A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes with high efficiency directly from 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R(2) = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings.     

A microfabricated electrical differential counter for the selective enumeration of CD4+ T lymphocytes

We have developed a microfabricated biochip that enumerates CD4+ T lymphocytes from leukocyte populations obtained from human blood samples using electrical impedance sensing and immunoaffinity chromatography. T cell counts are found by obtaining the difference between the number of leukocytes before and after depleting CD4+ T cells with immobilized antibodies in a capture chamber. This differential counting technique has been validated to analyze physiological concentrations of leukocytes with an accuracy of ～9 cells per μL by passivating the capture chamber with bovine serum albumin. In addition, the counter provided T cell counts which correlated closely with an optical control (R(2) = 0.997) for CD4 cell concentrations ranging from approximately 100 to 700 cells per μL. We believe that this approach can be a promising method for bringing quantitative HIV/AIDS diagnostics to resource-poor regions in the world.     

Controlled viable release of selectively captured label-free cells in microchannels

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.     

Magnetic bead based immunoassay for enumeration of CD4 T lymphocytes on a microfluidic device

Human immunodeficiency virus (HIV) diagnostics are urgently needed in resource-scarce settings. Monitoring of HIV-infected patients requires accurate counting of CD4⁺ T lymphocytes. However, the current methods for enumeration of CD4⁺ T lymphocytes are of high cost, technically complex and time-consuming. In this paper, we developed a simple, rapid and inexpensive one-step immunomagnetic method for separating and counting CD4⁺ T lymphocytes on microfluidic devices with enlarged reaction chambers. CD4⁺ T lymphocytes were successfully separated and captured from the cell suspension obtained from mouse thymus. CD4 counts were determined under an optical microscope in a rapid and simple format. In order to acquire the maximum efficiency of cell capture, relative parameters were investigated, including section area of the reaction chamber and injection flow rate of the cell suspension. The enlarged reaction chamber with two symmetrical cone-shaped ends was helpful for cell capture, and the maximum capability of captured CD4⁺ T lymphocytes was about 700 cells μL⁻¹. Our investigations avoided the complex sample pre-treatment, and the entire analysis time was significantly reduced to 15 min. This CD4 counting microdevice had the potential to reduce the cost for HIV diagnosis in resource-limited settings.     

Modification of a commercial automatic alpha counter for 2-π alpha counting

A 2-π proportional counting chamber is substituted for the end-window chamber in an automatic α/β counter, to achieve automated α-counting without sacrificing the efficiency and accuracy offered by manually operated 2-π proportional counters. A 2-π chamber was fitted with a thin metal base plate with a hole in the center. Appropriate sample inserts were made, and a mechanism was fabricated for raising the sample insert flush with the hole. A manifold and solenoids mounted in a double-wide NIM module are used to control counter gas purging of the chamber and normal gas flow during counting. The counter operations are controlled by modified logic circuits, also located in the module. During ca. 8 months of operation, the system has performed well.     

Analysis of peripheral T cells and the CC chemokine receptor (CCR5) delta32 polymorphism in prostate cancer patients treated with carboxymethyl-glucan (CM-G)

β-Glucan, derived from Saccharomyces cerevisiae, is a biological response modifier which affects the innate and adaptive immune responses. The CCR5 chemokine receptor is crucial for immune cell responses. In this study, the effects of the carboxymethylated form of β-glucan (CM-G) on the lymphocyte population of CCR5 genotype patients with prostate cancer (PCa), undergoing androgen deprivation therapy (ADT) was assessed. The CCR5 genotype and lymphocyte population was investigated by cytometry flow in 30 Brazilian patients with advanced PCa who were treated with CM-G for 28 days. The analysis of the CCR5 chemokine receptor revealed that the wild-type genotype Wt/Wt was present in 80% of patients, while the heterozygotic genotype Wt/delta32 was present in 20% of patients. After CM-G administration, a significant increase in CD3(+), CD4(+) and CD8(+) T lymphocytes was observed in patients who displayed the wild-type genotype for the CCR5 chemokine receptor. No association was found between patient's age or length of ADT and increase in T lymphocyte cells. The results demonstrated the ability of CM-G to stimulate CD4(+) and CD8(+) T cells in the peripheral blood of patients carrying a wild-type CCR5 genotype, suggesting an interaction between immunomodulation by CM-G and the CCR5 receptor.     

