Slight Hemolysis Research Articles

Sucrose based ionic liquid colloidal microgels in separation of biomacromolecules

Abstract Sucrose microgels were synthesized by using divinyl sulfone as crosslinker with a high yield, almost 100%. Sucrose microgels in the size range of 0.5–20 μm as revealed by SEM images were obtained. The prepared sucrose microgels were chemically modified with polyethyleneimine (PEI) that contain branched amine functional groups. Then, these modified (M-) Sucrose microgel were protonated with HCl to render further positive charges to enable anion exchange reaction with sodium dicyanamide (SDC), ammonium hexafluorophosphate (AHFP), sodium tetrafluoroborate (STFB) to generate ionic liquid colloid (ILC). IL in colloidal form can enable the advantages of natural polymeric colloids and IL. Upon modification, the negatively charged sucrose microgels with zeta potential (ZP) value of −30.2 ± 2 mV was increased to +30.1 ± 2 mV, and the ILC forms of Sucrose microgels were also measured as highly positively charged particles with about ZP value of +35 mV. The characterization of the ILC Sucrose microgels were evaluated by employing various techniques such as scanning electron microscope (SEM), FT-IR spectroscopy, thermogravimetric analysis, and zeta potential measurements. Interestingly, quaternized (Q-) and ILC forms of Sucrose microgels were found very effective antibacterial agents against Eschericia coli ATCC 8739 (gram negative) and Staphylococcus aureus ATCC 6538 (gram positive) microorganisms owing to their cationic nature. Furthermore, Sucrose based microgels demonstrated good blood compatibility with the exception of Q-Sucrose microgels with slight hemolysis ratio and highly blood clotting index. It was also revealed that SDC-Sucrose ILC microgels can be used in adsorbent material with high amount of protein absorbing capabilities e.g., 22.3 ± 6.2 lysozyme and 22.1 ± 1.1 mg/g bovine serum albumin (BSA) in PBS at pH 7.4.

Cycloartane-type glycosides from Astragalus brachycalyx FISCHER and their effects on cytokine release and hemolysis

Two new tridesmosidic cycloartane-type triterpene glycosides (1 and 2) were isolated from the methanolic extract of the roots of Astragalus brachycalyx FISCHER (A. brachycalyx) along with ten (3–12) known cycloartane-type triterpene glycosides. Structures of the new compounds were established as 3-O-β-d-xylopyranosyl-6-O-β-d-glucopyranosyl-16-O-β-d-glucopyranosyl-3β,6α,16β,24(S)-25-pentahydroxycycloartane (1), 3-O-[α-l-arabinopyranosyl-(1→2)-β-d-xylopyranosyl]-6-O-β-d-glucopyranosyl-16-O-β-d-glucopyranosyl-3β,6α,16β,24(S)-25-pentahydroxycycloartane (2), by using 1D and 2D-NMR techniques and mass spectrometry.In vitro immunomodulatory effects and hemolytic activities of the new saponins (1 and 2) and acetylated form of 1 (1a) were studied together with the BuOH and MeOH extracts of Astragalus brachycalyx. The results have proven that tridesmosidic Astragalus cycloartanes are noteworthy immunomodulatory compounds via induction of cytokine production, namely IL-2 and IFN-γ. The test compounds also resulted slight hemolysis at very high doses substantiating a safer profile compared to the positive control QS-21.

Effects of hemolysis and lipemia interference on kaolin-activated thromboelastography, and comparison with conventional coagulation tests

The effects of hemolysis and lipemia on thromboelastography (TEG) analysis have been scarcely evaluated in human samples, and neglected in clinical practice. We aimed to investigate the effects of in vitro mechanical hemolysis and lipemia on TEG analysis and conventional coagulation tests. Twenty-four healthy volunteers were enrolled in the study. Besides the controls, three groups with slight, moderate and severe mechanical hemolysis were constituted according to free hemoglobin (Hb) concentrations of 0.5–1.0, 2.0–6.0 and 7.0–13.0 g/L, respectively; and three groups with mild, moderate and high lipemia were established according to triglyceride concentrations of ∼6.0, ∼12.0, and ∼18.0 mmol/L, respectively. Four TEG parameters, reaction time (R), coagulation time (K), angle (α), and maximum amplitude (MA), were measured alongside conventional plasma tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (FIB) by mechanical method, and platelet count by optical method. Results showed that the median R and MA values at moderate and severe hemolysis and K at severe hemolysis exceeded respective reference intervals, and were considered unacceptable. Median values of TEG parameters in lipemic samples were all within reference intervals. Bias values of conventional plasma tests PT, APTT and FIB in hemolyzed or lipemic samples were all lower than the Clinical Laboratory Improvement Amendments (CLIA) allowable limits. Bias values of platelet count at moderate to severe hemolysis and lipemia exceeded the CLIA allowable limits. In conclusion, the detection of TEG was in general more affected by mechanical hemolysis than plasma coagulation tests. Pre-analytical variables should be taken into account when unexpected TEG results are obtained.

