Individual Plasma Samples Research Articles

Sensibilidad del equipo Cobas AmpliScreen™ HIV-1 Test, v1.5, para la detección de HIV-1

The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants.

Low‐abundance plasma protein differences between African‐American and Caucasian early pubertal females following supplementation with 9 mg Zn (1043.10)

The purpose of this research was to determine whether supplementation with oral zinc levels near the RDA for 4 wks would affect plasma protein levels in African‐American (AA) and Caucasian (C) early pubertal females. The subjects were given either 9 mg elemental zinc (n=75; 40 C, 35 AA) or placebo (n=71; 40 C, 31 AA) to consume for 4 wks. Plasma was collected from subjects at baseline (BL) and 4 wks, and was analyzed for zinc by atomic absorption. Plasma from subjects that were most responsive to zinc supplementation (BL to 4wk; n=5), or exhibited the least difference between BL and 4wk (placebo; n=5) were analyzed. Individual plasma samples were prefractionated on immunoaffinity columns to remove 14 high‐abundant plasma proteins. Effluent proteins were separated by two‐dimensional gel electrophoresis (400 µg; 15% acrylamide 2nd dimension). The gels were stained with Coomassie blue G‐250 and analyzed by densitometry. Significant differences were determined by Student’s t‐test. Comparisons were made between untreated (UnTrt; n=15) vs. zinc‐treated (ZnTrt; n=5) plasma. Two proteins were affected by zinc consumption, 33.8kDa/pI 5.4 (UnTrt C: 0.70±0.11 (SEM), ZnTrt C: 1.56±0.46 (SEM; p=0.02); UnTrt AA: 0.73±0.10 (SEM), ZnTrt AA: 0.64±0.24 (SEM; p=0.67) and 32.4kDA/pI 5.8 (UnTrt C: 1.14±0.22 (SEM), ZnTrt C: 1.58±0.18 (SEM; p=0.16); UnTrt AA: 1.97±0.22(SEM), ZnTrt AA: 1.02±0.28 (SEM; p=0.03). These zinc‐responsive proteins were affected by the race of the supplemented individuals, suggesting there are subtle differences in the zinc metabolism of individuals from different racial or cultural backgrounds.Grant Funding Source: Supported by USDA Hatch GEO00682

Assessment of Reproductive Function in Southern African Spiny Mice (Acomys spinosissimus) using Faeces as Hormone Matrix

For spiny mice, detailed long-term data on reproductive endocrine function is lacking, as limited blood volumes make frequent collections of individual plasma samples for hormone monitoring impractical. To provide the necessary tools in order to fill this gap, this study examined the suitability of two enzyme immunoassays for monitoring male and female reproductive function in the southern African spiny mouse (Acomys spinosissimus) based on faecal hormone analysis. Fourteen non-pregnant and one pregnant female and 24 male spiny mice were wild-caught and subsequently monitored under controlled conditions. Thirteen of the 14 non-pregnant females revealed elevated faecal progestagen metabolite concentrations with eight individuals showing indications of a luteal phase. Two females showed two post-ovulatory luteal phases with estimated cycle lengths of 16 and 18 days, respectively. The pregnant female had an elevated (231%) mean faecal progestagen metabolite level compared with the overall mean baseline hormone concentration determined for the 14 non-pregnant females. Males exposed to a long photoperiod, simulating summer-related breeding activity, exhibited a 47.8% increase in faecal androgen metabolite levels compared with males exposed to a short photoperiod. Collectively, the data demonstrated that reproductive endocrine function can be monitored in male and female spiny mice by measuring respective faecal hormone metabolites.

