Inkjet-Printing of Nanoparticle Gold and Silver Ink on Cyclic Olefin Copolymer for DNA-Sensing Applications.

Inkjet technology as a maskless, direct-writing technology offers the potential for structured deposition of functional materials for the realization of electrodes for, e.g., sensing applications. In this work, electrodes were realized by inkjet-printing of commercial nanoparticle gold ink on planar substrates and, for the first time, onto the 2.5D surfaces of a 0.5 mm-deep microfluidic chamber produced in cyclic olefin copolymer (COC). The challenges of a poor wetting behavior and a low process temperature of the COC used were solved by a pretreatment with oxygen plasma and the combination of thermal (130 °C for 1 h) and photonic (955 mJ/cm²) steps for sintering. By performing the photonic curing, the resistance could be reduced by about 50% to 22.7 µΩ cm. The printed gold structures were mechanically stable (optimal cross-cut value) and porous (roughness factors between 8.6 and 24.4 for 3 and 9 inkjet-printed layers, respectively). Thiolated DNA probes were immobilized throughout the porous structure without the necessity of a surface activation step. Hybridization of labeled DNA probes resulted in specific signals comparable to signals on commercial screen-printed electrodes and could be reproduced after regeneration. The process described may facilitate the integration of electrodes in 2.5D lab-on-a-chip systems.

Review: Electrochemical DNA sensing - Principles, commercial systems, and applications.

Driven by the vision of robust and portable, yet sensitive DNA detection systems for point-of-need applications, the development of electrochemical DNA sensing principles has been of high interest. Many different principles have been developed and these are regularly reviewed. However, the maturity of electrochemical principles and their ability to produce competitive real-world applications is rarely assessed. In this review, general electrochemical DNA sensing principles are briefly introduced and categorized into heterogeneous vs. homogeneous approaches, and then the subcategories label-free vs. labeled and reagent-less vs. reagent-dependent principles. We then focus on reviewing the electrochemical sensing principles implemented in DNA detection systems, which are commercially available or close to market entry, considering the complete analysis process, automation and the field of application. This allows us to outline and discuss which principles have proved suitable for which kinds of applications, as well as the stage of integration and automation. Examples from all the identified categories of electrochemical DNA sensing principles have found application in commercial detection systems or advanced prototypes. Various applications have already been demonstrated, ranging from on-site skin care testing, to food safety to the most frequent in vitro diagnostic tests, partially conducted in automated sample-to-answer devices. Our review is intended to enable researchers in areas related to electrochemistry, biochemistry or microfluidics to assess the commercial state of the art of electrochemical nucleic acid testing, and the interdisciplinary challenges for further improvements.

Practical Strategies to Improve the Clinical Utility of the Dermatopathology Report.

CONTEXT: -Dermatologists and subspecialty dermatopathologists, working together over many years, develop a common understanding of clinical information provided on the requisition and of terminology used in the pathology report. Challenges arise for pathologists without additional subspecialty training in dermatology/dermatopathology, and for any pathologist reporting skin biopsies for nondermatologists such as general practitioners or surgeons. OBJECTIVE: -To provide practical strategies to improve efficiency of dermatopathology sign-out, at the same time providing the clinician with clear diagnostic and prognostic information to guide patient management. DATA SOURCES: -The information outlined in this review is based on our own experiences with routine dermatopathology and dermatology practice, and review of English-language articles related to the selected topics discussed. CONCLUSIONS: -Using generic diagnoses for some benign lesions, listing pertinent negatives in the pathology report, and using logical risk management strategies when reporting on basal cell carcinoma, partial biopsies, or specimens with incomplete clinical information allow the pathologist to convey relevant and useful diagnostic information to the treating clinician.

Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes.

Multiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2-plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays.

Cellular Blue Nevomelanocytic Lesions: Analysis of Clinical, Histological, and Outcome Data in 37 Cases.

