Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Quality control of multichannel hematology analyzers. Bivariate on-line comparison of MCV and MCH values for the detection of random errors.

Volume and hemoglobin content of red blood cells are physiologically interdependent variables but are measured independently in multichannel hematologic analyzers. Therefore correlation of these results to each other can be used as an additional step in internal quality control. To facilitate precise correlation, data were collected from 44,873 consecutive routine determinations of red blood cell parameters. Gaussian distributions were fitted to the original distributions and bivariate elliptical acceptance limits were calculated for simultaneous evaluation of mean corpuscular volume and mean corpuscular hemoglobin results. With the proposed method, rare combinations of the three directly measured red blood cell parameters (hemoglobin, red blood cell count, and mean corpuscular volume) can be identified for the detection of random analytical or preanalytical errors. This mean corpuscular volume-mean corpuscular hemoglobin rule could be added to internal quality-control programs of hematologic analyzers to improve detection of unusual results and possible random errors.

Software for illustrative presentation of basic clinical characteristics of laboratory tests--GraphROC for Windows.

GraphROC for Windows is a program for clinical test evaluation. It was designed for the handling of large datasets obtained from clinical laboratory databases. In the user interface, graphical and numerical presentations are combined. For simplicity, numerical data is not shown unless requested. Relevant numbers can be "picked up" from the graph by simple mouse operations. Reference distributions can be displayed by using automatically optimized bin widths. Any percentile of the distribution with corresponding confidence limits can be chosen for display. In sensitivity-specificity analysis, both illness- and health-related distributions are shown in the same graph. The following data for any cutoff limit can be shown in a separate click window: clinical sensitivity and specificity with corresponding confidence limits, positive and negative likelihood ratios, positive and negative predictive values and efficiency. Predictive values and clinical efficiency of the cutoff limit can be updated for any prior probability of disease. Receiver Operating Characteristics (ROC) curves can be generated and combined into the same graph for comparison of several different tests. The area under the curve with corresponding confidence interval is calculated for each ROC curve. Numerical results of analyses and graphs can be printed or exported to other Microsoft Windows programs. GraphROC for Windows also employs a new method, developed by us, for the indirect estimation of health-related limits and change limits from mixed distributions of clinical laboratory data.

Estimation of reference change limits using patient data.

Two approaches for deriving reference change limits from patient data are described. In the direct method, hospital database information is used for the selection of appropriate reference groups. If database information is not sufficient or reliable enough, but still most of the source data can be considered as health-related, an indirect method can be applied in the calculation of rough estimates for reference change limits. A computer program developed by us, GraphROC for Windows, includes both methods for the estimation of change limits from patient data. Time between specimen collections should be included as one classifying factor in the selection of source data. When only one previous result is available for comparison, change limits based on the reference sample group form the only available guide for clinical interpretation. However, when several previous results are available and the within-subject variances for the considered analyte are known to be heterogeneous between individuals, the clinical interpretation should rather be based on application of time series analysis.

Reliability and adequacy of discharge diagnosis databases in the production of reference values.

Discharge diagnoses provide a possibility to select patients individually and then to establish reference values for both "pathological" and control groups. Currently, the available diagnostic information is still at its infancy and should be carefully evaluated before the reference values based on those groups are utilized. It is anticipated that electronic storage of diagnostic and therapeutic information will be applied more commonly in the future as the development of computers makes it easier. The advanced utilization of laboratory data challenges physicians both in the clinical and laboratory side to participate in this development in order to make the information systems serve their actual needs more closely.

Effect of platelet count on serum and plasma potassium: evaluation using database information from two hospitals.

The availability of retrospective data from potassium (K+) analyses from two hospitals, one using serum and the other plasma for electrolyte measurements, offered us the possibility to investigate the effect of blood platelet count on serum and plasma K+ concentrations. A weak correlation between plasma K+ and platelet count was observed. The in vitro increase of serum K+ in proportion to the platelet count has clinical significance in conditions, where it may impede the detection of an underlying true K+ disorder. Nomograms and correction factors, based on the correlation between platelet count and serum K+, have been suggested also in some recent reports. In the present study unselected routine patient data was used as source data. The effect of platelet count on the concentration of K+ in serum was lower than reported in previous studies, as indicated by the regression analysis. An increase of 1000 x 10(9)/l in the blood platelet count would cause an increase of about 0.7 mmol/l in the serum K+ concentration (p < 0.0001, r = 0.155). The weak correlation between platelet count and serum K+ does not support the application of platelet-count-based correction of serum K+ level in thrombocytosis. The laboratory should notify the clinician of the significance of the in vitro increase of K+ caused by increased platelet count. K+ should be measured from plasma in such cases.

Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma.

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6-10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities.

Development of standardized approaches to reporting of minimal residual disease data using a reporting software package designed within the European LeukemiaNet.

Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL+ chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFß-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL+ MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data.

Hippocampal volume, brain atrophy, and APOE genotype after traumatic brain injury.

OBJECTIVE: To examine the association between hippocampal volumes, general brain atrophy, and apolipoprotein E (APOE) polymorphism in patients with a remote traumatic brain injury (TBI). METHODS: MRI-based volumetric analyses of the hippocampus and lateral ventricles were performed in 58 patients with TBI of varying severity on average 31.3 years after the trauma. The APOE genotype was determined using standard methods and correlated with the MRI volumetric measurements. RESULTS: Hippocampal or lateral ventricle volumes did not differ significantly in those patients with the APOE-epsilon4 allele (APOE4) vs those without this allele. CONCLUSIONS: The APOE-epsilon4 allele was not associated with the development of hippocampal or ventricular atrophy after traumatic brain injury. If the APOE-epsilon4 allele is associated with an unfavorable outcome after traumatic brain injury as proposed, this association may involve mechanisms other than those responsible for the development of brain atrophy.

