Quantitative T2 mapping during follow-up after matrix-associated autologous chondrocyte transplantation (MACT): full-thickness and zonal evaluation to visualize the maturation of cartilage repair tissue

The purpose of this article was to evaluate the potential of in vivo zonal T2-mapping as a noninvasive tool in the longitudinal visualization of cartilage repair tissue maturation after matrix-associated autologous chondrocyte transplantation (MACT). Fifteen patients were treated with MACT and evaluated cross-sectionally, with a baseline MRI at a follow-up of 19.7 +/- 12.1 months after cartilage transplantation surgery of the knee. In the same 15 patients, 12 months later (31.7 +/- 12.0 months after surgery), a longitudinal 1-year follow-up MRI was obtained. MRI was performed on a 3 Tesla MR scanner; morphological evaluation was performed using a double-echo steady-state sequence; T2 maps were calculated from a multiecho, spin-echo sequence. Quantitative mean (full-thickness) and zonal (deep and superficial) T2 values were calculated in the cartilage repair area and in control cartilage sites. A statistical analysis of variance was performed. Full-tickness T2 values showed no significant difference between sites of healthy cartilage and cartilage repair tissue (p < 0.05). Using zonal T2 evaluation, healthy cartilage showed a significant increase from the deep to superficial cartilage layers (p < 0.05). Cartilage repair tissue after MACT showed no significant zonal increase from deep to superficial cartilage areas during baseline MRI (p > 0.05); however, during the 1-year follow-up, a significant zonal stratification could be observed (p < 0.05). Morphological evaluation showed no significant difference between the baseline and the 1-year follow-up MRI. T2 mapping seems to be more sensitive in revealing changes in the repair tissue compared to morphological MRI. In vivo zonal T2 assessment may be sensitive enough to characterize the maturation of cartilage repair tissue.

Real-time quantitative PCR to determine chlamydial load in men and women in a community setting

We used a PCR method to quantify the loads of Chlamydia trachomatis organisms in self-collected urine and vulvovaginal swab (VVS) samples from 93 women and 30 men participating in the Chlamydia Screening Studies Project, a community-based study of individuals not seeking health care. For women, self-collected VVS had a higher mean chlamydial load (10,405 organisms/ml; 95% confidence interval [95% CI], 5,167 to 21,163 organisms/ml) than did first-void urines (FVU) (503 organisms/ml; 95% CI, 250 to 1,022 organisms/ml; P < 0.001). Chlamydial loads in female and male self-collected FVU specimens were similar (P = 0.634). The mean chlamydial load in FVU specimens decreased with increasing age in females and males. There was no strong statistical evidence of differences in chlamydial load in repeat male and female FVU specimens taken when patients attended for treatment a median of 23.5 (range, 14 to 62) and 28 (range, 13 to 132) days later, respectively, or in VVS taken a median of 35 (range, 14 to 217) days later. In this study, chlamydial load values for infected persons in the community who were not seeking treatment were lower than those published in other studies involving symptomatic patients attending clinical settings. This might have implications for estimates of the infectiousness of chlamydia. The results of this study provide a scientific rationale for preferring VVS to FVU specimens from women.

Quantitative assessment of oral orbicular muscle deformation after cleft lip reconstruction: an ultrasound elastography study