Antitumor immunity induced by tumor cells engineered to express a membrane-bound form of IL-2

Transduction of cytokine gene into tumor cells is a promising method of tumor therapy, but the value is limited by accompanying side effects. To focus antitumor immune response to tumor antigen-specific CTL, we developed an antitumor vaccine by transfecting modified IL-2 gene in a membrane-bound form (mbIL-2) into B16F10 melanoma cells. The mbIL-2 clone showed reduced tumorigenicity and metastatic ability, and inhibited metastasis and prolonged the survival of mice against B16F10 cells. The inhibition of B16F10 metastasis by mbIL-2 was accompanied by the increment of CD8(+) T cells. The metastasis of mbIL-2 clone was significantly increased in the CD8(+) T cell-depleted mice, but not in CD4(+) T cell depleted mice. Spleen cells immunized with the mbIL-2 clone showed higher CTL activity towards B16F10 cells than those immunized with control cells. The size of CD8(+) T cell population in the lung of mice injected with the mbIL-2 clone was markedly greater than that of mice injected with B16F10 cells, but there was no detectible change in CD4(+) and CD8(+) T cell populations of lymph nodes and spleen. These results suggest that when the mbIL-2 clone is introduced into the blood stream, it migrates mainly to lung and activates CD8(+) T cells in situ, possibly by direct priming. Such a tumor vaccine may ameliorate the toxic side effects encountered with conventional cytokine gene therapy.     

Covalently Bound Antibody on Polystyrene Latex Beads: Formation, Stability, and Use in Analyses of White Blood Cell Populations

CD4 or CD8 antibodies were covalently bound to latex beads by reaction of activated CD4 or CD8 monoclonal antibodies with 2-μm-diameter, 1,3-diaminopropane (DAP) coupled, polystyrene aldehyde/sulfate latex beads. Spectrophotometric analyses of the filtrates of the antibody-bead conjugation mixtures for unreacted antibody allowed construction of binding curves of antibody for the polystyrene bead surface and evaluation of binding constants for association of antibody with bead, ranging from 1.5x10(7) to 1.6x10(7) M(-1) for CD4 and CD8 antibodies. The reaction of the antibody thiol group with the activated maleimide group on the bead at pH 7.2-7.3 was complete within 10-15 min. The kinetics of CD4 or CD8 monoclonal antibody displacement from the surface of covalently conjugated antibody-polystyrene latex beads was followed as a function of temperature (5, 22, and 37 degrees C) and the nature of the final diluent for the antibody-coated beads by measuring the concentration of antibody in the filtrates of conjugated beads by an ELISA (enzyme-linked immunosorbent assay). The displacement reaction showed a pseudo-zero-order dependence of the rate, with constants, k(1), ranging from 0.65x10(-17) to 270x10(-17) M s(-1). The functionality of antibody-coated beads suspended in various media was also monitored in a biological cell assay with whole blood. The cell assay depends on forming a layer of beads around targeted lymphocytes to distinguish them from nontargeted lymphocytes by differences in dc or rf conductivity or median angle light scatter. Covalently bound CD4 and CD8 antibody beads stored in one set of media at 5, 22, and 37 degrees C over a period of 16 weeks showed excellent results in the STKS assay with various blood donors, which correlated well (correlation coefficients of 0.99 for CD4 data and 0.93 for CD8 data) with reference results obtained with fluorescent markers by flow cytometry. Covalently bound CD4/CD8 beads stored for 2 weeks in BSA buffer at 5-37 degrees C performed equally well in providing accurate values of the percentage of CD4- or CD8-positive cells in the total white blood cell population, whereas the same beads stored in the 47-50 degrees C range showed some failures in performance. Comparison with antibody concentrations in filtrates of adsorbed antibody-bead suspensions showed 2- to 10-fold greater amounts of free antibody at comparable elapsed time, media, and temperature conditions. A threshold of 1-2 μg/mL of free antibody was necessary before adverse effects on the biological cell assay were noticeable. Copyright 2001 Academic Press.     