Assessment of reproductive function in southern African spiny mice (Acomys spinosissimus) using faeces as hormone matrix

For spiny mice, detailed long-term data on reproductive endocrine function is lacking, as limited blood volumes make frequent collections of individual plasma samples for hormone monitoring impractical. To provide the necessary tools in order to fill this gap, this study examined the suitability of two enzyme immunoassays for monitoring male and female reproductive function in the southern African spiny mouse (Acomys spinosissimus) based on faecal hormone analysis. Fourteen non-pregnant and one pregnant female and 24 male spiny mice were wild-caught and subsequently monitored under controlled conditions. Thirteen of the 14 non-pregnant females revealed elevated faecal progestagen metabolite concentrations with eight individuals showing indications of a luteal phase. Two females showed two post-ovulatory luteal phases with estimated cycle lengths of 16 and 18 days, respectively. The pregnant female had an elevated (231%) mean faecal progestagen metabolite level compared with the overall mean baseline hormone concentration determined for the 14 non-pregnant females. Males exposed to a long photoperiod, simulating summer-related breeding activity, exhibited a 47.8% increase in faecal androgen metabolite levels compared with males exposed to a short photoperiod. Collectively, the data demonstrated that reproductive endocrine function can be monitored in male and female spiny mice by measuring respective faecal hormone metabolites.

Role of beta‐defensin 2 and interleukin‐4 receptor as stroke outcome biomarkers

Acute ischemic stroke is a complex disease with huge interindividual evolution variability that makes challenging the prediction of an adverse outcome. Our aim was to study the association of bloodstream signatures to early neurological outcome after stroke, by combining a subpooling of samples strategy with protein array discovery approach. Plasma samples from 36 acute stroke patients (< 4.5 h from onset) were equally pooled within outcome groups: worsening, stability, and improvement (n = 3 pools of four patients each, for each outcome group). These nine pools were screened using a 177 antibodies library, and 35 proteins were found altered regarding outcome classification (p < 0.1). Processes of inflammation, immune response, coagulation, and apoptosis were regulated by these proteins. Ten representative candidates, mainly cytokines and chemokines, were assayed for replication in individual baseline plasma samples from 80 new stroke patients: β-defensin2, MIP-3b, plasminogen activator inhibitor 1 active, β-cell-attracting chemokine 1, Exodus-2, interleukin-4 receptor (IL-4R), IL-12p40, leukemia inhibitor factor, MIP-1b, and tumor necrosis factor-related weak inducer of apoptosis. Multivariate logistic regression analysis showed β-defensin 2 (ORadj 4.87 [1.13-20.91] p = 0.033) and IL-4R (ORadj 3.52 [1.03-12.08] p = 0.045) as independent predictors of worsening at 24 h after adjustment by clinical variables. Both biomarkers improve the prediction by 19% as compared to clinical information, suggesting a potential role for risk stratification in acute thrombolyzed stroke patients. Early neurological deterioration after stroke is not easily predictable. The use of blood biomarkers might help in decision-making processes regarding this complication. By combining a sub-pooling of samples strategy with protein array discovery approach, we have found two new biomarkers: beta-defensin-2 and interleukin-4 receptor. Both biomarkers improve the prediction of poor-outcome over clinical variables in the acute phase of stroke.

Abstract C196: EGFR mutation detection in ctDNA isolated from NSCLC patient plasma; a cross-platform comparison of leading technologies.

Abstract Emerging evidence suggests that circulating tumor DNA (ctDNA) provides an alternative diagnostic sample when surgical biopsies are inaccessible, and could enable disease diagnosis, frequent assessment of disease progression, and almost real-time monitoring of the emergence of resistance mutations. We hypothesized that novel highly sensitive technologies enable EGFR T790M mutation detection in ctDNA from patients that had previously been treated with and become resistant to EGFR-TKIs. We have used 3 technologies to assess T790M mutation detection rate. Samples comprised patients with sensitizing EGFR mutations and wild type EGFR patients who had been previously treated with EGFR TKI therapies. In approximately 50% of EGFR mutation positive cases, acquired resistance to EGFR-TKIs is mediated by an EGFR T790M mutation. Reference data for T790M mutations from tumor material was unavailable.We undertook a comprehensive cross-technology comparison of three technology platforms: ARMS based detection using the Roche cobas EGFR mutation detection kit; digital droplet PCR using the BioRad ddPCR instrument (by MolecularMD) and bead based digital PCR using the Inostics BEAMing technology for the detection of T790M mutations. In total, 135 frozen plasma samples, obtained from 2 clinical studies, were used to estimate T790M detection rates in ctDNA. Within the study group, 72 samples were taken from EGFR mutation positive patients, of which around 36 (expected range 28-44) would be expected to have T790M mutations, the remaining samples would be expected to be T790M negative. Each individual patient plasma sample was split and evaluated across multiple platforms. In addition, the suitability of 3 ctDNA preparation kits (cobas Circulating DNA Isolation kit, QIAamp Circulating Nucleic Acid kit and QIAamp DNA Mini kit) in terms of DNA yield was evaluated. The QIAamp Circulating Nucleic Acid kit and cobas Circulating DNA Isolation kit were equally effective in preparing ctDNA from plasma samples. Overall, concordance of EGFR T790M mutation status was high between all 3 methods. In addition, the false positive rate in the known EGFR mutation negative cases was low for all 3 methods. However, differences were seen in the rate of false negative results (assay sensitivity) between methods. DNA preparation kits specifically designed for use with ctDNA samples are preferable to other methods. Current technologies for the detection of T790M mutations in ctDNA, where patient tumor material is not available, still require further development to increase detection rates in these samples. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C196. Citation Format: Helen Brown, Roz Brant, Simon Dearden, Suzanne Jenkins, Kenneth Thress, Alain Horvais, Ruth March, J. Carl Barrett. EGFR mutation detection in ctDNA isolated from NSCLC patient plasma; a cross-platform comparison of leading technologies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C196.

