Small Volumes Of Peripheral Blood Research Articles

Design and validation of novel flow cytometry panels to analyze a comprehensive range of peripheral immune cells in mice.

The use of flow cytometry in mice is constrained by several factors, including the limited availability of mouse-specific antibodies and the need to work with small volumes of peripheral blood. This is particularly challenging for longitudinal studies, as serial blood samples should not exceed 10% of the total blood volume in mice. To address this, we have developed two novel flow cytometry panels designed to extensively analyze immune cell populations in mice during longitudinal studies, using only 50 µL of peripheral blood per panel. Additionally, a third panel has been designed to conduct a more detailed analysis of cytotoxic and inhibitory markers at the end point. These panels have been validated on a lipopolysaccharide (LPS)-induced lung inflammation model. Two experiments were conducted to 1) validate the panels' sensitivity to immune challenges (n=12) and 2) to assess intrinsic variability of measurements (n=5). In both experiments, we collected 50 µL of peripheral blood for each cytometry panel from the maxillary venous sinus. All antibodies were titrated to identify the optimal concentration that maximized the signal from the positive population while minimizing the signal from the negative population. Samples were processed within 1 hour of collection using a MACSQuant Analyzer 16 cytometer. Our results demonstrate that these immunological panels are sensitive enough to detect changes in peripheral blood after LPS induction. Moreover, our findings help determine the sample size needed based on the immune population variability. In conclusion, the panels we have designed enable a comprehensive analysis of the murine immune system with a low blood volume requirement, enabling the measure of both absolute values and relative percentages effectively. This approach provides a robust platform for longitudinal studies in mice and can be used to uncover significant insights into immune responses.

Strategy for Microscale Extraction and Proteome Profiling of Peripheral Blood Mononuclear Cells.

Peripheral blood mononuclear cells (PBMCs) play vital roles in physiological and pathological processes and represent a rich source for disease monitoring. Typical molecular profiling on PBMCs involves the sorting of cell subsets and thus requires a large volume of peripheral blood (PB), which impedes the clinical practicability of omics tools in PBMC measurements. It would be clinically invaluable to develop a convenient approach for preparing PBMCs from small volumes of PB and for deep proteome profiling of PBMCs. To this end, here, we designed an apparatus (PBMC-mCap) for microscale enrichment and proteome analysis of PBMCs, which pushed the needed PB volume from the normal 2 mL or higher to 100 μL or lower, comparable to the volume of a drop of finger blood. A PBMC-specific mass spectra library containing 8869 proteins and 121,956 peptides was further built, which, in combination with the optimized data-independent acquisition strategy, helped to identify 6000 and 6500 proteins from PBMCs with 100 μL and 1 mL of PB as initial materials, respectively. Further application of the strategy for PBMC proteomes revealed a steady difference between gender (male vs female) and upon stimulus (COVID-19 vaccination). For the latter, we observed differentially expressed genes and pathways involving the activation of immune cells, including the NF-κB pathway, inflammation response, and antiviral response. Our strategy for the proteome analysis of microscale PBMCs may provide a convenient clinical toolkit for disease diagnosis and healthy state monitoring.

An ultra-thin silicon nitride membrane for label-free CTCs isolation from whole blood with low WBC residue

Microfiltration is the most straightforward and effective method to enrich circulating tumor cells (CTCs) from large numbers of background blood cells in the peripheral blood. It is still very challenging to achieve both high recovery of CTCs and low residue of white blood cells (WBCs) through one-time whole blood filtration. In this study, through a simulation, we revealed that reducing the thickness of the filter significantly reduces the WBC residue. A filter device was created using a 400-nm-thick Si3N4 membrane. The recovery rates of A549 and MCF-7 tumor cell lines were 82.04 ± 1.19% and 86.53 ± 3.27%, respectively, while the residual number of WBCs was largely reduced from 6.84 × 106 cells/mL cells to 604 ± 33.74 cells/mL. In addition, the assembled device could process whole blood without pretreatment at a high throughput of 1 mL/min. Utilizing as small volume of peripheral blood as 1 mL, CTCs were enriched by our device in four of six lung cancer patients and four of five breast cancer patients. The lower WBC residue is advantageous in CTC enumeration and subsequent proteomic or genomic characterization. Therefore, this technology enables label-free CTC isolation with a low WBC residue from whole blood, paving the way for a CTC analysis in clinical applications.

Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells

BackgroundPatients affected by aggressive B-cell malignancies who are resistant to primary or salvage chemoimmunotherapy have an extremely poor prognosis and limited therapeutic options. Promising therapeutic success has been achieved with...

Culturing adequate CAR-T cells from less peripheral blood to treat B-cell malignancies.

Objective:Chimeric antigen receptor-modified T (CAR-T) cells have shown impressive results against relapsed/refractory B cell malignancies. However, the traditional manufacture of CAR-T cells requires leukapheresis to isolate large amounts of peripheral blood T cells, thus making some patients ineligible for the procedure.Methods:We developed a simple method for CAR-T cell preparation requiring small volumes of peripheral blood. First, CD3+ T cells isolated from 50 mL peripheral blood from patients (B-cell malignancies) were stimulated with immobilized anti-CD3/RetroNectin in 6-well plates and then transduced with CAR-expressing lentiviral vector. After 4 d, the T cells were transferred to culture bags for large-scale CAR-T cell expansion. In vitro and animal experiments were performed to evaluate the activity of the manufactured CAR-T cells. Finally, 29 patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 patients with B-cell lymphoma were treated with the CAR-T cells.Results:The CAR-T cells were expanded to 1–3 × 108 cells in 8–10 d and successfully killed B cell-derived malignant tumor cells in vitro and in vivo. For patients with B-ALL, the complete remission rate was 93% 1 month after CAR-T cell infusion; after 12 months, the overall survival (OS) and leukemia-free survival rates were 69% and 31%, respectively. For patients with lymphoma, the objective response rate (including complete and partial remission) was 78% 2 months after CAR-T cell infusion, and after 12 months, the OS and progression-free survival rates were 71% and 43%, respectively. Cytokine-release syndrome (CRS) occurred in 65.51% and 55.56% of patients with B-ALL and B-cell lymphoma, respectively; severe CRS developed in 20.69% of patients with B-ALL and in no patients with lymphoma.Conclusions:Our novel method can generate sufficient numbers of CAR-T cells for clinical use from 50–100 mL peripheral blood, thus providing an alternative means of CAR-T cell generation for patients ineligible for leukapheresis.

A simple and effective method for the isolation and culture of human monocytes from small volumes of peripheral blood

Innate immune cell defects contribute to severe autoimmunity and the pathogenesis of inflammatory disease. Monocyte-derived macrophages typically retain disease related signatures and represent an excellent in vitro model to uncover and validate mechanisms contributing to specific pathological states. Monocyte isolation procedures vary widely in terms of purity, yield, cost, degree of technical difficulty and volume of peripheral blood needed. This paper outlines a novel isolation method that yields monocytes through density gradient centrifugation (Ficoll® and hyperosmotic Percoll®). The protocol has been optimised for small volumes of blood (42 ml) and is simple, reproducible and inexpensive compared to other methods. Monocyte recovery is 70% (relative to monocyte numbers within the buffy coat) and the highly functional macrophages produced are characterised by excellent purity (98.6 ± 0.6%) and intact activation and phagocytic capacities. The method is well suited to investigations involving patient populations where a particular subset of immune cells is known to contribute to the pathogenesis of a specific disease or is aberrant as a consequence of that disease.

Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.

Malaria pathogenesis is caused by the replication of Plasmodium parasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long-term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by both P. vivax and P. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood-stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.

Ex vivo expanded natural killer cells from breast cancer patients and healthy donors are highly cytotoxic against breast cancer cell lines and patient-derived tumours

BackgroundNatural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours.MethodsWe used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro.ResultsAfter 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro.ConclusionNK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.

