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PubMed1998

Inter- and intraindividual reproducibility of anorectal manometry.

BACKGROUND: This study investigates the inter- and intraindividual variability of normal values and, thus, the reproducibility of anorectal manometry. MATERIALS AND METHODS: Following a standardized protocol, three anorectal manometries were performed 4 h apart on 2 days of investigation, with an interval of 4 weeks, in ten healthy volunteers. Measured parameters in all 60 manometries were: sphincter length (SL), resting pressure (RP), maximum squeeze pressure (MSP), relaxation of the internal anal sphincter (RIAS), and rectal compliance (RC). Interindividual variability was expressed as standard deviation from calculated mean values and intraindividual variability was tested with Wilcoxon's test for tied samples and Spearman's rank correlation test. RESULTS: A large interindividual variability was found for all measured parameters, except for SL, reflecting the extensive absolute range of measured values. Median intraindividual variability among the six individual measurements and between both measurement days revealed that MSP, RIAS and RC are parameters which were not reproducible in this volunteer study. A significant correlation between the results of the repetitive measurements and, thus, a good reproducibility was only found for the parameters SL and RP. CONCLUSIONS: Anorectal manometry has only limited diagnostic value; although rather exact quantifications of individual parameters can be achieved, the impact of these measurements should be regarded rather critically, since only SL and RP appeared to be reproducible parameters.

Adult↗

PubMed1980

Immunoglobulin-producing cells in jejunal mucosa of children with coeliac disease on a gluten-free diet and after gluten challenge.

The jejunal Ig-producing cell populations in a group of 12 children with established coeliac disease (CD) in remission on a gluten-free diet were compared with those of a group of 10 patients in clinical relapse after gluten challenge. Quantification of the various immunocyte classes was performed by paired immunofluorescence staining in an individually defined mucosal tissue unit constituting a 6-micron-thick and 500-micron-wide block of tissue, including the mucosa at full height from the muscularis mucosae. In the treated group the percentage IgA/IgM/IgG cell ratios were, on the average, 82.6:12.9:4.5. There were no differences in these ratios or in the absolute immunocyte numbers when compared with a group of six control patients without CD. After gluten challenge the corresponding ratios were 77.2:17.2:5.6. The median cell number per mucosal tissue unit in the IgA, IgM, and IgG class was raised 2.1, 3.8 and 2.9 times, respectively. These increases were statistically significant. Moreover, there was a significant negative correlation between the time to clinical relapse and the number of IgG cells per tissue unit after challenge. This finding indicates that locally produced antibodies of the IgG class are especially involved in the pathogenesis of CD.

Antibody-Producing Cells↗

PubMed1998

Microquantification of cellular and in vitro F-actin by rhodamine phalloidin fluorescence enhancement.

Based on the enhancement of rhodamine phalloidin fluorescence after its binding to actin filaments we have developed a technique to quantify F-actin, drastically (>> 100 times) reducing consumption of the expensive fluorescent dye and sample material in comparison to previous methods. Depolymerization of F-actin is prevented by utilizing short incubation times and stabilization of the filaments by actin-binding proteins or formaldehyde. Equilibrium and kinetic mathematical models relating rhodamine fluorescence with F-actin concentrations were used to predict the optimal assay conditions. The method has been applied to measure relative and absolute F-actin concentrations in cytosolic fractions and stimulus-induced actin polymerization in neutrophils. The cells were lysed with octy1-beta-D-glucopyranoside, which is compatible with the assay due to its high critical micelle concentration. As the assay takes less than 1 h and eliminates all previously required washing or extraction steps, it is faster and much simpler than any other presented up to now for quantification of filamentous actin. Moreover, the method is unique for reliable and easy F-actin measurements in cell-free systems.

Actins↗

PubMed2006

pH dependence of the dissociation of multimeric hemoglobin probed by high hydrostatic pressure.

