Performance of robust regression methods in real-time polymerase chain reaction calibration.

The ordinary least squares (OLS) method is routinely used to estimate the unknown concentration of nucleic acids in a given solution by means of calibration. However, when outliers are present it could appear sensible to resort to robust regression methods. We analyzed data from an External Quality Control program concerning quantitative real-time PCR and we found that 24 laboratories out of 40 presented outliers, which occurred most frequently at the lowest concentrations. In this article we investigated and compared the performance of the OLS method, the least absolute deviation (LAD) method, and the biweight MM-estimator in real-time PCR calibration via a Monte Carlo simulation. Outliers were introduced by replacement contamination. When contamination was absent the coverages of OLS and MM-estimator intervals were acceptable and their widths small, whereas LAD intervals had acceptable coverages at the expense of higher widths. In the presence of contamination we observed a trade-off between width and coverage: the OLS performance got worse, the MM-estimator intervals widths remained short (but this was associated with a reduction in coverages), while LAD intervals widths were constantly larger with acceptable coverages at the nominal level.

An Italian program of External Quality Control for chromogranin A (CgA) assay: performance evaluation of CgA determination.

BACKGROUND: Chromogranin A (CgA) is an acidic glycoprotein produced by many neuroendocrine cells and neurons. Currently, two different methods for assaying CgA, immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA), are widely used in routine practice. Within the framework of a Ministry of Health project, an External Quality Control program was developed to investigate the state of the art of CgA determination in Italy and to monitor the performance of laboratories carrying out this assay. This paper reports the results regarding laboratory performance. METHODS: A total of 43 laboratories participated in this program, in which 21 used the ELISA method and 22 the IRMA method. Each laboratory received six samples, three aliquots of serum and three of plasma, at high, intermediate and low concentrations. The results provided by the two assay methods were analyzed separately using two statistical approaches, the principal component analysis and the control chart method. RESULTS: For the IRMA method, questionable results for all samples were obtained by two laboratories, while in two other laboratories performance was questionable for only one sample. For the ELISA method, questionable performances were obtained in only one laboratory for the low and intermediate concentration samples, whereas in three laboratories performance was questionable for only one sample. Interestingly, the coefficients of variation increased approximately five-fold when shifting from the IRMA to the ELISA method. CONCLUSIONS: This program demonstrated both the requirement and demand for external quality assessment of CgA assay.

[Statistical notes. Statistical significance and clinical relevance: are they the two sides of the same medal?]. / Note statistiche. Significatività statistica e rilevanza clinica: due facce della stessa medaglia?

The aim of this statistical note is to draw the attention of the cardiologists to the aspects pertinent to the clinical relevance of the result of a clinical controlled randomized trial. The difference between clinical relevance and statistical significance is shown by using the results of GISSI and GUSTO III clinical controlled randomized trials.

EQUAL-qual: a European program for external quality assessment of genomic DNA extraction and PCR amplification.

BACKGROUND: Despite the rapid transition into routine clinical practice of molecular techniques based on PCR, external quality assessment (EQA) is still not widely available. The European Union and European Communities Confederation of Clinical Chemistry have supported the EQUAL project as a series of 3 different EQA programs for the assessment of molecular methods independently from analytes. We present the results from the EQUAL-qual program designed to evaluate the analytical aspects of DNA analysis by means of a conventional qualitative PCR experiment. METHODS: The EQUAL-qual program provided DNA, blood samples, and primer sets to participant laboratories to assess DNA extraction and PCR amplification. We have developed statistical procedures to identify laboratories performing poorly in DNA extraction (quality and quantity), PCR efficiency, and data interpretation after electrophoresis. RESULTS: An application to participate was obtained from 213 laboratories (from 25 countries), and 175 (82%) of laboratories submitted results for assessment. Questionable results in terms of quality and/or quantity of DNA derived from blood extractions were returned by 27% of laboratories (46 of 166). PCR efficiency showed high variability, with 3% of laboratories (5 of 163) showing a consistently low rate of amplification and 10% (18 of 175) not reporting the expected number of bands of the amplified targets. CONCLUSIONS: The results showed considerable variability in all phases of the experiment. The approach confirms the validity of EQA as a method for evaluating analytical aspects of PCR-based tests.

[What does "p" mean at conclusion of a test of hypothesis in a randomized controlled clinical trial of superiority?]. / Note statistiche. Cosa significa "p"a conclusione di un test d'ipotesi in una sperimentazione clinica controllata di superiorità?

The aim of this statistical note, the sixth in the series, is to introduce the rationale of the test of hypothesis suitable for comparing the effect of two treatments in a randomized controlled clinical trial of superiority. The presentation takes advantage of the analogy with a criminal trial debate based upon circumstantial evidence in an Italian Court. The results of three randomized controlled clinical trials: ISIS-1, AIMS and RESTORE are introduced and proper ways for their interpretation are suggested.

Axillary lymph node nanometastases are prognostic factors for disease-free survival and metastatic relapse in breast cancer patients.

PURPOSE: Early breast cancer presents with a remarkable heterogeneity of outcomes. Undetected, microscopic lymph node tumor deposits may account for a significant fraction of this prognostic diversity. Thus, we systematically evaluated the presence of lymph node tumor cell deposits

[Statistical notes. Which risks do the patient incur if his own cardiologist accepts to be involved in a clinical trial without deep consideration of the a priori available evidence?]. / Note statistiche. A quali rischi espone i propri pazienti il cardiologo che accetta di partecipare ad una sperimentazione clinica senza un'approfondita riflessione sulle evidenze disponibili a priori?

The aim of this statistical note is to describe the results of the randomized controlled clinical trial TARGET, which compared the effect of tirofiban (new treatment) and abciximab (standard treatment) in patients who were expected to undergo coronary stenting. Primary aim of TARGET was to evaluate the non-inferiority of tirofiban with respect to abciximab, but it concluded in favor of superiority of the standard treatment. The authors of this study point out that a deep consideration regarding a priori available evidence could have avoided to expose 2398 patients randomized to tirofiban to the risk of death or non-fatal myocardial infarction.

EQUAL-quant: an international external quality assessment scheme for real-time PCR.

BACKGROUND: Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR. METHODS: The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number. RESULTS: The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants. CONCLUSIONS: This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.