Invasive Sampling Techniques Research Articles

An Anaplastic Lymphoma Kinase (ALK) Immunohistochemical Pitfall: ALK-positive Histiocytosis versus Angiomatoid Fibrous Histiocytoma

Abstract Introduction/Objective The 5th Edition WHO classification expanded the histiocytic/dendritic cell neoplasms to include ALK-positive histiocytosis, which can present as multisystem or single-site disease, affecting various age groups. These tumors lack high-grade cytologic features but express ALK by immunohistochemistry, typically indicating ALK locus rearrangements. In this case study, a cutaneous mass initially suspected as ALK-positive histiocytosis was found to be angiomatoid fibrous histiocytoma (AFH) with ALK expression, highlighting the potential diagnostic pitfall due to overlapping histomorphology and ALK expression. Methods/Case Report A previously healthy twelve-year-old boy sought evaluation for a growing and ulcerating mass on his left arm, which developed gradually over a year post-injury. Laboratory tests showed elevated inflammatory markers and mild anemia. A biopsy of the ulcer revealed Staphylococcus aureus infection. Initial histological examination revealed mixed inflammatory infiltrates with focal necrosis, prominent histiocytes, and giant cells. Despite favoring an infectious cause, immunostains were performed. AE1/AE3, ERG, CD34, CD30, S100, SOX10, SMA, and MyoD1 were negative, while INI1 was retained. However, ALK was strongly positive in some histiocytoid cells, suggesting ALK-positive histiocytosis. Surgical resection was subsequently performed to manage the abscess and characterize the lesion. Histologic examination revealed a solid nodule containing spindled and epithelioid cells with a bland histiocytoid appearance, arranged in a compact syncytial growth pattern within the dermis and subcutis. Surrounding the tumor, there was a distinct cuffing of fibrosis with organized lymphoplasmacytic cells. Break apart fluorescence in situ hybridization studies showed rearrangement of EWSR1 but not ALK. The diagnosis of angiomatoid fibrous histiocytoma was rendered. Results (if a Case Study enter NA) NA Conclusion This case highlights the importance of understanding immunohistochemical features, especially those that are unusual and unexpected in rare diseases. With the growing use of minimally invasive sampling techniques, this case study underscores the significance of integrating molecular diagnostics with clinical, histological, and immunophenotypic evaluations to achieve an accurate diagnosis in surgical pathology.

Minimally Invasive Sampling of Mediastinal Lesions

This narrative review examines the existing literature on minimally invasive image-guided sampling techniques of mediastinal lesions gathered from international databases (Medline, PubMed, Scopus, and Google Scholar). Original studies, systematic reviews with meta-analyses, randomized controlled trials, and case reports published between January 2009 and November 2023 were included. Four authors independently conducted the search to minimize bias, removed duplicates, and selected and evaluated the studies. The review focuses on the recent advancements in mediastinal sampling techniques, including EBUS-TBNA, EUS-FNA and FNB, IFB, and nodal cryobiopsy. The review highlights the advantages of an integrated approach using these techniques for diagnosing and staging mediastinal diseases, which, when used competently, significantly increase diagnostic yield and accuracy.

Bronchoscopic Diagnosis of Severe Respiratory Infections

The diagnosis of severe respiratory infections in intensive care remains an area of uncertainty and involves a complex balancing of risks and benefits. Due to the frequent colonisation of the lower respiratory tract in mechanically ventilated patients, there is an ever-present possibility of microbiological samples being contaminated by bystander organisms. This, coupled with the frequency of alveolar infiltrates arising from sterile insults, risks over-treatment and antimicrobial-associated harm. The use of bronchoscopic sampling to obtain protected lower respiratory samples has long been advocated to overcome this problem. The use of bronchoscopy further enables accurate cytological assessment of the alveolar space and direct inspection of the proximal airways for signs of fungal infection or alternative pathologies. With a growing range of molecular techniques, including those based on nucleic acid amplification and even alveolar visualisation and direct bacterial detection, the potential for bronchoscopy is increasing concomitantly. Despite this, there remain concerns regarding the safety of the technique and its benefits versus less invasive sampling techniques. These discussions are reflected in the lack of consensus among international guidelines on the topic. This review will consider the benefits and challenges of diagnostic bronchoscopy in the context of severe respiratory infection.

Quantitative determination by UHPLC-MS/MS of 18 common drugs of abuse and metabolites, including THC and OH-THC, in volumetric dried blood spots: a sustainable method with minimally invasive sampling

The increased use of drugs of abuse urges forensic toxicologists to create quick, simple, minimally invasive sampling techniques for biological fluids combined with analytical methods assuring accurate results. To this purpose, a method was developed aimed at quantifying 18 drugs of abuse and metabolites in DBS. Validation of the method was conducted by spiking blank whole blood with the analytes on Capitainer® B cards. The extracts were analyzed by a targeted UHPLC-MS/MS method. Linear calibration was achieved in the range of 1–100 ng/mL for: amphetamine, MDA, MDMA, methamphetamine, cocaine, codeine, benzoylecgonine, cocaethylene, morphine, 6-MAM, buprenorphine, methadone, EDDP, ketamine, norbuprenorphine norketamine, THC, and OH-THC. Experimental LOD was found at 0.5 ng/mL for all analytes except for norbuprenorphine, THC and THC-OH which yielded a LOD of 1 ng/mL. Intra- and inter-day accuracy was satisfactory with bias% resulting within 5%. Evaluation of intra- and inter-day precision yielded CV% values within 20%, for all compounds except EDDP. Average extraction recovery calculated at low (2 ng/mL) and high (75 ng/mL) concentration levels was 63% while average matrix effect determined at the same levels was found to be within 85% − 115% for all analytes except from codeine (70%) and MDMA (131%). The method was applied to authentic blood samples spotted onto the DBS card and the minimum value detected was 1.3 ng/mL. HPLC-MS/MS proved capable to identify all the targeted analytes at low concentrations in the small blood volumes obtainable from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices.

Using Fin Ray Elemental Signatures and Growth Zone Width to Estimate Onset of Sexually Maturity in Lake Sturgeon (Acipenser fulvescens)

Characterizing inter- and intra-population levels of variability in age at sexual maturation for long-lived fishes provides insight into year-class strength and recruitment dynamics, allowing for more effective management practices. Here we analyzed the ontogenetic chronology of pectoral fin ray annuli trace elemental concentration profiles as well as changes in annuli growth in Lake Sturgeon (Acipenser fulvescens) to determine onset of sexual maturity (OSM). Elemental concentrations and growth-zone width were used to build a random forest classification model to discriminate year-specific signatures to before or after OSM from 98 individual fin rays from both sexes across multiple populations and watersheds. The model demonstrated an overall accuracy of 98.8%. Ba was the most important variable related to OSM discrimination success followed by zone width, Pb, Mn, Mg, Zn, Cu, and Sr. Fin ray elemental concentrations began to increase at approximately age 24 (± 4.7) in females and 15 (± 3.1) in males while zone width decreased. This study implies that new applications for fin ray microchemistry are possible and could benefit future fisheries management strategies specifically by adding nonlethal or less invasive sampling techniques for evaluating OSM in sturgeon.

THE APPLICATION OF BIOANALYTICAL METHOD OF TAMOXIFEN AND ITS ACTIVE METABOLITES FOR THERAPEUTIC DRUG MONITORING IN BREAST CANCER PATIENTS: A REVIEW

Breast cancer is the most common cancer around the world and in Indonesia. The most widely used agent for breast cancer treatment is tamoxifen, with a fixed dose of 20 mg per day. Tamoxifen is metabolized by cytochrome P450 3A4 (CYP3A4) and 2D6 (CYP2D6) to endoxifen and 4-hydroxytamoxifen, which have 30-to 100-fold more potent antiestrogenic activity than tamoxifen. High variations of CYP3A4 and CYP2D6 genes can lead to interpatient variability in its metabolites concentration. The dose can be increased to 40 or 60 mg per day based on individual needs. Therapeutic drug monitoring (TDM) is required to measure the concentration of tamoxifen and its metabolites to decide the individualized dose. The measurement of drug levels should use a sensitive, selective, accurate, precise, and reliable bioanalytical method. Various bioanalytical methods have been developed in several matrices: urine, scalp hair, serum, plasma, dried blood spot (DBS), and volumetric absorptive microsampling (VAMS) samples, with different sample preparations, and frequently using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The bioanalytical method of tamoxifen and its metabolites in the DBS sample was more suitable in the TDM application due to the low invasive sampling technique, more stable sample, and rapid sample preparation. Therefore, it is more time-and cost-efficient than the other methods.

Development and validation of a GT-seq panel for genetic monitoring in a threatened species using minimally invasive sampling.

Minimally invasive samples are often the best option for collecting genetic material from species of conservation concern, but they perform poorly in many genomic sequencing methods due to their tendency to yield low DNA quality and quantity. Genotyping-in-thousands by sequencing (GT-seq) is a powerful amplicon sequencing method that can genotype large numbers of variable-quality samples at a standardized set of single nucleotide polymorphism (SNP) loci. Here, we develop, optimize, and validate a GT-seq panel for the federally threatened northern Idaho ground squirrel (Urocitellus brunneus) to provide a standardized approach for future genetic monitoring and assessment of recovery goals using minimally invasive samples. The optimized panel consists of 224 neutral and 81 putatively adaptive SNPs. DNA collected from buccal swabs from 2016 to 2020 had 73% genotyping success, while samples collected from hair from 2002 to 2006 had little to no DNA remaining and did not genotype successfully. We evaluated our GT-seq panel by measuring genotype discordance rates compared to RADseq and whole-genome sequencing. GT-seq and other sequencing methods had similar population diversity and F ST estimates, but GT-seq consistently called more heterozygotes than expected, resulting in negative F IS values at the population level. Genetic ancestry assignment was consistent when estimated with different sequencing methods and numbers of loci. Our GT-seq panel is an effective and efficient genotyping tool that will aid in the monitoring and recovery of this threatened species, and our results provide insights for applying GT-seq for minimally invasive DNA sampling techniques in other rare animals.

African Swine Fever Diagnosis in Africa: Challenges and Opportunities.

The global spread of African swine fever (ASF) in recent decades has led to the need for technological advances in sampling and diagnostic techniques. The impetus for these has been the need to enable sampling by lay persons and to obtain at least a preliminary diagnosis in the field for early control measures to be put in place before final laboratory confirmation. In rural Africa, rapid diagnosis is hampered by challenges that include lack of infrastructure as well as human and financial resources. Lack of animal health personnel, access to affordable means to transport field samples to a laboratory, and lack of laboratories with the capacity to make the diagnosis result in severe under-reporting of ASF, especially in endemic areas. This review summarizes the challenges identified in gap analyses relevant to low- and middle-income countries, with a focus on Africa, and explore the opportunities provided by recent research to improve field diagnosis and quality of diagnostic samples used. Sampling techniques include invasive sampling techniques requiring trained personnel and non-invasive sampling requiring minimal training, sampling of decomposed carcass material, and preservation of samples in situations where cold chain maintenance cannot be guaranteed. Availability and efficacy of point-of-care (POC) tests for ASF has improved considerably in recent years and their application, as well as advantages and limitations, are discussed. The adequacy of existing laboratory diagnostic capacity is evaluated and opportunities for networking amongst reference and other laboratories offering diagnostic services are discussed. Maintaining laboratory diagnostic efficiency in the absence of samples during periods of quiescence is another issue that requires attention, and the role of improved laboratory networking is emphasized. Early diagnosis of ASF is key to managing the disease spread. Therefore, the establishment of the Africa Chapter of the Global African Swine Fever Research Alliance (GARA) increases opportunities for collaboration and networking among the veterinary diagnostic laboratories in the region.

Comprehensive Genomic Testing: Tissue Stewardship and Best Practices

Biomarker-driven targeted therapies have shaped the oncology treatment landscape for patients with advanced solid tumors over the past decade. Comprehensive genomic profiling (CGP) has played a key role in precision medicine as it enables simultaneous identification of multiple biomarkers to guide cancer diagnosis, therapy selection, and prognostication. As a result, tissue stewardship for successful CGP testing is paramount. In addition, widespread adoption of less invasive sampling techniques leaves less diagnostic tissue for additional or future testing as smaller biopsies are acquired. To help oncology care practitioners overcome these challenges, this paper provides an overview of current genomic testing methodologies and offers guidelines on best practices for tissue stewardship and preanalytic practices for successful CGP testing and efficient turnaround times for laboratory tests.

Successful DNA amplification of DNA from non-destructive buccal swabbing in Vespertilionid and Rhinolophid bats

AbstractAcquiring DNA from wild bats (Mammalia: Chiroptera) is typically undertaken utilizing highly invasive (but non-lethal) sampling techniques comprising wing biopsies and occasional blood samples. While non-invasive sampling is possible through the extraction of DNA from faecal samples, it is not always possible to acquire samples from individual bats whilst conducting fieldwork, and as such, this method is primarily applicable to roost occupancy identification. Similarly, wing swabbing is liable to cross-contamination from roost mates. Here we present the first use of oral (buccal) swabbing for successful, species-resolution DNA sequencing of Vespertilionidae and Rhinolophidae in 10 bat species (nine Vespertilionidae and one Rhinolophidae) from the UK.

Advancing Global Health Surveillance of Mycotoxin Exposures using Minimally Invasive Sampling Techniques: A State-of-the-Science Review.

Mycotoxins are a heterogeneous group of toxins produced by fungi that can grow in staple crops (e.g., maize, cereals), resulting in health risks due to widespread exposure from human consumption and inhalation. Dried blood spot (DBS), dried serum spot (DSS), and volumetric tip microsampling (VTS) assays were developed and validated for several important mycotoxins. This review summarizes studies that have developed these assays to monitor mycotoxin exposures in human biological samples and highlights future directions to facilitate minimally invasive sampling techniques as global public health tools. A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted. Key assay performance metrics were extracted to provide a critical review of the available methods. This search identified 11 published reports related to measuring mycotoxins (ochratoxins, aflatoxins, and fumonisins) using DBS/DSS and VTS assays. Multimycotoxin assays adapted for DBS/DSS and VTS have undergone sufficient laboratory validation for applications in large-scale population health and human biomonitoring studies. Future work should expand the number of mycotoxins that can be measured in multimycotoxin assays, continue to improve multimycotoxin assay sensitivities of several biomarkers with low detection rates, and validate multimycotoxin assays across diverse populations with varying exposure levels. Validated low-cost and ultrasensitive minimally invasive sampling methods should be deployed in human biomonitoring and public health surveillance studies to guide policy interventions to reduce inequities in global mycotoxin exposures.

Dermal microdialysis: A method to determine drug levels in the skin of patients with Post kala-azar dermal leishmaniasis (PKDL)

ObjectivesPost-kala-azar-dermal leishmaniasis (PKDL) is an infectious skin disease that occurs as sequela of visceral leishmaniasis (VL) and causes cutaneous lesions on the face and other exposed body parts. While the first-line drug miltefosine is typically used for 28 days to treat VL, 12 weeks of therapy is required for PKDL, highlighting the need to evaluate the extent of drug penetration at the dermal site of infection. In this proof-of-concept study, we demonstrate the use of a minimally invasive sampling technique called microdialysis to measure dermal drug exposure in a PKDL patient, providing a tool for the optimization of treatment regimens. Methods and materialsOne PKDL patient receiving treatment with miltefosine (50 mg twice daily for 12 weeks) was recruited to this proof-of-concept study and consented to undergo dermal microdialysis. Briefly, a μDialysis Linear Catheter 66 for skin and muscle, a probe with a semi-permeable membrane, was inserted in the dermis. A perfusate (a drug-free physiological solution) was pumped through the probe at a low flow rate, allowing miltefosine present in the dermis to cross the membrane and be collected in the dialysates over time. Protein-free (dialysates) and total (blood and skin biopsies) drug concentrations were analysed using LC-MS/MS. Resultsand conclusions: Using microdialysis, protein-free miltefosine drug concentrations could be detected in the infected dermis over time (Cmax ≈ 450 ng/ml). This clinical proof-of-concept study thus illustrates the potential of dermal microdialysis as a minimally invasive alternative to invasive skin biopsies to quantify drug concentrations directly at the pharmacological site of action in PKDL.

Use of volumetric absorptive microsampling and parallel reaction monitoring mass spectrometry for tacrolimus blood trough measurements at home in pediatric heart transplant patients

BackgroundMeasurement of trough levels for calcineurin inhibitors by venipuncture sampling is a mainstay of patient management in solid organ transplant recipients but challenging in pediatric patients. Volumetric Absorptive Microsampling (VAMS) is a patient-friendly, minimally invasive sampling technique to accurately collect blood. An assay for measurement of tacrolimus in blood using VAMS, coupled with parallel reaction monitoring (PRM) mass spectrometry, was validated in pediatric heart transplant patients. MethodsTacrolimus was measured by a newly developed high-resolution PRM assay and compared with low-resolution tandem mass spectrometry (MRM). Dried blood samples were collected from pediatric heart transplant patients (n = 35) using VAMS devices and a satisfaction survey was completed by patients/guardians. Tacrolimus concentrations were compared across whole liquid blood, dried blood spots, and capillary blood, and shipping stability determined. ResultsThe PRM assay was linear over a range 1–50 ng/mL, similar to MRM but had greater specificity due to reduced background noise. No significant differences in tacrolimus concentrations were observed between VAMS and venous blood. Tacrolimus dried on VAM tips was stable for 14 days and concentrations were unaffected by postal shipping. The variability in two simultaneously collected at-home patient samples was minimal – average concentration difference was 0.12 ± 0.94 ng/mL (p = 0.6) between paired samples. ConclusionA high resolution PRM mass spectrometry assay was developed for home-based dried blood collections for therapeutic monitoring of tacrolimus. The advantage of PRM was enhanced specificity and the VAMS devices provided a simple and convenient approach to blood sampling at home in pediatric heart transplant patients.

A new sex-specific genetic marker (fshr 1834G>T) for flathead grey mullet, Mugil cephalus, in Queensland, Australia

In this study, genetic sex marker candidates from northern hemisphere Mugil cephalus were tested to see if they could be used to sex a population originating from south-east Queensland, Australia. As such, a region of the follicle stimulating hormone receptor (fshr) gene was sequenced but did not contain previously published single nucleotide polymorphisms (SNPs). However, further screening of the sequenced fshr region revealed a promising sex marker candidate for Queensland M. cephalus, fshr 1834 G>T, which was accurate in 100% of fish tested (excluding intersex fish, which had the female genotype). While all females tested were homozygous G/G, males presented as either G/T (common) or T/T (lower frequency). Subsequently, a real-time high-resolution melt was developed to facilitate rapid and accurate genotyping of M. cephalus based on the fshr 1834 G>T SNP. Initial results suggest that fshr 1834 G>T is a useful SNP that can reduce the need for more invasive sampling techniques such as gonadal biopsy, provide information relating to the sex of captive stock prior to gonadal maturation, and may prove useful in wild population surveys and stock assessment.

Initial characterization of mitochondrial DNA control region haplotypes of the Antillean manatee (Trichechus manatus manatus, Sirenia:Trichechidae) in Guatemala

Introduction: Small populations are at risk of losing genetic variability much faster than large populations; this subsequently decreases their ability to adapt when facing environmental changes. A small population of the endangered Antillean manatee (Trichechus manatus manatus) has been identified in Guatemala.
 Objective: This study explored the genetic diversity of the Antillean manatee in Guatemala by analysing mitochondrial DNA control region haplotypes in the two most important habitats for the species, Bahía La Graciosa, a coastal bay and Bocas del Polochic, a coastal wetland, both located in the Izabal State.
 Methods: Genetic samples were collected using non or minimally invasive sampling techniques: scraping of epidermal tissue, collection of floating feces, and collection of tissue from carcasses. DNA extractions, DNA amplification using polymerase chain reaction (PCR), and sequencing of the control D-loop region were used to process and analyse the samples.
 Results: Seven mitochondrial DNA sequences were obtained from 36 samples collected (minimum of four and maximum of seven individuals). Four haplotypes were identified, A01, A03, A04, and J01. No other Central American country has reported this number of haplotypes in a manatee population, and it is the first time that haplotype A01 has been reported for the region. The Guatemalan manatee population comprises at least two genetic lineages, the Florida/Greater Antilles lineage (haplotypes A01, A03, and A04) and the Mesoamerican lineage (J01).
 Conclusion: Further studies, with the use of nuclear markers, are necessary to understand the population dynamics between Bahia La Graciosa and Bocas del Polochic to identify the number of management units present in the country; also, the degree of relatedness with the Belizean population needs to be established to better coordinate conservation efforts.

Risk of mother-to-child transmission after amniocentesis in pregnant women with hepatitis B virus: a retrospective cohort study

Amniocentesis is the most widely used invasive prenatal diagnostic sampling technique. However, whether this increases the risk of mother-to-child transmission of infectious diseases remains controversial. This study aimed to determine whether amniocentesis increases the risk of hepatitis B virus infection in infants who received standard prophylaxis, and to assess the related risk factors for mother-to-child transmission in women who underwent amniocentesis during pregnancy. This retrospective analysis used the clinical data of pregnant women with hepatitis B virus infection at West China Second University Hospital, Sichuan University in 2019. After meeting the inclusion criteria, the participants were divided into 2 groups on the basis of whether they had undergone amniocentesis during pregnancy. The infant hepatitis B virus serologic status was followed 1 to 6 months after completion of immunization. The infant testing positive for hepatitis B surface antigen and negative for Hepatitis B surface antibody indicated mother-to-child transmission of hepatitis B virus. In total, 1764 pregnant women with hepatitis B virus infection were enrolled. Of these, 846 underwent amniocentesis during pregnancy and 918 did not. All offspring received a standardized immunoprophylaxis schedule. The overall mother-to-child transmission rate for hepatitis B virus was 0.6% (5/846) in the amniocentesis group and 0.4% (4/918) in the control group (P=.745). Subgroup analysis showed that the mother-to-child transmission rate in hepatitis B e antigen-positive women was 1.8% (2/111) in the amniocentesis group and 1.0% (2/209) in the control group (P=.612). In women with high viral load, the mother-to-child transmission rate was 1.3% (1/78) vs 0.9% (1/107) (amniocentesis group vs control group; P=1.000). In the amniocentesis group, 31 amniotic fluid specimens had an abnormal appearance (bloody or brown). Univariate analysis showed that the mother-to-child transmission rates of these mothers were statistically higher than those of mothers with pale yellow or transparent amniotic fluid (2/31 vs 3/815; relative risk, 17.527 [3.037-101.151]; P=.012). Amniocentesis did not increase the risk of mother-to-child transmission of hepatitis B virus in infants who received a standardized immunoprophylaxis schedule, including those with mothers who were hepatitis B e antigen-positive or had a high viral load. However, the abnormal appearance (bloody or brown) of the amniotic fluid obtained during amniocentesis may indicate increased risk of mother-to-child transmission for hepatitis B virus.

394 Gene expression based molecular test proves clinical validity as diagnostic aid for the differential diagnosis of psoriasis and eczema in formalin fixed and paraffin embedded tissue

Abstract Highly specific and efficient drugs have been developed to treat noncommunicable chronic inflammatory skin diseases (ncISD). Due to their specificity, these drugs require precise diagnostics to attribute the most efficient treatment to each patient. Diagnosis is complicated by the complex pathogenesis of ncISD and their clinical and histological overlap. Especially, precise diagnosis of psoriasis and atopic eczema is difficult in special cases, and molecular tools need to be developed to support gold standard diagnosis. Psoriasis and eczema are among the most prevalent skin diseases worldwide with a combined incidence rate of at least 2%. Although more than a dozen of immune-modulating therapies, particularly monoclonal antibodies targeting specific cytokines or their receptors, have become available over the last decade allowing highly effective treatment, the ultimate breakthrough would include accurate diagnostic tools. Diagnosis of both diseases largely depends on a subjective visual exam and histopathological examination. Due to phenotypical overlaps of both diseases, up to 50% of cases with a palmoplantar localization are misdiagnosed. To close this diagnostic gap, a gene expression-based classifier using NOS2 and CCL27 has been proposed and its clinical validity examined and proven in various patient cohorts. This study aims to develop a real-time based molecular classifier (MC) to distinguish psoriasis from atopic eczema in formalin-fixed, paraffin-embedded (FFPE)-fixed skin samples for diagnostic measures and to evaluate the potential of minimally invasive microbiopsies and noninvasive tape strips or sampling disks for molecular diagnosis. The FFPE, micro-(Ø1mm) and macrobiopsies (Ø4–6 mm), and tape strips were collected from psoriasis and eczema lesions and analysed by real-time polymerase chain reaction (PCR) for the expression of NOS2 and CCL27. A molecular classifier (MC) was established using a linear regression model. We transferred the RNAprotect-based molecular classifier to FFPE samples and were able to produce comparable results. Whereas the RNAprotect based MC distinguished psoriasis from eczema with a sensitivity and specificity for psoriasis of 97.7% and 100%, respectively, and an AUC of 0.99, the FFPE-based classifier determined probabilities for psoriasis with a sensitivity and specificity and of 92% and 100%, respectively, and an AUC of 0.97. To test if microbiopsies are also adequate tissue samples for the MC, we analysed gene expression in 83 pairs of macro- and microbiopsies by real-time quantitative-PCR. Delta-CT values showed no significant difference between macro- and microbiopsies. The MC led to reliable results in the vast majority of samples (91.57%) independent of biopsy size, with a small increase of misclassification (4.98%). The FFPE-based molecular classifier (MC) precisely separates eczema from psoriasis and efficiently identifies subtypes of both diseases. The reliability of the two markers NOS2 and CCL27 has been shown in FFPE tissue facilitating a possible implementation of this molecular diagnostic aid in routine clinical pathological practice. We further show that the gene expression profile of RNA later fixed microbiopsies is comparable to standard 4–6 mm biopsies and that microbiopsies are equally suited for the MC. We demonstrate the potential of tape strips of inflamed epidermis for molecular diagnostics of ncISD. Due to the minimally invasive sampling procedure, tape stripping may be particularly useful for the testing of visible and sensitive skin areas and in children as well as for repetitive sampling procedures necessary to monitor treatment responses over time. In summary, the MC discriminates psoriasis from eczema producing meaningful results in a broad range of skin tissue derived from invasive to minimally invasive sampling techniques. Further efforts are required to simplify and shorten the labor- and cost-intensive procedures of RNA isolation and real-time PCR. One possible solution is a fully automated and closed system, which would allow the integration of molecular diagnostics into the routine patient management at the point of care.