We investigated the thermodynamic features of the classic alkaline dissociation of multimeric hemoglobin (3.1 MDa) from Glossoscolex paulistus (Annelidea) using high hydrostatic pressure. Light scattering measurements up to microscopic thermodynamic equilibrium indicated a high pH dependency of dissociation and association. Electron microscopy and gel filtration corroborated these findings. The volume change of dissociation decreased in absolute values from -48.0 mL/mol of subunit at pH 6.0 to -19.2 mL/mol at pH 9.0, suggesting a lack of protein interactions under alkaline conditions. Concomitantly, an increase in pH reduced the Gibbs free energy of dissociation from 37.7 to 27.5 kJ/mol of subunit. The stoichiometry of proton release calculated from the pressure-induced dissociation curves was +0.602 mol of H(+)/mol of subunit. These results provide a direct quantification of proton participation in stabilizing the aggregated state of the hemoglobin, and contribute to our understanding of protein-protein interactions and of the surrounding conditions that modulate the process of aggregation.

Dimerization↗

PubMedDate not supplied

Estimation of regional cerebral blood flow levels in ischemia using [(15)O]water of [(11)C]flumazenil PET without arterial input function .

Determination of residual flow within the ischemic brain may be important in patients eligible for thrombolytic therapy. Conventional cerebral blood flow (CBF) quantification often cannot be achieved in these cases because arterial blood sampling required for the input function is prohibited. Quantitative CBF was measured in 20 patients with ischemic stroke using [(15)O]water and [(11)C]flumazenil PET with arterial blood sampling and compared with normalized nonquantified data of the same patients. For both comparisons, percentiles were derived, allowing the determination of a normalized blood flow level corresponding to an absolute CBF value below which 95% of all pixels are located. These normograms can be used to derive absolute CBF levels in milliliters per 100 g per minute from normalized data without the need for measuring an arterial input function.

Aged↗

PubMed1996

Isometric force and maximal shortening velocity of single muscle fibers from elite master runners.

Single chemically permeabilized gastrocnemius fibers from six elite endurance-trained master runners (RUN group) and five age-matched sedentary controls (SED group) were mounted between a force transducer and a position motor, studied under conditions of maximal and submaximal Ca2+ activation, and subsequently electrophoresed on 5% polyacrylamide gels to determine myosin heavy chain (MHC) composition. For the SED group, peak isometric tension (Pzero) averaged 143 +/- 3, 156 +/- 4, and 170 +/- 4 kN/m2 and maximal shortening velocity (Vzero) averaged 0.43 +/- 0.01, 1.90 +/- 0.08, and 5.59 +/- 0.40 fiber lengths/s for fibers expressing type I, IIa, and IIx MHC, respectively (all comparisons, P < 0.05). Hill plot analysis of relative forces during submaximal Ca2+ activation indicated no SED vs. RUN differences in Ca2+ sensitivity or in the cooperativity of Ca2+ activation. However, at maximal Ca2+ activation, RUN type I and IIa fibers produced 15% less peak absolute force than SED fibers (P < 0.05). This reduction in fiber force was a direct result of the smaller diameter of the RUN fibers (P < 0.05), because Pzero, peak elastic modulus (Ezero), and Pzero/Ezero were not different between SED and RUN groups. RUN type I fibers also displayed a mean Vzero that was 19% higher than the average Vzero of the SED type I fibers (P < 0.05). In separate experiments, quantification of relative myosin light chain (MLC) isoform content revealed a 28% greater ratio of MLC3 to MLC2 in single type I fibers from the RUN group (P < 0.05), suggesting that the elevated Vzero of the RUN type I fibers was related to a greater expression of MLC3. In conclusion, the single fibers from the elite master runners displayed specific morphological and contractile properties that may enhance the performance of these athletes during prolonged muscular activity.

Adult↗

PubMed1998

Effect of genotypes on the quantification of hepatitis C virus (HCV) RNA in clinical samples using the Amplicor HCV Monitor Test and the Quantiplex HCV RNA 2.0 assay (bDNA).

