Sensitive Quantitative Assessment Research Articles

Cefoperazone rapidly and sensitive quantitative assessment via a validated RP-HPLC method for different dosage forms, in-use stability, and antimicrobial activities

Cefoperazone (Cfz) is a member of the third generation of parenteral cephalosporin antibiotics. It is used on a wide scale in prescribed antibiotic drugs as anti-infection, especially for Gram-negative and also against Gram-positive microorganisms. The current study aimed to find a rapid RP-HPLC method of Cfz analysis with high linearity, repeatability, sensitivity, selectivity, and inexpensive. In our developed method, there is no need to use special chemical reagents, a high percentage of organic solvent, a high flow rate, further guard column. The chromatographic system comprises an ODS column (150 mm × 4.6 mm × 5 μm). The mobile phase was prepared by mixing KH2PO4 solution: acetonitrile (80:20) with a flow rate of 1.0 mL/min at detection wavelength 230 nm, at room temperature using injection volume 20 μL. The method manifested a satisfied linearity regression R2 (0.9993) with a good repeatability range (0.34–0.92%) with LOD and LOQ; 4.04 μg/mL and 12.24 μg/mL respectively. The method proved its efficiency via system suitability achievement in the robustness and ruggedness conduction according to the validation guidelines. The shorter analysis time makes the method very valuable in quality control to quantify the commercial Cfz in pharmaceutical preparations. This improved HPLC method has been successfully applied for Cfz analysis for Peracef and Peractam vials in our routine finished and stability studies testing laboratories. Additionally, the detection limit of Cfz has been tested in our quality control lab to detect the smallest amount of traces that may be present after the cleaning process of the production machines for cephalosporins preparations. In a precedent for the first time, we were able to use the current analysis method to determine the minimum inhibitory concentration (MIC) and minimum bacteriostatic concentration (MBC). The conventional broth micro-dilution tube method was used to determine MIC at 250 µg/mL and MBC at 125 µg/mL of Cfz against the standard strain of Burkholderia cepacia (B. cepacia) ATCC 25416 as Gram-negative bacteria in vitro.

Rapidly, sensitive quantitative assessment of thiopental via forced stability indicating validated RP-HPLC method and its in-use stability activities

Thiopental sodium (Tho) is an intravenous anesthetic. The current study aimed to find a rapid RP-HPLC method of Tho analysis with high linearity, repeatability, sensitivity, selectivity, and inexpensive. In our developed method, there is no need to use special chemical reagents, a high percentage of organic solvent, a high flow rate, or a further guard column. The chromatographic system consists of an ODS column (150 mm × 4.6 mm × 5 μm). The mobile phase was prepared by mixing KH2PO4 solution: methanol (40:60) with a flow rate of 1.2 mL/min at a detection wavelength of 230 nm, at room temperature using an injection volume of 10 μL. The method manifested a satisfied linearity regression R2 (0.9997) with a good repeatability precision range (0.16–0.47%) with LOD and LOQ; 14.4 μg/mL and 43.6 μg/mL respectively. Additionally, the method proved its efficiency via system suitability achievement in robustness and ruggedness, according to the validation guidelines. The shorter analysis time makes the method very valuable in quality control to quantify the commercial Tho in pharmaceutical preparations. This improved HPLC method has been successfully applied for Tho analysis for Thiopental UP Pharma 500 mg vials and Thiopental Eipico 1.0 g vials in our routine finished and stability studies testing laboratories. Additionally, the detection limit of Tho has been tested in our quality control lab to detect the smallest amount of traces that may be present after the cleaning process of the production machines for cephalosporins preparations. The method has shown positive results for Tho in low-level raw materials and pharmaceutical formulations.

The Multiple Object Test as a performance-based tool to assess the decline of ADL function in Parkinson's disease.

