High-Throughput Cell Concentration Using A Piezoelectric Pump in Closed-Loop Viscoelastic Microfluidics.

Cell concentration is a critical process in biological assays and clinical diagnostics for the pre-treatment of extremely rare disease-related cells. The conventional technique for sample preconcentration and centrifugation has the limitations of a batch process requiring expensive and large equipment. Therefore, a high-throughput continuous cell concentration technique needs to be developed. However, in single-pass operation, the required concentration ratio is hard to achieve. In this study, we propose a closed-loop continuous cell concentration system using a viscoelastic non-Newtonian fluid. For miniaturized and integrated systems, two piezoelectric pumps were adopted. The pumping capability generated by a piezoelectric pump in a microfluidic channel was evaluated depending on the applied voltage, frequency, sample viscosity, and channel length. The concentration performance of the device was evaluated using 13 µm particles and white blood cells (WBCs) with different channel lengths and voltages. In the closed-loop system, the focused cells collected at the center outlet were sent back to the inlet, while the buffer solution was removed to the side outlets. Finally, to expand the clinical applicability of our closed-loop system, WBCs in lysed blood samples with 70% hematocrit and prostate cancer cells in urine samples were used. Using the closed-loop system, WBCs were concentrated by ~63.4 ± 0.8-fold within 20 min to a final volume of 160 µL using 10 mL of lysed blood sample with 70% hematocrit (~3 cP). In addition, prostate cancer cells in 10 mL urine samples were concentrated by ~64.1-fold within ~11 min due to low viscosity (~1 cP).

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool.

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2-6.7% for Plasmodium knowlesi and 0.3-4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson's correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r2=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.

Platelet thrombus formation by upstream activation and downstream adhesion of platelets in a microfluidic system.

Platelet activation causes platelet aggregation and their adhesion to the vascular wall. In the circulatory environment, platelet activation and adhesion might not occur at the same site. In this study, we developed a microfluidic platform to examine platelet adhesion and aggregation under pathophysiological shear flow. Upstream platelet activation was conducted either using agonists or by shear flow, whereas downstream platelet adhesion was induced using collagen-coated microbeads packed in a tube. Adopting microbeads, activated platelets led to rapid occlusion and blood flow arrest. The degree of platelet adhesion and aggregation was monitored by measuring the blood migration distance, allowing a flow-through in the microchannel until it was blocked. Downstream platelet adhesion was strongly dependent on the upstream activation parameters including shear rate ranges between 754 and 2400 s-1, shearing time greater than 10 s, and incubation time greater than 20 s. Furthermore, through the integration of various leading-edge technical elements, the present system produced comprehensive real-time results of platelet-associated thrombus formation. Thus, this disposable device might help examine platelet dysfunction for preoperative patients and antiplatelet therapy in the clinic.

Recent advances in microfluidic platelet function assays: Moving microfluidics into clinical applications.

The analysis of platelet aggregation and thrombosis kinetics has significantly advanced with progress in microfluidic technology. However, the results of platelet aggregation tests do not fully reflect the observed clinical outcomes. To address the present unmet clinical needs, the basic but essential biology of platelets should be reconsidered in relation to the characteristics of microfluidic systems employed for platelet tests. To this end, the present article provides an overview of commercially available point of care devices and focuses on recent microfluidic studies, describing their measurement principles. We critically discuss the characteristics of the microfluidics systems used to evaluate the complex processes underlying platelet aggregation, and that are specifically designed to mimic the pathophysiological environment of blood vessels, including hemodynamic factors as well as blood vessel injury. To this end, we summarize unsolved issues related to the application of platelet function tests based on microfluidics. Overall, we confirm that platelet function tests based on microfluidics provide a versatile platform that encompasses a variety of basic research, as well as clinical diagnostic applications.

The Effects of Oxygen and Treatments in Hypoxic Conditions in SH-SY5Y Cells.