The Amplicor HCV Monitor test and the Quantiplex HCV RNA 2.0 (bDNA) assay are two commercially available assays for the quantification of hepatitis C virus (HCV) RNA in clinical samples. A direct comparison of the two assays was carried out using sera frozen previously from patients known to be chronically infected with HCV. Overall, 61 samples from 51 patients were tested simultaneously by the two methods: 67% (28/42) of the patients were infected by HCV genotype/serotype 1, 10% (4/42) with type 2, and 24% (10/42) with type 3. When the absolute value from each assay was examined, the Quantiplex assay gave a consistently higher reading and the mean logarithmic difference between the two assays was 1.4 (1.0 in type 1, 2.0 in type 2, and 2.2 in type 3). When analyzed according to genotype, strong correlation was observed between the two assays for type 1 (r = 0.83, 95% CI 0.63-0.93, P < 0.01), but not for nontype 1 samples. Despite the difference in absolute level reported by the two assays, there was a consistent trend of change in HCV RNA concentration by both assays in patients whose consecutive samples were analyzed and the differences between the two assays in consecutive samples were within 0.4 log of each other. The results suggested that with samples containing genotype 1, the Amplicor assay was more sensitive than the Quantiplex assay by about one log. However, the sensitivities of the two assays with nontype 1 samples were much closer probably due to the failure of the Amplicor assay to quantify nontype 1 genotypes effectively.

Adolescent↗

PubMed1999

[The quantification of energy-rich phosphates in healthy and damaged heart muscle by SLOOP 31P-MR spectroscopy. Spatial localization with optimal pointspread function].

PURPOSE: A quantitative 31P-MR-spectroscopic technique was used to assess the energy metabolism in healthy and diseased myocardium. METHODS: 31P spectra were acquired on a 1.5 T scanner using a 3D-chemical shift imaging technique. Based on the anatomical information provided by 1H images, SLOOP (Spatial Localization with Optimal Pointspread Function) allows to obtain spectra from defined compartments. With SLOOP a free voxel shape with adaption to anatomic structures, e.g. the myocardium, is possible. Absolute values for phosphocreatine (PCr) and adenosine triphosphate (ATP) were determined using an external standard. RESULTS: 31P-spectra showed only minimal contamination by surrounding tissue. The standard deviation for the determined values of healthy volunteers was low. Compared to healthy volunteers, reduced PCr and ATP concentrations were seen for dilative cardiomyopathies and coronary artery disease and unchanged concentrations were observed for hypertensive heart disease. CONCLUSION: 31P-MR spectroscopy with SLOOP allows a non-invasive, quantitative analysis of cardiac energy metabolism.

Adenosine Triphosphate↗

PubMed2000

Quantification of homoplasy for nucleotide transitions and transversions and a reexamination of assumptions in weighted phylogenetic analysis.

Nucleotide transitions are frequently down-weighted relative to transversions in phylogenetic analysis. This is based on the assumption that transitions, by virtue of their greater evolutionary rate, exhibit relatively more homoplasy and are therefore less reliable phylogenetic characters. Relative amounts of homoplastic and consistent transition and transversion changes in mitochondrial protein coding genes were determined from character-state reconstructions on a highly corroborated phylogeny of mammals. We found that although homoplasy was related to evolutionary rates and was greater for transitions, the absolute number of consistent transitions greatly exceeded the number of consistent transversions. Consequently, transitions provided substantially more useful phylogenetic information than transversions. These results suggest that down-weighting transitions may be unwarranted in many cases. This conclusion was supported by the fact that a range of transition: transversion weighting schemes applied to various mitochondrial genes and genomic partitions rarely provided improvement in phylogenetic estimates relative to equal weighting, and in some cases weighting transitions more heavily than transversions was most effective.

Base Sequence↗

PubMed1984

Improved SPECT quantification using compensation for scattered photons.

