Search

Background Tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) in the blood of metastatic cancer patients associate with poor outcomes. In this study, we explored the human epidermal growth factor receptor 2 (HER2) expression on CTCs and tdEVs of metastatic breast cancer patients. Methods Blood samples from 98 patients (CLCC-IC-2006-04 study) were originally processed with the CellSearch® system using the CTC kit and anti-HER2 as an additional marker in the staining cocktail. CTCs and tdEVs were automatically enumerated from the generated CellSearch images using the open-source ACCEPT software. Results CTCs and tdEVs were subdivided based on their cytokeratin (CK) and HER2 phenotype into CK+HER2−, CK−HER2+, and CK+HER2+. The inclusion of anti-HER2 increased the percentage of informative samples with ≥ 1 detectable CTC from 89 to 95%. CK− CTCs and tdEVs correlated equally well with the clinical outcome as CK+ CTCs and tdEVs. Inter- and intra-patient heterogeneity was found for the CTC/tdEV phenotypes, and the presence of 2 or 3 classes of CTCs/tdEVs was associated with worse prognosis compared to a uniform CTC/tdEV phenotype present (1 class). The use of ≥ 7% HER2+CK+ tdEVs can predict HER2 expression of the tissue with 74% sensitivity and specificity using the HER2 amplification status of the primary tumor as a classification variable. Conclusions HER2 can be detected on CTCs and tdEVs not expressing CK, and these CK− CTCs/tdEVs have similar clinical relevance to CTCs and tdEVs expressing CK. tdEVs perform better than CTCs in predicting the HER2 status of the primary tissue. CTC and tdEV heterogeneity in the blood of patients is inversely associated with overall survival.

The presence of high expressing epithelial cell adhesion molecule (EpCAMhigh) circulating tumor cells (CTC) enumerated by CellSearch® in blood of cancer patients is strongly associated with poor prognosis. This raises the question about the presence and relation with clinical outcome of low EpCAM expressing CTC (EpCAMlow CTC). In the EU-FP7 CTC-Trap program, we investigated the presence of EpCAMhigh and EpCAMlow CTC using CellSearch, followed by microfiltration of the EpCAMhigh CTC depleted blood. Blood samples of 108 castration-resistant prostate cancer patients and 22 metastatic breast cancer patients were processed at six participating sites, using protocols and tools developed in the CTC-Trap program. Of the prostate cancer patients, 53% had ≥5 EpCAMhigh CTC and 28% had ≥5 EpCAMlow CTC. For breast cancer patients, 32% had ≥5 EpCAMhigh CTC and 36% had ≥5 EpCAMlow CTC. 70% of prostate cancer patients and 64% of breast cancer patients had in total ≥5 EpCAMhigh and/or EpCAMlow CTC, increasing the number of patients in whom CTC are detected. Castration-resistant prostate cancer patients with ≥5 EpCAMhigh CTC had shorter overall survival versus those with

Background: For rational decision making, assessing the cost-effectiveness and budget impact of new drugs and comparing the costs of drugs already on the market is required. In addition to value frameworks, such as the American Society of Clinical Oncology Value Framework and the European Society of Medical Oncology–Magnitude of Clinical benefit Scale, this also requires a transparent overview of actual drug prices. While list prices are available, evidence on treatment cost is not. This paper aims to synthesise evidence on the reimbursement and costs of high-cost breast cancer drugs in The Netherlands (NL). Methods: A literature review was performed to identify currently reimbursed breast cancer drugs in the NL. Treatment costs were determined by multiplying list prices with the average length of treatment and dosing schedule. Results: Comparing list prices to the estimated treatment cost resulted in substantial differences in the ranking of costliness of the drugs. The average mean treatment length was unknown for 11/31 breast cancer drugs (26.2%). The differences in the 15 highest-cost drugs were largest for Bevacizumab, Lapatinib and everolimus, with list prices of €541, €158, €1,168 and estimated treatment cost of €174,400, €18,682 and €31,207, respectively. The lowest-cost (patented) targeted drug is €1,818 more expensive than the highest-cost (off-patent) generic drug according to the estimated drug treatment cost. Conclusions: A lack of evidence on the reimbursement and cost of high-cost breast cancer drugs complicates rapid and transparent evidence synthesis, necessary to focus strategies aiming to limit the increasing healthcare costs. Interestingly, the findings show that off-patent generics (such as paclitaxel or doxorubicin), although substantially cheaper than patented drugs, are still relatively costly. Extending standardisation and increasing European and national regulations on presenting information on costs per cancer drug is highly recommended.