A stable isotope-labeled internal standard is essential for correcting for the interindividual variability in the recovery of lapatinib from cancer patient plasma in quantitative LC–MS/MS analysis

The development and validation of a LC-MS/MS method is often performed using pooled human plasma, which may fail to account for variations in interindividual matrices. Since calibrator standards and quality control samples are routinely prepared in pooled human plasma, variations in the extraction recovery and/or matrix effect between pooled plasma and individual patient plasma can cause erroneous measurements. Using both pooled human plasma as well as individual healthy donor and cancer patient plasma samples, we evaluated the analytical performance of two classes of internal standards (i.e., non-isotope-labeled and isotope-labeled) in the quantitative LC-MS/MS analysis of lapatinib. After exhaustive extraction with organic solvent, the recovery of lapatinib, a highly plasma protein-bound drug, varied up to 2.4-fold (range, 29-70%) in 6 different donors of plasma and varied up to 3.5-fold (range, 16-56%) in the pretreatment plasma samples from 6 cancer patients. No apparent matrix effects were observed for lapatinib in both pooled and individual donor or patient plasma samples. The calibration curve range was 5-5000ng/ml of lapatinib in plasma. Both the non-isotope-labeled (zileuton) and isotope-labeled (lapatinib-d3) internal standard methods showed acceptable specificity, accuracy (within 100±10%), and precision (<11%) in the determination of lapatinib in pooled human plasma. Nevertheless, only the isotope-labeled internal standard could correct for the interindividual variability in the recovery of lapatinib from patient plasma samples. As inter- and intra-patient matrix variability is commonly presented in the clinical setting, this study provides an example underscoring the importance of using a stable isotope-labeled internal standard in quantitative LC-MS/MS analysis for therapeutic drug monitoring or pharmacokinetic evaluation.

Normal prion protein is expressed on exosomes isolated from human plasma

Ritchie, Adam J Crawford, Damian M Ferguson, David J P Burthem, John Roberts, David J England Br J Haematol. 2013 Dec;163(5):678-80. doi: 10.1111/bjh.12543. Epub 2013 Sep 30.

Liquid chromatography–mass spectrometric determination of rufinamide in low volume plasma samples

Quantification of rufinamide in plasma was achieved using a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method. The chromatographic separation was achieved on a reversed phase column (Zorbax SB-C18 100mm×3mm, 3.5μm) under isocratic conditions. The mobile phase consisted of a mixture of water containing 0.1% formic acid and methanol (50:50, v/v). The mass spectrometric detection of the analyte was in multiple reaction monitoring mode (MRM) using an electrospray positive ionization (ESI positive). The monitored ions were 127m/z derived from 239m/z rufinamide and 108m/z derived from 251m/z the internal standard (lacosamide). Protein precipitation with methanol was applied for sample preparation using only 50μl aliquots. The concentration range was 40-2000ng/ml for rufinamide in plasma. The limit of detection was 1.25ng/ml and the lower limit of quantification was established at 5ng/ml rufinamide concentration. Selectivity and matrix effect was verified using individual human, rat and rabbit plasma samples. Short-term, post-preparative and freeze-thaw stability was also investigated. The proposed method provides accuracy, precision and high-throughput (short runtime 4.5min) for quantitative determination of rufinamide in plasma. This is the first reported liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for analysis of rufinamide from low volume plasma samples. The LC-MS/MS method was validated according to the current official guidelines and can be applied to accurately measure rufinamide level of large number of plasma samples from clinical studies or therapeutic drug monitoring.