IntroductionAs cognitive-driven worsening of activities of the daily living (ADL) in Parkinson’s disease (PD) is the core feature of PD dementia (PDD), there is great need for sensitive quantitative assessment. Aim of our study was the evaluation of cognitive-driven worsening of ADL by the performance-based Multiple Object Test (MOT), offering an essential clinical advantage as it is quick and easy to apply in a clinical context even on severely impaired patients.Methods73 PD patients were assessed longitudinally over a period of 37 (6–49) months. According to their neuropsychological profile the sample was divided into two groups: PD patients with (n = 34, PD-CI) and without cognitive impairment (n = 39, PD-noCI). The MOT comprises five routine tasks (e.g. to make coffee) quick and easy to apply. Quantitative (total error number, processing time) and qualitative parameters (error type) were analyzed using non-parametric test statistic (e.g.Wilcoxon signed-rank test, binary logistic regression).ResultsMedian number of total errors (p = 0.001), processing time (p<0.001), perplexity (p = 0.035), and omission errors (p<0.001) increased significantly from baseline to follow-up in the total sample. Worsening of MOT performance was correlated to cognitive decline in the attention/ executive function and visuo-constructive domain. PD-CI showed an increase in omission errors (p = 0.027) compared to PD-noCI over time. This increase in omission errors between visits was further identified as a risk marker for PDD conversion.ConclusionThe MOT, especially frequency of omission errors, is a promising tool to rate PD patients objectively and might help to identify patients with a high risk for having mild cognitive impairment or dementia.

Quantification of Bacterial Uropathogens in Preclinical Samples Using Real-Time PCR Assays

Uropathogenic Escherichia coli (UPEC) and Staphylococcus saprophyticus (S. saprophyticus) are responsible for the majority of community-acquired urinary tract infections (UTI). Agar plating, a gold standard for detection of bacterial uropathogens, is labor intensive, limited for distinguishing between environmental contaminants and pathogens, and fails to effectively detect mixed infections. A reliable method for specific and sensitive quantitative assessment of infections would allow cost-effective evaluation of large numbers of experimental samples. A methodology such as quantitative PCR (qPCR) addresses the limitations of agar plating. We developed and validated highly specific and sensitive qPCR assays to assist researchers in the evaluation of potential vaccines and interventions in preclinical models of UPEC and S. saprophyticus UTI. The developed UPEC PCR targeted a highly conserved region of the UPEC hemolysin D (hlyD) gene that reproducibly detected type strains CFT073 and J96 over a 9 log range with high precision. To quantify S. saprophyticus genomes, a separate qPCR assay targeting the Trk transport gene was developed with an 8 log range. Neither assay detected bacterial species predicted to be sample contaminants. Using our optimized workflow that includes automated steps, up to 200 urine or tissue samples can be processed in as few as 3h. Additionally, sequence comparisons of our primers and probe to other UTI bacterial strains indicated the broad applicability of these assays. These optimized qPCR assays provide a cost-effective and time-saving method for quantification of bacterial burdens in tissues and body fluids to assess the effectiveness of candidate vaccines or interventions.

Appraisal of IgM Kappa/IgM Lambda Variations Using HevyLite® After Rituximab As Consolidation Therapy in Patients with Waldenström’s Macroglobulinemia