We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates.

Cancer cells can enter the bloodstream, form distant metastases, and ultimately lead to death. A study by Allard and colleagues, which was published in the October 15, 2004, issue of Clinical Cancer Research, concluded that the CellSearch system could be used as a reliable tool to investigate circulating tumor cells and their clinical utility, and it spurred a still-growing interest in the field. Clin Cancer Res; 21(13); 2883–5. ©2015 AACR. See related article by Allard et al., Clin Cancer Res 2004;10(20) October 15, 2004;6897–904

Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on the surface of cancer cells and specific ligands deposited on sensor chips using an IBIS MX96 SPR imager (SPRi). As a model system, cells from the breast cancer cell line HS578T, SKBR3 and MCF7 were used. SPRi responses to Epithelial Cell Adhesion Molecule (EpCAM) antibody and other ligands coated on the sensor chips were measured. SPR curves show a response attributable to the sedimentation of the cells and a specific binding response on top of the initial response, the magnitude of which is dependent on the ligand density and the cell type used. Comparison of SPRi with flow cytometry showed similar EpCAM expression on MCF7, SKBR3 and HS578T cells.

Circulating tumor cells (CTCs) are very attractive surrogate markers for systemic cancer. Currently, major efforts are being made to use these rare cells in the sense of a liquid biopsy to gain molecular information for rational therapeutic decision-making. The advancements in molecular analyses of CTCs down to the single-cell level have been significant in recent years and some applications are ready to be used in clinical studies. As discussed in this review, a major challenge for translating such molecular CTC-based assays into the clinic is the extremely low frequency of CTCs and the associated problems of their reliable detection and isolation. A potential solution to overcome the low CTC frequency is the recently introduced diagnostic leukapheresis that permits screening of liters of blood. Discussed here are the challenges as well as the current efforts implementing this method into clinical workflows to realize more reliable liquid biopsies.

Circulating tumor cells (CTCs) are tumor cells shed into the peripheral blood of cancer patients. The increasing number of treatment options for patients with metastatic carcinomas has created a concomitant need for new methods to establish which therapy will be effective and to monitor their use. Detection and characterization of CTCs is important not only to guide therapy, but also to increase our fundamental understanding of tumor progression and the formation of distant metastasis in which CTCs play a crucial role. However, identification of CTCs is quite challenging and different definitions lead to a large variation of CTC counts that will have different clinical implications. Here we will review the challenges in defining a CTC and data that have been obtained using CTCs in clinical studies emphasizing their importance as a prognostic and predictive biomarker. Furthermore, we summarize reported microfluidic platforms for CTC isolation, enumeration, and characterization developed to overcome technical challenges with current CTC detection platforms.

The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free and overall survival for patients with metastatic and newly diagnosed breast cancer. The present study was undertaken to explore whether the presence of CTC before and during follow-up after surgery is associated with recurrence free survival (RFS) and overall survival (OS). In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 403 stage I-III patients before undergoing surgery for breast cancer (A) and if available 1 week after surgery (B), after adjuvant chemo- and/or radiotherapy or before start of long-term hormonal therapy (C), one (D), two (E) and three (F) years after surgery. Patients were stratified into unfavorable (CTC ≥1) and favorable (CTC=0) prognostic groups. >1 CTC in 30 ml blood was detected in 75/403 (19%) at A, 66/367 (18%) at B, 40/263 (15%) at C, 30/235 (12%) at D, 18/144 (11%) at E and 11/83 (13%) at F. RFS and OS was significantly lower for unfavorable CTC as compared to favorable CTC before surgery (p=0.022 and p=0.006), after adjuvant therapy (p

Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch(R) system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

The increasing number of treatment options for patients with metastatic carcinomas has created an accompanying need for methods to determine if the tumor will be responsive to the intended therapy and to monitor its effectiveness. Ideally, these methods would be noninvasive and provide quantitative real-time analysis of tumor activity in a variety of carcinomas. Assessment of circulating tumor cells shed into the blood during metastasis may satisfy this need. Here we review the CellSearch technology used for the detection of circulating tumor cells and discuss potential future directions for improvements.

