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PubMed2006

Comparison of derivative preprocessing and automated polynomial baseline correction method for classification and quantification of narcotics in solid mixtures.

This work offers a real-world comparison of derivative preprocessing and a new polynomial method described by Lieber and Mahadevan-Jansen (LMJ) for baseline correction of Raman spectra with widely varying backgrounds. This comparison is based on their outcomes in factor analysis, analyte discrimination, and quantification. Both correction methods are applied to a Raman spectra data set taken from 85 solid samples of illegal narcotics diluted with various materials. It is found that neither approach outperforms the other, as they give similar principal component analysis (PCA) models and quantification errors: cocaine and heroin show cross-validation errors of approximately 8%, while MDMA is quantified to a cross-validation error of approximately 3-4%. The LMJ method does offer several other advantages, the most significant being the retention of original peak shapes after the correction, which simplifies the interpretation of the preprocessed spectra. The LMJ method is therefore recommended for use as a baseline correction method in future research with Raman spectroscopy.

Algorithms↗
PubMed2004

Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation.

BACKGROUND: Detection of fetal DNA in maternal plasma is achievable at 5 weeks of gestation, but few large-scale studies have reported circulating fetal and maternal DNA across all trimesters. METHODS: Blood samples were collected from 201 women between 5 and 41 weeks of pregnancy. Quantitative PCR was used to assess total and fetal DNA concentrations, and allelic discrimination analysis was investigated as a route to detecting specifically fetal DNA. RESULTS: Male fetuses were detectable from 5 weeks amenorrhea with increasing fetal DNA concentrations across gestation. The sensitivity of fetal male gender determination in pregnancies with live birth confirmation was 99%, with 100% specificity. Total DNA concentrations did not correlate with gestational age, but appeared slightly higher in the first and third trimesters than in mid-pregnancy. Analysis of short tandem repeats demonstrated that significant improvements in the detection limit are required for specific detection of fetal DNA. CONCLUSIONS: The high sensitivity of PCR-based detection, together with quantification provided by real-time DNA analysis, has clear potential for clinical application in noninvasive prenatal diagnosis. However, accurate quantification using best-fit data analysis, standardization of methods, and performance control indicators are necessary for robust routine noninvasive diagnostics.

Age Factors↗
PubMed2006

Newborn screening for hepatorenal tyrosinemia: Tandem mass spectrometric quantification of succinylacetone.

BACKGROUND: False-positive and false-negative results occur in current newborn-screening programs for hepatorenal tyrosinemia, which measure tyrosine concentrations in blood spots, sometimes in combination with other metabolites, including succinylacetone. We present our experience with a newly described method for succinylacetone quantification in routine newborn screening. METHODS: Succinylacetone was extracted from blood spots that had already been extracted with absolute methanol for acylcarnitine and amino acid analysis. The solvent was acetonitrile-water (80:20 by volume) containing formic acid, hydrazine hydrate, and 100 nmol/L 5,7-dioxooctanoic acid as internal standard. Analysis was performed by tandem mass spectrometry in a separate run. RESULTS: Of 61,344 samples, 99.6% had succinylacetone concentrations < or =5 micromol/L. With a cutoff of 10 micromol/L, no false-positive results were obtained. In 2 patients, the succinylacetone concentrations in the dried blood spots from the 36th and 56th hours of life were 152 and 271 micromol/L, respectively, and the tyrosine concentrations were 54 and 129 micromol/L. Hepatorenal tyrosinemia was subsequently confirmed in both patients. Retrospective analysis of the neonatal screening samples of 2 additional known patients revealed increased succinylacetone concentrations of 46 and 169 micromol/L, respectively. CONCLUSIONS: Tandem mass spectrometric quantification directly from residual blood spots is a useful method for the early detection of hepatorenal tyrosinemia in newborn-screening programs.

Blood Specimen Collection↗
PubMed2006

Rapid and simultaneous quantification of 4 urinary proteins by piezoelectric quartz crystal microbalance immunosensor array.