AbstractAbstract 4879 Background:In Waldenström’s Macroglobulinemia (WM), response rates to rituximab (RTX) range from 30% to 75%. RTX is also known to generate a transient IgM flare in some patients. Our study aimed to characterize this phenomenon using the newly Hevylite® assay developed by The Binding Site that specifically quantifies IgM-kappa(K) and IgM-lambda(L). Methods:Between 2007 and 2009, 33 WM patients received a 6-course fludarabine-based regimen, 32 as first and 1 as second line. Partial response (PR), defined as a reduction ≥50% but <90% in baseline monoclonal peak (BMP) on serum protein electrophoresis (SPE), was the minimal goal. Two of the 32 patients treated at first line needed further treatment with CHOP regimen to reach the minimal PR. All received 4 consecutive weekly IV injections of 375 mg/m2 of RTX as consolidation. All were tested using the SPAplus analyzer for residual IgM-K and IgM-L at first and fourth injection. Monoclonal IgM-K or IgM-L level variations and IgM-K/IgM-L ratios were analyzed. SPE and immunofixation (IF) were performed 3 months after the fourth RTX injection to evaluate the impact of consolidation on response. Results:Of the 33 tested, at a variation threshold ≥20% relative to baseline IgM levels, 3 subgroups were defined: 15 or 45.5%, the no-flare patients, showed an increase or a decrease <20%, 11 or 33.3%, the flare patients, showed an increase ≥20% while 7 or 21.2%, the anti-flare patients, showed a decrease ≥20%. In the no-flare subgroup, there was no significant difference in monoclonal K (9) and L (6) light chains while in the 18 but 1 patients of the merged flare and anti-flare subgroups, monoclonal K light chain was overrepresented as there were 17 K vs 1 L (p<0.05). A highest initial IgM-K/L ratio (p<0.005) characterized the flare subgroup. No association was seen with demographic and hematologic characteristics and RTX pharmacokinetics. Regarding Fc gamma receptor (FcγR) IIIa-158 polymorphism, 10 patients were defined as VF and 5 as FF in the no-flare subgroup, 3 as VF, 3 as FF and 1 as VV in the anti-flare subgroup, 3 as VF and 8 as FF in the flare subgroup, with no statistically significant difference between the last 2 subgroups (p=0.22).We also analyzed the data without defining a critical threshold in monoclonal IgM level variations. Patients were, then, separated in 2 subgroups: 17 flare patients and 16 anti-flare patients. Monoclonal K light chain was overrepresented in the 2 subgroups, with 13 K vs 4 L in flare patients and 13 K vs 3 L in anti-flare patients; no statistically significant difference was found between them regarding monoclonal K and L light chains (p=0.32). No association was seen with demographic and hematologic characteristics and RTX pharmacokinetics. Regarding FcgR IIIa-158 polymorphism, there was a statistically significant difference (p=0.03) as 12 patients were of the FF subtype and 5 of the VF one in the flare subgroup while 4 were of the FF subtype, 11 of the VF one and 1 of the VV one in the anti-flare subgroup.At response evaluation 3 months after the fourth injection of RTX, all patients were screened for SPE and 22 for IF. When we compared the 17 patients of the flare subgroup to the 16 ones of the anti-flare subgroup, no patient vs 1 reached complete remission defined as disappearance of BMP on SPE and negativity of IF, 1 vs 6 advanced to very good partial response defined as a decrease ≥90% in BMP on SPE and positivity of IF and the remaining 16 vs 9 improved their partial response by a mean additional reduction of 20% (range: 0–30%) vs 18.75% (range: 0–50%) in BMP, but there was no statistically significant difference in this regard (p=0.79). Conclusions:Using Hevylite® assay, monoclonal IgM up and down variations of at least 20% following RTX occurred in up to 54.5% of our patients and this raised to 100% when no threshold value was considered. Such assay provides a reliable and highly sensitive quantitative assessment, especially in patients whose monoclonal IgM was at low concentration.Patients of the flare subgroup were predominantly of the FF subtype of FcgR IIIa-158 polymorphism while patients of the anti-flare subgroup were predominantly of VF subtype, which was statistically significant. It is tempting to hypothesize a difference in RTX mechanism of action, with ADCC predominating in the anti-flare subgroup and CDC in the flare subgroup. It would be of interest to assess such an hypothesis on a larger cohort and to analyze its impact on response profile. Disclosures:Pietrantuono:The Binding Site: Employment.

Sensitive Monitoring of Minimal Residual Disease Status in CEBPA-Mutated Acute Myeloid Leukemia Using Amplicon Deep-Sequencing