A Novel Soluble Form of Tim-3 Associated with Severe Graft-versus-Host Disease

The T cell Ig and mucin domain 3 (Tim-3) receptor has been implicated as a negative regulator of adaptive immune responses. We have utilized a proteomic strategy to identify novel proteins associated with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Mass spectrometry analysis of plasma from subjects with mid-gut and upper-gut GVHD compared with those without GVHD identified increased levels of a protein identified with high confidence as Tim-3. A follow-up validation study using an immunoassay to measure Tim-3 levels in individual plasma samples from 127 patients demonstrated significantly higher plasma Tim-3 concentrations in patients with the more severe mid-gut GVHD, compared with those with upper-gut GVHD (P = .005), patients without GVHD (P = .002), and normal controls (P < .0001). Surface expression of Tim-3 was increased on CD8+ T cells from patients with grade 2 to 4 acute GVHD (P = .01). Mass spectrometry–based profiling of plasma from multiple subjects diagnosed with common diseases provided evidence for restricted release of soluble Tim-3 in the context of GVHD. These findings have mechanistic implications for the development of novel strategies for targeting the Tim-3 immune regulatory pathway as an approach to improving control of GVHD.

AB0303 Development and optimization of sensitive peptide elisa for autoantibody testing in early and seronegative rheumatoid arthritis

BackgroundUsing a cDNA phage display approach, 14 novel autoantibody markers were previously discovered that can be detected in early and seronegative (rheumatoid factor-negative, anti-cyclic-citrullinated-peptides-negative) RA-patients1. The level of autoantibody reactivity...

Alterations in molecular status of plasma fibronectin associated with aging of normal human individuals

ObjectivesSenescence, progressive deterioration of many bodily functions might be associated with age-dependent alterations of plasma fibronectin (FN) molecular status (i.e., domain, glycotope, and molecular form expressions). Design and methodsFN molecular status was analyzed in 127 plasma samples of healthy individuals in groups of newborns, and subjects aged 3–14, 15–39, 41–59, and 60–82years by FN-ELISA, lectin-FN-ELISA, and immunoblotting using a set of domain-specific monoclonal antibodies, specific lectins, and monoclonal antibody to FN, respectively. ResultsDuring the first four decades of human life the levels of cell-binding-, carboxyl-terminal-, collagen-, heparin-, and fibrin-domains of plasma FN gradually increased.In subjects aged up to 82years the cell-binding and carboxyl-terminal FN domain concentrations did not change, while the heparin, fibrin, and collagen domains significantly increased.The relative reactivity of plasma FN with Maackia amurensis lectin, specific to α2,3-linked sialic acid, significantly decreased after birth, reaching a stable level in the subsequent life period, whereas with Sambucus nigra lectin, specific to α2,6-linked sialic acid, it significantly decreased in the 60–82year old group. Moreover, the appearance of 280-kDa and 320-kDa FN bands, absent in young and mature healthy individuals, was found in the groups of 41–59 and 60–82year olds. ConclusionsThe alterations of FN molecular status throughout growth, maturation and senescence might be associated not only with disturbances in the balance of FN production rate and degradation, but concomitantly with conformational rearrangements of FN and its engagement in age-related vascular remodeling processes.