Individualized correction of insulin measurement in hemolyzed serum samples.

Insulin measurement plays a key role in the investigation of patients with hypoglycemia, subtype classification of diabetes mellitus, insulin resistance, and impaired beta cell function. However, even slight hemolysis can negatively affect insulin measurement due to RBC insulin-degrading enzyme (IDE). Here, we derived and validated an individualized correction equation in an attempt to eliminate the effects of hemolysis on insulin measurement. The effects of hemolysis on insulin measurement were studied by adding lysed self-RBCs to serum. A correction equation was derived, accounting for both percentage and exposure time of hemolysis. The performance of this individualized correction was evaluated in intentionally hemolyzed samples. Insulin concentration decreased with increasing percentage and exposure time of hemolysis. Based on the effects of hemolysis on insulin measurement of 17 donors (baseline insulin concentrations ranged from 156 to 2119pmol/L), the individualized hemolysis correction equation was derived: INScorr=INSmeas/(0.705lgHbplasma/Hbserum-0.001Time-0.612). This equation can revert insulin concentrations of the intentionally hemolyzed samples to values that were statistically not different from the corresponding insulin baseline concentrations (p=0.1564). Hemolysis could lead to a negative interference on insulin measurement; by individualized hemolysis correction equation for insulin measurement, we can correct and report reliable serum insulin results for a wide range of degrees of sample hemolysis. This correction would increase diagnostic accuracy, reduce inappropriate therapeutic decisions, and improve patient satisfaction with care.

Complement Genotype and Multiparametric Laboratory Analysis to Predict Response to Eculizumab and to Adjust Treatment in Patients with Paroxysmal Nocturnal Hemoglobinuria

▪Background: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disorder caused by the proliferation of hematopoietic stem cells carrying a somatic mutation in the gene PIG-A, which is necessary for the biosynthesis of the glycosyl phosphatidyl-inositol anchor. As a consequence, PNH erythrocytes lack, among other proteins, the complement regulators CD55 and CD59 and become susceptible to complement-mediated intravascular hemolysis. Before Eculizumab, a monoclonal antibody that prevents C5 cleavage, life expectancy of PNH patients was about 10-15 years from diagnosis, being thrombotic events the main cause of death. Despite the unquestionable success of Eculizumab therapy to treat PNH, the responses among PNH patients show heterogeneity, with some patients remaining transfusion-dependent for reasons not completely understood.Methods: To get further insight into the different responses to Eculizumab treatment among patients, we have generated a PNH cohort including 12 PNH patients treated with Eculizumab. All patients included in the study had previously a minimum of ≥12 months on treatment with the drug (ten receiving 900 mg every 14 days, and two patients on 1200 mg every 14 days because breakthrough hemolysis). We have collected samples during two Eculizumab cycles (4 weeks) at different points during treatment: before and after Eculizumab injection and in the intermediate week. We have searched for correlations between clinical and hematological parameters, complement genetic findings, and deposition of C3 fragments on erythrocytes and plasma levels of complement activation components.Results: We report the CR1 genotypes, complement and hematological determinations in 12 PNH patients during their treatment with Eculizumab. We have established that concentrations of free Eculizumab above 110 µg/mL completely inhibit serum hemolytic activity. Consistent with this, in our patient cohort, most patients with levels of free Eculizumab close or below 110 µg/mL exhibited traces of C5b9 and/or CH50. In general, lowest levels of free Eculizumab correlate with higher levels of C5b9. Our results indicate that proper monitoring of PNH patients would be useful to optimize Eculizumab dosage. We have 3 patients that showed symptoms of intravascular hemolysis (high LDH and hemosiderinuria) despite having a negative CH50 accompanied with sufficient free Eculizumab levels. Further studies are currently being done to elucidate the causes. We have found a correlation between CR1 haplotype, red cellsopsonization and C3 plasma levels: H/L individuals present higher % of C3 positive erythrocytes and low C3 plasma levels. Two of these patients also had low C4 plasma levels, one of them presenting also low levels of FH. These individuals are currently being studied to determine whether these low levels are due to genetic factors or consumption. We found a correlation between levels of bilirubin and % of reticulocytes; patients who had more than the 30% of their erythrocytes opsonized presented higher percentage of reticulocytes and bilirubin in plasma, which could be taken as indicatives of slight extravascular hemolysis. In this regard, individuals carrying the CR1 L allele might require special attention. DisclosuresOjeda:Alexion Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Fores:Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Villegas-Martinez:Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Rodriguez-Cordoba:Alexion Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Hip septic arthritis diagnosed during rehabilitation of a trans-tibial amputation due to a diabetic foot ulcer: a case report