Reconstruction of a cleft lip leads inevitably to scar tissue formation. Scar tissue within the restored oral orbicular muscle might be assessed by quantification of the local contractility of this muscle. Furthermore, information about the contraction capability of the oral orbicular muscle is crucial for planning the revision surgery of an individual patient. We used ultrasound elastography to determine the local deformation (strain) of the upper lip and to differentiate contracting muscle from passive scar tissue. Raw ultrasound data (radio-frequency format; rf-) were acquired, while the lips were brought from normal state into a pout condition and back in normal state, in three patients and three normal individuals. During this movement, the oral orbicular muscle contracts and, consequently, thickens in contrast to scar tissue that will not contract, or even expand. An iterative coarse-to-fine strain estimation method was used to calculate the local tissue strain. Analysis of the raw ultrasound data allows estimation of tissue strain with a high precision. The minimum strain that can be assessed reproducibly is 0.1%. In normal individuals, strain of the orbicular oral muscle was in the order of 20%. Also, a uniform strain distribution in the oral orbicular muscle was found. However, in patients deviating values were found in the region of the reconstruction and the muscle tissue surrounding that. In two patients with a successful reconstruction, strain was reduced by 6% in the reconstructed region with respect to the normal parts of the muscle (from 22% to 16% and from 25% to 19%). In a patient with severe aesthetical and functional disability, strain decreased from 30% in the normal region to 5% in the reconstructed region. With ultrasound elastography, the strain of the oral orbicular muscle can be quantified. In healthy subjects, the strain profiles and maximum strain values in all parts of the muscle were similar. The maximum strain of the muscle during pout was 20% +/- 1%. In surgically repaired cleft lips, decreased deformation was observed.

Comparison of calcium analysis, longitudinal microradiography and profilometry for the quantitative assessment of erosion in dentine

Erosion of dentine causes mineral dissolution, while the organic compounds remain at the surface. Therefore, a determination of tissue loss is complicated. Established quantitative methods for the evaluation of enamel have also been used for dentine, but the suitability of these techniques in this field has not been systematically determined. Therefore, this study aimed to compare longitudinal microradiography (LMR), contacting (cPM) and non-contacting profilometry (ncPM), and analysis of dissolved calcium (Ca analysis) in the erosion solution. Results are discussed in the light of the histology of dentine erosion. Erosion was performed with 0.05 M citric acid (pH 2.5) for 30, 60, 90 or 120 min, and erosive loss was determined by each method. LMR, cPM and ncPM were performed before and after collagenase digestion of the demineralised organic surface layer, with an emphasis on moisture control. Scanning electron microscopy was performed on randomly selected specimens. All measurements were converted into micrometres. Profilometry was not suitable to adequately quantify mineral loss prior to collagenase digestion. After 120 min of erosion, values of 5.4 +/- 1.9 microm (ncPM) and 27.8 +/- 4.6 microm (cPM) were determined. Ca analysis revealed a mineral loss of 55.4 +/- 11.5 microm. The values for profilometry after matrix digestion were 43.0 +/- 5.5 microm (ncPM) and 46.9 +/- 6.2 (cPM). Relative and proportional biases were detected for all method comparisons. The mineral loss values were below the detection limit for LMR. The study revealed gross differences between methods, particularly when demineralised organic surface tissue was present. These results indicate that the choice of method is critical and depends on the parameter under study.

Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab microL(-1) milk and 0.4 ng mL(-1) Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n = 8) or non-transgenic (n = 7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Mineralogy-based quantitative precipitation and temperature reconstructions from annually laminated lake sediments (Swiss Alps) since AD 1580

[1] We present quantitative autumn, summer and annual precipitation and summer temperature reconstructions from proglacial annually laminated Lake Silvaplana, eastern Swiss Alps back to AD 1580. We used X-ray diffraction peak intensity ratios of minerals in the sediment layers (quartz qz, plagioclase pl, amphibole am, mica mi) that are diagnostic for different source areas and hydro-meteorological transport processes in the catchment. XRD data were calibrated with meteorological data (AD 1800/1864–1950) and revealed significant correlations: mi/pl with SON precipitation (r = 0.56, p < 0.05) and MJJAS precipitation (r = 0.66, p < 0.01); qz/mi with MJJAS temperature (r = −0.72, p < 0.01)and qz/am with annual precipitation (r = −0.54, p < 0.05). Geological catchment settings and hydro-meteorological processes provide deterministic explanations for the correlations. Our summer temperature reconstruction reproduces the typical features of past climate variability known from independent data sets. The precipitation reconstructions show a LIA climate moister than today. Exceptionally wet periods in our reconstruction coincide with regional glacier advances.