SPECT images are degraded by the inclusion of Compton-scattered photons within the pulse-height window. Phantom and patient studies with Tc-99m were used to evaluate a compensation method that consists of subtracting a fraction of the image reconstructed using events recorded within a secondary pulse-height window (92-125 keV) from that derived from the photopeak pulse-height window (127-153 keV). Images of line sources in air and in a water-filled phantom were stored. The compensated line spread functions (LSFs) were evaluated. In water, the absolute counting rates for the SPECT LSFs were within 10% of the rates measured in air. The phantom consisted of six solid acrylic spheres (diameters 10, 13, 16, 19, 25, 32 mm) placed within a cylindrical (22 cm diam) distribution of Tc-99m. For sphere diameters greater than 25 mm, the measured image contrasts were within 8% of the true uptake ratios. Our results have shown that high-quality, camera-based SPECT systems can reconstruct artifact-free images by making additional use of projection data acquired in a pulse-height window other than that over the primary photopeak. This compensation method results in qualitative and quantitative improvements for the limited source geometries investigated. Further studies are required to optimize this heuristic approach for other source geometries.

Humans↗

PubMed1993

Quantification of menstrual and diurnal periodicities in rates of cholesterol and fat synthesis in humans.

The mass isotopomer distribution analysis (MIDA) technique is applied here in men and menstruating women to quantify periodicities in the biosynthesis of serum cholesterol and very low density lipoprotein (VLDL)-palmitate. The isotopic enrichment of the true biosynthetic precursor (intracellular acetyl-CoA) during oral or intravenous administration of sodium[1-13C]- or [2-13C]acetate was calculated from mass isotopomer fractional abundances in free cholesterol and VLDL-palmitate, determined by gas chromatography-mass spectrometry (GC-MS). To convert fractional into absolute cholesterol synthesis rates, decay rate constants of plasma cholesterol were determined from the die-away curves of endogenously labeled high-mass isotopomers. Oral [13C]acetate was a 3-4 times more efficient means of labeling the precursor pool for VLDL-palmitate than was intravenous [13C]acetate, consistent with a splanchnic site of VLDL-fatty acid synthesis, whereas the precursor for free cholesterol had an intermediate enrichment, suggesting a contribution from extra-splanchnic tissues as well. Endogenous synthesis of serum cholesterol was 8-11 mg/kg per day (an estimated 65-75% of input into serum cholesterol); it was 1.5- to 3-fold higher at night than during the day (37-49 mg/h at night compared to 9-23 mg/h during the day) and did not vary over the menstrual cycle (608-697 mg/day). In contrast, endogenous synthesis of fatty acids made a relatively minor contribution to body fat pools (1/10-1/20) of input into VLDL-palmitate) compared to dietary fat intake; it was greater in the day-time, and was influenced by menstrual cycle (3-fold elevated in the follicular phase compared to the luteal phase), and body composition (higher in obese men than normal weight men, r2 = 0.59 for lipogenesis vs. body mass index). Factors responsible for periodicities in endogenous lipid synthesis can be studied in humans using this approach.

Acetates↗

PubMed2000

Simultaneous quantification of tacrolimus and sirolimus, in human blood, by high-performance liquid chromatography-tandem mass spectrometry.

In this paper the authors present a validated method for the simultaneous analysis of tacrolimus and sirolimus in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry. Blood samples (500 microL) were prepared by C18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring. The assay was linear for both compounds over the range 0.25-100 microg/L (r2 > 0.996, n = 7). At the limit of quantification (0.25 microg/L), for both sirolimus and tacrolimus, the interday imprecision was < 3% and the analytical recovery was between 97.0% and 102%, respectively. The interbatch and intrabatch coefficients of variation of the method for both analytes, at the three quality control concentrations (0.5, 20, and 80 microg/L), were < 16% and < 10%, respectively. The analytical recovery, at the three control concentrations, ranged from 99.2% to 104% of the nominal concentration. The mean absolute recovery (+/- standard deviation) of tacrolimus, sirolimus, and internal standard was 82 +/- 7%, 89 +/- 12%, and 77 +/- 8%, respectively (n = 12). In conclusion, the method presented can be used for simultaneous determination of tacrolimus and sirolimus and will aid in pharmacokinetic studies and therapeutic drug monitoring of these drugs. Furthermore, this method has economic benefits in the clinical setting where these drugs are coadministered.