肺心病患者全血锌、铜和外周血T淋巴细胞亚群的关系探讨

对28例住宅的肺心病患者和40例健康人分别进行了全血锌、铜的检测,并对28例肺心病患者全血锌、铜与其T淋巴细胞亚群（CD3、CD4、CD8,CD4／CD8）分别做线性相关分析。结果表明：（1）肺心病组全血锌低于健康人组（P＜0．01）,全血铜高于健康人组（P＜0．05）。（2）全血锌与T淋巴细胞亚群CD3、CD4、CD4／CD8呈明显正相关（P＜0．05）,与CD8呈负相关（P＜0．05）。提示慢性肺心病急性期患者全血锌下降,造成T淋巴细胞免疫功能低下,导致肺心病患者易合并肺部感染,使肺心病病情加重。认为慢性肺心病急性加重期患者在予以积极抗感染、对症的同时适当补充锌剂,能改善外周血T淋巴细胞亚群功能,提高机体免疫力,有助于慢性肺心病急性期患者的康复。     

Detecting interferon-gamma release from human CD4 T-cells using surface plasmon resonance

Cytokine secretion by leukocytes is an important indicator of immune response to pathogens and therefore has significant implications in disease diagnostics. Given heterogeneity of leukocyte subsets and the ability of multiple cell subsets to secrete the same cytokines, connecting cytokine production to a specific leukocyte subset is a distinct challenge. In the present paper we describe a strategy combining antibody (Ab)-based affinity cell separation and surface plasmon resonance (SPR) for capturing human CD4 T-cells and for label-free detection of cell-secreted interferon (IFN)-γ – an important inflammatory cytokine. Human blood was introduced into a flow chamber modified with anti-CD4 Abs resulting in capture of CD4⁺ T-cells. After mitogenic activation of cells inside the flow chamber, culture medium was routed onto an SPR chip modified with monoclonal IFN-γ Abs. SPR signal observed in this experiment correlated with cytokine production by T-cells. The strategy of combining SPR detection with cell purification may be used in the future for label-free, sensitive detection of multiple cytokines or proteins secreted by the desired cell subset.     

Constitutive expression of 4-1BB on T cells enhances CD4+ T cell responses

4-1BB, a transmembrane molecule, member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule in the immune response, plays a key role in the clonal expansion and survival of CD8(+) T cells. In this study, we investigated 4-1BB regulation of CD4(+) T cell responses using 4-1BB transgenic (TG) mice that constitutively expressed 4-1BB on mature T cells. We first showed that CD4(+) T cells of 4-1BB TG mice had more sustained proliferative capacity in response to TCR/4-1BB stimulation in vitro compared to WT mice. Secondly, 4-1BB TG mice exhibited a more elevated contact hypersensitivity (CHS) response mediated by CD4+ Th1 cells due to more vigorous expansion of and apoptotic inhibition of CD4(+) T cells. Finally, CD4(+) T cells of 4-1BB TG mice had a heightened capacity for T cell priming. Overall, our results demonstrate the involvement of 4-1BB in CD4(+) Th1 cell responses by regulating the clonal expansion and survival of CD4(+) T cells as seen in CD8(+) T cells.     

In vivo ligation of glucocorticoid-induced TNF receptor enhances the T-cell immunity to herpes simplex virus type 1

GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4(+)CD25(+) regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4(+) and CD8(+) T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4(+) or CD8(+) T cells with a CD4(+) or CD8(+) T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.     

