomics Suite™, version 6.6 (http://www.Partek.com). Affymetrix .cel files were RMA background corrected, subjected to quantile normalization and scaled to a mean of 150. A custom Entrez gene-based CDF file was used for probe set condensation and annotation (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/genomic_curated_CDF.asp). Cell type-specific gene signatures included those genes with a signal intensity of 200 or more in at least all but one sample of one cell type, but less than 100 in at least all but one of the samples of the other cell types excluding stabilized whole blood. The gene expression databases BioGPS (http://www.BioGPS.org) and Genevestigator (http://www.Genevestigator.com) were consulted for confirmation of cell type specific gene expression data. Gene groups for the perturbation score analysis were downloaded from http://www.SABiosciences.com. Unigene identifiers were mapped to Entrez identifiers, resulting in 502 probes of the custom, Entrezgene-based CDF file (Additional file 2: Supplementary table). Graphical presentation of data was performed using Prism GraphPad version 6.03 (http://www.graphpad.com).
Blood samples of a subset of subjects (10 females, 13 males) were analyzed on a Siemens ADVIA 120 analyzer (Siemens Healthcare Diagnostics, Erlangen, Germany). Time lag between venipuncture and analysis is considered not to affect cell counts ,. All cell counts were within the expected range for female and male healthy volunteers without overt pathological conditions affecting blood cell composition (http://www.Siemens.com/diagnostics) (Additional file 1: Table S2).
Cell sorting experiments - total RNA yield and quality
Total RNA yield and quality were assessed for stabilized whole blood samples and for isolated cell populations. Cell separation from K2-EDTA anticoagulated whole blood samples started within 30 minutes after venipuncture.
Overall, the total RNA yields and quality were sufficient for all different cell types tested (Additional file 1: Figure S1). The highest mean RIN value was 9.65 ± 0.18 for CD14+ cells, the lowest was 8.62 ± 0.65 for stabilized whole blood. The lowest RIN value was 6.6 for one of 58 whole blood samples. All total RNA samples were processed further in Affymetrix DNA microarray experiments. The mean quantity of extracted RNA per 6 mL ranged from 0.35 ± 0.12 μg for CD56+ cells to 9.8 ± 5.12 μg for whole blood. The minimum amount of extracted total RNA was 0.113 μg, the maximum was 24.6 μg for a CD15+ samples and a blood sample, respectively.
Cell sorting experiments - total RNA stability
The total RNA integrity of all other isolated cell types could be preserved, when stored at 4°C for up to 7 days in an EDTA-based additive (Figure 2B). The RIN value remained consistent for total RNA of CD19+ and CD14+ cells, but decreased slightly for CD8+ and CD15+ cells whilst remaining at a value, which is still acceptable for gene expression experiments. While the RNA integrity remained at a high level over a period of 7 days, the RNA yield decreased, as shown in Figure 2C. CD4+ and CD14+ cell preparations gave the highest total RNA yields, CD19+ and CD15+ the lowest.
Experimental procedures do not activate cells
Enrichment of cell types by MACS cell separation
Blood samples and corresponding sorted cells from a subset of donors (10 females, 13 males) were stored at 4°C until analysis within 6 hours after venipuncture on a MACSQuant Flow Cytometer (Miltenyi Biotec GmbH). The purity of the sorted cells was within the specifications of the vendor (Additional file 1: Table S3).
Gene expression analysis of isolated cell types
Despite methodological improvements in the development of qualified, clinically relevant genomics biomarkers with non- or minimally invasive technologies -, the path from discovery and validation, to regulatory approval, acceptance, and clinical application remains a challenge -. The analysis of genomics data of blood samples is demanding, due to various potentially confounding factors which need to be taken into account during the analytical procedure, including i) relative blood cell-type composition, ii) variation in cell-type gene expression signatures, iii) abundance of globin mRNA which interferes with microarray signal intensities, and iv) physiological condition of the individual ,,, to name only some. The present project aimed to evaluate whether gene expression analysis of cells separated directly from whole blood by magnetic cell sorting (MACS Technology) was feasible. After evaluating the effects of several experimental conditions on RNA quality, and yield, followed by gene expression analysis of eight separated human blood cell populations, we concluded that MACSCell Separation can be combined with gene expression profiling and is well suited for processing of clinical trial samples. We found that EDTA-based anticoagulants perform much better than formaldehyde-based additives in terms of total RNA yield, integrity, and stability. The EDTA blood collection tubes for molecular assays gave the best results in terms of total RNA quality and quantity. In our hands, one of the formaldehyde-based tube-type, designed for stabilizing white blood cells for FACS analysis, gave suboptimal total RNA quality scores independent of the storage temperature and therefore we no longer evaluated those collection tubes for our purpose.
