Cytokine Balance in Human Malaria: Does Plasmodium vivax Elicit More Inflammatory Responses than Plasmodium falciparum?

Background: The mechanisms by which humans regulate pro-and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum. Methodology/Principal Findings: We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-alpha receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85), P. falciparum (n = 30), or both species (n = 12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-alpha, IL-10/interferon (IFN)-gamma, IL-10/IL-6 and sTNFRII/TNF-alpha ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-alpha receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species. Conclusions: Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.

Drug-targeting in combined cancer chemotherapy: tumor growth inhibition in mice by association of paclitaxel and etoposide with a cholesterol-rich nanoemulsion

Lipid nanoemulsions (LDE) may be used as carriers of paclitaxel (PTX) and etoposide (ETP) to decrease toxicity and increase the therapeutic action of those drugs. The current study investigates the combined chemotherapy with PTX and ETP associated with LDE. Four groups of 10-20 B16F10 melanoma-bearing mice were treated with LDE-PTX and LDE-ETP in combination (LDE-PTX + ETP), commercial PTX and ETP in combination (PTX + ETP), single LDE-PTX, and single LDE-ETP. PTX and ETX doses were 9 mu mol/kg administered in three intraperitoneal injections on three alternate days. In two control groups mice were treated with saline solution or LDE alone. Tumor growth, metastasis presence, cell-cycle distribution, blood cell counts and histological data were analyzed. Toxicity of all treatments was evaluated in mice without tumors. Tumor growth inhibition was similarly strong in all treatment groups. However, there was a greater reduction in the number of animals bearing metastases in the LDE-PTX + ETP group (30 %) in comparison to the PTX + ETP group (82 %, p < 0.05). Reduction of cellular density, blood vessels and increase of collagen fibers in tumor tissues were observed in the LDE-PTX + ETP group but not in the PTX + ETP group, and in both groups reduced melanoma-related anemia and thrombocytosis were observed. Flow cytometric analysis suggested that LDE-PTX + ETP exhibited greater selectivity to neoplastic cells than PTX-ETP, showing arrest (65 %) in the G(2)/M phase of the cell cycle (p < 0.001). Toxicity manifested by weight loss and myelosuppression was markedly milder in the LDE-PTX + ETP than in the PTX + ETP group. LDE-PTX + ETP combined drug-targeting therapy showed markedly superior anti-cancer properties and reduced toxicity compared to PTX + ETP.

Synovial fluid chondroitin sulphate indicates abnormal joint metabolism in asymptomatic osteochondritic horses

Reasons for performing study: Alternative methods to evaluate the joint condition in asymptomatic osteochondrosis dissecans (OCD) and other joint diseases may be useful. Objectives: To investigate possible changes in synovial fluid composition that may lead to joint conditions in asymptomatic OCD, in mature horses. Methods: Animals aged >2 years, of different breeds, with OCD in the intermediate ridge of distal tibia, symptomatic or not, were studied. Synovial fluid samples (10 healthy; 11 asymptomatic OCD; 25 symptomatic OCD) were collected by arthroscopy from 29 horses. Glycosaminoglycans (GAGs) were analysed by a combination of agarose gel electrophoresis and enzymatic degradation with specific GAG lyases. The viscosity, white blood cell (WBC) count, protein concentration and hyaluronic acid (HA) molecular weight were also determined. Results: The method used here to analyse synovial fluid GAGs is reliable, reproducible and specific. The main synovial fluid GAGs are HA and chondroitin sulphate (CS), 93% and 7% respectively in normal horses. In symptomatic OCD, the concentrations of both increased (expressed as GAG/urea ratios), but CS increased more. The CS increased also in asymptomatic OCD. An inflammatory reaction was suggested by the increased WBC counts in OCD. The molecular weight of the synovial fluid HA was reduced in OCD, explaining the lower viscosity observed. Conclusions: The increased CS in synovial fluid of OCD joints in mature horses suggests that the synovial fluid CS and the WBC count are good markers of the joint conditions, allowing the identification of pathological phase in joint diseases. Potential relevance: The analysis of synovial fluid GAGs shows that cartilage damage occurs even in asymptomatic OCD, implying that arthroscopic removal of osteochondral fragments should be performed even in asymptomatic OCD.