Viral Hepatitis and HIV Infection Among Injection Drug Users in a Central Iranian City

This study aimed to determine the prevalence of serological markers for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and occult HBV infection among injection drug users (IDUs) with isolated anti-hepatitis B core (anti-HBc). A total of 153 male IDUs were tested for anti-hepatitis B surface (anti-HBs), hepatitis B surface antigen (HBsAg), anti-HBc, anti-HCV, and anti-HIV. The presence of HBV-DNA was determined in plasma samples of individuals with isolated anti-HBc (HBsAg negative, anti-HBs negative, and anti-HBc positive) by polymerase chain reaction (PCR). The prevalence of markers for viral hepatitis and HIV infections was 59.5% for anti-HCV, 44.4% for anti-HBs, 22.9% for anti-HBc, 7.2% for HBsAg, and 5.9% for anti-HIV. Several markers for coinfection, including HBV-HCV (5.9%), HCV-HIV (5.2%), HBV-HIV (2.0%), and HBV-HCV-HIV (1.3%), were present. Of the 7.2% of IDUs with isolated anti-HBc, all were anti-HCV positive and 18.2% were anti-HIV positive; however, no cases had detectable HBV-DNA as a marker of occult infection. Markers for HCV, HBV, HIV, and combinations of these infections were common among IDUs in a city of central Iran. Isolated anti-HBc was associated with HCV but not with occult HBV infection in this sample. The 10-fold higher prevalence of HCV than HIV infection may be a harbinger of increasing HIV among IDUs in this area.

Parvovirus B19V DNA contamination in Chinese plasma and plasma derivatives

BackgroundTo ensure the safety of plasma derivatives, screening for human parvovirus B19V genomic DNA in donated plasma using a pooling strategy is performed in some countries. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Chinese plasma pools, plasma derivatives and plasma donations to evaluate the risk posed by B19V.MethodsUsing a Q-PCR assay developed in-house, we tested for B19V genomic DNA in 142 plasma pools collected between January 2009 and June 2011 from two Chinese blood products manufacturers. Plasma derivatives collected between 1993–1995 (10 batches of albumin, 155 batches of intravenous immunoglobulin, IVIG) and 2009–2011 (50 batches of albumin, 54 batches of IVIG, 35 batches of factor VIII, 7 batches of fibrinogen, and 17 batches of prothrombin complex concentrate, PCC) were also tested for B19V contamination. In addition, B19V genome prevalence in minipools(including 90 individual donations) of 49680 individual plasma samples collected between August 2011 and March 2012 by a single Chinese manufacturer was investigated. IgM/IgG was also investigated in plasma pools/derivatives and in minipools with B19V-DNA titers above 1x104 and 1x106 geq/mL using B19 ELISA IgM/IgG assay(Virion-Serion, Würzburg, Germany), respectively.ResultsB19V-DNA was detected in 54.2% of plasma pools from two Chinese blood product manufacturers; among recently produced blood products, B19V was detected in 21/54 IVIG samples, 19/35 factor VIII samples, 6/7 fibrinogen samples, and 12/17 PCC samples, but not in albumin samples. The levels of B19V-DNA in these samples varied from 102-107 geq/mL. In samples with >104 geq/mL genome DNA, B19V-specific IgG was also found in all corresponding plasma pools and IVIG, whereas none was detected in the majority of other plasma derivatives. Screening of plasma donations indicated that most minipools were contaminated with B19V-DNA (102-108 geq/mL) and one donation had 1.09 × 1010 geq/mL B19V genomic DNA along with a non-classical IgG/IgM profile.ConclusionsDespite the implementation of some inactivation/removal methods designed to prevent viral contamination, B19V DNA was detectable in Chinese plasma pools and plasma derivatives. Thus, the introduction of B19V screening and discard donation with high viramic concentration for Chinese plasma donors would be desirable.

Quinine interactions with tryptophan and tyrosine in malaria patients, and implications for quinine responses in the clinical setting

Recent work with the yeast model revealed that the antiprotozoal drug quinine competes with tryptophan for uptake via a common transport protein, causing cellular tryptophan starvation. In the present work, it was hypothesized that similar interactions may occur in malaria patients receiving quinine therapy. A direct observational study was conducted in which plasma levels of drug and amino acids (tryptophan, tyrosine and phenylalanine) were monitored during quinine treatment of malaria patients with Plasmodium falciparum infections. Consistent with competition for uptake from plasma into cells, plasma tryptophan and tyrosine levels increased ≥2-fold during quinine therapy. Plasma quinine levels in individual plasma samples were significantly and positively correlated with tryptophan and tyrosine in the same samples. Control studies indicated no effect on phenylalanine. Chloroquine treatment of Plasmodium vivax-infected patients did not affect plasma tryptophan or tyrosine. During quinine treatment, plasma tryptophan was significantly lower (and quinine significantly higher) in patients experiencing adverse drug reactions. Plasma quinine levels during therapy are related to patient tryptophan and tyrosine levels, and these interactions can determine patient responses to quinine. The study also highlights the potential for extrapolating insights directly from the yeast model to human malaria patients.