Background: Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood. - Methods: Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels. - Results: A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained. - Conclusion: We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.

We have developed a platform for cell analysis based on immunomagnetic selection and magnetic alignment of cells in combination with an epi-illumination tracking and detection system. Whole blood was labeled with ferromagnetic nanoparticles and fluorescent probes, and placed in a magnetic field in a chamber. Cells labeled with ferromagnetic nanoparticles moved upward and aligned along ferromagnetic lines deposited by lithographic techniques on an optically transparent surface of the chamber. An epi-illumination system using a 635 nm laser diode as a light source scanned the lines and measured signals obtained from the aligned cells. The cell counts per unit of blood volume obtained with the system correlated well with those obtained from the counts from a standard hematology analyzer and flow cytometer. The cell analysis platform is significantly less complex and more sensitive than current cell analysis equipment and provides additional functionality through its ability to subject the cells to repeated and varied analyses while they remain in a natural environment (i.e., whole blood).

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5 center dot 10(4):10(2):1 for the apparent viscosities of 15 mu m diameter MDA-231 cells, 10 mu m white cells and 90 fl red cells passing through a 5 mu m pore. Fixation increases the pressure needed to pass cells through 8 mu m pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of similar to 10 mbar for a 1 cm(2) track-etched filter with 5 mu m pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45-DNA+CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 mm track-etched filter and the 5 mm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching>80% of cells from whole blood. TEM grids had poor recovery of similar to 25%. Median diameter of cell lines ranged from 10.9-19.0 mu m, compared to 13.1, 10.7, and 11.0 mu m for breast, prostate and colorectal CTC, respectively. The 11.4 mu m COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 mu m pores for 1 ml of blood with a

Circulating Tumor Cells (CTC) are rare cells originated from tumors that travel into the blood stream, extravasate to different organs of which only a small fraction will develop into metastasis. The presence of CTC enumerated with the CellSearch system is associated with a relative short survival and their continued presence after the first cycles of therapy indicates a futile therapy in patients with metastatic carcinomas. Detailed characterization of CTC holds the promise to enable the choice of the optimal therapy for the individual patients during the course of the disease. The phenotype, physical and biological properties are however not well understood making it difficult to assess the merit of recent technological advancements to improve upon the capture of CTC or to evaluate their metastatic potential. Here we will discuss the recent advances in the classification of CTC captured by the CellSearch system, the implications of their features and numbers. Latest capture platforms are reviewed and placed in the light of technology improvements needed to detect CTC. Physical properties, phenotype, viability and proliferative potential and means to assess their proliferation and metastatic capacity will be summarized and placed in the context of the latest CTC capture platforms.

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.

Background In current practice cancer tissue is taken at diagnosis to assess the presence of treatment targets. This however is suboptimal since tumor cells evolve due to genomic instability. Assessment of the genotype and phenotype of the CTCs will provide insights into which treatments would be most beneficial for the individual patient. Feasibility to detect treatment targets in CTCs has been demonstrated (Meng, Tripathy et al. 2004; de Bono, Attard et al. 2007; Rossi, Basso et al. 2010; Wang, Pfister et al. 2010). In this context, the development of a cytogenetic assay for CTCs will be crucial for successful molecular targeted therapy in cancer patients. Genetic characterization of CTCs is expected to gather new knowledge on the mechanism of metastasis and on potential targets of novel therapeutic strategies. Patients and methods Assay optimization for analysis of genetic aberrations of CTCs was performed with cells from tumor cell lines. Tumor cell lines with known chromosomal aberrations and mosaicism were spiked into 7.5mL whole blood samples, at numbers similar to those observed in-vivo in cancer patients. Tumor cells were enriched by CellSearch System and off-line purified and individually analyzed for chromosomal alterations. The procedure was next validated by using blood samples collected from cancer patients. Results/Conclusions A robust protocol for the isolation of individual CTCs followed by DNA extraction and amplification after CellSearch enrichment was established. The number and the quality of CTCs needed to obtain an informative analysis of chromosomal aberrations were set up. Accrual of cancer patients for individual CTC isolation and molecular characterization is ongoing. Updated data including evaluations on prognostic value of the genetic aberrations analysis of the CTCs and correlation with clinical stage, tumor burden, metastatic sites and survival will be available and presented at the meeting. Disclosure All authors have declared no conflicts of interest.

Minimal residual cancer is defined as “the presence of tumor cells that are not detectable by the current routine diagnostic procedures used for tumor staging in cancer patients after surgical removal of the primary tumor.” Data from European and North American groups have demonstrated the prognostic impact of disseminated tumor cells (DTCs)9 in the bone marrow of breast cancer patients. Circulating tumor cell (CTC) detection and enumeration in peripheral blood have been examined in prospective multicenter studies of metastatic breast, colorectal, and prostate cancers and have been associated with decreased progression-free and overall survival. An increasing number of clinical research studies are validating these observations and extending them to other cancers and to earlier disease stages. CTCs are highly heterogeneous, and their molecular characterization is important, not only to confirm their malignant origin but also to follow immune-phenotypic changes with tumor progression and identify diagnostically and therapeutically relevant targets that will help stratify cancer patients for individualized therapies. The rarity of CTCs—and thus the very limited amount of available sample—presents a formidable analytical and technical challenge. Recent technical advances in CTC detection and characterization include reverse-transcription quantitative PCR (RT-qPCR) methods, image-based approaches, and microfilter and microchip devices. CTCs represent a promising new diagnostic field for advanced-stage patients in that the sensitive CTC-detection platforms allow monitoring of disease and treatment efficacy. The development of single-cell technologies might allow profiling of these cells for the purpose of adapting treatment regimens. CTC-detection and- characterization techniques hold promise for playing a role as a “liquid biopsy” that will allow physicians to follow cancer changes over time and to tailor treatment. Current research on CTCs is focusing on the identification of novel diagnostic and therapeutic biomarkers produced by these cells. CTCs are promising as novel tumor biomarkers because they are well-defined targets for understanding …

Item does not contain fulltext BACKGROUND: We demonstrated that circulating tumor cell (CTC) number at baseline and follow-up is an independent prognostic factor in metastatic colorectal cancer (mCRC). This analysis was undertaken to explore whether patient and treatment characteristics impact the prognostic value of CTCs. PATIENTS AND METHODS: CTCs were enumerated with immunomagnetic separation from the blood of 430 patients with mCRC at baseline and on therapy. Patients were stratified into unfavorable and favorable prognostic groups based on CTC levels of > or = 3 or /=65 years (P = 0.0007), and ECOG PS of zero (P = 0.04), unfavorable baseline CTC was associated with inferior PFS. CONCLUSION: Baseline CTC count is an important prognostic factor within specific subgroups defined by treatment or patient characteristics.

Background: We developed a volumetric single platform image cytometer (SP ICM) that is dedicated to count CD4+ and CD8+ T lymphocytes for HIV monitoring in resource-constrained settings. The instrument was designed to be low-cost, yet reliable, easy-to-use, and robust. Methods: Whole blood is incubated with CD3-magnetic nanoparticles, CD4-phycoerythrin (PE), and CD8-peridinin-chlorophyll-protein complex (PerCP). The CD3 cells are immunomagnetically attracted to an analysis surface, where fluorescence images of CD4+ and CD8+ T lymphocytes are recorded and analyzed, respectively. We compared CD4, CD8 counts, and CD4/CD8 ratio obtained by the SP ICM with those from a SP flow cytometer (FCM) tetraCXP method on blood samples from 145 patients. Results: Good correlations were obtained (R: 0.96–0.99) between the SP ICM and the SP FCM. There was ∼10% CD8 undercount in the SP ICM, which could be partly caused by CD8+dim T lymphocytes that were not detected by the instrument or not counted by the image analysis due to the cross-talk from the CD4-PE signal in the CD8-PerCP image. Conclusions: The SP ICM is a good candidate for HIV monitoring in point-of-care settings of resource-constrained countries. © 2008 Clinical Cytometry Society

8000 Background: CP-751,871, a fully human IgG2 subtype monoclonal antibody, is a potent and specific inhibitor of the insulin-like growth factor type I receptor (IGF-IR). Methods: A comprehensive evaluation of blood and tissue markers of the IGF-IR pathway was undertaken during the conduct of phase I/II studies to evaluate the safety and efficacy of the combination of paclitaxel (T), carboplatin (C) and CP-751,871 (I) as first line treatment of advanced non-small cell lung cancer (NSCLC) patients (pts). Expression of IGF- IR and its ligands was investigated further in archival samples using tissue arrays and AQUA technology. IGF1R gene scanning was also performed. Circulating Tumor Cells (CTCs) expressing the IGF-IR were enumerated using CellTracks. Serum markers investigated included cleaved circulating IGF-IR, total and free IGF-I, IGFBP-3 and ALS. Glycemia was evaluated using fasting glucose. hGH and insulin levels were determined in a cohort of pts treated with single agent CP-751,871. Results: Blood...

Background: Induction of apoptosis is a common effect of many new and existing drugs, and Bcl-2 expression is associated with resistance to apoptosis. CTCs are a minimally invasive blood test and are a source of tumor cells that can be obtained serially during therapy. Monitoring Bcl-2, apoptosis, and other markers in CTCs may be useful endpoints in trials of new and targeted agents. The goal of this pilot study is to estimate Bcl-2 expression and apoptosis in CTCs from pts being treated for metastatic breast cancer (MBC). Methods: Whole blood was collected from pts initiating therapy for MBC at baseline, post-treatment (24, 48, or 72 hours), and 3–4 weeks. Samples were split into 3 × 7.5ml fractions. CTCs were isolated and enumerated using the CellTracks and CellSpotter systems. CTCs were defined as DAPI+, cytokeratin+, CD45-, with a cellular morphology. Apoptosis defined by positive staining with MAb M30. Bcl-2 status defined by staining with MAb Bcl-2–100. Results: 39/81 patients (49%) had ≥5 CTCs at baseline. 25% had ≥5 CTCs at 3–4 weeks. 35% of pts with CTCs had positive Bcl-2 staining in 91–100% of their CTCs (highest decile). Other pts were equally distributed across the remaining Bcl-2 deciles. At baseline, apoptosis correlates with CTC number. In samples with 1–4 CTCs, 80% of cells were apoptotic. For 5–49 CTCs, 48% were apoptotic; for 50–99, 34% were apoptotic; for 100–500, 28% were apoptotic; and for >500, 22% were apoptotic. Bcl-2 staining was inversely correlated with apoptosis staining. Pts were risk stratified by changes in CTC number after one cycle of therapy, and apoptosis increased with improving prognostic groups. One pt had markedly elevated CTCs of >20,000. The cells were remarkably 100% Bcl-2 positive and M30 negative (1% staining). Conclusions: These data confirm the prevalence of CTCs seen in the pivotal trial of CTCs in MBC (Cristofanilli NEJM 2004) and provide novel observations for Bcl-2 expression and apoptosis in CTCs. Higher Bcl-2 expression appears to correlate with decreased apoptosis. High fractions of apoptotic cells correlate with lower numbers of CTCs, which is associated with a better prognosis. Analysis of the post-treatment (24, 48, or 72 hour) samples and correlation of markers with progression free survival are ongoing and will be presented.

Simplification of cell enumeration technologies is necessary, especially for resource-poor countries, where reliable and affordable enumeration systems are greatly needed. In this paper, an immunomagnetic single-platform image cytometer (SP ICM) for cell enumeration based on antibody specificity is reported. A chamber/magnet assembly was designed such that the immunomagnetically labeled, acridine orange-stained cells in a blood sample moved to the surface of the chamber, where a fluorescent image was captured and analyzed for cell enumeration. The system was evaluated by applying one kind of antibody to count leukocytes and one kind for each leukocyte subpopulation: CD45 for leukocytes, CD3 for T lymphocytes, and CD19 for B lymphocytes. Excellent precision and linearity were achieved. Moreover, these cell counts, each from blood specimens of 42 to 52 randomly selected patients, were compared with those obtained by SP (TruCount) and dual-platform (DP) flow cytometry (FCM) technologies. The cell counts obtained by our system were in between those obtained from the TruCount and DP FCM methods; and good correlations were achieved ( R ≥ 0.95). For CD4 + counts, as we expected, the cell count by our system was significantly higher than the CD4 + T-lymphocyte counts obtained by SP and DP FCM methods. Immunophenotyping of the immunomagnetically selected CD4 + cells showed that, besides CD4 + T lymphocytes, a proportion of the CD4 + dim monocytes was also selected. Our system is a simple immunomagnetic SP ICM, which can potentially be used for enumeration of CD3 + CD4 + T lymphocytes in resource-poor countries if an additional CD3 immunofluorescent label is applied.

Background: For resource-poor countries, affordable methods are required for enumeration of CD4+ T lymphocytes of HIV-positive patients. For infants, additional determination of CD4/CD8 ratio is needed. - Methods: We determine the CD4+ and CD8+ T lymphocytes as the CD3+CD4+ and CD3+CD8+ population of blood cells. Target cells are CD3-immunomagnetically separated from the whole blood, and CD4-Phycoerythrin and CD8-PerCP immunofluorescently labeled. A point-of-care single platform image cytometer was developed to enumerate the target CD3+CD4+ and CD3+CD8+ populations. It has light-emitting diodes illumination, is fully computer-controlled, operates from a 12 V battery, and was designed to be cheap and easy-to-handle. Target cells are imaged on a CCD camera and enumerated by an image analysis algorithm. The cytometer outputs the absolute number of CD4+ and CD8+ T lymphocytes/l and CD4/CD8 ratio. - Results: The quality of the cell images obtained with the cytometer is sufficient for a reliable enumeration of target cells. The image cytometer achieves an accuracy of better than 10% in the range of 50-1700 cells/l. Analysis of blood samples from HIV patients yields a good agreement with the TruCount method for CD4 and CD8 count and CD4/CD8 ratio. - Conclusions: The image cytometer is affordable (component costs $3,000), compact (25 × 25 × 20 cm3), and uses disposable test materials, making it a good candidate to monitor progression of immunodeficiency disease in resource-poor settings.

The presence of circulating tumor cells (CTCs) in the blood of cancer patients may guide the use of therapy. We investigated how to evaluate a reduction in the number of CTCs after administration of therapy. CTCs were enumerated with the CellSearch system in 111 metastatic breast and 185 metastatic prostate cancer patients before start of a new line of chemotherapy and after initiation of therapy. Different means to express changes in CTC counts were evaluated with respect to overall survival (OS). A static CTC cutoff is the best method to determine whether a therapy is effective. This is exemplified by the highest Cox hazard ratio of 2.1 for OS; three methods to express relative differences performed worse. A lookup table is provided from which the significance of a change in CTCs can be derived. The aim of therapy should be the elimination of all CTCs. A period of 10 to 12 weeks of therapy is needed to reach the treatment effect on CTCs.

A method for the determination of circulating immune complexes (CIC) was adapted for flow cytometric analysis. Human granulocytes were used to phagocytose IgG-bearing CIC of serum from systemic lupus erythematosus (SLE) patients. A method for labeling the phagocytosed CIC with FITC-conjugated anti-human IgG was developed where the granulocytes remain in suspension during fixation and labeling. The fluorescence per cell, measured with a flow cytometer, is a measure of the total amount of the phagocytosed IgG. The results indicate that a rapid and quantitative method for the detection and measurement of phagocytosed CIC is possible using the flow cytometer.

Orthogonal and forward light-scattering properties of lymphocytes were measured from patients with different lymphocytic diseases in order to determine the potential value of light scattering as a screening device. Monitoring of orthogonal light scattering of lymphocytes of a B-cell chronic lymphocytic leukemia patient during splenic irradiation (SI) revealed the selective decrease of malignant cells and the fact that the major part of the residual lymphocytes were cytotoxic lymphocytes. By combining forward and orthogonal light scattering it was shown that lymphocytes from a patient with T gamma lymphocytosis were abnormal. Orthogonal light scattering also showed an increase in cytotoxic lymphocytes in a patient with mononucleosis infectiosa and in a splenectomized patient. Orthogonal light scattering of lymphocyte subpopulations showed that the leu8+ population of a patient with mononucleosis infectiosa was bidisperse. For elderly donors the occurrence of CD3+, CD4+, CD8+, and HNK-1+ lymphocytes with a large orthogonal light scattering varied considerably. The CD8+ lymphocytes of these donors consisted mainly of cytotoxic lymphocytes. These results show that determination of light-scattering properties of lymphocytes may yield important diagnostic information and can indicate when further investigation of the lymphocytes by means of immunofluorescence is necessary.

Light-scattering properties of human T-lymphocyte subpopulations selected by immunofluorescence were studied. Based on differences in orthogonal light scattering, two subpopulations of T8-positive cells can be distinguished. The first population (T8a) has the same orthogonal light-scattering properties as T4-positive cells, whereas the orthogonal light scattering of the second population (T8b) was about 70% larger. Orthogonal light scattering of Leu7-positive lymphocytes resembles that of the T8b population. We have studied the occurrence of the subpopulation in healthy individuals and we discuss their possible functional identification. Light-scattering properties of lymphocyte subpopulations in two patients with B-cell chronic lymphatic leukemia suggest that this observation is of clinical interest.

Light scattering properties of human lymphocyte subpopulations selected by immunofluorescence were studied with a flow cytometer. Regulatory and B-lymphocytes showed a low orthogonal light scatter signal, whereas cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15 revealed a large orthogonal light scatter signal. Two populations in light scatter histograms could be observed with monoclonal antibodies directed against determinants present on both regulatory and cytotoxic lymphocytes. By analysis of the lymphocytes of 16 individuals we found a linear relation between the number of cells with a large orthogonal light scattering and the number of cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15. These observations demonstrate physical differences between cytotoxic lymphocytes and regulatory and B lymphocytes. Moreover, the results suggest a method to estimate the amount of cytotoxic lymphocytes without using monoclonal antibodies.

We describe the effect of splenic irradiation (SI) (0,5–1 Gy weekly) on lymphocyte subpopulations for 7 patients with progressive B chronic B-lymphocytic leukemia (B-CLL). Using specific cellular characteristics we could distinguish normal from abnormal cells. The irradiation resulted in a decrease of lymph node size, reduction in spleen volume and decrease in peripheral blood lymphocytes. The one exception was a patient with a prolymphocytoid transformation of B-CLL. For 3 patients SI had to be interrupted or stopped because of severe cytopenia. Quantitation of malignant B cells and normal T lymphocytes revealed that the total irradiation dose which resulted in a specific decrease of malignant lymphocytes varied from patient to patient. Normal T-cell subpopulations, which were increased before SI, decreased to normal or abnormally low values during SI. In previously untreated patients, natural killer (NK) cell numbers decreased more rapidly than T-cell subpopulations. For 2 patients refractory to chemotherapy an increase of NK cells was observed upon SI.

Measurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinophilic granulocytes from neutrophilic granulocytes. We use this method to perform a four-parameter differential white blood cell analysis. A simple flow cytometer was built equipped with a 5-mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0 degrees and 2.6 degrees and angles between 3.0 degrees and 11.0 degrees. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering. Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H-6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes.

Background Tumor cells in the blood of patients with metastatic carcinomas are associated with poor survival. Knowledge of the cells’ genetic make-up can help to guide targeted therapy. We evaluated the efficiency and quality of isolation and amplification of DNA from single circulating tumor cells (CTC). Methods The efficiency of the procedure was determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system, followed by single cell sorting by fluorescence-activated cell sorting (FACS) and whole genome amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells was exome sequenced and the DNA quality analyzed. Single CTC from patients with lung cancer were used to demonstrate the potential of single CTC molecular characterization. Results The overall efficiency of the procedure from spiked cell to amplified DNA was approximately 20%. Losses attributed to the CellSearch system were around 20%, transfer to FACS around 25%, sorting around 5% and DNA amplification around 25%. Exome sequencing revealed that the quality of the DNA was affected by the fixation of the cells, amplification, and the low starting quantity of DNA. A single fixed cell had an average coverage at 20× depth of 30% when sequencing to an average of 40× depth, whereas a single unfixed cell had 45% coverage. GenomiPhi-amplified genomic DNA had a coverage of 72% versus a coverage of 87% of genomic DNA. Twenty-one percent of the CTC from patients with lung cancer identified by the CellSearch system could be isolated individually and amplified. Conclusions CTC enriched by the CellSearch system were sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data showed that this DNA could be used for variant calling, but not for quantitative measurements such as copy number detection. Close to 55% of the exome of single SKBR-3 cells were successfully sequenced to 20× depth making it possible to call 72% of the variants. The overall coverage was reduced to 30% at 20× depth, making it possible to call 56% of the variants in CellSave-fixed cells.