Pooled Human Plasma Research Articles

Long-Term Stability of Human Plasma Metabolites during Storage at −80 °C

Prolonged storage of biospecimen can lead to artificially altered metabolite concentrations and thus bias data analysis in metabolomics experiments. To elucidate the potential impact of long-term storage on the metabolite profile, a pooled human plasma sample was aliquoted and stored at -80 °C. During a time period of five years, 1012 of the aliquots were measured with the Biocrates AbsoluteIDQ p180 targeted-metabolomics assay at 193 time points. Modeling the concentration courses over time revealed that 55 out of 111 metabolites remained stable. The statistically significantly changed metabolites showed on average an increase or decrease of +13.7% or -14.5%, respectively. In detail, increased concentration levels were observed for amino acids (mean: + 15.4%), the sum of hexoses (+7.9%), butyrylcarnitine (+9.4%), and some phospholipids mostly with chain lengths exceeding 40 carbon atoms (mean: +18.0%). Lipids tended to exhibit decreased concentration levels with the following mean concentration changes: acylcarnitines, -12.1%; lysophosphatidylcholines, -15.1%; diacyl-phosphatidylcholines, -17.0%; acyl-alkyl-phosphatidylcholines, -13.3%; sphingomyelins, -14.8%. We conclude that storage of plasma samples at -80 °C for up to five years can lead to altered concentration levels of amino acids, acylcarnitines, glycerophospholipids, sphingomyelins, and the sum of hexoses. These alterations must be considered when analyzing metabolomics data from long-term epidemiological studies.

Got a Leak! Letʼs Glue It

Introduction: The procedure of choice for management of acute cholecystitis is laparoscopic cholecystectomy (LC). LC has it's distinct advantages; however, post-operative refractory bile leaks present a significant therapeutic challenge. Herein, we report a case of endoscopic occlusion of high grade bile leak from the gall bladder remnant that failed all standard endoscopic maneuvers via the use of human pooled plasma fibrinogen and thrombin (HPF) (Tissel, Baxter, CA) as a sealant. Case: A 65-year-old male presented with abdominal pain and was found to have CT and HIDA scan findings consistent with acute cholecystitis. Laparoscopic evaluation revealed severely inflamed and gangrenous gall bladder, hence he underwent an open subtotal cholecystectomy. Post operatively, patient had increased bilious output from the JP drain, therefore, a CT scan abdomen was done which revealed a biloma in the gall bladder fossa. ERCP revealed a high grade bile leak from the gall bladder remnant and suggestion of ampullary stenosis. Biliary sphincterotomy was performed and a 10 Fr x 5 cm plastic stent was placed in the common bile duct (CBD). Due to persistent high output from the JP drain, a repeat ERCP was done 4 weeks later; occlusion cholangiogram revealed low grade bile leak from the gall bladder remnant. Balloon sweep with clearance of biliary sludge was performed and the plastic stent was interchanged with a 10 mm x 6 cm fully covered self-expanding metal stent (FC-SEMS). Six weeks after placement of the FC-SEMS, patient still had persistent bilious drainage from the JP drain therefore a third ERCP was done. The previously placed biliary stent was removed and cholangiogram revealed a high grade bile leak. No filing defects or stricture were noted in the CBD. The CBD was dilated to 10 mm and the cystic duct stump was successfully cannulated and injected with 3 ml of HPF for the purpose of sealing the leak. Repeat cholangiogram demonstrated no leak. A 10 mm x 80 mm FC-SEMS was placed in the common bile duct. Follow up ERCP 3 months later revealed normal cholangiogram with no evidence of ongoing bile leak and the FC-SEMS was removed. Discussion: To our knowledge this a first report demonstrating use of HPF endoscopically for successful management of refractory bile leak. HPF should note be injected into vascular structures due to concern for vascular thrombosis. Further studies and reports are needed to evaluate the safety and efficacy of this technique.