Many patients are admitted to the emergency department due to trauma. Patients with massive hemorrhage and respiratory failure can fall into hypovolemic shock. Thereafter, oxygen is an essential part of the treatment of trauma patients, but the mechanisms of its effects in the management of trauma patients remain unknown. Therefore, we conducted an experiment to apply hypoxia, hyperoxia, and other treatment with the goal of decreasing hypoxic neuronal cells damage, as reflected by cell survival, apoptosis, hydrogen peroxide (H2O2) production, and hypoxia-inducible factor 1α (HIF) expression in SH-SY5Y cells. Under hypoxic insults, cell survival percentages decreased and apoptosis was seen with increased necrotic cell death. High-pressure oxygen (80% O2) had no effect compared with normal-pressure oxygen (20% O2). After exposure to hypoxia, H2O2 production and levels of HIF significantly increased compared with normoxia. However, when pentoxifylline (PTX), steroid, and hypertonic saline (HTS) were added after exposure to hypoxic conditions, the production of H2O2 and HIF levels significantly decreased in the groups treated with PTX and HTS. That is, the neuroprotective effect of PTX and HTS alleviated the impacts of hypoxic insulted on neuronal cells.

The inflammatory response of neutrophils in an in vitro model that approximates the postcardiac arrest state.

PURPOSE: Postcardiac arrest syndrome (PCAS) shares many features with sepsis including plasma cytokine elevation with dysregulation of cytokine production, and the presence of endotoxin in plasma. PCAS is closely related to ischemia-reperfusion injury. During ischemia-reperfusion injury, neutrophil, which is the first line of innate immunity, plays a major role. In this study, we investigated the inflammatory response of human neutrophils in an in vitro model which we simulated with hypoxia-normoxia and hypoxia-hyperoxia environments. METHODS: After separation of neutrophils from the whole blood, they were divided into 3 experimental groups: normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia groups. The production of H2O2, the expression of Toll-like receptor 4 (TLR4) receptor, and the extent of apoptosis of the neutrophils were checked. RESULTS: The in vitro hypoxia-normoxia and -hyperoxia models, which simulated the PCAS, showed initiation of the neutrophils' inflammatory reaction by hypoxia insult. Lipopolysaccharide amplifies such inflammation; therefore, prevention of secondary infection may be critical in postresuscitation patients. Temporary hyperoxia following hypoxic insult showed no difference in inflammatory reaction compared with hypoxia-normoxia. Rather, temporary hyperoxia may suppress or minimize inflammation by attenuation of TLR4 receptor. CONCLUSION: It is well known that continuous hyperoxygenation after successful cardiac arrest harms patients, but temporary hyperoxygenation with 100% O2 in a clinical situation may be helpful.

Variable number of tandem repeats of 9 Plasmodium vivax genes among Southeast Asian isolates.

The variable number of tandem repeats (VNTRs) provides valuable information about both the functional and evolutionary aspects of genetic diversity. Comparative analysis of 3 Plasmodium falciparum genomes has shown that more than 9% of its open reading frames (ORFs) harbor VNTRs. Although microsatellites and VNTR genes of P. vivax were reported, the VNTR polymorphism of genes has not been examined widely. In this study, 230 P. vivax genes were analyzed for VNTRs by SERV, and 33 kinds of TR deletions or insertions from 29 P. vivax genes (12.6%) were found. Of these, 9 VNTR fragments from 8 P. vivax genes were used for PCR amplification and sequence analysis to examine the genetic diversity among 134 isolates from four Southeast Asian countries (China, Republic of Korea, Thailand, and Myanmar) with different malaria endemicity. We confirmed the existence of extensive polymorphism of VNTR fragments in field isolates. This detection provides several suitable markers for analysis of the molecular epidemiology of P. vivax field isolates.

A simple method for activating the platelets used in microfluidic platelet aggregation tests: Stirring-induced platelet activation.

High-shear stimulation is well known as one of the key factors affecting platelet activation and aggregation, which can lead to the formation of a thrombus. In one of our previous studies, we introduced migration distance-based platelet function analysis in a microfluidic system. In this study, we set out to examine the effects of stirring on shear-induced platelet activation and aggregation in a chamber system by using a rotating stirrer. We found that the rotating stirrer caused not only rotational shear flow but also a strong radial secondary flow. The latter flow led to efficient mixing in the chamber. Moreover, the rotational flow led to the generation of shear stress, the magnitude of which can be controlled to activate the platelets. Activated platelets tend to aggregate themselves. The maximum platelet aggregation was observed at a critical shear rate of 3100 s-1, regardless of the stirrer shape. Furthermore, the time taken to attain maximum aggregation was significantly shortened when using a wide stirrer (30 s) instead of a narrow one (180 s). When using a flat stirrer, the non-uniform shear field in the chamber system was resolved with the radial secondary flow-induced mixing; thus, most of the platelets were homogenously activated. The stirring-induced platelet activation mechanism was experimentally confirmed in a microfluidic system for a platelet aggregation test while monitoring the migration distance until the microfluidic channel is occluded. Our findings indicate that the present system, consisting of a rotating stirrer and a confined chamber, provides effective shear stimulation for activating platelets and inducing platelet aggregates.