Dengue virus inactivation by minipool TnBP/Triton X‐45 treatment of plasma and cryoprecipitate

A minipool solvent/detergent (S/D; 1% TnBP/1% Triton X-45; 31°C) process was developed for viral inactivation of plasma and cryoprecipitate used for transfusion. The goal of this study was to determine the rate and extent of inactivation of dengue virus (DENV) during this process. DENV-1 was propagated using C6/36 mosquito cells to an infectivity titre close to 9 log and spiked (10% v/v) into individual plasma and cryoprecipitate samples from two distinct donors. Samples were taken right after spiking and during viral inactivation treatment by 1% TnBP-1% Triton X-45 at 31°C. DENV-1 infectivity was assessed on Vero E6 cells by a focus-forming assay (FFA). Culture medium and complement-inactivated plasma were used as experimental controls. Experiments were done in duplicate. DENV-1 infectivity was 7·5 log in spiked plasma and 7·1 and 7·3 log in spiked cryoprecipitate. There was no loss of DENV-1 infectivity in the spiked materials, nor in the controls not subjected to S/D treatment. No infectivity was found in plasma and cryoprecipitate subjected to S/D treatment at the first time-point evaluated (10 min). DENV-1 was strongly inactivated in plasma and cryoprecipitate, respectively, within 10 min of 1% TnBP/1% Triton X-45 treatment at 31°C. These data provide a reassurance of the safety of such S/D-treated plasma and cryoprecipitate with regard to the risk of transmission of all DENV serotypes and other flaviviruses.

Specificity Characteristics of 7 Commercial Creatinine Measurement Procedures by Enzymatic and Jaffe Method Principles

Standardized calibration does not change a creatinine measurement procedure's susceptibility to potentially interfering substances. We obtained individual residual serum or plasma samples (n = 365) from patients with 19 different disease categories associated with potentially interfering substances and from healthy controls. Additional sera at 0.9 mg/dL (80 μmol/L) and 3.8 mg/dL (336 μmol/L) creatinine were supplemented with acetoacetate, acetone, ascorbate, and pyruvate. We measured samples by 4 enzymatic and 3 Jaffe commercially available procedures and by a liquid chromatography/isotope dilution/mass spectrometry measurement procedure against which biases were determined. The number of instances when 3 or more results in a disease category had biases greater than the limits of acceptability was 28 of 57 (49%) for Jaffe and 14 of 76 (18%) for enzymatic procedures. For the aggregate group of 59 diabetes samples with increased β-hydroxybutyrate, glucose, or glycosylated hemoglobin (Hb A(1c)), the enzymatic procedures had 10 biased results of 236 (4.2%) compared with 89 of 177 (50.3%) for the Jaffe procedures, and these interferences were highly procedure dependent. For supplemented sera, interferences were observed in 11 of 24 (46%) of groups for Jaffe and 8 of 32 (25%) of groups for enzymatic procedures and were different at low or high creatinine concentrations. There were differences in both magnitude and direction of bias among measurement procedures, whether enzymatic or Jaffe. The influence of interfering substances was less frequent with the enzymatic procedures, but no procedure was unaffected. The details of implementation of a method principle influenced its susceptibility to potential interfering substances.