Cellular blue nevomelanocytic lesions (CBNLs) frequently pose diagnostic problems to pathologists, and their biological potential may be difficult to establish. In this study, the authors have analyzed the clinical, histological, and outcome data of 37 cellular blue nevomelanocytic lesions and the molecular characteristics of 4 lesions. The cohort of cases comprised 8 cellular blue nevi (CBNs), 17 atypical cellular blue nevi (ACBNs), and 12 blue-nevus-like melanomas (BNLMs) with a mean follow-up of 5 years. The average age at diagnosis was 25.9 years for patients with ACBN, versus 30.4 years for CBN, and 44.6 years for BNLM. Both CBN and ACBN occurred most frequently on the trunk or extremities, whereas BNLM primarily involved the scalp. Histologically, CBN and ACBN were characterized by a mean diameter of <1 cm, absence of necrosis, low mitotic rate (mean: 1-2 mitotic figures/mm), little or no infiltrative properties, and usually low-grade cytologic atypia. In contrast, BNLM had a mean diameter of 1.6 cm, necrosis, tissue infiltration, greater mitotic activity (mean: 6 mitotic figures/mm), and high-grade cytologic atypia. ACBNs often were larger, more densely cellular, exhibited higher mitotic counts, and were cytologically more atypical than CBN. Seven CBN cases with follow-up had a benign clinical course (average follow-up of 4.7 years). Among 6 patients with ACBN who underwent sentinel lymph node (SLN) biopsy, 3 were positive, and a single additional case had 1 positive non-SLN (this patient did not have a SLN biopsy performed). All 14 cases of ACBN with follow-up were alive and without recurrence with mean follow-up of 5 years. Of the 9 melanoma cases with follow-up, 3 patients with SLN and non-SLN involvement died from their disease (average follow-up of 4.8 years). Array comparative genomic hybridization was performed on 2 ACBNs and 1 BNLM: One of the 2 ACBNs showed chromosomal aberrations and 1 BNLM showed multiple chromosomal gains and losses. Multiplex polymerase chain reaction was performed on 1 ACBN, and no mutations were found. From these results, the authors conclude that ACBN occupy an intermediate position within the spectrum of CBN and BNLM, yet many lesions cannot be reliably distinguished from either CBN or BNLM because of overlapping histologic features. However, in general, ACBNs seem to aggregate more closely with CBN in terms of clinical, histological, molecular profile (limited data), and biological behavior.

Digital droplet PCR on disk.

Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis.

Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA.

Aqueous microdroplets provide miniaturized reaction compartments for numerous chemical, biochemical or pharmaceutical applications. We introduce centrifugal step emulsification for the fast and easy production of monodisperse droplets. Homogenous droplets with pre-selectable diameters in a range from 120 µm to 170 µm were generated with coefficients of variation of 2-4% and zero run-in time or dead volume. The droplet diameter depends on the nozzle geometry (depth, width, and step size) and interfacial tensions only. Droplet size is demonstrated to be independent of the dispersed phase flow rate between 0.01 and 1 µl s(-1), proving the robustness of the centrifugal approach. Centrifugal step emulsification can easily be combined with existing centrifugal microfluidic unit operations, is compatible to scalable manufacturing technologies such as thermoforming or injection moulding and enables fast emulsification (>500 droplets per second and nozzle) with minimal handling effort (2-3 pipetting steps). The centrifugal microfluidic droplet generation was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). It was used for absolute quantification of Listeria monocytogenes DNA concentration standards with a total analysis time below 30 min. Compared to digital droplet polymerase chain reaction (ddPCR), with processing times of about 2 hours, the overall processing time of digital analysis was reduced by more than a factor of 4.

Drug-induced subacute cutaneous lupus erythematosus associated with docetaxel chemotherapy: a case report.

BACKGROUND: Drug-induced subacute cutaneous lupus erythematosus is an uncommon disorder associated with the use of pharmacological agents including systemic chemotherapy. CASE PRESENTATION: We report a case of docetaxel-induced subacute cutaneous lupus erythematosus in a 60-year-old Caucasian female with Sjögren's syndrome diagnosed 2 months after receiving docetaxel as part of the adjuvant FEC-D (5-fluorouracil, epirubicin, cyclophosphamide, docetaxel) chemotherapy protocol for early stage breast cancer. Although the exact mechanisms behind the autoimmune response elicited by docetaxel are unclear, the involvement of anti-SSA/Ro antibodies has been implicated. CONCLUSION: This case highlights the symptom severity and clinical course of docetaxel-induced subacute cutaneous lupus erythematosus, and highlights the importance of recognizing this uncommon but potentially severe chemotherapy-associated cutaneous reaction.

Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer.

INTRODUCTION: Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals. METHODS: Twenty-eight lung adenocarcinomas with known ALK status were used as blinded study samples. Thirteen laboratories performed IHC using locally developed staining protocols for 5A4, ALK1, or D5F3 antibodies; results were assessed by H-score. Twelve centers conducted FISH using protocols based on Vysis' ALK break-apart FISH kit. Initial IHC results were used to optimize local IHC protocols, followed by a repeat IHC study to assess the results of standardization. Three laboratories conducted a prospective parallel IHC and FISH analysis on 411 consecutive clinical samples using post-validation optimized assays. RESULTS: Among study samples, FISH demonstrated 22 consensus ALK+ and six ALK wild type tumors. Preoptimization IHC scores from 12 centers with 5A4 and the percent abnormal cells by FISH from 12 centers showed intraclass correlation coefficients of 0.83 and 0.68, respectively. IHC optimization improved the intraclass correlation coefficients to 0.94. Factors affecting FISH scoring and outliers were identified. Post-optimization concurrent IHC/FISH testing in 373 informative cases revealed 100% sensitivity and specificity for IHC versus FISH. CONCLUSIONS: Multicenter standardization study may accelerate the implementation of ALK testing protocols across a country/region. Our data support the use of an appropriately validated IHC assay to screen for ALK+ lung cancers.