A major part of lower limb amputations concerns patients with diabetes, and is related to the appearance of a diabetic foot ulcer, that can cause serious complications if it becomes septic. A 64-year-old woman with diabetes mellitus, socially isolated, is hospitalized, being in a hyperosmolar coma; her diabetes was decompensated by a toxic shock due to a necrotic diabetic foot ulcer, requiring right trans-tibial amputation. Then she was admitted to a rehabilitation unit, where she was suffering from increasing left hip pains (strongly limiting her capacity to walk), in a clinical and biological inflammatory context, that led us to make a MRI. A liquid collection into left ilio-psoas muscle associated to a rapidly destructive left hip arthritis were diagnosed. PET-scan was in favour of a septic arthritis. Surgery enabled to make biological samples (finding a Bacteroides fragilis and meticillin-resistant Staphylococcus aureus infection), and a to implant a hip spacer, pending a hip prosthetic replacement. The most likely etiologic diagnosis–although not certain–seems to be an infected psoas hematoma (the first hip pains she notified were accompanied by a slight hemolysis), that caused septic hip arthritis by spreading by contiguity. Among anaerobic hip infections, Gram-positive cocci are most frequently found in patients having been treated by surgery (such as arthroplasties), whereas Gram-negative bacterias are more related to debilitated patients who had not had any hip surgical treatment [1] . In case of diabetic foot ulcer infection, we should always think about the risk of a systemic infectious complications. Atypical left hip pains can be the only symptom of an infected psoas hematoma in debilitated patients as described in this observation.

Enhanced Eryptosis Following Auranofin Exposure

Background/Aims: The antiinflammatory, antimicrobial and anticancer drug auranofin has previously been shown to trigger apoptosis, the suicidal death of nucleated cells. Side effects of the drug include anaemia. At least in theory the anaemia could result from stimulation of suicidal death of erythrocytes or eryptosis, which involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Methods: Stimulators of eryptosis include oxidative stress and increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). In the present study, phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca²⁺]_i from Fluo3-fluorescence. Results: A 24 hours exposure of human erythrocytes to auranofin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells (from 2.2 ± 0.5 to 17.4 ± 1.5%), significantly decreased forward scatter and significantly enhanced ROS. At higher concentrations (10 µg/ml) auranofin triggered slight hemolysis (from 2.1 ± 0.2 to 3.2 ± 0.3%). Conclusions: Auranofin stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress.

Anti-plasmodial activity of aroylhydrazone and thiosemicarbazone iron chelators: Effect on erythrocyte membrane integrity, parasite development and the intracellular labile iron pool

Iron chelators inhibit the growth of the malaria parasite, Plasmodium falciparum, in culture and in animal and human studies. We previously reported the anti-plasmodial activity of the chelators, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), 2-hydroxy-1-naphthylaldehyde 4-methyl-3-thiosemicarbazone (N4mT), and 2-hydroxy-1-naphthylaldehyde 4-phenyl-3-thiosemicarbazone (N4pT). In fact, these ligands showed greater growth inhibition of chloroquine-sensitive (3D7) and chloroquine-resistant (7G8) strains of P. falciparum in culture compared to desferrioxamine (DFO). The present study examined the effects of 311, N4mT and N4pT on erythrocyte membrane integrity and asexual parasite development. While the characteristic biconcave disk shape of the erythrocytes was unaffected, the chelators caused very slight hemolysis at IC50 values that inhibited parasite growth. The chelators 311, N4mT and N4pT affected all stages of the intra-erythrocytic development cycle (IDC) of P. falciparum in culture. However, while these ligands primarily affected the ring-stage, DFO inhibited primarily trophozoite and schizont-stages. Ring, trophozoite and schizont-stages of the IDC were inhibited by significantly lower concentrations of 311, N4mT, and N4pT (IC50=4.45±1.70, 10.30±4.40, and 3.64±2.00μM, respectively) than DFO (IC50=23.43±3.40μM). Complexation of 311, N4mT and N4pT with iron reduced their anti-plasmodial activity. Estimation of the intracellular labile iron pool (LIP) in erythrocytes showed that the chelation efficacy of 311, N4mT and N4pT corresponded to their anti-plasmodial activities, suggesting that the LIP may be a potential source of non-heme iron for parasite metabolism within the erythrocyte. This study has implications for malaria chemotherapy that specifically disrupts parasite iron utilization.