A lectin-coupled, multiple reaction monitoring based quantitative analysis of human plasma glycoproteins by mass spectrometry

Aberrant protein glycosylation may be closely associated with cancer pathology. To measure the abundance of protein glycoforms with a specific glycan structure in plasma samples, we developed a lectin-coupled multiple reaction monitoring (MRM)-based mass spectrometric method. It was confirmed that the method could provide reproducible results with precision sufficient to distinguish differences in the abundance of protein glycoforms between individuals. Plasma samples prepared from hepatocellular carcinoma (HCC) patients without immuno-depletion of highly abundant plasma proteins were fractionated by use of fucose-specific aleuria aurantia lectin (AAL) immobilized on magnetic beads by use of a biotin-streptavidin conjugate. The lectin-captured fractions were digested by trypsin and profiled by tandem mass spectrometry. From the proteomic profiling data, target glycoproteins were selected and analyzed quantitatively by MRM-based analysis. The reproducibility of MRM-based quantification of the selected target proteins was reliable, with precision (CV; ≤14% for batch-to-batch replicates and ≤19% for replicates over three days) sufficient to distinguish differences in the abundance of AAL-captured glycoforms between individual plasma samples. This lectin-coupled, MRM-based method, measuring only lectin-captured glycoforms of a target protein rather than total target protein, is a tool for monitoring differences between individuals by measuring the abundance of aberrant glycoforms of a target protein related to a disease. This method may be further applied to rapid verification of biomarker candidates involved in aberrant protein glycosylation in human plasma.

Plasma Biomarkers for Detecting Hodgkin's Lymphoma in HIV Patients

The lifespan of people with human immunodeficiency virus (HIV) infection has increased as a result of effective antiretroviral therapy, and the incidences of the AIDS-defining cancers, non-Hodgkin's lymphoma and Kaposi sarcoma, have declined. Even so, HIV-infected individuals are now at greater risk of other cancers, including Hodgkin's lymphoma (HL). To identify candidate biomarkers for the early detection of HL, we undertook an accurate mass and elution time tag proteomics analysis of individual plasma samples from either HIV-infected patients without HL (controls; n = 14) and from HIV-infected patient samples with HL (n = 22). This analysis identified 60 proteins that were statistically (p<0.05) altered and at least 1.5-fold different between the two groups. At least three of these proteins have previously been reported to be altered in the blood of HL patients that were not known to be HIV positive, suggesting that these markers may be broadly useful for detecting HL. Ingenuity Pathway Analysis software identified “inflammatory response” and “cancer” as the top two biological functions associated with these proteins. Overall, this study validated three plasma proteins as candidate biomarkers for detecting HL, and identified 57 novel candidate biomarkers that remain to be validated. The relationship of these novel candidate biomarkers with cancer and inflammation suggests that they are truly associated with HL and therefore may be useful for the early detection of this cancer in susceptible populations.

Quantitative Mass Spectrometry Analysis Using PAcIFIC for the Identification of Plasma Diagnostic Biomarkers for Abdominal Aortic Aneurysm

BackgroundAbdominal aortic aneurysm (AAA) is characterized by increased aortic vessel wall diameter (>1.5 times normal) and loss of parallelism. This disease is responsible for 1–4% mortality occurring on rupture in males older than 65 years. Due to its asymptomatic nature, proteomic techniques were used to search for diagnostic biomarkers that might allow surgical intervention under nonlife threatening conditions.Methodology/Principal FindingsPooled human plasma samples of 17 AAA and 17 control patients were depleted of the most abundant proteins and compared using a data-independent shotgun proteomic strategy, Precursor Acquisition Independent From Ion Count (PAcIFIC), combined with spectral counting and isobaric tandem mass tags. Both quantitative methods collectively identified 80 proteins as statistically differentially abundant between AAA and control patients. Among differentially abundant proteins, a subgroup of 19 was selected according to Gene Ontology classification and implication in AAA for verification by Western blot (WB) in the same 34 individual plasma samples that comprised the pools. From the 19 proteins, 12 were detected by WB. Five of them were verified to be differentially up-regulated in individual plasma of AAA patients: adiponectin, extracellular superoxide dismutase, protein AMBP, kallistatin and carboxypeptidase B2.Conclusions/SignificancePlasma depletion of high abundance proteins combined with quantitative PAcIFIC analysis offered an efficient and sensitive tool for the screening of new potential biomarkers of AAA. However, WB analysis to verify the 19 PAcIFIC identified proteins of interest proved inconclusive save for five proteins. We discuss these five in terms of their potential relevance as biological markers for use in AAA screening of population at risk.