The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum

BackgroundAt a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study.ResultsA pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-119, MSP-142, MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule.ConclusionAnalysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross-comparisons of results of vaccine trials performed in different centres/with different products; (2) to facilitate standardization and harmonization of immunological assays used in epidemiology research; and (3) to allow optimization and validation of immunological assays used in malaria vaccine development.

A targeted proteomic assay for the measurement of plasma proteoforms related to human aging phenotypes.

Circulating polypeptides and proteins have been implicated in reversing or accelerating aging phenotypes, including growth/differentiation factor 8 (GDF8), GDF11, eotaxin, and oxytocin. These proteoforms, which are defined as the protein products arising from a single gene due to alternative splicing and PTMs, have been challenging to study. Both GDF8 and GDF11 have known antagonists such as follistatin (FST), and WAP, Kazal, immunoglobulin, Kunitz, and NTR domain-containing proteins 1 and 2 (WFIKKN1, WFIKKN2). We developed a novel multiplexed SRM assay using LC-MS/MS to measure five proteins related to GDF8 and GDF11 signaling, and in addition, eotaxin, and oxytocin. Eighteen peptides consisting of 54 transitions were monitored and validated in pooled human plasma. In 24 adults, the mean (SD) concentrations (ng/mL) were as follows: GDF8 propeptide, 11.0 (2.4); GDF8 mature protein, 25.7 (8.0); GDF11 propeptide, 21.3 (10.9); GDF11 mature protein, 16.5 (12.4); FST, 29.8 (7.1); FST cleavage form FST303, 96.4 (69.2); WFIKKN1, 38.3 (8.3); WFIKKN2, 32.2 (10.5); oxytocin, 1.9 (0.9); and eotaxin, 2.3 (0.5). This novel multiplexed SRM assay should facilitate the study of the relationships of these proteoforms with major aging phenotypes.

Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

Phenotypic and phylogenetic relatedness of selected Staphylococcus aureus strains cultured from HIV seropositive mothers and their neonate's pair

Background: Staphylococcus aureus is a major pathogen to man and is invasive when the skin is breached as well as in trauma. The frequency and transmission of S. aureus was determined in high vaginal swabs (HVS), oropharynx, and breast milk of human immunodeficiency virus (HIV)-seropositive women to their neonates at healthcare centres, Ondo State, Nigeria between November 2014 and December 2015. Materials and Methods: A total of 114 HIV pregnant women were screened for S. aureus isolates in the HVS, oropharynx, and breast milk employing sterile cotton tipped applicator. The swabs were subsequently introduced into sterile thioglycollate medium and incubated at 37°C for 24 h. When growth was noticed, a loopful was introduced onto freshly prepared mannitol salt agar (MSA). Colonies of cocci that fermented mannitol on MSA were presumptively deemed as S. aureus, but confirmed as S. aureus isolates by the slide and tube agglutination tests in pooled human plasma. HIV seropositivity was determined by the HIV1/2 strip (Determine Test, Alere, London, England, UK) and confirmed as such by enzyme-linked immunosorbent assay (Abbot laboratories, Chicago, USA). Phenotypes were determined by susceptibility testing and phylogenetic relatedness by random amplified polymorphic DNA polymerase chain reaction. Results: Results showed that 50 (43.85%) were colonized by S. aureus; however, 4 (8%) showed mother-to-child transmission from the three sites. Although all isolates tested were resistant to nalidixic acid and penicillin, 93.75% of isolates were sensitive to imipemen. Conclusion: The study revealed a high frequency of colonization of S. aureus in HVS (64%), oropharynx (20%), and breast milk (16%). Horizontal transmission from mother to neonate was 8% while phylogroups showed similarities in genetic relatedness.

Specificity of B-Type Natriuretic Peptide Assays: Cross-Reactivity with Different BNP, NT-proBNP, and proBNP Peptides.