Individualized correction of neuron-specific enolase (NSE) measurement in hemolyzed serum samples

BackgroundAccuracy of serum neuron-specific enolase (NSE) measurement is paramount, particularly in the context of neurological outcome prognostication. However, NSE measurements are compromised by even slight hemolysis, as it is abundant in red blood cells (RBCs). We derived and validated an individualized hemolysis correction equation in an attempt to reduce the current rejection rate of 14% at our institution. MethodsIntracellular NSE was measured in RBC lysates to determine concentration variability. A correction equation was derived, accounting for both RBC-derived NSE false-elevation and hemoglobin-derived signal quenching. The performance of this individualized correction was evaluated in intentionally hemolyzed samples and accuracy was compared to a generalized correction. ResultsSignificant inter-individual variability of RBC NSE was observed, with an almost two-fold range (15.7–28.5ng NSE/mg Hb, p<0.001); intra-individual variability was insignificant. The individualized hemolysis correction equation derived: NSEcorr=NSEmeas−(Hbserum)(NSERBCs/Hb)+0.0844(Hbserum)+1.1 corrected 95% of the intentionally hemolyzed samples to within ±5ng/ml of corresponding baseline NSE concentrations, compared to 74% using a generalized formula. ConclusionsThe individualized hemolysis correction provides increased accuracy in the estimation of true serum NSE concentrations for hemolyzed samples, compared to a generalized approach, by accounting for inter-individual RBC NSE variability. Incorporating this correction should reduce sample rejection rates and overall health care costs.

Stimulation of Suicidal Erythrocyte Death by Increased Extracellular Phosphate Concentrations

Background/Aim: Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, and [Ca²⁺]_i from Fluo3-fluorescence. Results: Following a 48 hours incubation, the percentage of phosphatidylserine exposing erythrocytes markedly increased as a function of extracellular phosphate concentration (from 0-5 mM). The exposure to 2 mM or 5 mM phosphate was followed by slight but significant hemolysis. [Ca²⁺]_i did not change significantly up to 2 mM phosphate but significantly decreased at 5 mM phosphate. The effect of 2 mM phosphate on phosphatidylserine exposure was significantly augmented by increase of extracellular Ca²⁺ to 1.7 mM, and significantly blunted by nominal absence of extracellular Ca²⁺, by additional presence of pyrophosphate as well as by presence of p38 inhibitor SB203580. Conclusion: Increasing phosphate concentration stimulates erythrocyte membrane scrambling, an effect depending on extracellular but not intracellular Ca²⁺ concentration. It is hypothesized that suicidal erythrocyte death is triggered by complexed CaHPO₄.

Induction of apoptotic erythrocyte death by rotenone

The pesticide rotenone stimulates apoptosis and rotenone intoxication has been considered a cause of Parkinson's disease. Rotenone further sensitizes tumor cells to cytotoxic drugs. The apoptotic effect of rotenone is at least partially due to mitochondrial injury. Even though lacking mitochondria and nuclei, erythrocytes may undergo eryptosis, an apoptosis-like suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and enhanced ceramide formation. The present study explored, whether rotenone elicits eryptosis. To this end, [Ca2+]i was estimated utilizing Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, ceramide utilizing fluorescence antibodies and hemolysis from hemoglobin release. A 48h exposure to rotenone significantly increased Fluo3-fluorescencei (≥1μM), increased ceramide abundance (10μM), decreased forward scatter (≥2.5μM) and increased annexin-V-binding (≥ 1μM). Rotenone exposure was further followed by slight but significant hemolysis. Rotenone-induced cell membrane scrambling was significantly blunted, but not completely abrogated by removal of extracellular Ca2+. The present observations disclose a novel effect of rotenone, i.e. triggering of erythrocyte shrinkage and cell membrane scrambling, an effect paralleled by and partially dependent on Ca2+-entry.

