An optimized method for the induction and purification of mouse bone marrow dendritic cells.

Dendritic cells (DCs) play an essential role in the initiation of adaptive immune responses, but they are rare in all organs. The traditional methods used to increase the yield and purity of DCs are the early removal of granulocyte culture medium and the isolation of high-purity DCs by magnetic-activated cell sorting (MACS). This study provides a more rapid and economical optimization method to obtain more high-purity DCs. (i) We harvested 18% more bone marrow (BM) cells by using forceps to crack the epiphysis instead of cutting it with scissors during BM cell extraction. (ii) When the cells in the culture medium that is discarded on day 3 in the traditional method were centrifuged and then added back to the petri dish, the DC yield on day 5 increased by 61%. (iii) On the third day, the addition of fresh medium and the retention of the original medium rather than discarding it increased the number of DCs harvested on the fifth day by 137%. (i-iii) The improved method cost an average of 74% less than the conventional method and yielded the same number and function of cells. (iv) The initial number of BM cells was increased by 15% in 4-week-old mice compared with 8-week-old mice. (v) The Percoll density centrifugation (PDS) method was used to purify DCs on day 6 after induction, and the purity of the DCs was greater than 90%, which showed no significant difference from the MACS method. However, the yield of the PDS method increased by 21%. In addition, the PDS method has a lower cost, with an average purification cost of 4 CNY ($0.58) compared with 648 CNY ($93.25) for MACS, reducing the cost by 99%. Therefore, high-purity and high-yield DCs can be rapidly obtained through a five-step improvement in the process of BM cell extraction, induction and purification.

A single-view field filter device for rare tumor cell filtration and enumeration.

In this work, we demonstrate a single-view field filter (SVFF) device for the efficient filtration and enumeration of rare tumor cells in the blood. In our device, the track-etched membrane is integrated within a low-cost polymer-film microfluidic chip, and multiplex microfiltration chambers are designed. Our device permits the performing of multiple sample tests on a single membrane and the dynamical observation of the entire filtration process in a single field of view. To characterize the device performance, our device is first tested using tumor cells, and three different cell behaviors are observed during the filtration process. Finally, we successfully apply our device for the separation of rare tumor cells from the lysed blood samples at various flow rates. The recovery rates of 93.3, 87.6, and 84.1% can be respectively achieved at the throughputs of 50, 100, and 150 µL/min. Our single-view field filter (SVFF) device offers the advantages of label-free filtration, efficient enumeration, easy integration, and low cost, and holds the potential to be used as an efficient tool for the filtration and enumeration of rare cells.

Isolation of a novel embryonic stem cell cord blood-derived population with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Recent studies highlight the existence of a population of cord blood (CB)-derived stem cells that bare embryonic features (very small embryonic-like stem cells [VSELs]) as the most primitive CB-stem cell population. In the present study, we present for the first time a novel and high purity isolation method of VSELs with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). METHODS: The experimental procedure includes isolation upon gradually increased centrifugation spins and chemotaxis to Stromal cell-derived factor 1a (SDF-1a). Τhis cell population is characterized with flow cytometry, alkaline phosphatase (ALP) staining and qRT-PCR. The functional role of the isolated VSELs is assayed following co-culture with WJ-MSCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs), whereas the stimulation of the quiescent VSEL population is verified via cell cycle analysis. The in vitro hematopoietic capacity is evaluated in methylcellulose cultures and also through induction of erythroid differentiation. RESULTS: The final isolated subpopulation is characterized as a small-sized CD45/Lineage-/CXCR4+/CD133+/SSEA-4+cell population, positive in ALP staining and overexpressing the Oct3/4, Nanog and Sox-2 transcription factors. Upon the co-culture with MSCs, a stimulation of the quiescent VSEL population is observed. An impressive increase in the co-expression of the CD34+/CD45+ markers is observed following the co-culture with the WJ-MSCs, which is confirmed by the intense clonogenic ability suggesting in vitro differentiation toward all of the hematopoietic cell lineages and successful differentiation toward erythrocytes. DISCUSSION: Conclusively, we propose a novel, rapid and rather simplified isolation method of CB-VSELs, capable of in vitro hematopoiesis.

Modeling biological and economic uncertainty on cell therapy manufacturing: the choice of culture media supplementation.