Data set for pathology reporting of cutaneous invasive melanoma: recommendations from the international collaboration on cancer reporting (ICCR).

An accurate and complete pathology report is critical for the optimal management of cutaneous melanoma patients. Protocols for the pathologic reporting of melanoma have been independently developed by the Royal College of Pathologists of Australasia (RCPA), Royal College of Pathologists (United Kingdom) (RCPath), and College of American Pathologists (CAP). In this study, data sets, checklists, and structured reporting protocols for pathologic examination and reporting of cutaneous melanoma were analyzed by an international panel of melanoma pathologists and clinicians with the aim of developing a common, internationally agreed upon, evidence-based data set. The International Collaboration on Cancer Reporting cutaneous melanoma expert review panel analyzed the existing RCPA, RCPath, and CAP data sets to develop a protocol containing "required" (mandatory/core) and "recommended" (nonmandatory/noncore) elements. Required elements were defined as those that had agreed evidentiary support at National Health and Medical Research Council level III-2 level of evidence or above and that were unanimously agreed upon by the review panel to be essential for the clinical management, staging, or assessment of the prognosis of melanoma or fundamental for pathologic diagnosis. Recommended elements were those considered to be clinically important and recommended for good practice but with lesser degrees of supportive evidence. Sixteen core/required data elements for cutaneous melanoma pathology reports were defined (with an additional 4 core/required elements for specimens received with lymph nodes). Eighteen additional data elements with a lesser level of evidentiary support were included in the recommended data set. Consensus response values (permitted responses) were formulated for each data item. Development and agreement of this evidence-based protocol at an international level was accomplished in a timely and efficient manner, and the processes described herein may facilitate the development of protocols for other tumor types. Widespread utilization of an internationally agreed upon, structured pathology data set for melanoma will lead not only to improved patient management but is a prerequisite for research and for international benchmarking in health care.

Utilization and utility of immunohistochemistry in dermatopathology.

Immunohistochemistry (IHC) is considered a valuable ancillary tool for dermatopathology diagnosis, but few studies have measured IHC utilization by dermatopathologists or assessed its diagnostic utility. In a regionalized, community-based dermatopathology practice, we measured IHC utilization (total requests, specific antibodies requested, and final diagnosis) over a 12-month period. Next, we assessed diagnostic utility by comparing a preliminary "pre-IHC" diagnosis based on routine histochemical staining with the final diagnosis rendered after consideration of IHC results. The dermatopathology IHC utilization rate was 1.2%, averaging 3.6 stains requested per case. Melanocytic, hematolymphoid, and fibrohistiocytic lesions made up 23%, 18%, and 16%, respectively, of the total cases requiring IHC. S100 and Melan A were the most frequently requested stains, ordered on 50% and 34% of IHC cases, respectively. The utility study revealed that IHC changed the diagnosis in 11%, confirmed a diagnosis, or excluded a differential diagnosis in 77%, and was noncontributory in 4% of cases. Where IHC results prompted a change in diagnosis, 14% were a change from a benign to malignant lesion, whereas 32% changed from one malignant entity to another. IHC is most commonly used in cutaneous melanocytic and hematolymphoid lesions. In 11% of dermatopathology cases in which IHC is used, information is provided that changes the H&E diagnosis. Such changes may have significant treatment implications. IHC is noncontributory in only a small percentage of cases.

Underrecognition of pathologist contributions to articles published in a major multidisciplinary medical journal.

The Canadian Medical Association Journal (CMAJ) is a high-impact multidisciplinary medical journal. We have observed instances in which a pathology diagnosis, documented with gross or microscopic images, forms an integral part of a CMAJ article, but a pathologist is neither an author nor acknowledged as a contributor. To examine the hypothesis that pathologist contributions are underrecognized and/or underdocumented, we reviewed all CMAJ articles over a 6-year period (September 2003-2009), and correlated the use of pathology images with pathologist authorship or contribution. For each article containing pathology images, department affiliations of authors were determined, and acknowledgments were assessed. Although only 1.7% of articles contained pathology images, 47% (26/55) of these articles did not include a pathologist as either an author or a contributor. We conclude that important intellectual contributions of pathologists are underrecognized and suggest that the scientific credibility of pathology data is in doubt when pathologists do not take on full responsibility of authorship or are not acknowledged as contributors.

Pemphigus vegetans associated with intranasal cocaine abuse.