Sulindac Sulfide – Induced Stimulation of Eryptosis

Background: Sulindac sulfide, a non-steroidal anti-inflammatory drug (NSAID), stimulates apoptosis of tumor cells and is thus effective against malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i) and ceramide formation. The present study explored, whether sulindac sulfide stimulates eryptosis. Methods: [Ca²⁺]_i was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from binding of fluorescent annexin-V, hemolysis from hemoglobin release, and ceramide abundance utilizing fluorescent antibodies. Results: A 48 h exposure to sulindac sulfide (≤ 20 µM) was followed by significant increase of [Ca²⁺]_i, enhanced ceramide abundance, decreased forward scatter and increased percentage of annexin-V-binding erythrocytes. Sulindac sulfide triggered slight but significant hemolysis. Removal of extracellular Ca²⁺ significantly blunted, but did not abrogate the effect of sulindac sulfide (20 µM) on annexin-V-binding. Conclusion: Sulindac sulfide stimulates the suicidal death of erythrocytes or eryptosis, an effect paralleled by Ca²⁺-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.

Enhanced Erythrocyte Membrane Exposure of Phosphatidylserine Following Sorafenib Treatment: An in vivo and in vitro Study

Background: Sorafenib (Nexavar^®), a polytyrosine kinase inhibitor, stimulates apoptosis and is thus widely used for chemotherapy in hepatocellular carcinoma (HCC). Hematological side effects of Nexavar^® chemotherapy include anemia. Erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine-exposure at the cell surface. Signaling leading to eryptosis include increase in cytosolic Ca²⁺activity ([Ca²⁺]_i), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. Methods: [Ca²⁺]_iwas estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide with antibody binding-dependent fluorescence, cytosolic ATP with a luciferin–luciferase-based assay, and oxidative stress from 2’,7’ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h exposure of erythrocytes to sorafenib (≥0.5 µM) significantly increased Fluo 3 fluorescence, decreased forward scatter, increased annexin-V-binding and triggered slight hemolysis (≥5 µM), but did not significantly modify ceramide abundance and cytosolic ATP. Sorafenib treatment significantly enhanced DCFDA-fluorescence and the reducing agents N-acetyl-L-cysteine and tiron significantly blunted sorafenib-induced phosphatidylserine exposure. Nexavar^® chemotherapy in HCC patients significantly enhanced the number of phosphatidylserine-exposing erythrocytes. Conclusions: The present observations disclose novel effects of sorafenib, i.e. stimulation of suicidal erythrocyte death or eryptosis, which may contribute to the pathogenesis of anemia in Nexavar^®-based chemotherapy.

Molecular Cloning of Novel Antimicrobial Peptide Genes from the Skin of the Chinese Brown Frog, Rana chensinensis

One species of the Chinese brown frog, Rana chensinensis, is widely distributed in north-central China. In this study, a cDNA library was constructed to clone the antimicrobial peptides' genes from the skin of R. chensinensis. Twenty-three prepropeptide cDNA sequences encoding twelve novel mature antimicrobial peptides were isolated and characterized. Six peptides belonged to three known families previously identified from other Ranid frogs: temporin (4 peptides), brevinin-2 (1 peptide), and palustrin-2 (1 peptide). The other six peptides showed little similarity to known antimicrobial peptides. According to the amino acid sequences, with or without α-helix structure, and either hydrophilic or hydrophobic, these were organized into four new families: chensinin-1 (3 peptides), chensinin-2 (1 peptide), chensinin-3 (1 peptide), and chensinin-4 (1 peptide). Five peptides from different families were chemically synthesized, and their antimicrobial, cytolytic, and hemolytic activities were evaluated. Of these, brevinin-2CE showed strongest antimicrobial activities against both the Gram-positive and Gram-negative bacteria with a slight hemolysis. Temporin-1CEe and palustrin-2CE also displayed a slight hemolysis, but they had different activities to prokaryotic cells. Temporin-1CEe showed higher antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria, whereas it was contrary to palustrin-2CE. Chensinin-1 CEb and chensinin-3CE only had moderate antimicrobial activity against microorganisms. In addition, the brevinin-2 peptides from different brown frogs were analyzed to reveal the taxonomy and phylogenetic relationships of R. chensinensis.