Differential roles of T-cell subsets in regulation of ultraviolet radiation induced cutaneous photocarcinogenesis

Ultraviolet (UV) radiation, in particular the midwavelength range (UVB; 290-320 nm), is one of the most significant risk factors for the development of nonmelanoma skin cancer. UVB radiation-induced immunosuppression, which occurs in both humans and laboratory animals, contributes to their pathogenesis. However, there are conflicting reports on the relative role of CD4(+) and CD8(+) T cells in UVB induced skin cancer. The purpose of this study was to delineate the contribution of these two cell subpopulations to UVB induced immunosuppression and tumor development using C3H/HeN (WT), CD4 knockout (CD4(-/-) ) and CD8 knockout (CD8(-/-) ) mice. We observed that UVB induced skin carcinogenesis was retarded in terms of number of tumors per group, tumor volume and percentage of mice with tumors, in mice deficient in CD4(+) T cells compared with wild-type mice, whereas significantly greater (P < 0.05) numbers of tumors occurred in CD8(-/-) mice. These results indicate that, CD4(+) T cells promote tumor development while CD8(+) T cells have the opposite effect. Further, we found that CD4(+) T cells from tumor-bearing mice produced interleukin (IL)-4, IL-10, and IL-17 whereas CD8(+) T cells produced interferon-γ. Manipulation of T-cell subpopulations that are induced by UVB radiation could be a means of preventing skin cancers caused by this agent.     

Low-CD4+ T-lymphocyte blood samples for external quality assessment of CD4 testing in resource-poor settings

External quality assessment (EQA) of CD4 testing is important for clinically monitoring of patients with HIV/AIDS. EQA materials are limited to blood samples from normal blood donors with normal CD4+ T-lymphocyte levels. This study aimed to develop low-CD4+ T-lymphocyte blood samples for CD4 EQA. CD4+ T-lymphocyte-depleted blood samples were prepared using a magnetic bead separation technique. These CD4+ T-lymphocyte-depleted blood samples were mixed with undepleted whole blood samples to obtain low-CD4+ T-lymphocyte blood samples. The percentage and the number of CD4+ T-lymphocytes were determined using a flow cytometer. An evaluation study of this low-CD4+ T-lymphocyte blood sample was performed by sending to participating laboratories to investigate the potential use as CD4 EQA material. Our results showed that a magnetic separation technique could be used to prepare low-CD4+ T-lymphocyte blood samples. CD4+ T-lymphocytes in the low-CD4+ T-lymphocyte blood samples ranged from 15 % to 18 % and 160–300 cell/μl, respectively. In addition, CD4 testing of our low-CD4+ T-lymphocyte blood samples could be achieved following both the single- and the dual-platform flow cytometric approaches. A pilot EQA investigation revealed that the CV values of the number and percentage of CD4+ T-lymphocytes were 14 % and 10 %, respectively. Having the low-CD4+ T-lymphocyte blood samples in our CD4 EQA program should ensure reliability and credibility of CD4 testing in Thailand and other resource-poor countries.     

A new device to study ex-vivo the effects of extracorporeal photochemotherapy on the immune system