ABO*Ael03/O genotype with ABO discrepancy: the first case in Korea.

The Ael subgroup expresses the least amount of A antigens and could only be detected by performing the adsorption-elution test. The frequency of the Ael subgroup is about 0.001% in Koreans, and the Ael02 allele, which originates from A102, is the most frequently identified allele in the Korean population. We report a Korean family with the Ael03 allele identified by molecular genetic analysis. To the best of our knowledge, this is the first such report in Korea to date.

Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid.

Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their facile manipulation and low costs. Although methods involving their magnetic separation have been extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired monodispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial microbeads (NucliSENS-easyMAG, BioMérieux). Moreover, the DMSA-MNPs were successfully employed in the isolation of genomic DNA from human blood. In addition, the resulting DNA-nanoparticle complex was directly subjected to PCR amplification without prior elution, which could eventually lead to simple, rapid, sensitive and integrated diagnostic systems.

Magnetic separation of malaria-infected red blood cells in various developmental stages.

Malaria is a serious disease that threatens the public health, especially in developing countries. Various methods have been developed to separate malaria-infected red blood cells (i-RBCs) from blood samples for clinical diagnosis and biological and epidemiological research. In this study, we propose a simple and label-free method for separating not only late-stage but also early-stage i-RBCs on the basis of their paramagnetic characteristics due to the malaria byproduct, hemozoin, by using a magnetic field gradient. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with a ferromagnetic wire fixed on a glass slide. To evaluate the performance of the microfluidic device containing the ferromagnetic wire, lateral displacement of NaNO2-treated RBCs, which also have paramagnetic characteristics, was observed at various flow rates. The results showed excellent agreement with theoretically predicted values. The same device was applied to separate i-RBCs. Late-stage i-RBCs (trophozoites and schizonts), which contain optically visible black dots, were separated with a recovery rate of approximately 98.3%. In addition, using an optimal flow rate, early-stage (ring-stage) i-RBCs, which had been difficult to separate because of their low paramagnetic characteristics, were successfully separated with a recovery rate of 73%. The present technique, using permanent magnets and ferromagnetic wire in a microchannel, can effectively separate i-RBCs in various developmental stages so that it could provide a potential tool for studying the invasion mechanism of the malarial parasite, as well as performing antimalarial drug assays.

Migration distance-based platelet function analysis in a microfluidic system.

Aggregation and adhesion of platelets to the vascular wall are shear-dependent processes that play critical roles in hemostasis and thrombosis at vascular injury sites. In this study, we designed a simple and rapid assay of platelet aggregation and adhesion in a microfluidic system. A shearing mechanism using a rotating stirrer provided adjustable shear rate and shearing time and induced platelet activation. When sheared blood was driven through the microchannel under vacuum pressure, shear-activated platelets adhered to a collagen-coated surface, causing blood flow to significantly slow and eventually stop. To measure platelet adhesion and aggregation, the migration distance (MD) of blood through the microchannel was monitored. As the microstirrer speed increased, MD initially decreased exponentially but then increased beyond a critical rpm. For platelet-excluded blood samples, there were no changes in MD with increasing stirrer speed. These findings imply that the stirrer provided sufficiently high shear to activate platelets and that blood MD is a potentially valuable index for measuring the shear-dependence of platelet activation. Our microfluidic system is quick and simple, while providing a precise assay to measure the effects of shear on platelet aggregation and adhesion.

Retinol binding protein-albumin domain III fusion protein deactivates hepatic stellate cells.

Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis.

Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells.

Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin(domain III) (R-III) and albumin(domain I)-RBP-albumin(III) (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti-fibrotic drug.

A case of therapy-related acute lymphoblastic leukemia with t(11;19)(q23;p13.3) and MLL/MLLT1 gene rearrangement.