Hematologic and Plasma Biochemical Values of Hyacinth Macaws (Anodorhynchus hyacinthinus)

The hyacinth macaw (Anodorhyncus hyacinthinus), considered the largest psittacine bird species in the world, is an endangered species, with a remaining population of approximately 6500 birds in the wild. To establish hematologic and plasma biochemical reference ranges and to verify differences related to sex, samples from 29 hyacinth macaws (14 males. 15 females) were obtained from birds apprehended from illegal wildlife trade and subsequently housed at the Sorocaba Zoo, Brazil. No significant differences in hematologic or plasma biochemical values were found between females and males. Compared with published reference values, differences were found in mean concentrations of total red blood cell count, corpuscular volume, corpuscular hemoglobin level, total white blood cell count, aspartate aminotransferase level, creatine kinase concentration, alkaline phosphatase concentration, and phosphorus level. Baseline hematologic and plasma biochemical ranges were established, which may be useful as reference values for clinicians working with this endangered species in captivity or rehabilitation centers.

Hematologic Reference Values for Clinically Healthy Captive Golden Conures (Guaruba guarouba)

Golden conures or ararajubas (Guaruba guarouba) are endangered parrots endemic to the Brazilian Amazon forest. Body mass, blood cell counts, and total plasma protein were determined for 70 clinically healthy golden conures captive at zoologic parks and private breeder facilities in Brazil. Hematologic results (mean +/- SD) were: Erythrocytes 3.6 +/- 0.5 x 10(6) cells/mm(3), hemoglobin 12.8 +/- 1.4 g/dl, packed cell volume 46 +/- 3.8%, mean corpuscular volume 132 +/- 20 fl, mean corpuscular hemoglobin (MCH) 36 +/- 5.7 pg, mean corpuscular hemoglobin concentration (MCHC) 28 +/- 3.5%, thrombocytes 26.3 +/- 9.3 x 10(3) cells/mm(3), leukocytes 11.9 +/- 4.5 x 10(3) cells/mm(3), heterophils 6284 +/- 2715 cells/mm(3), lymphocytes 5473 +/- 2408 cells/mm(3), monocytes 113 +/- 162 cells/mm(3), eosinophils 10 +/- 42 cells/mm(3), basophils 27 +/- 64 cells/mm(3). Body mass was 254 +/- 24.9 g and total plasma protein (TPP) was 3.54 +/- 0.58 g/dl. No statistical differences were observed between genders within age groups. Differences between juveniles (J) and adults (A) were identified for TPP < A), MCH (J > A), and MCHC (J > A). These results provide reliable reference values for the clinical interpretation of hematologic results for the species. Hematology may be an important tool for population health investigations on free-ranging golden conure populations and will also be essential to survey the health of release candidates in future reintroduction programs.

Hemolytic activity of alternative complement pathway as an indicator of innate immunity in pacu (Piaractus mesopotamicus)

The objective of this study was to evaluate the methodology to establish the hemolytic activity of alternative complement pathway as an indicator of the innate immunity in Brazilian fish pacu (Piaractus mesopotamicus), in addition to verifying the influence of beta-glucan as an immunostimulant. Fish were fed with diets containing 0, 0.1 and 1% beta-glucan, during seven days, and then inoculated with Aeromonas hydrophila. Seven days after the challenge, they were bled for serum extraction. The methodology consisted of a kinetic assay that allows calculating the required time for serum proteins of the complement to promote 50% lysis of a rabbit red blood cell suspension. The method developed in mammals was successfully applied for pacu and determined that the hemolytic activity of the proteins of the complement system (alternative pathway) increased after the pathogen challenge, but was not influenced by the beta-glucan treatment.