Extracorporeal photochemotherapy (ECP) is a medical procedure effective in the treatment of several different T-cell mediated diseases such as cutaneous T-cell lymphoma and Graft-versus-Host Disease. During ECP treatment the patient's blood is processed by means of a cell separator to collect leukocytes (leukapheresis), mostly lymphocytes and monocytes, which are then incubated with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused to the patient. It has been suggested that during ECP not only UV-A irradiation but also changes in the environmental condition may be relevant. Although ECP has been shown to have an in-vivo immunomodulatory effect, the mechanisms through which ECP exerts its effect remain elusive. One of the reasons for this incomplete knowledge is the absence of a reliable model for ECP. In order to investigate the effect of ECP on the peripheral immune system, we developed a new device which mimics the complete ECP cycle including blood transit through the cell separator. Peripheral blood samples (50ml) were obtained from volunteers and processed using a peristaltic pump. Peripheral blood mononuclear cells (PBMC) were then collected and treated with 8-MOP and UV-A under the same conditions used for the patients' therapy. Using this strategy we investigated 8-MOP, UV-A and their combined effect on the production of the pro-inflammatory cytokines interferon-gamma (IFN-gamma), interleukine-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) in PBMC with and without polyclonal stimulation. We firstly demonstrated that our device does not affect total red and white blood cell counts. After 8-MOP and UV-A irradiation a significant decrease was observed in both activated CD4(+) and CD8(+) T lymphocytes producing IFN-gamma, IL-2 and TNF-alpha. Our findings are in line with those previously obtained in humans after complete ECP treatment, thus suggesting that our newly developed device is suitable for investigating the mechanism of action of ECP ex-vivo.     

Concurrent hypermulticolor monitoring of CD31, CD34, CD45 and CD146 endothelial progenitor cell markers for acute myocardial infarction

The circulating endothelial progenitor cells (EPCs) in blood of acute myocardial infarction (AMI) patient have been monitored in many previous studies. The number of circulating EPC increases in the blood of patients at onset of the AMI. EPC is originated from bone marrow. It performs vessel regeneration. There are many markers used for detecting EPC. Four of these markers, CD31, CD34, CD45, and CD146, were concurrently detected at the single cell level for the identification of EPC in the present preliminary study. The CD45 negative cell sorting was performed to peripheral blood mononuclear cells (PBMCs) acquired from four AMI patients with a magnetic bead sorter, since, EPCs expressed CD45 negative or dim. The resultant PBMC eluents were treated with quantum-antibody conjugates for the probing four different markers of EPCs and then applied to a high-content single cell imaging cytometer using acousto-optical tunable filter (AOTF). The use of quantum dot, with narrow emission wavelength range and AOTF enabling cellular image at a particular single wavelength, is very advantageous for accurate high-content AMI diagnosis based on simultaneous monitoring of many markers. The number of EPC increased as compared with control in three of four AMI patients. In this approach, two EPC subtypes were found, CD31(+), CD34(+), CD45(−/dim), CD146(−) as early outgrowth EPCs and CD31(+), CD34(+), CD45(−/dim), CD146(+) as late outgrowth EPCs. Patient 1 had CD31(+), CD34(+), CD45(−/dim), CD146(+) cells whose percentage was 4.21% of cells. Patient 2 had 2.38% of CD31(+), CD34(+), CD45(−/dim), CD146(−) cells and patient 3 had 4.28% of CD31(+), CD34(+), CD45(−/dim), CD146(+) cells.     

Hematocrit analysis through the use of an inexpensive centrifugal polyester-toner device with finger-to-chip blood loading capability

Hematocrit (HCT) measurements are important clinical diagnostic variables that help physicians diagnose and treat various medical conditions, ailments, and diseases. In this work, we present the HCT Disc, a centrifugal microdevice fabricated by a Print, Cut and Laminate (PCL) method to generate a 12-sample HCT device from materials costing <0.5 USD (polyester and toner or PeT). Following introduction from a drop of blood (finger stick), whole blood metering and cell sedimentation are controlled by centrifugal force, only requiring a CD player motor as external hardware and, ultimately, a cell phone for detection. The sedimented volume from patient blood in the HCT Disc was analyzed using a conventional scanner/custom algorithm for analysis of the image to determine a hematocrit value, and these were compared to values generated in a clinical laboratory, which correlated well. To enhance portability and assure simplicity of the HCT measurement, values from image analysis by a cell phone using a custom application was compared to the scanner. Fifteen samples were analyzed with cell phone image analysis system and were found to be within 4% of the HCT values determined in the clinical lab. We demonstrate the feasibility of the PeT device for HCT measurement, and highlight its uniquely low cost (<0.5 USD), speed (sample-to-answer <8 min), multiplexability (12 samples), low volume whole blood requirement (<3 μL), rotation speeds (<4000 rpm) needed for effective measurement as well as the direct finger-to-chip sample loading capability.     