B-type natriuretic peptides (BNPs) are used clinically to diagnose and monitor heart failure and are present in the circulation as multiple proBNP-derived fragments. We investigated the specificity of BNP immunoassays with glycosylated and nonglycosylated BNP, N-terminal proBNP (NT-proBNP), and proBNP peptides to probe the cross-reactivity of each assay. Nine B-type natriuretic peptides were studied,including synthetic and recombinant BNP (Shionogi, Scios, Mayo), human and synthetic glycosylated and nonglycosylated NT-proBNP (HyTest, Roche Diagnostics), and human glycosylated and nonglycosylated proBNP (HyTest, Scios). Five BNP [Abbott, Abbott POC, Alere, Beckman Coulter, Siemens (Centaur)], 9 NT-proBNP [Ortho-Clinical Diagnostics, Roche, Response, bioMerieux, Siemens (Dimension, Immulite, Stratus CS), Mitsubishi] and 3 research-use-only proBNP immunoassays [Biosite (Alere), Bio-Rad, Goetze] were evaluated. Specificity was assessed by calculating the recovery between baseline and peptide-spiked human plasma pools at target concentrations of 100 ng/L BNP, 300 ng/L proBNP, or 450 ng/L NT-proBNP. All assays were performed in duplicate. BNP and NT-proBNP assays demonstrated substantial cross-reactivity with proBNP peptides. NT-proBNP assays do not detect glycosylated forms of either NT-proBNP or proBNP. proBNP assays preferentially detect the BNP 1-32 peptide and have minimal cross-reactivity with BNP peptides and glycosylated proBNP. BNP or NT-proBNP results are not transferable among the current existing immunoassays owing to their differences in cross-reactivity and ability to detect various glycosylated forms of proBNP-derived fragments. Opportunities remain to standardize and harmonize BNP and NT-proBNP assays, as well as to develop specific proBNP assays, to widen their clinical scope of use.

A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-β 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.

BackgroundAmyloid-β 1–42 peptide (Aβ1–42) is associated with plaque formation in the brain of patients with Alzheimer’s disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aβ1–42 in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents.MethodsA sensitive digital ELISA for measurement of Aβ1–42 using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aβ1–42 in clinical samples from patients treated with the β-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721.ResultsThe prototype assay measured Aβ1–42 with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aβ1–42 peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aβ1–42 in all of the 84 clinical samples tested. A rapid reduction in levels of Aβ1–42 was detected within 1 h after drug treatment, and a dose-dependent decrease of Aβ1–42 levels was also observed over the time course of sample collection.ConclusionsThis digital ELISA has potential utility in clinical applications for quantification of Aβ1–42 in plasma where high sensitivity and precision are required.

A highly sensitive assay of human plasma xanthine oxidoreductase activity using stable isotope-labeled xanthine and LC/TQMS.

Associations between elevated plasma xanthine oxidoreductase (XOR) activity and various pathologies have been widely reported. However, it has been difficult to accurately measure human plasma XOR activity because the XOR activity of humans is lower than that of animals such as mouse. We developed a highly sensitive assay for XOR activity utilizing a combination of [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. In the present study, we established and validated a novel human plasma XOR activity assay utilizing this technique. The calibration curve of [13C2,15N2]uric acid showed linearity over the range of 4-4000nM (r2>0.995) with a lower limit of quantitation of 4nM which corresponds to an XOR activity of 6.67pmol/h/mL plasma. Intra- and inter-assay coefficients of variation of pooled human plasma XOR activity were 6.5% and 9.1%, respectively. Plasma XOR activities of 20 healthy volunteers ranged from 32.8 to 227pmol/h/mL (mean±SD=89.1±55.1, n=20), which correlated with alanine transaminase (r=0.827), aspartate transaminase (r=0.487), and uric acid (r=0.502). The established assay is expected to be useful for investigating the function of XOR and the effect of its inhibitors in various diseases.

Multiple Time-of-Flight/Time-of-Flight Events in a Single Laser Shot for Improved Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry Quantification.