Efficient and reliable establishment of lymphoblastoid cell lines by Epstein-Barr virus transformation from a limited amount of peripheral blood

Lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV) serve as an unlimited resource of human genomic DNA. The protocol that is widely used to establish LCLs involves peripheral blood mononuclear cell isolation by density gradient centrifugation, however, that method requires as much as 5 ml of peripheral blood. In this study, in order to provide a more simple and efficient method for the generation of LCLs, we developed a new protocol using hemolytic reaction to enrich white blood cells for EBV transformation and found that the hemolytic protocol successfully generated LCLs from a small volume (i.e., 0.1 ml) of peripheral blood. To assess the quality of genomic DNA extracted from LCLs established by the hemolytic protocol (LCL-hemolytic), we performed single nucleotide polymorphism (SNP) microarray genotyping using the GeneChip® 100 K Array Set (Affymetrix, Inc.). The concordances of the SNP genotyping resulting from genomic DNA from LCL-hemolytic (99.92%) were found to be as good as the technical replicate (99.90%), and Kappa statistics results confirmed the reliability. The findings of this study reveal that the hemolytic protocol is a simple and reliable method for the generation of LCLs, even from a small volume of peripheral blood.

Functional osteoclastogenesis: the baseline variability in blood donor precursors is not associated with age and gender.

Mononuclear osteoclast precursors circulate in the monocyte fraction of peripheral blood and form multinuclear cells with all osteoclastic phenotypic characteristics when cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kB ligand (RANKL). The method to obtain osteoclast precursors from peripheral blood is simple but the number of recovered osteoclasts is often largely insufficient for functional analyses. The original aim of this study was to develop a rapid and efficient method that could overcome the donor variability and enrich the osteoclast precursors from a small volume of peripheral blood as a basis for future clinical studies to correlate the differentiation potential of circulating osteoclast precursors with bone lesions in cancer patients. We improved the efficiency of osteoclastogenesis by reducing isolation and purification times and overcame the use of flow cytometry and immunomagnetic purification procedures. In our culture system the osteoclast number was increased several-fold and the precursors were able to reach a full differentiation within seven days of culture. Both age as well as gender differences in osteoclastogenesis efficiency were no longer evident by processing limited volume blood samples with this simple and rapid method.

Massive, Clinical Grade Expansion Of Polyclonal T Cells Using Blinatumomab For Adoptive Autologous Cellular Therapy Of CLL Patients