Inhibition of the Growth ofPlasmodium falciparumin Culture by Stearylamine-Phosphatidylcholine Liposomes

We have examined the effect of stearylamine (SA) in liposomes on the viability of Plasmodium falciparum in culture by studying the inhibition of incorporation of [3H]-hypoxanthine in the nucleic acid of parasites. Stearylamine in liposomes significantly inhibits the growth of the parasites depending on the phospholipids composition. The maximum inhibition was observed when SA was delivered through Soya phosphatidylcholine (SPC) liposomes. The chain length of alkyl group and density of SA in liposomes play a significant role in inhibiting the growth of the parasites. Incorporation of either cholesterol or Distearylphosphatidylethanolamine−Methoxy-Polyethylene glycol-2000 (DSPE-mPEG-2000) in Soya phosphatidylcholine-stearylamine (SPC-SA) liposomes improves the efficacy. Intraerythrocytic entry of intact SPC-SA liposomes into infected erythrocytes was visualized using fluorescent microscopy. No hemolysis was observed in uninfected erythrocytes, and slight hemolysis was noted in infected erythrocytes at high concentrations of SPC-SA liposomes. Overall, our data suggested SA in SPC-liposomes might have potential application in malaria chemotherapy.

Evaluation of performance of a rapid immunofluorescence method for assaying plasma NT-proBNP

Objective To analyze the performance of a rapid immunofluorescence assay for plasma NT-proBNP. Methods Human plasma NT-proBNP was measured by RAMP in 264 healthy cases and 78 patients with heart failure. The precision, stability, linearity and interference factors of RAMP were evaluated according to the protocold from America CLSI. Meanwhile, the results were compared with those obtained by Elecsys. Results Functional sensitivity of RAMP in 20% CV and 10% CV were 48 ng/L and 57 ng/L respectively. The linear range was 18-8 000 ng/L. NT-proBNP in plasma samples detected by RAMP were stable at room temperature for 24 hours, 4 ℃ for 3 days and - 20 ℃ for 20 days. No influences on results were observed throughout three freeze-thaw cycles. The results measured by RAMP were compared between EDTA-K2 anticoagulant plasma and heparin anticoagulant plasma, which showed that YEDTA-K2 =0. 953 9 Xheparin + 0. 365 2 ( R2 = 0. 982, P < 0. 01, n = 40 ). The results of EDTA-K2 and heparin anticoagulant plasma using RAMP and Elecsys showed no significant difference( P ＞0. 05). Slight hemolysis( Hb 2 g/L)had small effect on the results of PAMP and Elecsys assays and deviations of them were below 5%. However median and heavy hemolysis( Hb 3-4 g/L )obviously influenced the results with deviation were more than 15%. The values measured by RMAP fell from 390 ng/L to 82 ng/L, and those measured by Elecsys method fell from 390 ng/L to 178 ng/L when 3 different concentrations of triacylglycerol were added, the values measured by two methods fell from 7 777 ng/L to 7 741 ng/L when bilirubin ( 16-330 μmol/L) was added. The anti-interference ability of RAMP method was similar to Elecsys. Results detected with two methods in 45 EDTA-K2 anticoagulant plasma samples were analyzed with Passing and Bablok regression.The regression equation was YRAMP = 0. 972 8XElecsys - 0. 035 2 (R2 = 0. 994, P ＞ 0. 05, n = 45 ). When heparin anticoagulant plasma samples increased to 78 samples, Passing and Bablok regression equation showed:YRAMP= 0. 983 2XElecsys - 0. 037 6 ( R2 = 0. 991, P ＞ 0. 05, n = 78 ). NT-proBNP levels in healthy adults were elevated along with age and related to the gender. The median value of female[105(60-120) ng/L] was higher than that of male [59 ( 44-91 ) ng/L, Z = 3. 264, P < 0. 01] significantly. Conclusions RAMP method has good repeatability, outstanding stability, and broad linear range. The results detected by RAMP is consistent with results detected by Elecsys. Key words: Peptide fragments; Natriuretic peptide, brain; Fluoroimmunoassay; Heparin; Evalution studies

