Results of the UK NEQAS for Molecular Genetics reference sample analysis

Aims: In addition to providing external quality assessment (EQA) schemes, United Kingdom National External Quality Assessment service (UK NEQAS) for Molecular Genetics also supports the education of laboratories. As an enhancement to the Molecular Pathology EQA scheme, a human cell-line reference sample, manufactured by Thermo Fisher Scientific (AcroMetrix), was provided for analysis. This contained many variants, present at frequencies between 1% and 17.9%. Methods: One hundred and one laboratories submitted results, with a total of 2889 test results on 53 genes being reported. Known polymorphisms, 46/2889 (1.59%) results, were excluded. Variants detected in the seven most commonly reported (and clinically relevant) genes, KRAS, NRAS, BRAF, EGFR, PIK3CA, KIT and PDGFRA, are reported here, as these genes fall within the scope of UK NEQAS EQA schemes. Results: Next generation sequencing (NGS) was the most commonly performed testing platform. There were between 5 and 27 validated variants in the seven genes reported here. Eight laboratories correctly reported all five NRAS variants, and two correctly reported all eight BRAF variants. The validated mean variant frequency was lower than that determined by participating laboratories, with single-gene testing methodologies showing less variation in estimated frequencies than NGS platforms. Laboratories were more likely to correctly identify clinically relevant variants. Conclusions: Over 100 laboratories took the opportunity to test the ‘educational reference sample’, showing a willingness to further validate their testing platforms. While it was encouraging to see that the most widely reported variants were those which should be included in routine testing panels, reporting of variants was potentially open to interpretation, thus clarity is still required on whether laboratories selectively reported variants, by either clinical relevance or variant frequency

Detection of MMPs and their inhibitors using a whole blood protease assay

The level of Matrix Metalloproteinases (MMPs) in whole blood has been investigated, with an emphasis on the effect of certain MMP inhibitors. A synthetically derived charge changing substrate, which has a cleavage site specific to enzymes MMP-2 and MMP-9, was used for detection. Presence of these enzymes was tested both with and without inhibitors, first in a buffer solution, and then in whole blood. The blood was taken from insulin resistant volunteers at different time points after the consumption of a meal rich in protein and fat. The volunteers were part of a twelve-week study performed to assess the change in levels of MMPs in the blood after being on a low-glycemic index diet. The presence of these enzymes in blood was assessed by reacting the blood with the synthetic substrate for a fixed period of time, and then using PAGE (polyacrylamide gel electrophoresis). After electrophoresis, the gel was imaged and enzyme levels quantified using a fluorescent detector. The gels were analyzed for intensity of fluorescence, which had a direct correlation with the amount of the MMP-2/9 present. Results indicated that there were slight changes in the levels of these enzymes after the 12-week period. However, the various MMP inhibitors such as grape seed extract (GSE), and doxycycline showed a definite decrease in enzyme activity, after reacting these inhibitors in whole blood in reaction tube