Chromosome microarray proficiency testing and analysis of quality metric data trends through an external quality assessment program for Australasian laboratories.

Chromosome microarrays are an essential tool for investigation of copy number changes in children with congenital anomalies and intellectual deficit. Attempts to standardise microarray testing have focused on establishing technical and clinical quality criteria, however external quality assessment programs are still needed. We report on a microarray proficiency testing program for Australasian laboratories. Quality metrics evaluated included analytical accuracy, result interpretation, report completeness, and laboratory performance data: sample numbers, success and abnormality rate and reporting times. Between 2009 and 2014 nine samples were dispatched with variable results for analytical accuracy (30-100%), correct interpretation (32-96%), and report completeness (30-92%). Laboratory performance data (2007-2014) showed an overall mean success rate of 99.2% and abnormality rate of 23.6%. Reporting times decreased from >90 days to <30 days for normal results and from >102 days to <35 days for abnormal results. Data trends showed a positive correlation with improvement for all these quality metrics, however only 'report completeness' and reporting times reached statistical significance. Whether the overall improvement in laboratory performance was due to participation in this program, or from accumulated laboratory experience over time, is not clear. Either way, the outcome is likely to assist referring clinicians and improve patient care.

High-throughput genotyping of human platelet antigens using the 5'-nuclease assay and minor groove binder probe technology.

BACKGROUND AND OBJECTIVES: Human platelet antigen (HPA) genotyping is a valuable tool for typing platelets to assist in the management of alloimmunized thrombocytopenic patients. We describe, for the first time, 5' nuclease assays (NA) to genotype HPA-5 and -15, and improved 5'-NA to genotype HPA-1, -2 and -3, by utilizing minor groove binder (MGB) and non-fluorescent quencher (NFQ) technology. Superior probe specificity and fluorescent performance is attained through MGB-NFQ probe modifications compared with previous 5'-NA designs. MATERIALS AND METHODS: Primers and dye-labelled MGB-NFQ probes were designed and synthesized to detect the single nucleotide polymorphism responsible for each HPA-1, -2, -3, -5 and -15. One-hundred blood samples were tested for the combinations of HPA genotypes 1, 2, 3 and 5 by our traditional sequence-specific primer-polymerase chain reaction (SSP-PCR) method, and 41 blood samples were tested for HPA-15 by SSP-PCR at an external laboratory. These results were then compared with those obtained by using the new 5'-NA. RESULTS: There was complete concordance of results for all samples tested by SSP-PCR and 5'-NA. The 5'-NA offers distinct advantages over non-fluorescent genotyping methods. DNA amplification and allele discrimination occurs in a single closed tube for each antigen with no post-PCR manipulation required. This minimizes the risk of cross-contamination and mislabelling of samples, as well as making the assay less time-consuming to perform. In comparison with other fluorescent assays, the 5'-NA has the highest sample throughput, resulting from the use of a 96-well platform, identical cycling conditions for all assays and the potential for automation. CONCLUSIONS: Genotyping for HPA-1, -2, -3, -5, and -15 by the 5'-NA is suitable for routine analysis. The latest 5'-NA design, using MGB probe technology, ensures superior detection of all alleles and is the most versatile fluorescent assay, ideal for both urgent clinical samples and large-scale screening programs.