BACKGROUND: Urinary proteins are predictive and prognostic markers for diabetes nephropathy. Conventional methods for the quantification of urinary proteins, however, are time-consuming, and most require radioactive labeling. We designed a label-free piezoelectric quartz crystal microbalance (QCM) immunosensor array to simultaneously quantify 4 urinary proteins. METHODS: We constructed a 2 x 5 model piezoelectric immunosensor array fabricated with disposable quartz crystals for quantification of microalbumin, alpha1-microglobulin, beta2-microglobulin, and IgG in urine. We made calibration curves after immobilization of antibodies at an optimal concentration and then evaluated the performance characteristics of the immunosensor with a series of tests. In addition, we measured 124 urine samples with both QCM immunosensor array and immunonephelometry to assess the correlation between the 2 methods. RESULTS: With the QCM immunosensor array, we were able to quantify 4 urinary proteins within 15 min. This method had an analytical interval of 0.01-60 mg/L. The intraassay and interassay imprecisions (CVs) were <10%, and the relative recovery rates were 90.3%-109.1%. Nonspecificity of the immunosensor was insignificant (frequency shifts <20 Hz). ROC analyses indicated sensitivities were > or =95.8% and, specificities were > or =76.3%. Bland-Altman difference plots showed the immunosensor array to be highly comparable to immunonephelometry. CONCLUSIONS: The QCM system we designed has the advantages of being rapid, label free, and highly sensitive and thus can be a useful supplement to commercial assay methods in clinical chemistry.

Alpha-Globulins↗
PubMed1996

Mass spectrometric quantification of neuropeptides.

The molecular specificity of MS/MS methods for the accurate quantification of endogenous neuropeptides, such as ME and BE, exceeds that of all other methods because: 1. MS/MS qualitative analysis first establishes the amino acid sequence of the endogenous peptide; 2. MS/MS establishes and maintains the structural link between the (M + H)+ ion and its corresponding amino acid sequence-determining ion(s) during quantification; and 3. A stable isotope-incorporated synthetic peptide internal standard is used. Endogenous ME and BE have been measured in human pituitary tissue by this quantitative analytical MS method.

Humans↗
PubMed2004

Quantification of ribozyme target RNA using real-time PCR.

An important part of the ribozyme efficiency-screening process is to have a fast and accurate way to measure steady-state levels of the target RNA. Here, we describe the use of real-time polymerase chain reaction (PCR) for quantification of ribozyme target transcripts. In contrast to classical quantitative PCR, real-time PCR does not require extensive manipulation or generation of relatively complex reagents, thus reducing the risk of contamination. PCR products generated by Taq polymerase in the presence of SYBR Green dye I can be monitored each cycle by collecting fluorescence signals emitted only as the double-stranded DNA is formed. The temperature at which the fluorescent data used for quantification are collected is based on the melting-curve analysis of the amplified product. After constructing a standard curve by plotting the log of the standards' copy number vs their fractional cycle number, the copy number of the unknown samples is automatically determined by interpolation of this curve. However it is very important to validate the melting curve profile with standard gel electrophoresis, particularly while setting up the technique. Real-time PCR is fast and reproducible. Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 h. The coefficient of variance is 15% in the range of 104-106 gene copies.

Base Sequence↗
PubMed2004

The determination and quantification of photosynthetic pigments by reverse phase high-performance liquid chromatography, thin-layer chromatography, and spectrophotometry.

Chorophylls and carotenoids are functionally important pigment molecules in photosynthetic organisms. Methods for the determination of chlorophylls a and b, beta-carotene, neoxanthin, and the pigments that are involved in photoprotective cycles such as the xanthophylls are discussed. These cycles involve the reversible de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin, as well as the reversible de-epoxidation of lutein-5,6-epoxide into lutein. This chapter describes pigment extraction procedures from higher plants and green algae. Methods for the determination and quantification using high-performance liquid chromatograpy (HPLC) are described as well as methods for the separation and purification of pigments for use as standards using thin-layer chromatography (TLC). In addition, several spectrophotometric methods for the quantification of chlorophylls a and b are described.

Arabidopsis↗
PubMed2005

DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

A protocol is described for the isolation of DNA and subsequent preparation of samples for the measurement of adduct levels by accelerator mass spectrometry (AMS). AMS is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. However, special precautions must be taken to avoid cross-contamination of isotope between samples and to produce a sample that is compatible with AMS. The DNA isolation method described is based on digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen DNA isolation columns. DNA is then precipitated with isopropanol, washed repeatedly with 70% ethanol to remove salt, and then dissolved in water. This method has been used to generate reliably good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. For quantification of adduct levels from 14C-labeled compounds, DNA samples are then converted to graphite, and the 14C content is measured by AMS.

Animals↗
PubMed2006

Overview of real-time RT-PCR strategies for quantification of gene rearrangements in the myeloid malignancies.