The occurrence of dominant spinocerebellar ataxias among 251 Finnish ataxia patients and the role of predisposing large normal alleles in a genetically isolated population.

OBJECTIVES: Frequency and distribution of dominant ataxias caused by dynamic mutations may vary in different populations, which has been explained on the basis of relative frequency of predisposing normal alleles. The aim of the study was to evaluate the occurrence of spinocerebellar ataxias (SCAs) and dentatorubral-pallidoluysian atrophy (DRPLA) in Finland, and to investigate the role of predisposing normal alleles in a genetically homogenous population. MATERIAL AND METHODS: Mutation analyses for SCA1, 2, 3, 6, 7, 8, 10, 12, 17, and DRPLA and frataxin genes were performed for 251 unrelated Finnish patients who presented with progressive ataxia disorder. RESULTS: Expansions of SCA1, SCA2, SCA6, SCA7, SCA8, and SCA17 genes were detected in 2, 1, 1, 7, 22, and 1 patients, respectively. Altogether, 39 and 7% of dominant and sporadic SCA patients, respectively, harboured expansions at some of the investigated loci. Normal variation, collected from 477 to 502 chromosomes at each disease loci, revealed that Finns were different from the Japanese but largely similar to other Caucasians. CONCLUSIONS: Lack of SCA3 and excess of SCA8 are characteristic to the Finnish population. Homozygosity for the SCA8 expansion increases penetrance. Frequencies of large normal alleles at the SCA loci predict poorly prevalence of the respective diseases in Finland. Prioritization in DNA testing, based on ethnic origin and geographical location, is recommendable in Finland, and analogous approach may be applied to other countries as well.

Regression-based reference limits: determination of sufficient sample size.

Regression analysis is the method of choice for the production of covariate-dependent reference limits. There are currently no recommendations on what sample size should be used when regression-based reference limits and confidence intervals are calculated. In this study we used Monte Carlo simulation to study a reference sample group of 374 age-dependent hemoglobin values. From this sample, 5000 random subsamples, with replacement, were constructed with 10-220 observations per sample. Regression analysis was used to estimate age-dependent 95% reference intervals for hemoglobin concentrations and erythrocyte counts. The maximum difference between mean values of the root mean square error and original values for hemoglobin was 0.05 g/L when the sample size was > or = 60. The parameter estimators and width of reference intervals changed negligibly from the values calculated from the original sample regardless of what sample size was used. SDs and CVs for these factors changed rapidly up to a sample size of 30; after that changes were smaller. The largest and smallest absolute differences in root mean square error and width of reference interval between sample values and values calculated from the original sample were also evaluated. As expected, differences were largest in small sample sizes, and as sample size increased differences decreased. To obtain appropriate reference limits and confidence intervals, we propose the following scheme: (a) check whether the assumptions of regression analysis can be fulfilled with/without transformation of data; (b) check that the value of v, which describes how the covariate value is situated in relation to both the mean value and the spread of the covariate values, does not exceed 0.1 at minimum and maximum covariate positions; and (c) if steps 1 and 2 can be accepted, the reference limits with confidence intervals can be produced by regression analysis, and the minimum acceptable sample size will be approximately 70.

Rapid single-tube screening of the C282Y hemochromatosis mutation by real-time multiplex allele-specific PCR without fluorescent probes.

BACKGROUND: An accurate determination of the major HFE mutation (C282Y), which is associated with hereditary hemochromatosis, is important in diagnosis and risk assessment for this disease. We report a single-tube high-throughput PCR method for the detection of C282Y. METHODS: We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches. RESULTS: PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished. CONCLUSIONS: This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.

Antimicrobial therapy of septicemic patients in intensive care units before and after blood culture reporting.

68 cases of positive blood cultures from 54 intensive care unit (ICU) patients were analyzed retrospectively. The empiric antimicrobial therapy was correct in 65% of the cases as judged by the species and sensitivity of the blood culture isolate. After initial Gram-staining results were known, coverage increased to 77%. After the final blood culture results the coverage was still only 81%. The bacteremia-related mortality was 13%. Although there was no significant difference between the occurrence of bacteremia-related and non-bacteremia-related deaths either in patients with correct or non-optimal empiric treatment, this study emphasizes the need for better utilization of culture reporting. A considerable part of the final blood culture results went unnoticed by the ICU physicians which stresses the importance of good communication between the laboratory and wards.

Reference intervals for 24-h urinary normetanephrine, metanephrine, and 3-methoxy-4-hydroxymandelic acid in hypertensive patients.

The urinary excretion of normetanephrine, metanephrine, and 3-methoxy-4-hydroxymandelic acid was quantified by HPLC in hypertensive patients who were being routinely investigated for the exclusion of pheochromocytoma. The data were used to calculate reference intervals for these analytes. Age- and sex-specific 95% reference intervals for 24-h urinary excretion were as follows (mumol/24 h): normetanephrine in men ages 16-35 years (n = 17) 0.7-3.4, in men older than 35 years (n = 121) 0.8-5.1, in women ages 16-35 years (n = 41) 0.5-2.1, and in women older than 35 years (n = 144) 0.6-3.3 mumol/24 h; metanephrine in men (n = 138) 0.3-2.0, and in women (n = 185) 0.2-1.3; and 3-methoxy-4-hydroxymandelic acid in men (n = 142) 10-54, and in women (n = 184) 9-38. Normetanephrine or metanephrine excretion rates or both exceeded the proposed reference intervals in seven of the eight patients with pheochromocytoma. Determination of 3-methoxy-4-hydroxymandelic acid excretion did not yield any additional diagnostic information.