Quantitative matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) approaches have historically suffered from poor accuracy and precision mainly due to the nonuniform distribution of matrix and analyte across the target surface, matrix interferences, and ionization suppression. Tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity as well as improve signal-to-noise ratios by eliminating interferences from chemical noise, alleviating some concerns about dynamic range. However, conventional MALDI TOF/TOF modalities typically only scan for a single MS/MS event per laser shot, and multiplex assays require sequential analyses. We describe here new methodology that allows for multiple TOF/TOF fragmentation events to be performed in a single laser shot. This technology allows the reference of analyte intensity to that of the internal standard in each laser shot, even when the analyte and internal standard are quite disparate in m/z, thereby improving quantification while maintaining chemical specificity and duty cycle. In the quantitative analysis of the drug enalapril in pooled human plasma with ramipril as an internal standard, a greater than 4-fold improvement in relative standard deviation (<10%) was observed as well as improved coefficients of determination (R2) and accuracy (>85% quality controls). Using this approach we have also performed simultaneous quantitative analysis of three drugs (promethazine, enalapril, and verapamil) using deuterated analogues of these drugs as internal standards.

Establishment of replacement International Standard 13/132 for human antibodies to Toxoplasma gondii

Sixteen laboratories carried out a collaborative study to validate 13/132 as a replacement International Standard (IS) for TOXM (3rd IS for anti-Toxoplasma Serum, Human, 1000 IU). 13/132 is a freeze dried preparation of pooled human plasma from six donors who experienced a recent Toxoplasma gondii infection. The potency of 13/132 was compared to TOXM and 01/600 (1st IS for anti-Toxoplasma IgG, Human, 20 IU). Samples were tested for IgA, IgG, IgG avidity and IgM in agglutination assays; enzyme linked immunosorbent assays (ELISA), enzyme linked fluorescent assays, immunoblots, immunofluorescence assays and the Sabin–Feldman dye test for Ig. 13/132 was strongly positive for Ig, IgA, IgG and IgM and the reproducibility was very good. 13/132 contains high levels of anti-Toxoplasma Ig, IgG and IgM and its potency falls between TOXM and 01/600. The avidity of IgG was found to be low, similar to the avidity of IgG from TOXM. 13/132 was established by the Expert Committee on Biological Standardization as the 4th IS for Antibodies, Human, to T. gondii with an assigned unitage of 160 IU per ampoule for Ig by dye test and 263 U per ampoule for IgG by ELISA.

Viral nucleic acids in human plasma pools.

The identification of viruses in human blood is required for epidemiologic surveillance and to detect potentially emerging threats to blood transfusion safety. Viral nucleic acids in plasma fractionation pools assembled from blood donors in the United States and Europe were analyzed by viral metagenomics. Anelloviruses were detected in each of the 10 plasma pools. Human pegivirus A (HPgV; GB virus type C) sequences were identified in eight of the 10 pools, more than 90% of which belong to Genotype 2. The recently described human HPgV2 in Flaviviridae was not detected. A small number of sequence reads of human papillomavirus were also detected in three pools. In one pool, two different gemycircularvirus genomes were identified and fully sequenced. The capsid protein of one gemycircularvirus shared 83% to 84% identity to those of genomes from human serum and sewage. The presence of the gemycircularvirus genomes in the plasma pool was independently confirmed and the viral concentration estimated by digital PCR at more than 10(6) copies/mL assuming their origin from single donors. Further research is required to elucidate whether gemycircularviruses can infect humans or are indicative of contamination occurring during phlebotomy, plasma pool processing, or ongoing donor fungal infections.

171. Quantification of Human FVIII and Estimation of Its Molecular Weight: Potential Applications in Gene Therapy for Hemophilia A