In acute myeloid leukemia (AML), molecular diagnosis for the optimal management of patients and for minimal residual disease (MRD) monitoring is of extreme importance. Cumulative data suggest that quantitative monitoring or MRD in AML with fusion transcripts corresponding to 5(I;21), inv(16), and t(15;17) is useful in distinguishing patients at high risk of relapse from those in durable remission. Real-time quantitative polymerase chain reaction (RQ-PCR) is by far the most sensitive assay in the context of MRD detection. We present herein an overview of the principles of RQ-PCR encompassing both the chemistries (double-stranded DNA detection or specific fragment detection) and the instruments. The absolute and relative quantification and the most commonly used methods for calculation of MRD results in absolute quantification are also described.

Computer Systems↗
PubMed2006

TaqMan reverse transcriptase-polymerase chain reaction coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type.

Real-time TaqMan polymerase chain reaction (PCR) assays allow quantification of the initial amount of target in a specimen, specifically, and reproducibly. The major limitation of TaqMan PCR assays is that they do not detect the size of the amplified target sequence. TaqMan PCR coupled with capillary electrophoresis is an alternative approach that can be used to circumvent this limitation. In this chapter, the utility of this approach in the identification and quantification of bcr-abl fusion transcripts produced as a result of t(9;22)(q34;q11) in chronic myelogenous leukemia is described. In this assay, abl primer labeled at its 5'-end with the fluorescent dye NED (Applied Biosystems [ABI], Foster City, CA) is incorporated into the bcr-abl fusion product during the real-time PCR. The incorporated NED fluorescent dye is then used subsequently to identify the specific fusion transcript present in a given specimen by high-resolution capillary electrophoresis and GeneScan (ABI) analysis. Knowledge of the type of fusion transcript present in a specimen is useful to rule out false-positive results and to compare clones before and after therapy.

Animals↗
PubMed2004

Bone marrow angiogenesis: methods of quantification and changes evolving in chronic myeloproliferative disorders.

Until now little information is available about bone marrow (BM) angiogenesis in chronic myeloproliferative disorders (CMPDs). Amongst the various immunohistochemical markers for endothelial cells CD34 and CD105 have proven to be most reliable since they exhibit no relevant co-staining. Determination of vascularity has to include pathophysiological aspects of perfusion. Therefore, quantification of the microvascular density (MVD) by the so-called hot spot method has to be improved by parameters that characterize blood flow more properly like microvessel area (luminal distension), shape (form factor), tortuosity, and branching (maximal vessel length). In comparison to the normal BM chronic myeloid leukemia (CML) revealed a significant increase in MVD which was functionally associated with elevated levels of angiogenic cytokines. Structure of vessels was significantly altered by showing an enhanced irregularity of shape and tortuosity and increase in fibers was conspicuously accompanied by a higher degree of MVD. Contrasting the group of patients with Imatinib (STI571) therapy interferon failed to reduce the number of vessels. Following bone marrow transplantation a significant enhancement of the MVD was found in the early post-transplant period, but after about 6 months normalization occurred. Anomalies of microvascular architecture were easily demonstrable by three-dimensional reconstruction and consisted of a complex branching network of irregular shaped sinuses. Chronic idiopathic myelofibrosis displayed a significant increase in the MVD only in the advanced fibrosclerotic stages. This feature was accompanied by an enhanced luminal distension and tortuosity, thus contrasting the prefibrotic and early fibrotic phases of this disorder. Similar to CML a relationship between evolving myelofibrosis and change in vascular architecture was encountered. This feature may present a possible target for future anti-angiogenic therapy. In essential thrombocythemia there is only a mild increase in MVD detectable while in polycythemia vera besides an enlarged number, a luminal dilation due to the densely packed erythrocytes is recognizable. In conclusion, contrasting the usually applied quantification technique more elaborate morphometrical methods are warranted to obtain a better insight into the vascular architecture of the BM. In CMPDs angiogenesis is significantly associated with the evolution of myelofibrosis and may be altered by therapeutic regimens probably due to changes in cytokine release.

Bone Marrow↗
PubMed2002

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis.

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3'-end of the allele-specific primer. Amplification is monitored by SYBR-Green I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (F-VIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (F-VIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.

Alleles↗
PubMed2001

Technique for the quantification of transient quadratic phase couplings between heart rate components.