WBC count and functional changes induced by co-administration of clofazimine and clarithromycin, in single and multiple doses, in Wistar rats

Clofazimine and clarithromycin are used to treat leprosy and infections caused by Mycobacterium avium complex. Little data on the toxicity of co-administration of these two drugs are available. Here we evaluated the potential adverse effects of polytherapy with these two drugs in male Wistar rats by determining WBCs counts and other blood cell counts, neutrophilic phagocytosis, and burst oxidative, by flow cytometry. We observed an increase in WBCs, in multiple-dose regimens, and in polymorphonuclear cells, in both single- clarithromycin only and multiple dose regimens. We also observed a reduction in mononuclear cell counts in single and multiple doses. The drugs seem to reverse the mononuclear and polymorphonuclear cell ratio. An increase in oxidative burst was observed in animals treated with the drugs administered either individually or combined. In conclusion, clofazimine and clarithromycin change WBCs counts. Our results may contribute for a better understanding of the mechanisms related to the effects of co-administrating the two drugs.

Differential MiRNA expression in childhood acute lymphoblastic leukemia and association with clinical and biological features

The present study aimed to analyze the expression profile of the microRNAs previously described as associated with childhood ALL, miR-92a, miR-100, miR-125a-5p, miR-128a, miR-181b, miR-196b and let-7e, and their association with biological/prognostic features in 128 consecutive samples of childhood acute lymphoblastic leukemia (ALL) by quantitative real-time PCR. A significant association was observed between higher expression levels of miR-196b and T-ALL, miR-100 and patients with low white blood cell count at diagnosis and t(12;21) positive ALL. These findings suggest a potential activity of these microRNAs in pediatric ALL biology. (C) 2011 Elsevier Ltd. All rights reserved.

Interaction among nitric oxide (NO)-related genes in migraine susceptibility

The pathogenic mechanisms involved in migraine are complex and not completely clarified. Because there is evidence for the involvement of nitric oxide (NO) in migraine pathophysiology, candidate gene approaches focusing on genes affecting the endothelial function have been studied including the genes encoding endothelial NO synthase (eNOS), inducible NO synthase (iNOS), and vascular endothelial growth factor (VEGF). However, investigations on gene-gene interactions are warranted to better elucidate the genetic basis of migraine. This study aimed at characterizing interactions among nine clinically relevant polymorphisms in eNOS (T-786C/rs2070744, the 27 bp VNTR in intron 4, the Glu298Asp/rs1799983, and two additional tagSNPs rs3918226 and rs743506), iNOS (C(-1026)A/rs2779249 and G2087A/rs2297518), and VEGF (C(-2578)A/rs699947 and G(-634)C/rs2010963) in migraine patients and control group. Genotypes were determined by real-time polymerase chain reaction using the Taqman(A (R)) allele discrimination assays or PCR and fragment separation by electrophoresis in 99 healthy women without migraine (control group) and in 150 women with migraine divided into two groups: 107 with migraine without aura and 43 with aura. The multifactor dimensionality reduction method was used to detect and characterize gene-gene interactions. We found a significant interaction between eNOS rs743506 and iNOS 2087G/A polymorphisms in migraine patients compared to control group (P < 0.05), suggesting that this combination affect the susceptibility to migraine. Further studies are needed to determine the molecular mechanisms explaining this interaction.

Probiotic cheese attenuates exercise-induced immune suppression in Wistar rats

Intense physical activity results in a substantial volume of stress and hence a significant probability of immunosuppression in athletes, with milk proteins being, perhaps, the most recommended protein supplements. Consumption of a probiotic cheese can attenuate immune suppression induced by exhausting exercise in rats. A popular Brazilian fresh cheese (Minas Frescal cheese) containing Lactobacillus acidophilus LA14 and Bifidobacterium longum BL05 was fed for 2 wk to adult Wistar rats, which then were brought to exhaustion on the treadmill. Two hours after exhaustion, the rats were killed and material was collected for the determination of serum uric acid, total and high-density lipoprotein cholesterol fraction, total protein, triacylglycerols, aspartate aminotransferase, alanine aminotransferase, creatine kinase, and blood cell (monocyte, lymphocyte, neutrophil, and leukocyte) counts. Exercise was efficient in reducing lymphocyte counts, irrespective of the type of ingested cheese, but the decrease in the group fed the probiotic cheese was 22% compared with 48% in the animals fed regular cheese. Monocyte counts were unaltered in the rats fed probiotic cheese compared with a significant decrease in the rats fed the regular cheese. Most importantly, ingestion of the probiotic cheese resulted in a >100% increase in serum high-density lipoprotein cholesterol and a 50% decrease in triacylglycerols. We conclude that probiotic Minas Reseal cheese may be a viable alternative to enhance the immune system and could be used to prevent infections, particularly those related to the physical overexertion of athletes.