An Acoustically Driven Microliter Flow Chamber on a Chip (μFCC) for Cell–Cell and Cell–Surface Interaction Studies

A novel method for pumping very small volumes of liquid by using surface acoustic waves is employed to create a microfluidic flow chamber on a chip. It holds a volume of only a few μl and its planar design provides complete architectural freedom. This allows for the reconstruction of even complex flow scenarios (e.g. curvatures, bifurcations and stenosis). Addition of polymer walls to the planar fluidic track enables cell culturing on the chip surface and the investigation of cell–cell adhesion dynamics under flow. We demonstrate the flexibility of the system for application in many areas of microfluidic investigations including blood clotting phenomena under various flow conditions and the investigation of different stages of cell adhesion.     

Simultaneous counting of two subsets of leukocytes using fluorescent silica nanoparticles in a sheathless microchip flow cytometer

A portable flow cytometer has been recognized as an important tool for many clinical applications such as HIV/AIDS screening in developing countries and regions with limited medical facilities and resources. Conventional flow cytometers typically require multiple detectors for simultaneous identification of multiple subsets of immune cell. To minimize the number of detectors toward portable flow cytometry or to analyze multi-parametric cellular information with minimum number of detectors in conventional flow cytometers, we propose a versatile multiplexed cell-counting method using functional silica nanoparticles (SiNPs). FITC-doped SiNPs, which are 100 times brighter than the FITC molecules itself, were used as new intensity-based fluorescent dye complexes to simultaneously measure two subsets of leukocytes using a single detector. CD45(+)CD4(+) cells tagged with these FITC-doped SiNPs were 50 times brighter than CD45(+)CD4(-) cells tagged only with FITC. To make the overall system compact, a disposable microchip flow cytometer that does not require sheath flow was developed. Combining these dye-doped SiNPs based detection schemes and the sheathless microchip flow cytometer scheme, we successfully identified and counted two subsets of leukocytes simultaneously (R(2) = 0.876). These approaches can be the building blocks for a truly portable and disposable flow cytometer for various clinical cytometry applications.     

Liquid‐phase microextraction combined with liquid chromatography‐mass spectrometry. Extraction from small volumes of biological samples

Liquid‐phase microextraction (LPME) is a sample preparation technique based on disposable polypropylene hollow fibres, which results in efficient sample clean‐up and high preconcentration. The present paper describes the combination of LPME with LC‐MS utilising electrospray ionisation for high sensitivity. Nine antidepressant drugs were extracted from 50 or 500 μL of plasma or whole blood samples, through a thin layer of dodecyl acetate immobilised in the pores of the hollow fibre, and into 15 μL of 200 mM formic acid as acceptor solution inside the hollow fibre. Analyte recoveries in the range 12–68% and 9–52% were obtained from 50 μL of plasma and whole blood respectively. The acceptor solution (15 μL) was diluted with 60 μL of 5 mM ammonium formate pH = 2.7 prior to injection into the LC‐MS system. The system was qualitatively investigated for matrix effects utilising a post‐column infusion system. Whole blood from 5 different persons was cleaned‐up by LPME and injected onto the analytical column while a solution of the 9 model compounds was continuously infused post‐column. No signs of ion suppression were seen for any of the model compounds. Limits of quantification (S/N = 10) were in the low ng/mL range for 6 of the 9 model compounds utilising a whole blood sample volume of only 50 μL. The repeatability of the extractions was investigated utilising paroxetine as internal standard. Acceptable RSDs (%) were obtained (< 20%) for 5 of the antidepressants. By increasing the sample volume from 50 to 500 μL of whole blood RSDs below 20% (3–16%) were observed for all 8 antidepressants.