BackgroundThe combined use of chemotherapy and monoclonal antibodies has proved highly effective for the treatment of CLL but often results in severe life threatening immunosuppression. The development of adoptive therapy with autologous T cells could be clinically relevant to overcome these problems. MethodsWe have devised a novel, simple and efficient method for ex vivo expansion of normal autologous T cells from the peripheral blood of CLL patients for adoptive therapy, using blinatumomab (CD3xCD19) and rhIL-2 in serum-free medium. The complete phenotype of in vitro expanded T cells was analyzed by flow cytometry and their cytotoxic activity by calcein release assays. ResultsWe performed 18 expansions of T cells, starting from a very small volume of peripheral blood from untreated CLL patients (mean 10.3 ml, range 2-30 ml) that contained a mean of 9.2x106 T cells (range 0.4 to 51x106)(Fig.1). This method allowed reproducible expansion in about 21 days of a mean 410x106 CD3+ T cells (range 71 to 2184x106). The mean fold expansion of T cells in about 3 weeks of in vitro culture was 224 (range 4.4-1326). The only significant contaminant in final Blinatumomab Expanded T cell cultures (BET) were NK cells (mean 18.5%). Indeed addition of blinatumomab and rhIL-2 to the cultures led to a rapid decrease in CLL B cells, which took place from days 7 to 14 onwards and resulted in their complete depletion within 3 weeks (mean 0.2% CLL B cells at days 18-25). Only in one case, a significant percentage of CLL B cells could be observed at the end of culture, but this was due to the particularly high percentage neoplastic cells in the starting population in this patient (98%), resulting in relatively late depletion of these cells, which took place between days 14 and 21, and therefore remained detectable in this case at day 24 (3.8% CLL B cells at day 24). Despite the very low percentage of starting T cells in this specific patient (1.2%), 152x106 T cells could be obtained, equivalent to a 42 fold expansion.In the 18 expansions performed, the resulting BET cells contained both CD4+ and CD8+ cells in varying proportions (median 46.2% and 44.4% respectively). Only in two cases the final product was composed predominantly of CD4+ cells (95%). Expanded T cells were polyclonal, as shown by TCR Vβ expression which was within the normal range by flow cytometry. Indeed CMV specific clones, detected by CMV peptide (pp65495-503)-loaded HLA-A*0201 tetramer, were expanded using this method and detected in equivalent proportion before and after expansion. Final T cells were composed predominantly of the effector and central memory subsets. Th1 were slightly prevalent over Th2 cells (means 20% and 10%, respectively), whereas Th17 and Treg were less than 1%.Since CLL derived T cells have been shown previously to have enhanced expression of the synapse regulators CD272 and CD279 compared to normal T cells, leading to impaired immunological synapse formation, we have analyzed these markers in both starting and BET cells from 4 patients. We observed that CD272 and CD279 diminished in BET compared to the starting CLL T populations (from 73% to 19% and 61% to 18%, respectively). These data suggest that stimulation and expansion with blinatumomab and rhIL-2 has normalized expression of these regulators on CLL T cells. Indeed BET were highly cytotoxic against CD19+ targets cell lines or primary CLL cells, with 70-90% lysis at a 3:1 effector target ratio in presence of blinatumomab.Finally BET were compared to Xcellerated cells expanded using anti-CD3/CD28 Dynabeads and rhIL-2. The expansion protocols using either blinatumomab or anti-CD3/CD28 Dynabeads showed equivalent efficiency and comparable cell composition at the end of culture. Further comparison of the T cell subsets present in BET or CD3/CD28 cultures is in progress. ConclusionsThese data altogether suggest that the use of blinatumomab and rhIL-2 provides a reproducible, simple and GMP-compliant protocol, allowing expansion of large numbers of autologous polyclonal T cells depleted of CLL cells, from relatively small volumes of peripheral blood from CLL patients. This approach is an attractive option for adoptive therapy in these patients after immunosuppressive treatments. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Platform Minigene AAVS1 Targeted Safe Harbor Approach For Genetic Correction Of iPSC Derived From Patients With Each Of The 5 Genetic Forms Of Chronic Granulomatous Disease

There are 5 genetic forms of chronic granulomatous disease (CGD), resulting from mutations in one of the CYBB, CYBA, NCF1, NCF2, or NCF4 genes encoding, respectively, the subunits of the phagocyte oxidase (phox) gp91phox, p22phox, p47phox, p67phox and p40phox. Phox is required for blood neutrophils and monocytes to produce microbicidal superoxide and hydrogen peroxide. We previously demonstrated zinc finger nuclease (ZFN) targeting of a corrective minigene encoding gp91phox to the AAVS1 safe harbor site of iPSC from a patient with X-linked CGD (XCGD), which restored phox activity in neutrophils differentiated from the corrected iPSC (Blood 2011, 117:5561). Subsequently, we have generated iPSC from the CD34+ hematopoietic stem cells (HSC) present in small volumes of peripheral blood from additional CGD patients to include those representing the other genetic forms of CGD (Blood 2013, 121:e98). We used our previously reported gp91phox AAVS1 targeting plasmid to create CGD genotype-specific targeting plasmids for correction of the other 4 genetic forms of CGD by replacing gp91 cDNA with cDNA for each of the autosomal recessive CGD genes. Using these genotype specific targeting plasmids with AAVS1 targeting ZFNs, this approach resulted in safe harbor minigene correction of iPSC for each of the 5 genetic forms of CGD, routinely achieving minigene insertion with constitutive expression of the specific desired transgene phox subunit protein in 70-80% percent of selected iPSC clones. Neutrophils or macrophages were generated from iPSC by first differentiating in STEMdiff APEL medium for 13 days to generate CD34+CD45+ cells, which were further expanded in HSC medium for 7 days. The expanded cells were differentiated either to neutrophils with G-CSF in the presence or absence of OP9 mouse stromal cells for 7 days, or to macrophages with M-CSF for 10 to 14 days. Full restoration of oxidase activity was observed in neutrophils or macrophages derived from minigene corrected CGD iPSC representing all the 5 genetic types of CGD. Furthermore, we demonstrated significant correction of bacterial growth inhibition function in corrected CGD iPSC derived neutrophils (Staphylococci) and macrophages (Granulibacter). Of particular note, the M-CSF differentiated macrophages derived from iPSC showed a significant increase in IL-10 production (p < 0.01) in response to UV-inactivated Listeria, with comparable levels to M-CSF derived macrophages differentiated from peripheral blood monocytes. This type of iPSC gene-correction approach with ex vivo production of autologous functional neutrophils or macrophages could be used for supportive autologous cell therapy in CGD patients with infections not responding to conventional treatment. Using a universal platform approach that combines iPSC generation from small volumes of peripheral blood and ZFN mediated AAVS1 safe harbor minigene targeting, we show it is possible to efficiently go from peripheral blood sample to genetically corrected iPSC to generation of functionally corrected neutrophils and macrophages using an identical approach for all 5 genetic forms of CGD. This platform approach is broadly applicable to a wide range of inherited single gene metabolic disorders. Disclosures:No relevant conflicts of interest to declare.