Paradoxical effects of brain death and associated trauma on rat mesenteric microcirculation: an intravital microscopic study

OBJECTIVE: Experimental findings support clinical evidence that brain death impairs the viability of organs for transplantation, triggering hemodynamic, hormonal, and inflammatory responses. However, several of these events could be consequences of brain death-associated trauma. This study investigated microcirculatory alterations and systemic inflammatory markers in brain-dead rats and the influence of the associated trauma. METHOD: Brain death was induced using intracranial balloon inflation; sham-operated rats were trepanned only. After 30 or 180 min, the mesenteric microcirculation was observed using intravital microscopy. The expression of P-selectin and ICAM-1 on the endothelium was evaluated using immunohistochemistry. The serum cytokine, chemokine, and corticosterone levels were quantified using enzyme-linked immunosorbent assays. White blood cell counts were also determined. RESULTS: Brain death resulted in a decrease in the mesenteric perfusion to 30%, a 2.6-fold increase in the expression of ICAM-1 and leukocyte migration at the mesentery, a 70% reduction in the serum corticosterone level and pronounced leukopenia. Similar increases in the cytokine and chemokine levels were seen in the both the experimental and control animals. CONCLUSION: The data presented in this study suggest that brain death itself induces hypoperfusion in the mesenteric microcirculation that is associated with a pronounced reduction in the endogenous corticosterone level, thereby leading to increased local inflammation and organ dysfunction. These events are paradoxically associated with induced leukopenia after brain damage.

Effetti clinici e biologici della terapia epigenetica nelle sindromi mielodisplastiche a basso rischio

Background: Nucleoside 5-Azacitidine (5-Aza) in high risk MDS patients (pts) at a dose of 75mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates (hematologic improvement (HI) 50-60%, complete remission (CR) 10-30%) and prolongation of survival (at 2 years 50,8%). Aim: The role of 5-Aza in low-risk MDS patients is not well defined but its use in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial a low-dose schedule of 5-Aza (75 mg/mq daily for 5 consecutive days every 28 days) was given to low-risk MDS pts in order to evaluate its efficacy and tolerability and to identify biological markers to predict the response. Methods: From September 2008 to February 2010, 34 patients were enrolled into the study. Fifteen patients had refractory anemia (RA), 5 patients refractory anemia with ringed sideroblasts (RARS), 7 patients refractory cytopenia with multilineage dysplasia (RCMD) and 7 patients refractory anemia with excess blasts-1 (RAEB-1). All patients failed previously EPO therapy and were in chronic red blood cell (RBC) supportive care with a median transfusions requirement of 4 units/monthly. The response treatment criteria was according to IWG 2006. Results: At present time 31 out of 34 pts are evaluable: 12/31 pts (39%) completed the treatment plan (8 courses), 7/31 pts (22%) performed the first 4 courses, 8/31 (26%) made 1 to 3 courses and 4/31 (13%) died during the treatment period. Out of 12 pts who completed the 8 courses of therapy 10 (83%) obtained an HI, 2/12 (17%) maintained a stable disease. Out of 10 pts who obtained HI, 4 pts (40%) achieved a CR. Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%) but they were transitory and usually no delay of treatment was necessary. 2/4 pts died early after the 1th cycle for septic shock and gastrointestinal hemorrage respectively whereas 2/4 pts died in a condition of stable disease after the 4th cycle for pneumonia and respiratory distress. Samples for biologic studies have been collected from the pts before starting the therapy and at the end of 4th and 8th course. Preliminary data on the lipid signalling pathways suggested a direct correlation between PI-PLC-β1 gene expression and 5-Aza responsiveness. Conclusion: Interim analysis of our study based on the small number of cases who completed the treatment program, shows that 83% of pts obtain an HI and 40% obtain a CR. 4 patients died during the treatment and even if the causes were reported as no related to the therapy it has been considered that caution has to be reserved in given 5-Aza in these pts who are elderly and frail. Preliminary data of PI-PLC-β1 gene expression suggest that this and probably other biological markers could help us to know a priori who are the patients who have more chances to respond.