A technique for the time-variant analysis of quadratic phase coupling (QPC) in heart rate data is introduced and tested in 6 human neonates during quiet sleep. The set up of the approach is based up on the assumption that QPCs in the heart rate variability (HRV) are related to amplitude modulation effects. The application of the biamplitude deals with the detection of the coupling pattern and the bicoherence is used for the statistical quantification of coupling. By means of the results of bispectral analysis the time-variant processing has been adapted. The frequency-selective complex demodulation of the HRV leads to the envelope of the respiratory sinus arrhythmia (RSA), this has been used as one input for a time-variant coherence analysis. The other input is the low-pass filtered 10-second-rhythm of the HRV. A time-continuous quantification of the QPC, caused by amplitude modulation (10-second-rhythm modulates the RSA), is possible using this approach. According to our observed results in neonatal HRV both a phase co-ordination between the 10-second-rhythm and RSA as well as a non-linear coupling (amplitude modulation) between these HRV components can be seen.

Electrocardiography↗
PubMed2003

Profiling and in vivo quantification of proteins by high resolution mass spectrometry: the example of goserelin, an analogue of luteinizing hormone-releasing hormone.

Proteins are essential biomolecules which are frequently involved in major pathological syndromes and are widely used as diagnostic markers or therapeutic agents. The emergence of proteomics will doubtless further increase the significance of proteins both in the clinic and in the life sciences in general. Our main objective is to offer innovative solutions to what we like to call the "post-proteomics era". To achieve our goal, we intend to develop novel approaches and technologies for in vivo metabolic studies of proteins using mass spectrometry (MS), focusing on pharmacokinetics and pharmacodynamics. Using goserelin as a model, we have successfully developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of an intact analogue of luteinizing hormone-releasing hormone (LHRH) in small volumes of rat plasma samples at concentrations ranging from 0.3 to 405.0 microg/l. To this end, a microbore reversed-phase-HPLC system was coupled on-line to a tandem high resolution quadrupole time-of-flight (Q-TOF) instrument fitted with an electrospray ion source and operated in LC-MS/MS mode. External calibration was used and the high resolution was crucial to discard contaminating signals, which would not have been possible with the more conventional triple quadrupole mass spectrometers operated in a static mode. For low sample amounts, calibration curves were constructed corresponding to rat plasma levels of 0.3 to 16.4 microg/l and found to be of third order with a coefficient of determination greater than 0.999. The relative standard deviation was found to be lower than 15%. A lower limit of detection (LLOD) of 0.17 microg/l and a lower limit of quantification (LLOQ) of 0.3 microg/l were determined.

Animals↗
PubMed2004

Isolation of plasma small-dense low-density lipoprotein using a simple air-driven ultracentrifuge and quantification using immunoassay of apolipoprotein B.

Small-dense low-density lipoprotein (SD-LDL) is associated with coronary heart disease risk. Current methods for its quantification are expensive, complex and time-consuming. Plasma was adjusted to a density (D) of 1.044 g/ml in a volume of 0.18 ml and centrifuged in a Beckman Airfuge at 160 000 x g for 3 h 7 min and apolipoprotein B (apoB) then determined in the infranatant. Results were compared with centrifugation of 5 ml of plasma at D = 1.044 g/ml at 144 000 x g for 18 h in a preparative ultracentrifuge (UC). We obtained blood samples from healthy subjects (n = 73) and from dyslipidaemic patients (n = 112). SD-LDL apoB levels as determined using the UC method ranged from 0.01-0.43 g/l and there was a good correlation with Airfuge results (r = 0.925; p < 0.0001; n = 185). There was a mean difference of 0.0125 g/l between methods. SD-LDL apoB levels as determined using the Airfuge showed a reasonable agreement with results obtained using density gradient ultracentrifugation (r = 0.773; p < 0.01; n = 12). Airfuge results correlated directly with triglyceride concentration, in both healthy men (r = 0.296; p < 0.05) and dyslipidaemic men (r = 0.520; p < 0.001) and also in dyslipidaemic women (r = 0.463; p < 0.005). Airfuge results correlated inversely with high-density lipoprotein-cholesterol (HDL-C) concentration, in both healthy men (r = -0.237; p < 0.05) and dyslipidaemic men (r = -0.293; p < 0.005). Using a micro-method, we obtained results which establish that the Airfuge provides a more rapid and less expensive method for quantification of SD-LDL.

Apolipoproteins B↗
PubMed2006

Real-time RT-PCR quantification of PRAME gene expression for monitoring minimal residual disease in acute myeloblastic leukaemia.

