Practices and Perceived Value of Proficiency Testing in Clinical Laboratories.

BACKGROUND: Proficiency testing (PT) can have regulatory and nonregulatory uses, providing an effective tool for quality improvement. Clinical laboratories were surveyed to determine how they perceive PT and how they use PT results and materials to improve laboratory testing quality. METHODS: All laboratories certified to perform nonwaived testing under the CLIA regulations expected to perform required PT were invited to participate in the survey. We examined respondents' use of PT from 5 laboratory types: hospital, independent, public health, physician office, and "all other." Respondents' awareness of resources about PT was also examined. Several questions allowed responses on a categorical scale. RESULTS: Varying proportions of the respondents (n = 769) used PT to identify problems in the preanalytic (48%), analytic (86%), and postanalytic (76%) phases of testing. Responses also showed that PT was important for demonstrating personnel competency (93%), inappropriate specimen handling (80%), incorrect result interpretation (84%), and other uses. Respodents purchased PT even when not required to do so (77%). Based on all responses, most considered PT worth the cost (65%). CONCLUSIONS: Laboratories, regardless of type, have found ways of using leftover PT samples and the information from PT event summaries to help improve laboratory quality. Our findings suggest many laboratories are not taking full advantage of PT to improve testing quality. Additionally, the study suggests a need to improve awareness of resources about PT.

Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study.

PURPOSE: Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology. METHODS: An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation. RESULTS: The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified. CONCLUSION: The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.

Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening.

BACKGROUND: CDC's Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. METHODS: Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. RESULTS: The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. CONCLUSIONS: Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays.

Assessment of DNA contamination from dried blood spots and determination of DNA yield and function using archival newborn dried blood spots.

BACKGROUND: Residual dried blood spots (DBS) from newborn screening programs are often stored for years and are sometimes used for epidemiological studies. Because there is potential for DNA cross-contamination from specimen-to-specimen contact, we determined contamination levels following intentional contact and assessed archival DBS DNA degradation after storage in an uncontrolled environment. METHODS: DBS from healthy adult females were rubbed with DBS from healthy or cystic fibrosis (CF)-affected adult males. Total human and male DNA was measured from the female DBS. Contamination levels were assessed using short tandem repeats (STRs). Female DBS contaminated with CF male DNA containing the F508del were analyzed for presence of this mutation. Archival DBS DNA amplification efficiency was determined using STR analysis. RESULTS: Most female DBS were contaminated, however only one specimen showed an incomplete STR profile consistent with contaminating CF-affected male DNA. Further testing by CF mutation screening was negative. DNA extracted from archival DBS showed robust amplification (range 100 bp-320 bp). CONCLUSIONS: Lightly abrasive contact between DBS resulted in DNA cross-contamination. The contaminating DNA did not interfere in CF-mutation tests; however this should be determined for individual assays. Since DNA from archival DBS robustly amplifies, newborn DBS could provide an invaluable resource for public health studies.

Development and characterization of dried blood spot materials for the measurement of immunoreactive trypsinogen.

OBJECTIVES: In response to increasing numbers of states in the US that test newborn babies for cystic fibrosis (CF), the Newborn Screening Quality Assurance Programme initiated a pilot proficiency testing programme for immunoreactive trypsinogen (IRT), the biomarker for CF. Dried blood spot specimens (DBS) were used to evaluate the performance of laboratories that screen babies for CF. METHODS: DBS were prepared from human whole blood enriched with physiologically relevant levels of IRT. Various methods of making IRT-enriched DBS were used to optimize the recovery and stability of the biomarker, including preparation of DBS from either intact or lysed red blood cells, varying the timing of IRT addition to blood before dispensing onto filter paper, adding a protease inhibitor cocktail, and treating serum with charcoal before IRT enrichment. The recovery and stability of IRT in DBS were assessed. Newborn screening laboratories were offered the opportunity to test blind-coded DBS in the pilot PT programme. RESULTS: IRT was stable in the filter paper matrix when stored for one year at either -20 degrees C or 4 degrees C. Fifty percent more IRT was recovered from DBS prepared with lysed red blood cells where the IRT was added to blood just before dispensing; however, protease inhibitors did not improve IRT recovery. CONCLUSIONS: IRT in the DBS matrix is stable and can be shipped worldwide under ambient conditions. Optimal IRT recovery was achieved by adjustment of DBS production practices. Laboratories receiving specimens accurately measured IRT by a variety of commercial and in-house methods.

Report from a workshop on multianalyte microsphere assays.

Multiplexed assays using fluorescent microspheres is an exciting technique that has been gaining popularity among researchers, particularly those in the public health field. Part of its popularity is due to its flexibility, as both immunoassays and oligonucleotide hybridization assays can be developed on this platform. This report summarizes a workshop held by the Centers for Disease Control and Prevention that discussed issues surrounding these assays and the Luminex 100 xMAP instrument. Topics included instrumentation, assay design, sample matrix and volume, quality control, and development of commercial applications.