Abstract 2517 Introduction:CEBPA (CCAAT/enhancer binding protein alpha) encodes a member of the basic region leucine zipper (bZIP) transcription factor family essential for myeloid differentiation. CEBPA mutations occur predominantly in AML with a normal karyotype and CEBPA mutated AML has been included as provisional entity in the WHO classification. Cases with biallelic mutations were reported as being associated with a favorable clinical outcome, thus patients are spared from allogeneic transplantation in first CR. Screening for CEBPA mutations in patients with AML is often performed applying a combination of fragment length analysis, DHPLC and subsequent direct sequencing using Sanger technique (conventional methods). Study Design: Next-generation amplicon deep-sequencing (454 Life Sciences, Branford, CT) is a more sensitive quantitative detection method than Sanger sequencing and thus was used to analyze 144 samples from 29 CEBPA mutated AML patients with a normal karyotype. For a longitudinal analysis starting at diagnosis and following the course of treatment bone marrow (n=134) or peripheral blood (n=10) samples were obtained between 5/2006 and 6/2011. The sequencing assay targeted the complete coding region of CEBPA, covered with 4 amplicons, and was performed using genomic DNA extracted from mononuclear cells. In median, 711 reads per amplicon were generated using the NGS assay, thereby allowing a sensitive quantitative assessment of the CEBPA mutational burden in order to monitoring minimal residual disease (MRD). In median, 4 time points per patient (range: 2–9) were included with a median time span of 9.5 months (range: 1–45 months). The median sampling interval was 2 months (range: 0.3–45 months). Results: First, we evaluated the concordance of mutation detection by comparing data from NGS and conventional methods using the samples at initial diagnosis. In all 29 AML patients NGS concordantly detected the mutations known from conventional methods, i.e. in total 26 frame-shifts, 15 in-frame alterations, 8 missense, and 2 nonsense mutations. Further, at initial diagnosis, deep-sequencing detected the mutations with a median burden of 44% sequencing reads (range 3%–88%) and thus already allowed a quantitative assessment of the mutational load. There was no difference observed for 6 patients with monoallelic vs. 21 cases with biallelic mutations (excluding 2 cases with homozygous alterations). We next investigated the distribution of clones and their underlying kinetics of clone size reduction during subsequent high-dose chemotherapy cycles. Overall, 26/29 cases were evaluable and the clone size was assessed by NGS at the second analysis point during course of disease–in median 63 days from time of diagnosis (range 10–215 days): (i) In 16/26 cases, deep-sequencing was not able anymore to detect the mutations as observed at diagnosis. 14 of these 16 negative cases stayed in complete molecular remission till the end of follow-up (median follow-up 6.5 months, range 1–34.2; 2/14 cases with allogeneic stem cell transplantation). (ii) Interestingly, in 4/26 cases residual disease with clones ranging from 8%–50% was indicative of non-response to treatment. In this subgroup 3/4 patients were characterized by resistant disease or early relapse (1 case excluded due to short follow-up). (iii) In the remainder group of 6/26 patients with mutations still detectable in a range of 0.12%–3.7%, complete molecular remission status was achieved at subsequent time points. However, in this group also 3 relapses were observed including 2 cases with allogeneic stem cell transplantation. Of note, in 3/6 cases from the latter group, NGS had outperformed conventional methods and was able to still detect residual clones enabling a superior monitoring of therapy response. In all cases with biallelic mutations both clones responded in parallel with similar kinetics. Moreover, 5 patients were investigated following relapse of AML or non-response to therapy. In all 5/5 analyses including 2 monoallelic and 3 biallelic alterations the same mutations as harbored at initial diagnosis remained detectable. Conclusion:CEBPA mutations provide increasing clinical utility for the detection of MRD. We here demonstrated that deep-sequencing is a suitable unbiased and robust method to accurately detect and quantify CEBPA aberrations enabling an individualized monitoring of disease status and treatment efficacy. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Grossmann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Stopp:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Native‐DIGE: A new look at the mitochondrial membrane proteome

Respiratory chain proteins play a pivotal role in mitochondrial metabolism and thereby in the aging process. Differential display of the mitochondrial proteome reveals the abundance changes occurring in proteins as response to complex events such as senescence and aging. However, there is an absolute need to implement a detection technique that could potentially encompass the hydrophobic and very basic membrane proteins, along with the soluble ones. It is also important to assess protein-protein interactions, besides changes in abundance. Native-difference gel electrophoresis (DIGE) is an approach that facilitates sensitive quantitative assessment of changes in membrane and soluble proteins. It stretches the boundaries of detecting abundance changes to protein-protein interactions for interpretation of a proteome in a more "meaningful" way. Here we evaluate the benefits of blue-native fluorescence DIGE as a method in differential quantitative proteomics with a focus on critical issues for application and experimental design.