Alkylantien induzierte Zytotoxizität, DNA-schadensinduzierte Reparaturkapazität und Apoptoseinduktion von Immunzellen

Monozyten und Monozyten-abgeleitete Dendritische Zellen (DCs) spielen eine bedeutende Rolle im Immunsystem. Da DCs bei der Tumorabwehr mitwirken, ist es wichtig, dass Monozyten als auch DCs sich gegenüber zytotoxischen Agenzien aus der Chemotherapie wehren können. Chemotherapeutika reagieren mit der DNA, jedoch die DNA-Reparaturkapazität von Monozyten und DCs wurde noch nicht untersucht. Dazu wurde die Sensitivität in Monozyten und DCs gegenüber verschiedene genotoxische Agenzien untersucht. Dabei wurde herausgefunden, dass Monozyten sensitiv auf methylierende Agenzien (MNNG, MMS und Temozolomid) reagieren und ein verstärktes Zellsterben und Apoptoseinduktion zeigen. Im Vergleich zu weiteren Zytostatika wie Fotemustin, Mafosfamid und Cisplatin reagierten Monozyten und DCs gleich sensitiv. Diese Ergebnisse weisen auf einen Defekt in der Reparatur von DNA-Methylierungsschäden in Monozyten hin. Da die Expression des Reparaturproteins O6-Methylguanin-DNA Methyltransferase (MGMT) in Monozyten höher war als in DCs und deren Inhibierung durch O6-Benzylguanin keinen Effekt auf die Sensitivität von Monozyten hatte, wurde der Reparaturweg der Basenexzisionsreparatur untersucht. Im Vergleich zu DCs waren die Monozyten unfähig die BER durchzuführen, welche durch Einzelzellgelelektrophorese gemessen wurde. Expressionsuntersuchungen ergaben, dass in Monozyten XRCC1 und Ligase IIIα fehlen im Vergleich zu DCs, Makrophagen, hämatopoetische Stammzellen und Lymphozyten, welche diese Proteine exprimieren. Diese Ergebnisse zeigen einen spezifischen DNA-Reparaturdefekt in einer bestimmten Blutzellpopulation. Durch den BER Defekt in Monozyten kann es durch methylierende Tumorwirkstoffe während einer Chemotherapie zur Depletion und zu einer abgeschwächten Immunantwort kommen.