Chromatography, High Pressure Liquid↗

PubMed2003

Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction.

Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1991

Platelet aggregation, storage pool deficiency, and protein phosphorylation in mice with Chediak-Higashi syndrome.

The beige (bgJ/bgJ) mouse is a well-described murine model of Chediak-Higashi syndrome. Platelet function was examined in normal and beige mice to better characterize the defective aggregation response in platelets from mice with Chediak-Higashi syndrome. Platelet aggregation after collagen, thrombin, and phorbol-12-myristate 13-acetate stimulation was significantly (P less than 0.025) decreased in platelets from beige mice, relative to platelets from normal mice. Compared with beige and normal mice, those heterozygous for the bg trait had intermediate responses to collagen and thrombin, but not phorbol-12-myristate 13-acetate. The defect(s) in aggregation of platelets from beige mice was associated with a dense granule storage pool deficiency and decreased stores of serotonin and adenine nucleotides in platelets. Mice heterozygous for the bg trait had normal platelet serotonin and adenine nucleotide concentrations. Platelets from beige mice were approximately 10 times more sensitive to prostacyclin inhibition of collagen-induced aggregation than were platelets from control mice. However, a significant difference in platelet cyclic AMP concentration was not apparent between beige and normal mice after prostacyclin stimulation. Platelet endoperoxide synthesis measured by quantification of thromboxane B2, was normal in beige mice. Protein phosphorylation patterns in mouse platelets were similar to those seen in human platelets. Thrombin and collagen-induced [32P] phosphorylation of 40- and 20-kD proteins in platelets from normal and beige mice was similar. Results indicate that the biochemical defect(s) in platelet function in beige mice is partially attributable to storage pool deficiency and does not result in an absolute defect in phosphorylation of 40- and 20-kD proteins.

Animals↗

PubMed1999

Single- versus dual-platform assays for human CD34+ cell enumeration.

We comparatively assessed CD34+ cell quantification by two of the recently available single platform assays, the IMAGN 2000 STELLer (Immucor, Lisbon, Portugal) microvolume fluorimetry and the ProCOUNT (BD-ENZIfarma, Lisbon, Portugal) flow cytometry, with our "in-house" dual-platform flow cytometric assay. The performance of the methods was evaluated by linearity and reproducibility tests. The linearity study, over a range of 0-1,200 CD34+ cell/microl, gave a good linear relationship for the three methods, with R(2) > 0.99. Precision tested at three different concentrations gave coefficients of variation ranging from 3.6-26.4% for the STELLertrade mark, 2.4-13.8% for the ProCOUNT, and 3.2-6.4% for flow cytometry. CD34+ cells were quantified in umbilical cord blood (UCB), UCB enriched-leukocyte buffy-coat (BC), mobilized peripheral blood (PB) and mobilized peripheral blood progenitor cells (PBPC) collected by leucapheresis, from a total of 72 samples. Flow cytometric results showed good linear correlation to the absolute counts obtained by the STELLer and ProCOUNT for all samples (R > 0.90 for all methods), with no differences when compared by paired tests (P > 0.05). Linear correlations between methods were also found when individually looking at the different cell sources: UCB or PB, BC, and PBPC, with low, intermediate and high CD34+ cell concentrations, respectively. Furthermore, with the exception of a significant difference between the ProCOUNT and STELLer results for UCB (P < 0.05), no other difference between methods was found for each of the individual populations (P > 0.05). To our knowledge, this is the first report in which the results are presented and analyzed according to each source of CD34+ cells. Our results show that the STELLer and the ProCOUNT are equally efficient for the dual-platform flow cytometric assay in CD34+ cell quantification.