AIM: To evaluate the cost-effectiveness of autologous cell therapy manufacturing in xeno-free conditions. MATERIALS & METHODS: Published data on the isolation and expansion of mesenchymal stem/stromal cells introduced donor, multipassage and culture media variability on cell yields and process times on adherent culture flasks to drive cost simulation of a scale-out campaign of 1000 doses of 75 million cells each in a 400 square meter Good Manufacturing Practices facility. RESULTS & CONCLUSION: Passage numbers in the expansion step are strongly associated with isolation cell yield and drive cost increases per donor of $1970 and 2802 for fetal bovine serum and human platelet lysate. Human platelet lysate decreases passage numbers and process costs in 94.5 and 97% of donors through lower facility and labor costs. Cost savings are maintained with full equipment depreciation and higher numbers of cells per dose, highlighting the number of cells per passage step as the key cost driver.

A Versatile, Low-Cost, Multiway Microfluidic Sorter for Droplets, Cells, and Embryos.

Partitioning and sorting particles, including molecules, cells and organisms, is an essential prerequisite for a diverse range of applications. Here, we describe a very economical microfluidic platform (built from parts costing about U.S. $6800 for a stand-alone system or U.S. $3700, when mounted on an existing fluorescence microscope connected to a computer) to sort droplets, cells and embryos, based on imaging data. Valves operated by a Braille display are used to open and close microfluidic channels, enabling sorting at rates of >2 Hz. Furthermore, we show microfluidic 8-way sorting for the first time, facilitating the simultaneous separation and collection of objects with diverse characteristics/phenotypes. Due to the high flexibility in the size of objects that can be sorted, the low cost, and the many possibilities enabled by imaging technology, we believe that our approach nicely complements existing FACS and µFACS technology.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations.

The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30-50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46±0.63×106 vs. 2.4±0.28×106, respectively, from 1.0×108 lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to our previous method (2.36±0.70×106), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward.

High-throughput, low-loss, low-cost, and label-free cell separation using electrophysiology-activated cell enrichment.

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.

FAST: Size-Selective, Clog-Free Isolation of Rare Cancer Cells from Whole Blood at a Liquid-Liquid Interface.

Circulating tumor cells (CTCs) have great potential to provide minimally invasive ways for the early detection of cancer metastasis and for the response monitoring of various cancer treatments. Despite the clinical importance and progress of CTC-based cancer diagnostics, most of the current methods of enriching CTCs are difficult to implement in general hospital settings due to complex and time-consuming protocols. Among existing technologies, size-based isolation methods provide antibody-independent, relatively simple, and high throughput protocols. However, the clogging issues and lower than desired recovery rates and purity are the key challenges. In this work, inspired by antifouling membranes with liquid-filled pores in nature, clog-free, highly sensitive (95.9 ± 3.1% recovery rate), selective (>2.5 log depletion of white blood cells), rapid (>3 mL/min), and label-free isolation of viable CTCs from whole blood without prior sample treatment is achieved using a stand-alone lab-on-a-disc system equipped with fluid-assisted separation technology (FAST). Numerical simulation and experiments show that this method provides uniform, clog-free, ultrafast cell enrichment with pressure drops much less than in conventional size-based filtration, at 1 kPa. We demonstrate the clinical utility of the point-of-care detection of CTCs with samples taken from 142 patients suffering from breast, stomach, or lung cancer.

Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient.

Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.

A non-invasive genomic diagnostic method for bladder cancer using size-based filtration and microchip electrophoresis.

Bladder cancer (BC) cells spontaneously exfoliated in the urine of patients with BC. Detection of exfoliated tumor cells has clinical significance in cancer therapy because it would enable earlier non-invasive screening, diagnosis, or prognosis of BC. In this research, a method for analyzing genetic abnormalities of BC cells collected from urine samples was developed. Target BC cells were isolated by filtration. To find conditions that achieve high cell recovery, we investigated the effects of filter type, concentration of fixative, and flow rate. Cells captured on the filter membrane were completely retrieved within 15s. Selected genes for genomic analysis, mutated genes (FGFR3, TERT and HRAS) and methylated genes (ALX4, RALL3, MT1A, and RUNX3) were amplified by polymerase chain reaction (PCR), and subsequently, were identified by microchip electrophoresis (MCE). Analysis by MCE reduces the risk of contamination, sample consumption, and analysis time. Our developed approach is economical, effectively isolates cancer cells, and permits flexible molecular characterization, all of which make this approach a promising method for non-invasive BC detection.

Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture.

Red blood cell depletion from bone marrow and peripheral blood buffy coat: a comparison of two new and three established technologies.

BACKGROUND: Red blood cell (RBC) depletion is a standard technique for preparation of ABO-incompatible bone marrow transplants (BMTs). Density centrifugation or apheresis are used successfully at clinical scale. The advent of a bone marrow (BM) processing module for the Spectra Optia (Terumo BCT) provided the initiative to formally compare our standard technology, the COBE2991 (Ficoll, manual, "C") with the Spectra Optia BMP (apheresis, semiautomatic, "O"), the Sepax II NeatCell (Ficoll, automatic, "S"), the Miltenyi CliniMACS Prodigy density gradient separation system (Ficoll, automatic, "P"), and manual Ficoll ("M"). C and O handle larger product volumes than S, P, and M. STUDY DESIGN AND METHODS: Technologies were assessed for RBC depletion, target cell (mononuclear cells [MNCs] for buffy coats [BCs], CD34+ cells for BM) recovery, and cost/labor. BC pools were simultaneously purged with C, O, S, and P; five to 18 BM samples were sequentially processed with C, O, S, and M. RESULTS: Mean RBC removal with C was 97% (BCs) or 92% (BM). From both products, O removed 97%, and P, S, and M removed 99% of RBCs. MNC recovery from BC (98% C, 97% O, 65% P, 74% S) or CD34+ cell recovery from BM (92% C, 90% O, 67% S, 70% M) were best with C and O. Polymorphonuclear cells (PMNs) were depleted from BCs by P, S, and C, while O recovered 50% of PMNs. Time savings compared to C or M for all tested technologies are considerable. CONCLUSION: All methods are in principle suitable and can be selected based on sample volume, available technology, and desired product specifications beyond RBC depletion and MNC and/or CD34+ cell recovery.

An ultra-high-throughput spiral microfluidic biochip for the enrichment of circulating tumor cells.

The detection and characterization of rare circulating tumor cells (CTCs) from the blood of cancer patients can potentially provide critical insights into tumor biology and hold great promise for cancer management. The ability to collect a large number of viable CTCs for various downstream assays such as quantitative measurements of specific biomarkers or targeted somatic mutation analysis is increasingly important in medical oncology. Here, we present a simple yet reliable microfluidic device for the ultra-high-throughput, label-free, size-based isolation of CTCs from clinically relevant blood volumes. The fast processing time of the technique (7.5 mL blood in less than 10 min) and the ability to collect more CTCs from larger blood volumes lends itself to a broad range of potential genomic and transcriptomic applications. A critical advantage of this protocol is the ability to return all fractions of blood (i.e., plasma (centrifugation), CTCs and white blood cells (WBCs) (size-based sorting)) that can be utilized for diverse biomarker studies or time-sensitive molecular assays such as RT-PCR. The clinical use of this biochip was demonstrated by detecting CTCs from 100% (10/10) of blood samples collected from patients with advanced-stage metastatic breast and lung cancers. The CTC recovery rate ranged from 20 to 135 CTCs mL(-1) and obtained under high purity (of 1 CTC out of every 30-100 WBCs which gives ∼4 log depletion of WBCs). They were identified with immunofluorescence assays (pan-cytokeratin+/CD45-) and molecular probes such as HER2/neu.

Comparison of human mesenchymal stem cells isolated by explant culture method from entire umbilical cord and Wharton's jelly matrix.

Adult stem cells are of particular importance for applications in regenerative medicine. Umbilical cord was established recently as an alternative source of mesenchymal stem cell (MSC) instead of bone marrow (BM) and is superior to BM and other adult tissues according to several MSC properties. Additionally, for the purpose of cell therapy in clinical scale, steps of cell isolation, expansion and culture required to be precisely adjusted in order to obtain the most cost-effective, least time-consuming, and least labor-intensive method. Therefore, in this study, we are going to compare two simple and cost-effective explant culture methods for isolation of MSCs from human umbilical cord. One of the methods isolates cells from entire cord and the other from Wharton's jelly matrix. Isolated cells then cultured in simple medium without addition of any growth factor. MSCs obtained via both methods display proper and similar characteristics according to morphology, population doubling time, post-thaw survival, surface antigenicity and differentiation into adipocytes, osteocytes, and chondrocytes. MSCs can easily be obtained from the entire cord and Wharton's jelly, and it seems that both tissues are appropriate sources of stem cells for potential use in regenerative medicine. However, from technical large-scale preview, MSC isolation from entire cord piece is less labor-intensive and time-consuming than from Wharton's jelly part of the cord.

Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate.

Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose explant culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by explant culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As explant culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.

Polymeric microfluidic devices exhibiting sufficient capture of cancer cell line for isolation of circulating tumor cells.

Here, we developed polymeric microfluidic devices for the isolation of circulating tumor cells. The devices, with more than 30,000 microposts in the channel, were produced successfully by a UV light-curing process lasting 3 min. The device surface was coated with anti-epithelial cell adhesion molecule antibody by just contacting the antibody solution, and a flow system including the device was established to send a cell suspension through it. We carried out flow tests for evaluation of the device's ability to capture tumor cells using an esophageal cancer cell line, KYSE220, dispersed in phosphate-buffered saline or mononuclear cell separation from whole blood. After the suspension flowed through the chip, many cells were seen to be captured on the microposts coated with the antibody, whereas there were few cells in the device without the antibody. Owing to the transparency of the device, we could observe the intact and the stained cells captured on the microposts by transmitted light microscopy and phase contrast microscopy, in addition to fluorescent microscopy, which required fluorescence labeling. Cell capture efficiencies (i.e., recovery rates of the flowing cancer cells by capture with the microfluidic device) were measured. The resulting values were 0.88 and 0.95 for cell suspension in phosphate-buffered saline, and 0.85 for the suspension in the mononuclear cell separation, suggesting the sufficiency of this device for the isolation of circulating tumor cells. Therefore, our device may be useful for research and treatments that rely on investigation of circulating tumor cells in the blood of cancer patients.

A much convenient and economical method to harvest a great number of microglia.

Microglia, implicating in such neuro-pathologies as brain inflammation, neurodegeneration, glioma, and neurogenesis, play an important role in central nervous system. Advanced research on microglia is crucial in exploring the neuro-pathology and neuro-physiology of these diseases, so how to culture large numbers of microglia in vitro becomes the base of a research. The wildly used method, at present, obtaining microglia from murine cannot fulfill the requirement of research, costing too much time and needing too many rats. We intend to introduce an optimized method that can harvest large quantities of microglia with high purity. Neonatal 2-3 days old Wistar rats were sacrificed and the cerebral cortices were trypsinized. We primarily cultured mixed cortical cells for 8-10 days. The microglia were harvested from the liquid supernatant; the left cells in the mixed cortical glial culture were passaged at a 1:2 density. After another 8-10 days of culture, microglia were collected again. And then, we passaged the left cells again for acquiring microglia from the third collection. We did not add additional mitogens in the experiment. At last, on average, 7.0 × 10(6) microglia were collected from one neonatal rat. By this modified method, much more microglia can be effectively and easily harvested comparing with the usual protocol before. We compared the characteristics of microglia harvested from these three passages, such as morphology, phenotype, purity, and abilities on proliferation, secretion, and phagocytosis. The cells presented typical microglia morphology, having phenotype markers of CD11b/c and CD45. The microglia from these three passages retained similar phagocytosis and secretion functions. Expanded population of microglia for investigation can be provided by this easy method in a short time with little cost and few rats.

Development of a low-cost magnetic microfluidic chip for circulating tumour cell capture.

The authors have developed a novel fabrication process for a selective micro-magnetic activated cell sorting (MACS) chip based on ferromagnetic material encapsulated micropillars (FMEMs), which is technically simple and low cost. The FMEM produces a high field gradient to magnetically attract, capture and hold cells on its interface. System test simulations were carried out to predict the efficacy of target capture and verify that the actual magnetic particles behaviour agreed well with model predictions. To determine the ability of the novel microMACS chip to capture circulating tumour cells (CTCs), SW620 human colon cancer cells were used in an in vitro flow model system and were able to be captured with the efficiency of 72.8%. The obvious accumulation of CTCs at a certain location on the chip suggested shear stress events at the pillar boundary may influence efficacy, and should be considered in further optimisation efforts.

Novel automated blood separations validate whole cell biomarkers.

BACKGROUND: Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples. METHODS AND FINDINGS: To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes. CONCLUSIONS: Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.