In vivo consequences of AML1-ETO fusion protein expression for hematopoiesis

The t(8;21) (q22;q22) translocation fusing the ETO (also known as MTG8) gene on human chromosome 8 with the AML1 (also called Runx1 or CBFα) gene on chromosome 21 is one of the most common genetic aberrations found in acute myeloid leukemia (AML). This chromosomal translocation occurs in 12 % of de novo AML cases and in up to 40 % of the AML-M2 subtype of the French-American-British classification. To date, the in vivo function of aberrant AML1-ETO fusion protein expression has been investigated by several groups. However, in these studies, controversial results were reported and some key issues remain unknown. Importantly, the consequences of aberrant AML1-ETO expression for self-renewing hematopoietic stem cells (HSCs), multipotent hematopoietic progenitors (MPPs) and lineage-restricted precursors are not known. rn The aim of this thesis was to develop a novel experimental AML1-ETO in vivo model that (i) overcomes the current lack of insight into the pre-leukemic condition of t(8;21)-associated AML, (ii) clarifies the in vivo consequences of AML1-ETO for HSCs, MPPs, progenitors and more mature blood cells and (iii) generates an improved mouse model suitable for mirroring the human condition. For this purpose, a conditional tet on/off mouse model expressing the AML1-ETO fusion protein from the ROSA26 (R26) locus was generated. rn Aberrant AML1-ETO activation in compound ROSA26/tetOAML1-ETO (R26/AE) mice caused high rates of mortality, an overall disruption of hematopoietic organs and a profound alteration of hematopoiesis. However, since the generalized activity of the R26 locus did not recapitulate the leukemic condition found in human patients, it was important to restrict AML1-ETO expression to blood cell lineages. Therefore, bone marrow cells from non-induced R26/AE mice were adoptively transplanted into sublethal irradiated RAG2-/- recipient mice. First signs of phenotypical differences between AML1-ETO-expressing and control mice were observed after eight to nine months of transgene induction. AML1-ETO-expressing mice showed profound changes in hematopoietic organs accompanied by manifest extramedullary hematopoiesis. In addition, a block in early erythropoiesis, B- and T-cell maturation was observed and granulopoiesis was significantly enhanced. Most interestingly, conditional activation of AML1-ETO in chimeric mice did not increase HSCs, MPPs, common lymphoid precursors (CLPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) but promoted the selective amplification of granulocyte-macrophage progenitors (GMPs). rn The results of this thesis provide clear experimental evidence how aberrant AML1-ETO modulates the developmental properties of normal hematopoiesis and establishes for the first time that AML1-ETO does not increase HSCs, MPPs and common lineage-restricted progenitor pools but specifically amplifies GMPs. The here presented mouse model not only clarifies the role of aberrant AML1-ETO for shaping hematopoietic development but in addition has strong implications for future therapeutic strategies and will be an excellent pre-clinical tool for developing and testing new approaches to treat and eventually cure AML.rn

Optimisation of chemical lysis protocol for point-of-care leukocyte differentiation

The full blood cell (FBC) count is the most common indicator of diseases. At present hematology analyzers are used for the blood cell characterization, but, recently, there has been interest in using techniques that take advantage of microscale devices and intrinsic properties of cells for increased automation and decreased cost. Microfluidic technologies offer solutions to handling and processing small volumes of blood (2-50 uL taken by finger prick) for point-of-care(PoC) applications. Several PoC blood analyzers are in use and may have applications in the fields of telemedicine, out patient monitoring and medical care in resource limited settings. They have the advantage to be easy to move and much cheaper than traditional analyzers, which require bulky instruments and consume large amount of reagents. The development of miniaturized point-of-care diagnostic tests may be enabled by chip-based technologies for cell separation and sorting. Many current diagnostic tests depend on fractionated blood components: plasma, red blood cells (RBCs), white blood cells (WBCs), and platelets. Specifically, white blood cell differentiation and counting provide valuable information for diagnostic purposes. For example, a low number of WBCs, called leukopenia, may be an indicator of bone marrow deficiency or failure, collagen- vascular diseases, disease of the liver or spleen. The leukocytosis, a high number of WBCs, may be due to anemia, infectious diseases, leukemia or tissue damage. In the laboratory of hybrid biodevices, at the University of Southampton,it was developed a functioning micro impedance cytometer technology for WBC differentiation and counting. It is capable to classify cells and particles on the base of their dielectric properties, in addition to their size, without the need of labeling, in a flow format similar to that of a traditional flow cytometer. It was demonstrated that the micro impedance cytometer system can detect and differentiate monocytes, neutrophils and lymphocytes, which are the three major human leukocyte populations. The simplicity and portability of the microfluidic impedance chip offer a range of potential applications in cell analysis including point-of-care diagnostic systems. The microfluidic device has been integrated into a sample preparation cartridge that semi-automatically performs erythrocyte lysis before leukocyte analysis. Generally erythrocytes are manually lysed according to a specific chemical lysis protocol, but this process has been automated in the cartridge. In this research work the chemical lysis protocol, defined in the patent US 5155044 A, was optimized in order to improve white blood cell differentiation and count performed by the integrated cartridge.