Development of Aβ terminal end-specific antibodies and sensitive ELISA for Aβ variant

Alzheimer’s disease (AD) is a neurodegenerative affliction associated with memory dysfunction. Senile plaques are a pathological hallmark of AD, and amyloid β (Aβ) peptides are a major component of these plaques. Aβ peptides are derived from proteolytic cleavage of the Aβ protein precursor (APP) by β- and γ-secretases to generate two principal species, Aβ1–40 and Aβ1–42. We have developed antibodies against the N- and C-termini of these peptides, and an ELISA for accurate and sensitive quantitative assessment. Sandwich ELISA composed of N-terminus (Aβ1) end-specific antibody, clone 82E1, and C-termini end-specific antibodies, and clones 1A10 and 1C3 for Aβ40 and Aβ42, respectively, detects full-length Aβ1–40 and 1–42 with a sensitivity in the sub single digit fmol/ml (equivalent to single digit pg/ml) range with no cross-reactivity to APP. A combination of C-termini antibodies and an antibody against the middle region of Aβ detects mouse Aβ in non-transgenic mouse brains.

Development of A$beta; terminal end-specific antibodies and sensitive ELISA for A$beta; variant

Alzheimer’s disease (AD) is a neurodegenerative affliction associated with memory dysfunction. Senile plaques are a pathological hallmark of AD, and amyloid β (Aβ) peptides are a major component of these plaques. Aβ peptides are derived from proteolytic cleavage of the Aβ protein precursor (APP) by β- and γ-secretases to generate two principal species, Aβ1–40 and Aβ1–42. We have developed antibodies against the N- and C-termini of these peptides, and an ELISA for accurate and sensitive quantitative assessment. Sandwich ELISA composed of N-terminus (Aβ1) end-specific antibody, clone 82E1, and C-termini end-specific antibodies, and clones 1A10 and 1C3 for Aβ40 and Aβ42, respectively, detects full-length Aβ1–40 and 1–42 with a sensitivity in the sub single digit fmol/ml (equivalent to single digit pg/ml) range with no cross-reactivity to APP. A combination of C-termini antibodies and an antibody against the middle region of Aβ detects mouse Aβ in non-transgenic mouse brains.

Quantitative Assessment of Cerebral Ventricular Volumes in Chronic Fatigue Syndrome

Previous qualitative volumetric assessment of lateral ventricular enlargement in chronic fatigue syndrome (CFS) has provided evidence for subtle structural changes in the brains of some individuals with CFS. The aim of this pilot study was to determine whether a more sensitive quantitative assessment of the lateral ventricular system would support the previous qualitative findings. In this study, we compared the total lateral ventricular volume, as well as the right and left hemisphere subcomponents in 28 participants with CFS and 15 controls. Ventricular volumes in the CFS group were larger than in control groups, a difference that approached statistical significance. Group differences in ventricular asymmetry were not observed. The results of this study provide further evidence of subtle pathophysiological changes in the brains of participants with CFS.

Computer assisted quantitation of terminal hepatic vein connective tissue in the rat

Computer assisted image analysis has been used to quantify the cellular and extracellular connective tissue component of rat liver terminal hepatic venules, in control animals and those exposed to 40% ethanol in drinking water. A significant relationship existed between the size of the terminal hepatic venule and the amount of connective tissue it contained in 14 of 15 controls and 17 of 18 ethanol exposed rats. Thickening of the terminal hepatic vein wall assessed to be present in ethanol treated rats by direct observation was confirmed by image analysis in all cases (p < 0.01). Significant differences between treated and control livers (p < 0.05) were detected by image analysis when not apparent to human observers. Sensitive quantitative assessment of terminal hepatic vein wall thickening was thus achieved by computerized analysis of liver sections.