Therapy-related ALL (t-ALL) is a rare secondary leukemia that develops after chemotherapy and/or radiotherapy for primary malignancies. Chromosomal 11q23 abnormalities are the most common karyotypic alterations in t-ALL. The t(11;19)(q23;p13) aberration is extremely rare and has not been confirmed at the molecular genetic level. Here, we report a case of t-ALL with t(11;19)(q23;p13.3) and MLL-MLLT1 (alias ENL) gene rearrangement confirmed by cytogenetic analysis, multiplex reverse transcription-PCR (multiplex RT-PCR), and DNA sequencing in a patient who had undergone treatment for breast cancer. A 40-yr-old woman developed acute leukemia 15 months after undergoing 6 cycles of adjuvant chemotherapy (doxorubicin 60 mg/m² and cyclophosphamide 600 mg/m²), radiation therapy (dose, 5,900 cGy), and anticancer endocrine therapy with tamoxifen. The complete blood cell counts and bone marrow examination showed increased blasts and the blasts showed B lineage immunophenotype (positive for CD19, CD34, and cytoplasmic CD79a). Cytogenetic analysis revealed the karyotype 47,XX,+X,t(11;19)(q23;p13.3)[4]/46,XX[16]. FISH analyses, multiplex RT-PCR, and DNA sequencing confirmed the MLL-MLLT1 gene rearrangement. The patient underwent induction chemotherapy with fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (Hyper-CVAD) and achieved complete remission. Subsequently, she underwent consolidation chemotherapy, but died of brain ischemia in the pons and the region of the middle cerebral artery. To our knowledge, this is the first case report of t-ALL with t(11;19)(q23;p13.3) and the MLL-MLLT1 gene rearrangement.

[Evaluation of various formulae for glomerular filtration rate estimation and proposal of new formulae for the Korean population].

BACKGROUND: Some researchers have questioned the necessity of adjusting glomerular filtration rate (GFR) by body surface area (BSA). We compared the relationship between estimated GFR (eGFR) and radionuclide GFR (rGFR) with or without BSA adjustment by comparing the results obtained using various formulae with those obtained using 2 new proposed formulae. METHODS: A retrospective study was performed using 204 Korean individuals whose GFR had been estimated by the (99m)Tc-diethylenetriaminepentaacetic acid method between March 2004 and July 2008. We used the modification of diet in renal disease (MDRD) II formula, Mayo clinic quadratic (MCQ) formula, Cockcroft-Gault (CG) formula, and lean body mass-adjusted CG formula. Two new formulae, skeletal muscle mass index (SMI)-adjusted CG formula and SMI × 3.4/SCr, were proposed by us. We analyzed each parameter with Pearson's correlation coefficient and also obtained the bias values. RESULTS: BSA did not satisfy the fundamental prerequisites of an adjustment factor for rGFR. MDRD II and MCQ GFR estimates demonstrated higher Pearson's correlation coefficient with BSA-unadjusted rGFR than they did with BSA-adjusted rGFR. The other GFR formulae estimates showed better correlation with rGFR and more favorable bias (P<0.001) when both GFR estimates and rGFR values were BSA-unadjusted. SMI-adjusted CG and SMI × 3.4/SCr GFR estimates demonstrated correlation with rGFR and bias values similar to those of the MDRD II and CG GFR estimates. CONCLUSIONS: We suggest that absolute, non-corrected GFR and GFR estimate be preferred in daily practice. The absolute, non-corrected GFR and GFR estimate are considered helpful for patients with eGFR ≤ 60 mL/min/1.73 m(2). We also recommend the clinical use of the new formulae, SMI-adjusted CG and SMI × 3.4/SCr (BSA-unadjusted).

Albumin expression is required for adipocyte differentiation of 3T3-L1 cells.

We have previously demonstrated that albumin is directly involved in the formation of cytoplasmic lipid droplets in pancreatic stellate cells and may act as a downstream effector of adipogenic transcription factors, PPAR-gamma and C/EBP-alpha. Here, we investigated the role of albumin in adipocyte differentiation using 3T3-L1 cells. Albumin expression was significantly increased at later stages of adipocyte differentiation, which was accompanied with increased C/EBP-beta binding to albumin promoter. Suppression of albumin expression using short-hairpin RNA (shRNA) during differentiation led to a considerable reduction in lipid droplet formation, whereas albumin overexpression was stimulatory. Furthermore, point mutation in its fatty acid-binding sites inhibited lipid droplet formation. Consistent with these in vitro finding, Nagase analbuminemic rats displayed reduced fat accumulation. Therefore, our findings suggest that albumin may play a distinct role in adipocyte differentiation by promoting lipid accumulation.