It is known that nucleic acids from formalin-fixed, paraffin-embedded (FFPE) tissues are usually significantly degraded and chemically modified by formaldehyde caused by the fixation and embedding conditions . Therefore, nucleic acids isolated from FFPE samples are often of a lower molecular weight than those obtained from fresh or frozen samples. The other evaluated collection tube, designed for stabilizing circulating tumor cells, gave good quality total RNA when stored at 4°C for 3 days but the total RNA yields decreased and the transcripts were not preserved as well as in the PAXgene Blood RNA Tubes. These collection tubes with formaldehyde-based additives were therefore not suitable for the purpose of stabilizing the total RNA in this context.
RNA quality and yield were stable at a high level when stored at 4°C throughout the observation period of up to 7 days. Neither EDTA nor the addition of magnetic beads activated the cells. Lyons et al. investigated the effect of positive and negative selection by MACS Cell Separation on the activation state of cells . They reported that positive selection did not activate the leukocyte subsets, supporting the notion presented here, that cells were not activated by storage or by MACS Cell Separation. We determined the status of activation by a series of experiments and the invention and application of a so called perturbation score, which compresses normalized signal intensities of pre-defined, study-relevant gene-groups into a single value. An unchanging perturbation score indicated lack of activation. The concept of gene scores to reflect severity of gene expression changes is not new. Adopted by others, Famulski et al. have successfully applied a gene score to clinical transplantation research and use it as molecular diagnostic tool -. Famulski’s gene score is based on fold change and is thus dependent on reference gene expression data, for example from control samples. In contrast, the perturbation score we applied in the present study is independent of comparator data. Thus, individual samples can be assessed for expression changes of predefined gene groups.
Computational methods offer alternatives to physical cell separation. However, changes in cell composition and microenvironmental changes may represent obstacles towards the investigation of gene expression changes within a sample . Several algorithms aiming to deconvolute cell composition have been developed -. Some algorithms rely on the a priori knowledge of either the cell type composition in a sample or on pre-determined gene expression profiles from isolated cell types ,. Recently, the possibility to reconstruct cell type composition in complex tissues without prior knowledge of either cell frequency or of pre-established cell type specific gene expression signatures was investigated on simulated data, awaiting further validation . Other efforts aim to deconvolute cell composition changes caused by micro-environmental changes such as cell infiltration during progression of some diseases, or change of gene expression profiles during disease treatment. Further advances in this area, including validation, using data derived from clinical samples, will be highly welcome. Physical cell separation has limitations of its own. MACS Cell Separation can only be performed on cells in suspension, and only by employing predefined cell surface markers or marker combinations, excluding the discovery of new cell populations in a sample. However, due to the high technical reproducibility and robustness, MACS Cell Separation coupled to transcriptomics presents a valuable platform for routine investigations in clinical trials.
Here we outline a workflow for magnetic bead-based, delayed cell sorting of CD3+ T-lymphocytes, CD4+ helper T-cells, CD8+ cytotoxic T-cells, CD14+ monocytes, CD15+ granulocytes, CD19+ B-lymphocytes, CD45+ leukocytes, and CD56+ NK cells, designed for transcriptome analysis of multi-center clinical trial samples. The observed cell-stability at 4°C provides the potential to uncouple the blood draw at the clinical site from MACS Cell Separation, e.g. at a central laboratory. Cells were not activated by the MACS process. This process facilitates the integration of cell sorting directly from whole blood coupled to transcriptomics in clinical trial protocols.
ML, AS wrote the manuscript, SSS, ML carried out the cell sorting and extraction experiments. NH carried out the gene expression experiments. MB carried out the hematology. FM carried out the FACS analysis and participated in the study design. AV carried out the FACS analysis. EL, MS and AS performed the statistical analysis. BG, UJ, EL, FS, ML participated in the study design. AV, NN and KJJ supported the project. All authors read and approved the final manuscript.
Chip description file
Fluorescence-activated cell sorting
RNA integrity number
Gina Cavelti, Urs Affentranger, Marc Altorfer, Jürg Eichenberger, and Lukas Baeriswyl for conduct of experiments and Daniel Wahl for technical support.
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