For cell line controls, healthy donor blood was collected in Cell-free DNA BCT™ (Streck) tubes, spiked with cell line cells, and the slides were prepared and stained as previously described [27 ]. For patient samples, peripheral blood samples from seven (7) patients with metastatic castration resistant prostate cancer (mCRPC) were collected in CellSave tubes (Janssen Diagnostics) and shipped to Epic Sciences (San Diego, CA) at ambient temperature, and processed onto slides as previously described [26 ,27 ]. Briefly, red blood cells were lysed, and all nucleated cells were deposited onto glass microscopy slides at a density of 3 x 106 cells/slide and stored at -80°C prior to staining. Three cell lines representative of prostate cancer with known genetic changes were used: VCaP (ATCC catalog #CRL-2876), LNCaP (ATCC catalog #CRL-1740), and PC3 (ATCC catalog #CRL-1435).
Cellsave tubes
Manufactured by Johnson & Johnson
Sourced in United States
The CellSave tubes are laboratory equipment designed for the collection and preservation of blood samples. They are used for the stabilization of circulating tumor cells (CTCs) in whole blood samples prior to analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cellsearch system, by Johnson & Johnson (2 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Cell free dna bct, by Streck (1 mentions)
Cellsearch, by Johnson & Johnson (1 mentions)
Edta collection tubes, by Sarstedt (1 mentions)
Countess cell counter, by Thermo Fisher Scientific (1 mentions)
15 protocols using cellsave tubes
1
Characterization of Prostate Cancer Cells
2
Optimized CTC Isolation from Whole Blood
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Whole blood samples, with three matched tubes for each blood draw, were collected in CellSave tubes (Janssen Diagnostics, LLC) from patients with RCC at Mayo Clinic and shipped to Creatv MicroTech for analysis. The concordance of the recovery was determined through processing of the matched tubes as with cryopreservation and without cryopreservation, respectively. Two of the tubes labeled as without cryopreservation (Tube 1 and Tube 2) were processed through CellSieve™ microfiltration within 24 h after blood draw. The third tube was processed through Ficoll separation, cryopreservation at −80 °C for 7 days, followed by thawing and CellSieve™ microfiltration, and antibody staining. The procedures of microfiltration and antibody staining were the same as used in the spiking experiments. The filter-captured cells from the Tubes 1 and 3 were stained with an antibody mixture consists of fluorescent dye-conjugated antibodies against CK8, 18, 19/FITC, vimentin/EF615 and CD45/Cyanine5. The filter-captured cells from the Tube 2 were stained with an antibody mixture consists of fluorescence-conjugated antibodies against PD-L1/Alexa Fluor 488, vimentin/EF615 and CD45/Cyanine5. The CTC was defined as a nucleated cell with positive staining for CKs and vimentin but negative staining for CD45 for Tubes 1 and 3. The CAML was defined according to criteria described previously [2 (link)].
3
CellSearch: Automated CTC Isolation and Identification
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For CellSearch analysis, 7.5 ml of blood was drawn into CellSave tubes (Janssen Diagnostics, LLC) and processed within 76 hours. The semi-automated analysis was performed as described elsewhere34 (link). In short, EpCAM coated ferro-fluids were used to isolate CTCs followed by a cytokeratin expression and DAPI based identification step and an automated scanning process. CD45 immunofluorescence staining was used to identify unspecifically enriched leukocyte fractions. Cells were evaluated by an experienced scientist (SR).
4
CTC Enumeration and Analysis Procedure
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CellSearch CTC analyses on n = 47 patients at both C1 and C3 were done on replicate 7.5 ml whole blood samples collected from the same vein puncture in 9 ml CellSave tubes (Janssen) and analyzed within a mean of 43 hours, range [3–100 h]. Due to technical issues, three replicate samples were scanned after up to 100 hours instead of the recommended 96 hours. The replicate samples were reported as mean of the two samples in the unit CTC/7.5 ml blood except when using the individual replicate sampling to calculate CV%(dd) for SCV-limits (Equation 3 ).
The CTC analyses were carried out on the CellSearch System (formerly owned by Veridex, now Janssen). Assumed CTC were quantified using the FDA approved CellSearch Epithelial Cell Kit, ref 7900000, Janssen based on immunomagnetic enrichment of cells of ephithelial origin and positive for the epithelial cell adhesion molecule (EpCAM). Positive identification of suspected CTC was done by staining with phycoerythrin (PE) conjugated antibodies specific for cytokeratins 8, 18, and 19 and an associated DAPI stained nuclei. Residual leucocytes with positive DAPI were negatively discriminated by anti-CD45 antibodies conjugated with allophycocyanin (APC). All CTC evaluations after the Veridex criteria were performed by a single certified operator to avoid inter analyst variation [17] (link).
The CTC analyses were carried out on the CellSearch System (formerly owned by Veridex, now Janssen). Assumed CTC were quantified using the FDA approved CellSearch Epithelial Cell Kit, ref 7900000, Janssen based on immunomagnetic enrichment of cells of ephithelial origin and positive for the epithelial cell adhesion molecule (EpCAM). Positive identification of suspected CTC was done by staining with phycoerythrin (PE) conjugated antibodies specific for cytokeratins 8, 18, and 19 and an associated DAPI stained nuclei. Residual leucocytes with positive DAPI were negatively discriminated by anti-CD45 antibodies conjugated with allophycocyanin (APC). All CTC evaluations after the Veridex criteria were performed by a single certified operator to avoid inter analyst variation [17] (link).