BACKGROUND: Specific gene rearrangements are used for minimal residual disease (MRD) assessment, but are frequently lacking in leukaemias. In these cases, the quantification of PRAME (preferentially expressed antigen of melanoma) transcripts could be useful. METHODS: PRAME transcripts were quantified by real-time RT-PCR in normal and leukaemic samples, and the results were compared with those of conventional RT-PCR. Basal expression of PRAME was determined in 25 blood samples and 25 bone marrow samples from healthy donors, as well as in 12 haematological cell lines (Jurkat, K562, HL60, DOHH2, IM9, Daudi, CEM, KG1, DG75, 8226, U937, Raji). RESULTS: In paediatric acute myeloid leukaemia (AML) (n=22) and acute lymphoblastic leukaemia (ALL) (n=17), and in adult AML (n=20), abnormal PRAME expression was found in 41%, 35% and 40% of cases, respectively. To assess the sensitivity of PRAME for monitoring MRD, PRAME-positive t(8;21) AML samples with detectable AML1/ETO expression by conventional RT-PCR (n=17) were assessed for quantitative expression of AML1/ETO and PRAME. The expression of these genes was closely correlated. To confirm that PRAME expression was correlated with clinical data, the expression of PRAME was also sequentially followed in patients (n=13) from onset to cytological remission or relapse. The cytological and molecular data were highly correlated in all patients. CONCLUSIONS: Our data confirm that PRAME quantification by real-time RT-PCR appears suitable for monitoring MRD in PRAME-positive leukaemia.

Adult↗
PubMed2004

Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein.

Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4'iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the donor in energy transfer studies. Phospholipids labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) were selected as the acceptors. The dependence of protein-lipid selectivity on both hydrophobic mismatch and headgroup family was determined. M13 major coat protein exhibited larger selectivity toward phospholipids which allow for a better hydrophobic matching. Increased selectivity was also observed for anionic phospholipids and the relative association constants agreed with the ones already presented in the literature and obtained through electron spin resonance studies. This result led us to conclude that fluorescence resonance energy transfer is a promising methodology in protein-lipid selectivity studies.

Algorithms↗
PubMed2002

Thyroglobulin mRNA quantification in the peripheral blood is not a reliable marker for the follow-up of patients with differentiated thyroid cancer.

BACKGROUND: The detection of serum thyroglobulin (Tg) by immunoassay is widely used to detect residual, recurring or metastatic thyroid carcinoma tIssue in patients with differentiated thyroid cancer (DTC) after total thyroidectomy and radioiodine therapy. However, this method requires thyroid hormone withdrawal to increase sensitivity and is limited by the interference of anti-Tg antibodies. To solve these problems, the detection of Tg mRNA from circulating thyroid cells by reverse transcription (RT)-PCR has been suggested as an alternative method. However, different previous reports show discrepant conclusions as to the clinical usefulness of Tg mRNA quantification. METHODS: We compared three methods of blood collection and RNA extraction, and quantified Tg mRNA (by real time RT-PCR) in the peripheral blood of a) probands without thyroid disease (n=42), patients with b) thyroid autonomy (n=15), c) Graves' disease (n=22), d) euthyroid goiter (n=6), and in DTC-patients after thyroidectomy and radioiodine therapy e) with (n=16) and f) without (n=37) metastasis. As the use of citrate blood in combination with a subsequent separation of mononuclear cells showed a significantly better RNA yield than the extraction of RNA from EDTA or citrate blood without the separation of mononuclear cells, this was the method used. Total RNA was reverse transcribed with random hexamer primers and Tg mRNA was amplified by real time RT-PCR using specific primers and hybridization probes. The Tg mRNA concentrations were normalized to beta-actin mRNA concentrations. RESULTS: Mean circulating Tg mRNA for each group detailed above, expressed as the ratio of Tg to beta-actin concentrations x 1000, were: a) 2.3 (range 0.03-70.89), b) 0.25 (range 0.02-0.55), c) 0.31 (range 0.05-1.36), d) 0.18 (range 0.08-0.35), e) 0.57 (range 0.03-3.03) and f) 0.17 (range 0.02-0.60). Furthermore, we found no correlation between serum Tg and Tg mRNA. CONCLUSIONS: In summary, our data do not show significant differences in Tg mRNA expression between the investigated groups. Therefore, the detection and quantification of Tg mRNA in peripheral blood is unlikely to be suitable for the follow-up of DTC.

Biomarkers, Tumor↗