Albumin mediates PPAR-gamma or C/EBP-alpha-induced phenotypic changes in pancreatic stellate cells.

Activation of quiescent hepatic stellate cells (HSCs) into myofibroblast-like cells is a key event of liver fibrosis, and adipogenic transcription factors, PPAR-gamma and C/EBP-alpha, reverse HSC activation. As albumin was reported to maintain the quiescent phenotype of stellate cells, we examined whether it plays a role in PPAR-gamma and C/EBP-alpha-mediated effects. Pancreatic stellate cells (PSCs) were isolated from rat pancreas and used in their culture-activated phenotype. Forced expression of PPAR-gamma or C/EBP-alpha in PSCs increased albumin mRNA and protein levels by >2.5-fold, which is accompanied with increased C/EBP-beta binding to albumin promoter. PPAR-gamma and C/EBP-alpha also induced a phenotypic switch from activated to quiescent cells and, interestingly, suppression of albumin using short-hairpin RNA (shRNA) blocked their effects. Therefore, our findings suggest that albumin may be a downstream effector of PPAR-gamma and C/EBP-alpha in PSCs and that it can be an attractive molecular target for anti-fibrotic therapies.

TGF-beta signaling preserves RECK expression in activated pancreatic stellate cells.

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis, but the detailed mechanism for dysregulated accumulation of extracellular matrix (ECM) remains unclear. Cultured rat PSCs become activated by profibrogenic mediators, but these mediators failed to alter the expression levels of matrix metalloproteinases (MMPs) to the endogenous tissue inhibitors of metalloproteinases (TIMPs). Here, we examined the expression of RECK, a novel membrane-anchored MMP inhibitor, in PSCs. Although RECK mRNA levels were largely unchanged, RECK protein expression was barely detected at 2, 5 days after plating PSCs, but appeared following continued in vitro culture and cell passage which result in PSC activation. When PSCs at 5 days after plating (PSCs-5d) were treated with pepstatin A, an aspartic protease inhibitor, or TGF-beta1, a profibrogenic mediator, RECK protein was detected in whole cell lysates. Conversely, Smad7 overexpression or suppression of Smad3 expression in PSCs after passage 2 (PSCs-P2) led to the loss of RECK protein expression. These findings suggest that RECK is post-translationally processed in pre-activated PSCs but protected from proteolytic degradation by TGF-beta signaling. Furthermore, collagenolytic activity of PSCs-5d was greatly reduced by TGF-beta1, whereas that of PSCs-P2 was increased by anti-RECK antibody. Increased RECK levels were also observed in cerulein-induced acute pancreatitis. Therefore, our results suggest for the first time proteolytic processing of RECK as a mechanism regulating RECK activity, and demonstrate that TGF-beta signaling in activated PSCs may promote ECM accumulation via a mechanism that preserves the protease inhibitory activity of RECK.

Prevalence of antibodies to the circumsporozite protein of Plasmodium vivax in five different regions of Korea.

Malaria has recently re-emerged in the Republic of Korea (ROK), but only few malaria seroprevalences were reported. We obtained 1014 serum samples from inhabitants of five regions of ROK during the high transmission season between June and August in 2001. The levels of anti-circumsporozoite protein (CSP) antibody were assessed in samples using an indirect enzyme-linked immunosorbent assay (ELISA). The highest IgG seroreactivity against Plasmodium vivax recombinant CSP antigen was found among male residents of Cheolwon gun (13.5%), then Incheon (4.7%). The IgG seroreactivity from other regions ranged from 0.0% to 2.0%. These epidemiological data of seroprevalence in five regions of Korea showed a similar pattern to the annual incidence of malaria in these respective regions. The prevalence of antibodies increased with age, suggesting that the age and area-related prevalence patterns reflected differences in the inoculation rates between age groups and geographic regions. Seroprevalence and annual incidence were positively correlated in some areas of Korea.