5
High-Grade Sarcoma Blood Collection
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Blood samples were collected under a protocol approved by the Johns Hopkins University Institutional Review Board, after obtaining informed consent. A total of 54 blood samples were collected from 36 patients who presented to Johns Hopkins University Sidney Kimmel Comprehensive Cancer Center with a diagnosis of high-grade sarcoma between February 2015 and November 2016. A total of 10 ml of blood was collected in either EDTA blood collection tubes or CellSave tubes (Janssen Diagnostics, Raritan, NJ) and was processed for CTC detection within 2 hours of collection in all cases.
6
CTC Detection and Somatostatin Receptor Expression
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For each patient, two 7.5 ml blood samples were collected into evacuated CellSave tubes (Janssen Diagnostics, Raritan, NJ, USA) and maintained at room temperature. All samples were processed within 96 h of collection. The CellSearch platform was used for detection and enumeration of CTCs as previously described (Khan et al, 2011b (link)). Analysis of SSTR2 and SSTR5 expression on CTCs was performed using Alexa Fluor 488-conjugated Somatostatin Receptor 2 antibody and Alexa Fluor 488-conjugated Somatostatin Receptor 5 antibody at pre-determined concentrations and exposure times. Cells were defined as positive for SSTR2 or SSTR5 when fourth channel staining was present. All evaluations regarding enumeration of CTCs and expression of SSTR2 and SSTR5 were made by two independent operators without knowledge of patient pathology.
7
Blood Sampling for Breast Cancer CTC Analysis
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Blood from healthy individuals and metastatic breast cancer patients was obtained from the Department of Transfusion Medicine and Department of Gynecology at the University Medical Center Hamburg-Eppendorf, respectively. All study participants gave written informed consent. The examination of blood from breast cancer patients was approved by the local ethics review board Aerztekammer Hamburg (OB/V/03). Breast cancer patients’ blood was sampled in EDTA collection tubes (01.1605.001, Sarstedt). Blood from healthy donors was collected either in EDTA or CellSave tubes (7900005, Janssen Diagnostics) and spiked with SK-BR-3 cells to simulate CTCs.
8
CellSearch Enumeration of Spiked Cells
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Blood samples were collected from a volunteer in Cellsave tubes (Janssen Diagnostics, Raritan, NJ, USA) one day before addition of cells. The tubes were weighed before and after sampling for calculation of blood volume and stored at room temperature.
The cells were grown until approximately 90% confluent, then trypsinized for exactly 7 min and counted four times or more in a Countess cell counter (Invitrogen, Carlsbad, CA, USA). After dilution to 20,000 cells/mL in complete medium, 10 µL (thus containing a calculated 200 cells) was added to each blood sample. CellSearch counting was performed within 3 hours from addition of cells.
The cells were grown until approximately 90% confluent, then trypsinized for exactly 7 min and counted four times or more in a Countess cell counter (Invitrogen, Carlsbad, CA, USA). After dilution to 20,000 cells/mL in complete medium, 10 µL (thus containing a calculated 200 cells) was added to each blood sample. CellSearch counting was performed within 3 hours from addition of cells.
9
CTC Isolation and Enumeration using CellSearch
10
Recurrent Glioblastoma Treatment Protocol
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This prospective study was a side study of the randomised multi-centre phase II trial from the Dutch Neuro-Oncology Group (LWNO) ‘BELOB' (Netherlands Trial Register ID NTR1929). In-depth information regarding eligibility criteria, treatment and outcome assessments were described in the paper regarding the primary clinical end points of the study (Taal et al, 2014 (link)). In brief, patients with recurrent glioblastoma were stratified according to centre, ECOG performance status and age, to be subsequently randomised between bevacizumab in combination with lomustine, bevacizumab single agent or lomustine single agent.
Lomustine was given orally every 6 weeks, for a maximum of six cycles. Bevacizumab was given intravenously every 2 weeks until disease progression. One treatment cycle was defined as 6 weeks. Overall survival (OS) was measured from the day of randomisation until death from any cause.
The central and local institutional review boards approved the protocol and all patients provided written informed consent. Peripheral blood samples for CEC analyses were acquired in CellSave tubes (Janssen Diagnostics, Raritan, NJ, USA) before the start of treatment (baseline) and after 4 and 6 weeks of treatment. Samples were maintained at room temperature and processed within 96 h of blood collection.
Lomustine was given orally every 6 weeks, for a maximum of six cycles. Bevacizumab was given intravenously every 2 weeks until disease progression. One treatment cycle was defined as 6 weeks. Overall survival (OS) was measured from the day of randomisation until death from any cause.
The central and local institutional review boards approved the protocol and all patients provided written informed consent. Peripheral blood samples for CEC analyses were acquired in CellSave tubes (Janssen Diagnostics, Raritan, NJ, USA) before the start of treatment (baseline) and after 4 and 6 weeks of treatment. Samples were maintained at room temperature and processed within 96 h of blood collection.
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