Current protocols for measurement of FVIII rely on chromogenic or coagulation assays, and are not designed for quantification of FVIII antigen. Development of assays for measuring FVIII antigen may be necessary and beneficial for determining the efficiency of newer therapeutic approaches for Hemophilia A such as gene therapy. Herein, we describe the development of proprietary assays for quantification of human FVIII in plasma matrices from heterologous species, and estimation of the human FVIII molecular weight.A variety of monoclonal antibodies against the FVIII heavy and light chain were screened for their ability to recognize human FVIII from normal human plasma pool, or CHO cell derived recombinant human FVIII-B domain deleted (FVIII-BDD) spiked in mouse and non-human primate (NHP) plasma. Based on the initial screen, a monoclonal each against the human FVIII heavy or the light chain was selected for enrichment, followed by detection using a sheep anti-FVIII polyclonal antibody. Using this sandwich ELISA procedure, a standard curve was generated for human FVIII-BDD antigen in the range of 100 ng/ml - 0.1 ng/ml, and minimal cross reactivity was observed when tested with mouse and NHP plasma pools containing endogenous FVIII antigen. Furthermore, individual mouse strain plasma pools, and individual NHP samples representing different genetic origins were also screened for cross-reactivity to endogenous FVIII antigen and found to be negative. The sandwich ELISA was used to quantify FVIII antigen in defined plasma samples from normal or Hemophilia A human subjects, and antigen levels correlated with the reported FVIII activity.The ELISA procedure was modified and adapted for estimation of the human FVIII antigen molecular weight. FVIII heavy and light chains, from normal human plasma pool or recombinant human FVIII-B domain deleted (FVIII-BDD), were enriched followed by resolution using a denaturing reducing PAGE and western blotting. The apparent migration of heavy and light chain was at the appropriate molecular weights; 90-185 kDa for heavy chain and 70 kDa for light chain. In case of heavy chain from normal human plasma pool, a ladder of differing molecular weights was seen and may be indicative of the differential furin and thrombin dependent processing of the B-domain.Thus, these newly developed assays for quantification of human FVIII and estimation of its molecular weights will be useful for further characterization of gene therapy for Hemophilia A.

Colloidal Confinement of Polyphosphate on Gold Nanoparticles Robustly Activates the Contact Pathway of Blood Coagulation.

Platelet-sized polyphosphate (polyP) was functionalized on the surface of gold nanoparticles (GNPs) via a facile conjugation scheme entailing EDAC (N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride)-catalyzed phosphoramidation of the terminal phosphate of polyP to cystamine. Subsequent reduction of the disulfide moiety allowed for anchoring to the colloidal surface. The ability of the synthesized polyP-GNPs to initiate the contact pathway of clotting in human pooled normal plasma (PNP) was then assayed by quantifying changes in viscous, mechanical, and optical properties upon coagulation. It is revealed that the polyP-GNPs are markedly superior contact activators compared to molecularly dissolved, platelet-sized polyP (of equivalent polymer chain length). Moreover, the particles' capacity to mobilize Factor XII (FXII) and its coactivating proteins appear to be identical to very-long-chain polyP typically found in bacteria. These data imply that nanolocalization of anionic procoagulants on colloidal surfaces, achieved through covalent anchoring, may yield a robust contact surface with the ability to sufficiently cluster active clotting factors together above their threshold concentrations to cease bleeding. The polyP-GNPs therefore serve as a promising foundation in the development of a nanoparticle hemostat to treat a range of hemorrhagic scenarios.

Apoptotic Platelet Events Are Not Observed in Severe von Willebrand Disease-Type 2B Mutation p.V1316M.

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.

IVIG therapy in pemphigus vulgaris has corticosteroid-sparing and immunomodulatory effects.

Intravenous immunoglobulin (IVIG) is a biological agent composed of polyclonal antibodies prepared from a large cohort of human plasma pools. IVIG is increasingly used for the treatment of various antibody-mediated diseases, including pemphigus vulgaris (PV). The aim of the study was to evaluate the benefit and safety profiles of high dose IVIG therapy in PV patients determined by clinical remission, corticosteroid-sparing and immunomodulatory effects, and adverse events at 12 months' follow up. Ten PV patients underwent 3-8 monthly cycles of IVIG therapy at a dose of 2 g/kg per cycle. The pemphigus disease area index (PDAI) score, direct immunofluorescence (DIF), indirect immunofluorescence (IIF), and corticosteroid dosage were evaluated before IVIG therapy, after each cycle, and at 6 and 12 months' follow up. The baseline PDAI score was 75.70 ± 21.0 and baseline prednisone dosage was 201.60 ± 71.7 mg/day. The PDAI score reduction of 98% was achieved at 12 months' follow up and a corticosteroid dose reduction of 90% corresponded to clinical improvement. The decrease in both values was statistically significant (P = 0.002, respectively). At 12 months' follow up, seven patients were shown to be negative on IIF, of whom three proved to be negative on DIF. Adverse events were mild and transient and did not require the cessation of IVIG therapy. IVIG induced long-term clinical remission, while displaying a corticocorticosteroid-sparing effect and evoking a long-standing immunomodulatory effect in PV patients. The safety profile of IVIG therapy was assessed as good.