Hemolysis correction factor in the measurement of serum neuron-specific enolase

Neuron-specific enolase (NSE) is comprised of two isoforms, ag and gg, of the glycolytic enzyme enolase. It is present almost exclusively in neurons and neuroendocrine tissues (1–3). The protein is a valuable tumor marker for monitoring the outcome and disease course in patients with cancer of the neuroendocrine type, in particular small cell lung cancer and neuroblastoma (4, 5). In addition, the concentration of NSE in biological fluids might be a useful parameter for the assessment of neural brain damage as NSE was shown to be released into cerebrospinal fluid and blood following cerebral injury (5, 6). However, the ag-isoform was also reported to be present in circulating blood cells, especially in erythrocytes and platelets (3, 7). As a consequence, slight hemolysis – even undetectable to the naked eye (3) – can produce falsely increased degrees of immunoreactivity (1). Considering the role of NSE as an important biomarker, interpretation of NSE results is unreliable unless accompanied by an estimate of red cell disruption (3). In the case of hemolysis, it is common practice to include a comment on the laboratory report and not give the NSE value itself. We examined the possibility of using data correction to report a clinically more reliable result when measuring NSE in hemolysed serum, instead of reporting a comment that the assay could not be performed. NSE was measured using a sandwich immunoassay with the electrochemiluminescence (ECL) technology installed on a Modular E170 (Roche Diagnostics, Mannheim, Germany). Evaluation of the magnitude of hemolysis was achieved using the hemolytic index (H). The serum index was routinely measured with a Cobas 6000 analyzer (Roche Diagnostics, Mannheim, Germany) using absorbance values of two bichromatic readings (570 and 600 nm, and 660 and 700 nm). The absorbance values are converted into semi-

A Novel Hemoglobin, Bonn, Causes Falsely Decreased Oxygen Saturation Measurements in Pulse Oximetry

A 4-year-old boy and his father exhibited low oxygen saturation measured transcutaneously by pulse oximetry, a finding that could not be confirmed by arterial blood gas analysis. Both patients exhibited slight hemolysis in their blood, and the boy had a microcytic anemia. There was no evidence of hypoxemia or methemoglobinemia. Despite the normal results from the arterial blood gas analysis, a right-to-left-shunt was assumed in the boy until a cardiology examination excluded this diagnosis. Sleep apnea syndrome was suspected in the father and treated with nocturnal positive pressure respiration based on the low oxygen saturation values obtained with pulse oximetry. Only after consultation with our laboratory was a hemoglobin variant suspected and investigated. We performed hemoglobin protein analysis by HPLC, electrophoretic separation, and spectrophotometry and DNA sequence analysis of the alpha-globin gene. Both HPLC chromatographic separation and alkaline electrophoresis revealed a unique hemoglobin peak. In both patients, alpha-globin gene sequencing revealed a mutation resulting in a histidine-to-aspartatic acid substitution at position alpha87. The low oxygen saturation measurement by pulse oximetry was due to hemoglobin Bonn oxyhemoglobin having an absorption peak at 668 nm, near the 660 nm measured by pulse oximeters. Hemoglobin Bonn is a novel hemoglobin variant of the proximal alpha-globin that results in falsely low oxygen saturation measurements with pulse oximetry.

Corneal abscess due to Suttonella indologenes

© 2007 Elsevier 0196-4399/00 (see frontmatter) 139 Case Report The patient was a 75-year-old female who was admitted to the hospital in December 2004 with a diagnosis of ocular cicatricial pemphigoid (ocular scar with blisters) (1). She presented with severe corneal lesions and palpebral (eyelid) lesions secondary to trichiasis (inversion of eyelid hairs, causing irritation) and symblepharon (adhesion of eyelid to eyeball) in both eyes, which were treated with 50 mg of azathioprine/ day. In June 2005, she returned to the ophthalmology service and a corneal abscess (0.5 cm by 1 cm) was diagnosed. Specimens for bacterial, mycobacterial, and fungal smears and cultures were obtained from the corneal scrapings under slit lamp examination. The scrapings were inoculated onto 5% Columbia sheep blood agar, chocolate agar, Sabouraud dextrose agar, and Lowenstein-Jensen agar using standard microbiological culture techniques. After 24 h of incubation in air supplemented with 5% CO2, small, smooth, and glistening colonies grew, which showed slight alpha hemolysis on sheep blood agar. The colonial appearance resembled Capnocytophaga sp. (Fig. 1). The colonies were gray with spreading edges, showing gliding motility. After 72 h of incubation, the colonies were larger, their diameters increasing by Case Report

Trypanocidal and leishmanicidal activities of different antimicrobial peptides (AMPs) isolated from aquatic animals

Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5 μM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100 μM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50 μM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent.