Real-life data from standardized preanalytical coding (SPREC) in tissue biobanking and its dual use for sample characterization and process optimization.

The standardized preanalytical code (SPREC) aggregates warm ischemia (WIT), cold ischemia (CIT), and fixation times (FIT) in a precise format. Despite its growing importance underpinned by the European in vitro diagnostics regulation or broad preanalytical programs by the National Institutes of Health, little is known about its empirical occurrence in biobanked surgical specimen. In several steps, the Tissue Bank Bern achieved a fully informative SPREC code with insights from 10,555 CIT, 4,740 WIT, and 3,121 FIT values. During process optimization according to LEAN six sigma principles, we identified a dual role of the SPREC code as a sample characteristic and a traceable process parameter. With this preanalytical study, we outlined real-life data in a variety of organs with specific differences in WIT, CIT, and FIT values. Furthermore, our FIT data indicate the potential to adapt the SPREC fixation toward concrete paraffin-embedding time points and to extend its categories beyond 72 h due to weekend delays. Additionally, we identified dependencies of preanalytical variables from workload, daytime, and clinics that were actionable with LEAN process management. Thus, streamlined biobanking workflows during the day were significantly resilient to workload peaks, diminishing the turnaround times of native tissue processing (i.e. CIT) from 74.6 to 46.1 min under heavily stressed conditions. In conclusion, there are surgery-specific preanalytics that are surgico-pathologically limited even under process optimization, which might affect biomarker transfer from one entity to another. Beyond sample characteristics, SPREC coding is highly beneficial for tissue banks and Institutes of Pathology to track WIT, CIT, and FIT for process optimization and monitoring measurements.

Correction: Rainer et al. The Influence of Above-Ground Herbivory on the Response of Arctic Soil Methanotrophs to Increasing CH4 Concentrations and Temperatures. Microorganisms 2021, 9, 2080.

The authors wish to make the following corrections to this paper [...].

The Influence of Above-Ground Herbivory on the Response of Arctic Soil Methanotrophs to Increasing CH4 Concentrations and Temperatures.

Rising temperatures in the Arctic affect soil microorganisms, herbivores, and peatland vegetation, thus directly and indirectly influencing microbial CH4 production. It is not currently known how methanotrophs in Arctic peat respond to combined changes in temperature, CH4 concentration, and vegetation. We studied methanotroph responses to temperature and CH4 concentration in peat exposed to herbivory and protected by exclosures. The methanotroph activity was assessed by CH4 oxidation rate measurements using peat soil microcosms and a pure culture of Methylobacter tundripaludum SV96, qPCR, and sequencing of pmoA transcripts. Elevated CH4 concentrations led to higher CH4 oxidation rates both in grazed and exclosed peat soils, but the strongest response was observed in grazed peat soils. Furthermore, the relative transcriptional activities of different methanotroph community members were affected by the CH4 concentrations. While transcriptional responses to low CH4 concentrations were more prevalent in grazed peat soils, responses to high CH4 concentrations were more prevalent in exclosed peat soils. We observed no significant methanotroph responses to increasing temperatures. We conclude that methanotroph communities in these peat soils respond to changes in the CH4 concentration depending on their previous exposure to grazing. This "conditioning" influences which strains will thrive and, therefore, determines the function of the methanotroph community.

Implementation of a 'lean' cytopathology service: towards routine same-day reporting.

OBJECTIVES: To systematically assess the effects of a Lean management intervention in an academic cytopathology service. METHODS: We monitored outcomes including specimen turnaround times during stepwise implementation of a lean cytopathology workflow for gynaecological and non-gynaecological cytology. RESULTS: The intervention resulted in a major reduction of turnaround times for both gynaecological (3rd quartile 4.1 vs 2.3 working days) and non-gynaecological cytology (3rd quartile 1.9 vs. 1.2 working days). Introduction of fully electronic reporting had additional effect over continuous staining of slides alone. The rate of non-gynaecological specimens reported the same day increased from 4.5% to 56.5% of specimens received before noon. CONCLUSIONS: Lean management principles provide a useful framework for organization of a cytopathology workflow. Stepwise implementation beginning with a simplified gynaecological cytology workflow allowed involved staff to monitor the effects of individual changes and allowed for a smooth transition.

Polystyrene-Divinylbenzene-Based Adsorbents Reduce Endothelial Activation and Monocyte Adhesion Under Septic Conditions in a Pore Size-Dependent Manner.

Endothelial activation with excessive recruitment and adhesion of immune cells plays a central role in the progression of sepsis. We established a microfluidic system to study the activation of human umbilical vein endothelial cells by conditioned medium containing plasma from lipopolysaccharide-stimulated whole blood or from septic blood and to investigate the effect of adsorption of inflammatory mediators on endothelial activation. Treatment of stimulated whole blood with polystyrene-divinylbenzene-based cytokine adsorbents (average pore sizes 15 or 30 nm) prior to passage over the endothelial layer resulted in significantly reduced endothelial cytokine and chemokine release, plasminogen activator inhibitor-1 secretion, adhesion molecule expression, and in diminished monocyte adhesion. Plasma samples from sepsis patients differed substantially in their potential to induce endothelial activation and monocyte adhesion despite their almost identical interleukin-6 and tumor necrosis factor-alpha levels. Pre-incubation of the plasma samples with a polystyrene-divinylbenzene-based adsorbent (30 nm average pore size) reduced endothelial intercellular adhesion molecule-1 expression to baseline levels, resulting in significantly diminished monocyte adhesion. Our data support the potential of porous polystyrene-divinylbenzene-based adsorbents to reduce endothelial activation under septic conditions by depletion of a broad range of inflammatory mediators.

Proper paraffin slide storage is crucial for translational research projects involving immunohistochemistry stains.

The use of paraffin slides and tissue microarrays (TMA) is indispensable for translational research. However, storage of paraffin slides over time has a substantial detrimental effect on the quality and reliability of immunohistochemistry stains. Particularly affected by this issue may be any collaborative efforts where paraffin slides or TMAs are shipped to central laboratories and then 'biobanked' for some time until use. This article summarizes some of the key issues affecting loss of antigenicity on paraffin slides and some simple storage solutions to help maintain high quality immunohistochemistry results when paraffin slides must be stored for a certain time prior to use.

Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy.

Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

The apoptotic and proliferation rate of tumour budding cells in colorectal cancer outlines a heterogeneous population of cells with various impacts on clinical outcome.

AIMS: In colorectal cancer (CRC), tumour buds represent an aggressive cell type at the invasive front with apparently low proliferation. The aim of this study was to determine proliferation and apoptotic rates of buds in comparison to tumour centre, front and mucosa. METHODS AND RESULTS: Whole tissue sections from 188 CRC patients underwent immunohistochemistry for Ki67. Ten high-power fields (HPFs) were evaluated in mucosa, tumour centre, tumour front and tumour buds (total = 40 HPFs/case). Caspase-3 and M30 immunohistochemistry were performed on a multipunch tissue microarray from the same cohort. Ki67, caspase-3 and M30 immunoreactivity were correlated with outcome. The average percentage of cells showing Ki67 positivity was 5.2% in mucosa, and was not significantly different between the centre and front of the tumour (38.2% and 34.9%; P < 0.0001); 0.3% of buds showed Ki67 positivity (P < 0.0001). Caspase-3 expression was similar in mucosa, tumour centre and tumour front, but lower in tumour buds (<0.1%; P < 0.0001). M30 staining in buds was decreased (0.01%; P < 0.0001) in comparison to other areas. Ki67 positivity in buds was detrimental to survival in univariate (P = 0.0352) and multivariate (P = 0.0355) analysis. Caspase-3-positive tumours showed better outcome than negative tumours (P = 0.0262); but tumours with caspase-3-positive buds showed a worse outcome than those with caspase-3-negative buds (P = 0.0235). CONCLUSIONS: Ki67, caspase-3 and M30 staining is absent in most tumour buds, suggesting decreased proliferation and apoptosis. However, the fact that Ki67 and caspase-3 immunoreactivity was associated with unfavourable prognosis points to a heterogeneous population of tumour buds.

Possible role of Cdx2 in the serrated pathway of colorectal cancer characterized by BRAF mutation, high-level CpG Island methylator phenotype and mismatch repair-deficiency.

Colorectal cancer is a heterogeneous disease at the histomorphological, clinical and molecular level. Approximately 20% of cases may progress through the "serrated" pathway characterized by BRAF mutation and high-level CpG Island Methylator Phenotype (CIMP). A large subgroup are additionally microsatellite instable (MSI) and demonstrate significant loss of tumor suppressor Cdx2. The aim of this study is to determine the specificity of Cdx2 protein expression and CpG promoter hypermethylation for BRAF(V600E) and high-level CIMP in colorectal cancer. Cdx2, Mlh1, Msh2, Msh6, and Pms2 were analyzed by immunohistochemistry using a multi-punch tissue microarray (TMA; n = 220 patients). KRAS and BRAF(V600E) mutation analysis, CDX2 methylation and CIMP were investigated. Loss of Cdx2 was correlated with larger tumor size (P = 0.0154), right-sided location (P = 0.0014), higher tumor grade (P < 0.0001), more advanced pT (P = 0.0234) and lymphatic invasion (P = 0.0351). Specificity was 100% for mismatch repair (MMR)-deficiency (P < 0.0001), 92.2% (P < 0.0001) for BRAF(V600E) and 91.8% for CIMP-high. Combined analysis of BRAF(V600E)/CIMP identified Cdx2 loss as sensitive (80%) and specific (91.5%) for mutation/high status. These results were validated on eight well-established colorectal cancer cell lines. CDX2 methylation correlated with BRAF(V600E) (P = 0.0184) and with Cdx2 protein loss (P = 0.0028). These results seem to indicate that Cdx2 may play a role in the serrated pathway to colorectal cancer as underlined by strong relationships with BRAF(V600E), CIMP-high and MMR-deficiency. Whether this protein can only be used as a "surrogate" marker, or is functionally involved in the progression of these tumors remains to be elucidated.

Hepatocellular and cholangiolar carcinoma-derived cell lines reveal distinct sets of chromosomal imbalances.

OBJECTIVES: Hepatocellular carcinoma (HCC) and cholangiolar carcinoma (CC) cell lines are used to analyze the basic mechanisms of carcinogenesis and target therapies. However, it is not yet clear which chromosomal aberrations are to be typically expected in such cell lines. It is also not clear whether there are prerequisites for in vitro growth on the genomic and/or expression level. We therefore analyzed HCC and CC cell lines for typical genetic settings. METHODS: The HCC cell lines HLE, HLF, Huh7, HepG2 and Hep3b and the CC cell lines EGI1, MzCha1 and TFK-1 were analyzed using high-density arrays for comparative genomic hybridization (aCGH; 244,000 oligonucleotides). Additional fluorescence in situ hybridization analyses were done to confirm the aCGH results and to add information regarding the aneuploidy of cell lines. RESULTS: The gain of 1q, in particular q21-22, was detected in all HCC cell lines also as a partial loss of 13q. In contrast, a loss of 8p in combination with a relative gain of 8q was seen in all CC but no HCC cell lines. Interestingly, a gain of 17q was seen in all cell lines. These aberrations are also well documented for surgical tumor specimens. Besides these imbalances, the cell lines revealed imbalances for 11p, 12p, 14q, 16p, 16q, 21q and 22q, respectively, only rarely seen in surgical tumor specimens. These aberrations could be of importance for the in vitro cultivation of tumor cells. Structural aberrations were accompanied by aneuploidy in 3 of 5 HCC cell lines and 2 of 3 CC cell lines. Ploidy status was not correlated to any of the imbalances mentioned above. CONCLUSIONS: HCC and CC cell lines revealed characteristic chromosomal imbalances similar to those seen in surgical tumor specimens including chromosomes 1, 8, 13 and 17, respectively. These aberrations are characteristic of the histogenetic origin of the tumor cells. However, the chromosomal imbalances that occurred probably led to the ability of tumor cells to grow in vitro.

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma.

The prognostic outcome for hepatocellular carcinoma (HCC) remains poor. Disease progression is accompanied by dedifferentiation of the carcinoma, a process that is not well understood. The aim of this study was to get more insight into the molecular characteristics of dedifferentiated carcinomas using high throughput techniques. Microarray-based global gene expression analysis was performed on five poorly differentiated HCC cell lines compared with non-neoplastic hepatic controls and a set of three cholangiolar carcinoma (CC) cell lines. The gene with the highest upregulation was HLXB9. HLXB9 is a gene of the homeobox genfamily important for the development of the pancreas. RT-PCR confirmed the upregulation of HLXB9 in surgical specimens of carcinoma tissue, suggesting its biological significance. Interestingly, HLXB9 upregulation was primary observed in poorly differentiated HCC with a pseudoglandular pattern compared with a solid pattern HCC or in moderate or well-differentiated HCC. Additional the expression of translated HLXB9, the protein HB9 (NCBI: NP_001158727), was analyzed by western blotting. Expression of HB9 was only detected in the cytoplasm but not in the nuclei of the HCC cells. For validation CC were also investigated. Again, we found an upregulation of HLXB9 in CC cells accompanied by an expression of HB9 in the cytoplasms of these tumor cells, respectively. In conclusion, homeobox HLXB9 is upregulated in poorly differentiated HCC with a pseudoglandular pattern. The translated HB9 protein is found in the cytoplasm of these HCC and CC. We therefore assume HLXB9 as a possible link in the understanding of the development of HCC and CC, respectively.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat.

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2.

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Immunoliposome targeting to mesangial cells: a promising strategy for specific drug delivery to the kidney.

Mesangial cell-mediated nephropathies are a frequent cause of ESRD. Specific drug delivery to mesangial cells might be more effective and better tolerated than existing systemic treatments. Rat mesangial cells are characterized by Thy1.1 antigen expression. Therefore, OX7-coupled immunoliposomes (OX7-IL) were prepared by coupling liposomes with F(ab') fragments of OX7 mAb directed against Thy1.1 antigen. As the glomerular endothelium is fenestrated and no basement membrane separates glomerular capillaries from the mesangium, mesangial cells represent a particularly suitable target for drug delivery by OX7-IL. Therefore, the targeting efficacy of OX7-IL to mesangial cells was investigated. Specific targeting in vitro was obtained, and intravenous injection of OX-7-IL to rats showed a specific targeting of all mesangial cells in both kidneys. OX7-IL showed marked accumulation in the cytoplasm of rat mesangial cells, both in vitro and in vivo. This renal targeting was blocked when free OX7 F((ab')2) fragments were co-administered with OX7-IL. Rats that were given a single intravenous injection of low-dose doxorubicin encapsulated in OX7-IL showed extensive glomerular damage, whereas other parts of the kidney and other organs were spared. Free doxorubicin and the liposomal formulation of this agent had no effect. Thus, immunoliposomes are a very promising delivery system for the treatment of kidney diseases.