A Novel Biopsy Needle with Double Concave-Curved Cutting Edges

BackgroundVacuum-assisted biopsy is a minimally invasive sampling technique that relies on rotational cutting as a major tissue retrieving method. A precise diagnosis of the disease requires large, uncrushed samples, which are impacted by the cutting force of the biopsy needle. ObjectivesThis study proposes a novel needle design with double-concave-curved cutting edges, which is more suited for rotational needle cutting. We aimed to optimize the design so that large, undamaged samples could be extracted with minimal cutting force. Materials and MethodsFive-factor experiments were designed using the Taguchi method. Experiments involving rotational needle insertion and tissue sampling were conducted to examine the effects of these variables on the cutting force and sampling quality, respectively. The relationship between the cutting force and sampling quality was analyzed. ResultsFor needle insertion, the optimal design within the design space demonstrated a marked improvement in cutting force from 1.2107 to 0.1888 N. Furthermore, the optimized double concave-curved needle outperformed the blunt needle under the same needle speeds, showing a 24.4% reduction in cutting force (0.1888 vs. 0.2496 N). Increasing rotation-translation ratio or insertion speed would allow for extracting a larger sample (increased up to 21.95% in weight and 17.21% in total length) but may also increase the rotation speed, resulting in sample fragmentation. To simultaneously improve sampling quality and cutting force, a higher K value, larger rotation-translation ratio, and slower insertion speed are suggested. Based on the conditions examined in this study, the optimal needle configuration should include a sharpened cutting edge with a K value of 0.2, a rotation-translation ratio of 8, and an insertion speed of 1 mm/s.

Updates in pathology and molecular diagnostics to inform the evolving landscape of thoracic surgery and oncology.

The pathologic assessment of lung cancers provides essential guidance to the surgeon and oncologist who are considering the best treatment strategies for patients with both early and advanced-stage disease. The management of patients with lung cancer is predicated first and foremost on access to an accurate diagnosis, even when the sample size is limited, as is often the case with use of modern, minimally invasive sampling techniques. Once the diagnosis and disease stage are established, predictive biomarker testing may be essential, particularly for those patients with nonsmall cell lung carcinoma (NSCLC) being considered for immunotherapy or genomic biomarker-driven targeted therapy. This review will discuss the best practices for the diagnosis of NSCLC using morphology and immunohistochemistry, thus providing the surgeon with needed information to understand and critically evaluate pathology reports. Controversial and evolving topics including tumor spread through airspaces,evaluation of multiple tumors, and staging based on invasive tumor size will be addressed. Clinical genomic profiling in NSCLC is driven by published guidelines and reflects evidence based on clinical trials and regulatory approvals. In this fast-moving space, surgeons should be aware of the critical immunohistochemical and genomic biomarkers that drive systemic therapy decisions and anticipate when such testing will be required, both to ensure adequate sampling and to advise the pathologist when tumor material will be required for biomarker analysis. The basic approaches to and sample requirements for molecular biomarker testing will be addressed. As biomarker testing moves exclusively from advanced-stage patients into earlier stage disease, the surgeon should be aware of the relevant markers and work with the pathologist and oncologist to ensure that this information is available to facilitate timely access to therapies not just in the advanced setting, but in consideration of neoadjuvant and adjuvant care.

Lipid profile variability in children at different ages measured in dried blood spots.

Dried blood spot (DBS) is a minimally invasive sampling technique that has several advantages over conventional venipuncture/arterial blood sampling. More recently, DBS has also been applied for lipidomics analysis, but this is an area that requires further research. The few works found in the literature on lipidomics of DBS samples performed the analysis in adult samples, leaving pediatric ages unmapped. The objective of this study was to assess the variability of the lipid profile (identified by high-resolution C18 RP-LC-MS/MS) of DBS at pediatric age (0-10 days, 2-18 months, and 3-13 years) and to identify age-related variations. The results revealed that the lipidomic signature of the three age groups is significantly different, especially for a few species of neutral lipids and phosphatidylcholines. The main contributors to the differentiation of the groups correspond to 3 carnitine (Car), 2 cholesteryl ester (CE), 2 diacylglycerol (DG), 2 triacylglycerol (TG), 3 phosphatidylcholine (PC), 1 ether-linked PC, 1 phosphatidylethanolamine (PE), 1 ether-linked PE and 1 phosphatidylinositol (PI) species, all with statistically significant differences. Additionally, lipid species containing linoleic acid (C18:2) were shown to have significantly lower levels in the 0-10 days group with a gradual increase in the 2-18 month, reaching the highest concentrations in the 3-13 year group. The results of this study highlighted the adaptations of the lipid profile at different pediatric ages. These results may help improve understanding of the evolution of lipid metabolism throughout childhood and should be investigated further.