Method Development, Validation and Pharmacokinetics for Ramipril and Hydrochlorothiazide in Human Plasma and Application in a Bioequivalence Study Based on Healthy Jordanian Volunteers

A simple liquid chromatography mass spectrometric method was developed for quantitative determination of Ramipril (RAM) and Hydrochlorothiazide (HCTZ) simultaneously in human plasma. RAM, HCTZ and the internal standards (RAM-D5, HCTZ-13C, D2) were extracted from human plasma by direct protein precipitation. The chromatographic separation was achieved using ACE 5 C8, (50 mm x 4.6 mm, 5µm) column with gradient elution mobile phase of 0.25 mM Ammonium chloride and methanol. The method validation intends to investigate sensitivity, linearity, precision and accuracy, recovery, matrix effect, specificity and stability according to the European Medicines Agency (EMA) guideline. Standard calibration levels were prepared in pooled human plasma to attain final dynamic range of 0.2 - 20.0 ng/mL for RAM and 1.0 - 150 ng/mL for HCTZ. Clinical bioequivalence study was successfully investigated by application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products for 5mg RAM/ 25mg Hydrochlorothiazide single dose.

6-Deoxy-6-aminoethyleneamino cellulose: synthesis and study of hemocompatibility

Hemocompatibility of aqueous solutions of antimicrobial 6-deoxy-6-aminoethyleneamino (AEA) cellulose with different degrees of substitution (DS, 0.54–0.92) was investigated in vitro. The AEA cellulose derivatives were synthesized by tosylation of cellulose and subsequent nucleophilic substitution with 1,2-diaminoethane. The structure was confirmed by elemental analysis as well as by FTIR and NMR spectroscopies. Markers for coagulation (thrombin generation, aPTT, PT, blood clotting, thrombocyte activation) and membrane integrity (hemolysis) were measured in human whole blood, human platelet-rich plasma, human pooled plasma, and erythrocytes suspension. AEA cellulose with a low DS of 0.54 showed the highest hemocompatibility in vitro, suggesting the possibility of biomedical applications.

Biomonitoring of perfluorinated compounds in a drop of blood.

Biomonitoring of pollutants and their metabolites and derivatives using biofluids provides new opportunities for spatiotemporal assessment of human risks to environmental exposures. Perfluorinated compounds (PFCs) have been used widely in industry and pose significant environmental concerns due to their stability and bioaccumulation in humans and animals. However, current methods for extraction and measurement of PFCs require relatively large volumes (over one hundred microliters) of blood samples, and therefore, are not suitable for frequent blood sampling and longitudinal biomonitoring of PFCs. We have developed a new microassay, enabled by our silicon microfluidic chip platform, for analyzing PFCs in small volumes (less than five microliters) of blood. Our assay integrates on-chip solid-phase extraction (SPE) with online nanoflow liquid chromatography-electrospray ionization-mass spectrometry (nanoLC-ESI-MS) detection. We demonstrated high sample recovery, excellent interday and intraday accuracy and precision, and a limit of detection down to 50 femtogram of PFCs, in one microliter of human plasma. We validated our assay performance using pooled human plasma and NIST SRM 1950 samples. Our microfluidic chip-based assay may enable frequent longitudinal biomonitoring of PFCs and other environmental toxins using a finger prick of blood, thereby providing new insights into their bioaccumulation, bioavailability, and toxicity.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

IntroductionFetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity.MethodsSCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation.ResultsSCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons.ConclusionsThe tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.