Large Scale Selective Ex Vivo Expansion of CD4+CD25+FOXP3+ Regulatory T Cells from Peripheral Blood.

CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. However, the frequencies of Treg in peripheral blood are very low. Here we attempted the ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. CD4+ T cells from peripheral blood of healthy donors or patients with chronic graft-versus-host disease (GVHD) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-b. After one week of culture, expanding cells were once detached from the plate and subjected to the fresh medium including rhIL-2 and rhTGF-b but not MAbs. After 2-weeks of culture, phenotypic and functional analyses were performed. Mixed lymphocyte reaction was done using CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations. Xenogeneic -GVHD in NOD-Scid mice was induced by the injection of human T cells expressing luciferace transgene, followed by in vivo bioluminescence imaging (BLI) analysis using a CCD camera.Our expansion procedure with TGF-b yielded 45-83% purity of Foxp3+CD25+CD4+Treg co-expressing CTLA-4, CD54 and GITR, while 8-42% purity without TGF-b(p<0.001). These cell populations also displayed CD45RO+CD45RA−CD26high+ memory phenotype. An average expansion rate of Treg was 62,200 fold (25,500–97,900) in healthy donors during the culture periods (n=5). Thus, we obtained an average of 4.7x108 Treg from the initial number of 5x105 CD4+ T cells in peripheral blood. Additionally, from peripheral CD4+ T cells in patients with chronic GVHD, Treg could be expanded equivalently to healthy donors. The resulting Treg-rich populations inhibited the proliferative response of CFSE-labeled T cells to autologous and allogeneic DC (Figure 1). The ex vivo expanded Treg-rich populations had the inhibitory effect on xeno-reactive T cells expressing luciferase transgene in a xenogeneic GVHD model (Figure 2). Our procedure has allowed efficient ex vivo expansion of Treg-rich populations from a small volume of peripheral blood, and will be applicable to clinical use. [Display omitted] [Display omitted]

Exploiting Cytokine Secretion to Rapidly Produce Multivirus-Specific T Cells for Adoptive Immunotherapy