BACKGROUND: Pemphigus vegetans is a rare variant of pemphigus vulgaris, comprising 1 to 2% of all pemphigus cases. Exposures to oral agents such as captopril and penicillamine and, less commonly, physical or chemical factors have been implicated in the development of pemphigus. METHODS: We report a 42-year-old white male with a 12-month history of hypertrophic, vegetative plaques affecting primarily his external nares and upper lips. The patient had a history of alcoholism and intermittent drug abuse, primarily intranasal cocaine, since his youth. He had been using cocaine heavily three to four times/week for 1 month prior to and 1 month following the onset of the eruption but has since ceased use. His clinical features and histopathologic findings were consistent with a diagnosis of pemphigus vegetans. Treatment with high-dose prednisone (80 mg/d) and mycophenolate mofetil (1.5 g/d) resulted in resolution of the lesions after 18 months. RESULTS AND CONCLUSIONS: To our knowledge, this is the second report proposing an association between intranasal cocaine use and the pemphigus family of disorders. Although the relationship between illicit drug use and the development of pemphigus is unclear, we postulate that intranasal cocaine abuse is operative in our patient's disease. Herein we discuss drug and other external precipitants of pemphigus and review previous case reports of pemphigus associated with illicit drugs.

Solid-phase PCR in a picowell array for immobilizing and arraying 100,000 PCR products to a microscope slide.

We present a method for performing highly parallel PCR reactions in a picowell array (PWA) simultaneously immobilizing generated PCR products in a covalent and spatially-resolved manner onto a microscope slide via solid-phase PCR (SP-PCR). This so called PWA-SP-PCR was performed in picowell arrays featuring 100,000 wells cm(-2) of 19 pL reaction volumes with a surface-to-volume ratio of 0.2 µm(-1). Positive signals were obtained in 97.2% of the 110,000 wells in an area of 110 mm(2). Immobilized DNA was either indirectly detected using streptavidin-Cy5 or directly by molecular hybridisation of Cy3- and/or Cy5-labelled probes. Amplification and immobilization was demonstrated for template DNA ranging from 100 bp up to 1513 bp lengths. Even single DNA molecules were successfully amplified and immobilized demonstrating digital solid-phase PCR. Compared to widely established emulsion based PCR (emPCR) approaches, leading to PCR products immobilized onto bead surfaces in a highly parallel manner, the novel technique results in direct spatial registration of immobilized PCR products in a microarray format. This enables the subsequent use for massively parallel analysis similar to standard microarrays.

Melanoma margin assessment.

Primary cutaneous melanoma is treated by excisional surgery and careful histologic assessment of the specimen margins is a crucial component of pathology reporting. Surgical margins may be assessed by conventional transverse (bread-loaf) vertical sections, by en face vertical sections, or by en face oblique sections. Transverse techniques only sample a small percentage of the surgical margin. En face techniques are technically challenging but allow assessment of close to 100% of the margin. Margin assessment for melanoma removed from chronically sun-damaged skin is difficult. Melanoma in situ shows contiguous melanocyte growth, nesting, or intraepidermal pagetoid spread. Pitfalls include solar melanocytic hyperplasia, solar lentigines, melanocytic hyperplasia secondary to previous biopsy, lichenoid reactions, and invasive melanoma mimicking scar or benign nevus. En face sections can be used to assess margins for melanoma on sun-damaged skin, and evidence suggests that frozen sections may also be employed by experienced clinicians. Immunohistochemistry is a useful ancillary technique, enabling more accurate identification of in situ melanoma within a surgical margin.

Immunohistochemistry utilization in autopsy pathology: a Canadian experience.

Immunohistochemistry (IHC) is an important part of the diagnostic work-up in surgical pathology, but the use of IHC in autopsy pathology is poorly defined. We measured IHC utilization by pathologists performing 609 consecutive non-medicolegal, hospital-based, adult autopsies over a three-year period. IHC requests on non-neurologic and neurologic material were analyzed separately. Total stains, number of tissue blocks, specific antibody requests, resident trainee involvement, and ordering pathologist were recorded. For all autopsies on which IHC was requested, the final autopsy report was reviewed. IHC was requested on 345 cases (57%), and a total of 4612 stains were performed (mean 13.5 per autopsy). For non-neurologic autopsy tissues, IHC was used most commonly for the accurate diagnosis of malignancy. For neuropathologic autopsy examinations, IHC was employed most commonly to exclude neurodegenerative conditions and correlate ante-mortem clinical neurologic findings. Resident involvement did not significantly impact utilization. Individual pathologists demonstrated a wide variation in IHC utilization. We conclude that IHC is used extensively in Canadian non-medicolegal autopsy pathology reflecting the complexity, extent, and severity of disease in patients dying in a tertiary-care, academic hospital setting. Utilization is strongly influenced by the neuropathology component of these autopsies. The results provide a point of reference for IHC utilization in autopsy pathology.