Past analysis of laboratory methods used for mycology specimens revealed significant variation in practices, many of which fell short of recommended procedures. In 2016 these findings led to a set of recommendations for laboratories to consider modification of their methods where appropriate, to analyse current laboratory methods used by participants in the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) Mycology module, and to compare these to the 2016 recommendations. Seven test items, with 105-107 participants each, were analysed. Several laboratories (7-12%) did not handle specimens as recommended in an appropriate biological safety cabinet. Direct microscopy was not performed on tissue specimens 23-25% of the time. The most used staining method was potassium hydroxide with an optical brightener for fluorescent microscopy (49%) followed by Gram stain (33%). While 17-25% of laboratories used three or more media, use of four or more was uncommon (<3%). Between 9-13% of participants used only a single non-inhibitory medium for cultures. Urine specimens were incubated longer than recommended with 57% of laboratories incubating for >7days and 24% >21 days. Duration of incubation was shorter than recommended for several specimen types with 36% of skin specimens and 37-48% of tissue specimens being kept ≤21 days. For cultures kept >7 days, 13% were inspected daily but for those incubating >14 days only 3%. The methods of several laboratories remain outside recommended practice. An updated set of recommendations are made.
The emergence of spatial profiling technologies in recent years has accelerated opportunities to profile in detail the molecular attributes of a wide range of tissue pathologies using archival specimens. However, tissue treatment for fixation and storage does not always support generation of high-quality genomic data. The purpose of this study was to investigate the impacts of Proteinase K (ProtK) treatment, as a way to increase target transcript exposure, on downstream sequencing data quality metrics for spatial transcriptomic data using formalin-fixed, paraffin-embedded samples. In a series of four independent assessments using different tissue types (nasal mucosa, tonsil, pancreas), varying concentrations of ProtK (ranging from 0.1 to 1 μg/mL) were used as part of the sample processing workflow to generate transcriptomic data using the Nanostring GeoMx DSP and Illumina NextSeq 2000 platforms. Use of higher concentrations of ProtK was generally found to increase total reads (2-4-fold). However, negative probe counts also tended to be increased (2-12-fold), resulting in reductions in the signal-to-noise ratio (10-70% lower) and the number of genes detected above background (50-80% lower). These effects were not seen in all tissues and impacts of tissue handling and processing, beyond ProtK treatment, on data quality metrics, also require consideration. Regardless, these observations highlight the need for careful consideration of a range of sample processing factors and benefits that may be achieved through the optimisation of sample processing workflows for specific tissues as a way to maximise the generation of quality data using spatial transcriptomic approaches.
Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) is a rare and highly aggressive lymphoma with characteristic ALK rearrangements. Various fusion genes involving ALK have been demonstrated, but the influence of the ALK fusion partners on ALK protein expression and the genetic characteristics of ALK+ LBCL remain relatively unknown. In this study, we conducted an extensive clinicopathological and molecular analysis on seven cases of ALK+ LBCL to explore the correlation between ALK fusion genes and ALK protein expression, thereby enriching the genetic characteristics of this tumour. We integrated the findings from clinical, histopathological/immunophenotypic, and molecular studies, including three samples subjected to next-generation sequencing, and six cases underwent RNA-based ALK fusion gene detection. We identified five distinct types of ALK fusion genes, including CLTC, NPM1, PABPC1, SEC31A, and TFG. Notably, only the NPM1::ALK fusion showed nuclear and cytoplasmic ALK staining, and the remaining four fusion genes resulted in cytoplasmic ALK staining. Our analysis revealed that the CLTC::ALK fusion resulted in a unique cytoplasmic perinuclear Golgi zone focal granular heterogeneous staining pattern of ALK. Additionally, we identified six potentially clinically significant gene mutations, including TET2, CHD2, DTX1, KMT2D, LRP1B, and XPO1. Furthermore, in all cases, the absence of 5-hydroxymethylcytosine (5hmC) was observed. We present seven cases of ALK+ LBCL, discussing the correlation between fusion genes and ALK protein expression, and enhancing our understanding of the genetic attributes of this tumour. This study also shows the loss of 5hmC in nearly all seven ALK+ LBCL cases, independently of TET2 mutations.