Antigens, CD34↗

PubMed2006

Model-based registration for dynamic cardiac perfusion MRI.

PURPOSE: To assess the accuracy of a model-based approach for registration of myocardial dynamic contrast-enhanced (DCE)-MRI corrupted by respiratory motion. MATERIALS AND METHODS: Ten patients were scanned for myocardial perfusion on 3T or 1.5T scanners, and short- and long-axis slices were acquired. Interframe registration was done using an iterative model-based method in conjunction with a mean square difference metric. The method was tested by comparing the absolute motion before and after registration, as determined from manually registered images. Regional flow indices of myocardium calculated from the manually registered data were compared with those obtained with the model-based registration technique. RESULTS: The mean absolute motion of the heart for the short-axis data sets over all the time frames decreased from 5.3+/-5.2 mm (3.3+/-3.1 pixels) to 0.8+/-1.3 mm (0.5+/-0.7 pixels) in the vertical direction, and from 3.0+/-3.7 mm (1.7+/-2.1 pixels) to 0.9+/-1.2 mm (0.5+/-0.7 pixels) in the horizontal direction. A mean absolute improvement of 77% over all the data sets was observed in the estimation of the regional perfusion flow indices of the tissue as compared to those obtained from manual registration. Similar results were obtained with two-chamber-view long-axis data sets. CONCLUSION: The model-based registration method for DCE cardiac data is comparable to manual registration and offers a unique registration method that reduces errors in the quantification of myocardial perfusion parameters as compared to those obtained from manual registration.

Algorithms↗

PubMed2005

Liquid chromatography-mass spectrometry and 15N metabolic labeling for quantitative metabolic profiling.

Metabolomics, i.e., the global analysis of cellular metabolites, is becoming a powerful tool for gaining insights into biological functions in the postgenomic context. However, absolute quantitation of endogenous metabolites in biological media remains an issue, and available technologies for the analysis of metabolome still lack robustness and accuracy. We describe here a new method based on liquid chromatography-mass spectrometry and (15)N uniform metabolic labeling of Saccharomyces cerevisiae for accurate and absolute quantitation of nitrogen-containing cell metabolites in metabolic profiling experiments. As a proof of concept study, eight sulfur metabolites involved in the glutathione biosynthesis pathway (i.e., cysteine, homocysteine, methionine, gamma-glutamylcysteine, cystathionine, reduced and oxidized forms of glutathione, and S-adenosylhomocysteine) were simultaneously quantified. The analytical method has been validated by studies of stability, selectivity, precision, and linearity and by the determination of the limits of detection and quantification. It was then applied to the analysis of extracts from cadmium-treated yeasts. In these conditions, the intracellular concentrations of most of the metabolites involved in the glutathione biosynthesis pathway were increased when compared to control extracts. These data correlate with previous proteomic results and also underline the importance of glutathione in cadmium detoxication.

Cadmium↗

PubMed1999

Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a 'real time' quantitative RT-PCR assay.

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the sensitivity of a serial dilution of total RNA from a positive cell line (K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ABL ratio in all samples. The two patients treated by IFN showed a progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10(-4). The four patients remained in clinical and cytogenetic CR. The seven other allografted patients were studied immediately after the procedure. Three of them showed a progressive decrease in the BCR-ABL/ABL ratio which reached 10(-4) 7 months after allo-BMT. The three patients remained in hematologic and cytogenetic CR. The remaining four allografted patients had progressive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR in the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round nested PCR. However, evolution changes in the results of real-time quantitative RT-PCR often preceded those of the conventional technique: a decrease of the BCR-ABL/ABL ratio preceded progression from first round to second round positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinical and cytogenetic evolution than conventional qualitative techniques and may help in making early therapeutic decisions in CML, especially after molecular relapse.

Adult↗