Viral infections remain a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT), and conventional small-molecule therapeutics often have modest benefit, high cost and adverse effects. Adoptive transfer of donor-derived, virus-specific T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) proved feasible and safe after HSCT, and reconstituted immunity to all 3 viruses. Current protocols to generate multivirus-specific T cells are lengthy, taking up to 12 weeks. Since viral infections often occur <30 days after HSCT, more rapid production is required. The IFN-γ capture (Miltenyi) and tetramer methods can select antigenspecific T cells directly from peripheral blood and have been used clinically to prevent infections with AdV and CMV respectively. Tetramer selection is limited to certain HLA class I molecules with known epitope specificities. By contrast, the IFN-γ capture assay can be used to select multi-epitope and multi-antigen, CD4+ and CD8+ T cells from any donor, irrespective of HLA type. We now describe a modified rapid selection method for production and characterization of CD4+ and CD8+ T cells specific for CMV, EBV and AdV in a single infusate using the IFN-γ capture assay. We could manufacture a specific product from small volumes of peripheral blood over a 48 hour time period. These cells are suited for infusion into immunocompromised patients. Activated PBMCs were transduced either with Ad5f35pp65 vector or Ad5f35LMP2 vector and then combined. After overnight culture, IFN-γ-secreting antigen-specific T cells were captured. The number of selected cells ranged from 0.06 ×106 to 0.36 ×106 cells (mean: 0.14 ± 0.06 ×106; n=5) per 20 × 106 PBMCs.Importantly, we demonstrate that the selected cells lack alloreactivity, indicating that the infused cells should be safe in vivo. By expanding a small fraction of the selected product we could characterize the phenotype, specificity and function of the infused product, to allow subsequent comparison with clinical outcome. All products were able to secrete IFN-γ in response to each antigen and kill CMV, EBV and AdV antigen-expressing targets. These results show that we can successfully generate trivirus-specific T cells for infusion within 3 days without sacrificing the product characterization required to correlate product with clinical outcome.

Circulating tumor cells in patients with castration-resistant metastatic prostate cancer

5076 Background: Blood-based assays are urgently needed to provide molecular information on the specific targets expressed in tumor cells to optimize treatment selection. Antibody-capture technologies have been applied to isolate circulating tumor cells (CTC) from small volumes of peripheral blood from patients with progressive castrate metastatic prostate cancer. It has been demonstrated previously that CTC isolated from these patients represent authentic prostate cancer cells. Methods: CTC, positive for EpCAM (epithelial cellular adhesion molecule) and nuclear DAPI, and CD45 negative, were isolated from 120 patients with clinical castrate metastatic disease. All patients had rising PSA levels and were on stable treatment regimens at the time of CTC sampling. We tested the association between CTC counts and PSA levels, and the extent of disease to bone, and soft tissue metastasis by Wilcoxon rank sum. Fluorescence in situ hybridization was performed for AR and ERBB2 genes by an adapted method in CTC. Results: The average age in this patient cohort was 69 years, and median PSA at the time when CTC were drawn was 111 ng/mL (range 0.86–12147 ng/mL). The patterns of metastatic spread included disease in soft tissue only in 12 patients (10%), in bone and soft tissue in 67 (56%), and in bone only in 41 patients (34%). CTC counts ranged from 0 to 1958 cells per 7.5 mL of blood. A large number of patients (54, 45%) had 10 or more circulating tumor cells, while only 33 patients (27.5%) had 1 or less CTC per sample of blood. Significantly higher numbers of CTC were detected in patients with bone metastasis compared to those without bone metastasis (11 vs. 2.5, p&lt;0.01). In patients with marked amplification of AR locus (five patients), tetraploidy was noted in the majority of cases (four cases). Two patients without AR amplification showed apparent tetraploidy, while no analyzed samples (nine) had amplification of ERBB2. Conclusions: The analysis of cancer-related gene alterations in CTC is feasible in a hospital-based laboratory. Further gene expression studies focused on the patients with higher numbers of CTC in correlation with clinical outcomes, as well as the investigation of CTC gene expression during specific treatments are under way. No significant financial relationships to disclose.

Circulating Tumor Cell Analysis in Patients with Progressive Castration-Resistant Prostate Cancer

To better direct targeted therapies to the patients with tumors that express the target, there is an urgent need for blood-based assays that provide expression information on a consistent basis in real time with minimal patient discomfort. We aimed to use immunomagnetic-capture technology to isolate and analyze circulating tumor cells (CTC) from small volumes of peripheral blood of patients with advanced prostate cancer. Blood was collected from 63 patients with metastatic prostate cancer. CTCs were isolated by the Cell Search system, which uses antibodies to epithelial cell adhesion marker and immunomagnetic capture. CTCs were defined as nucleated cells positive for cytokeratins and negative for CD45. Captured cells were analyzed by immunofluorescence, Papanicolau staining, and fluorescence in situ hybridization. Most patients (65%) had 5 or more CTCs per 7.5 mL blood sample. Cell counts were consistent between laboratories (c = 0.99) and did not change significantly over 72 or 96 h of storage before processing (c = 0.99). Their identity as prostate cancer cells was confirmed by conventional cytologic analysis. Molecular profiling, including analysis of epidermal growth factor receptor (EGFR) expression, chromosome ploidy, and androgen receptor (AR) gene amplification, was possible for all prostate cancer patients with >or=5 CTCs. The analysis of cancer-related alterations at the DNA and protein level from CTCs is feasible in a hospital-based clinical laboratory. The alterations observed in EGFR and AR suggest that the methodology may have a role in clinical decision making.

Blood-sample processing for the study of age-dependent gene expression in peripheral blood mononuclear cells.

Although most new biogerontological studies seeking to identify longevity candidate genes and factors involved in successful human aging are population based, and likely to involve the collection of blood from extremely old individuals, to our knowledge no unified protocols have yet been published to describe a methodology permitting the simultaneous generation of different kinds of biological specimens derived from a single source of a very small volume of peripheral blood. Here we describe a method permitting the simultaneous generation of plasma, RNA, DNA, protein, fixed lymphocytes, and frozen blood aliquots from a single 10- to 30-ml blood sample obtained from donors of any age (10-102 years old), and we show that the quality and quantity of DNA, RNA, protein, and fixed lymphocytes obtained do not vary significantly with age. As is frequently observed, the older individuals have higher plasma proportions.

Analysis of IgE heavy chain sequences from a small volume of peripheral blood from a mildly atopic individual

Analysis of IgE heavy chain sequences from a small volume of peripheral blood from a mildly atopic individual

Uptake and killing ofListeria monocytogenesby normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting

Cellular components of innate immunity (NK cells, monocytes and granulocytes) play an important role in early resistance to Listeria monocytogenes in the mouse model. Minimally invasive methods of measuring the bacteriocidal capacity of these cells may be useful as a biomarker of susceptibility in humans. A technique was developed whereby the uptake and survival of L. monocytogenes could be measured in human granulocytes and monocytes using small volumes of peripheral blood. This method used flow cytometry to detect the presence of PKH-2-labeled bacteria within these cells. Survival of bacteria was determined by sorting of infected cells based on a combination of fluorescence and light scattering properties. Considerable variation in bacterial recovery was seen between normal volunteers. There was consistently greater survival of a fully virulent strain of L. monocytogenes within monocytes and granulocytes compared with an isogenic strain lacking the hemolysin, listeriolysin O, when measured at baseline. There was no evidence of longer-term bacterial survival or growth at 2 or 24 h. This technique may be useful for assessment of both host resistance and pathogen virulence.

Uptake and killing of Listeria monocytogenes by normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting

Cellular components of innate immunity (NK cells, monocytes and granulocytes) play an important role in early resistance to Listeria monocytogenes in the mouse model. Minimally invasive methods of measuring the bacteriocidal capacity of these cells may be useful as a biomarker of susceptibility in humans. A technique was developed whereby the uptake and survival of L. monocytogenes could be measured in human granulocytes and monocytes using small volumes of peripheral blood. This method used flow cytometry to detect the presence of PKH-2-labeled bacteria within these cells. Survival of bacteria was determined by sorting of infected cells based on a combination of fluorescence and light scattering properties. Considerable variation in bacterial recovery was seen between normal volunteers. There was consistently greater survival of a fully virulent strain of L. monocytogenes within monocytes and granulocytes compared with an isogenic strain lacking the hemolysin, listeriolysin O, when measured at baseline. There was no evidence of longer-term bacterial survival or growth at 2 or 24 h. This technique may be useful for assessment of both host resistance and pathogen virulence.