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.

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.

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.

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.

Atypical cellular blue nevi (cellular blue nevi with atypical features): lack of consensus for diagnosis and distinction from cellular blue nevi and malignant melanoma ("malignant blue nevus").

The distinction of cellular blue nevi (CBN) with atypical features ["atypical" CBN (ACBN)] from conventional CBN and malignant melanomas related to or derived from CBN remains a difficult problem. Here, we report on the diagnosis of various cellular blue melanocytic neoplasms by 14 dermatopathologists who routinely examine melanocytic lesions. Three parameters were assessed: (1) for between rater analyses, we calculated interobserver agreement by the kappa statistic (regardless of whether the diagnosis was correct). (2) For each individual lesion, we reported whether a majority agreement (>50%) was reached and, if so, whether the majority agreed with the gold standard diagnosis, derived from standardized histopathologic criteria for melanoma, definitive outcome such as metastatic event or death of disease, or disease-free follow-up for > or =4 years. (3) For the individual pathologists, we calculated sensitivity and specificity for each type of lesion. The study set included 26 melanocytic lesions: (1) 6 malignant melanomas developing in or with attributes of CBN; (2) 11 CBN with atypical features and indeterminate biologic potential (ACBN); (3) 8 conventional CBN; and (4) 1 common BN. The kappa values for interrater agreement varied from 0.52 (95% confidence interval 0.45, 0.58) for melanoma to 0.02 (0.05, 0.08) for ACBN and 0.20 (0.13, 0.28) for CBN. The kappa for all lesions was 0.25 (0.22, 0.28). The pathologists' sensitivities were 68.6% (61.0%, 76.1%) for melanoma, 33.1% (21.0%, 45.2%) for ACBN, and 44.6% (29.0%, 60.3%) for CBN. The specificities were 65.7% (55.8%, 75.6%) for melanoma, 84.7% (77.3%, 92.2%) for ACBN, and 89.9% (82.7%, 97.1%) for CBN. Overall, greater than 50% of the pathologists agreed and were correct in their diagnosis 38.5% (10 lesions) of the time. There was a majority agreement, but with an incorrect diagnosis, another 26.9% (7 lesions) of the time. Six of the 7 majority agreements with an incorrect diagnosis were for ACBN lesions. In summary, the results of our study indicate that there is substantial confusion and disagreement among experienced histopathologists about the definitions and biologic nature of cellular blue melanocytic neoplasms particularly those thought to have atypical features ("atypical" CBN).

Anatomic location of hyaluronic acid filler material injected into nasolabial fold: a histologic study.

BACKGROUND Products instilled within or beneath the skin to improve its physical features are known as fillers. The position of the filler within the skin is one determinant of the end cosmetic result. OBJECTIVE The objective was to histologically determine the anatomic location of injected hyaluronic acid (HA) filler within nasolabial fold (NLF) skin. METHODS AND MATERIALS Sixteen patients (12 females, 4 males; median age, 59 years) undergoing Mohs micrographic surgery for basal cell carcinoma of the NLF area consented to injection of Burow's triangle or dog-ear redundant skin with HA gel (Juvederm), ex vivo, in vivo, or in vivo with delayed (1-4 weeks) removal. Sections of alcohol-fixed, paraffin-embedded tissue specimens were stained with hematoxylin and eosin and with Hale's colloidal iron for detection of acid mucins. Dermal thickness was measured and HA distribution assessed. RESULTS NLF dermal thickness was 1.37+/-0.27 mm (mean+/-SD), with a range of 1.04 to 1.86 mm. All 16 patients showed HA filler localized to the subcutis. In 9/16 tissue samples, some HA was present in the deep dermis, but filler was only observed in more superficial dermis in 1 patient. The thickness of injected filler was 2.11+/-0.63 mm, but filler was often transected at the specimen base. CONCLUSION The predominant localization of injected HA filler is within the subcutis. A relatively thin NLF dermal thickness, typically <1.50 mm, likely precludes accurate injection of filler into dermal collagen. The results suggest that dermal localization of HA filler products is not required for an excellent cosmetic result.

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.

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.

Are en face frozen sections accurate for diagnosing margin status in melanocytic lesions?

To assess the diagnostic accuracy of margin evaluation of melanocytic lesions using en face frozen sections compared with standard paraffin-embedded sections, we studied 2 sets of lesions in which en face frozen sections were used for analysis of surgical margins (13 from malignant melanomas [MMs] and 10 from nonmelanocytic lesions [NMLs]). Routine permanent sections were cut after routine processing. The slides were mixed and coded randomly. Fifteen dermatopathologists examined the cases separately. Margin status was categorized as positive, negative, or indeterminate. Kappa statistics were calculated per dermatopathologist and per case. One case from each group was excluded because epidermis was not available in the routine sections. Of 330 evaluations (22 cases, 15 dermatopathologists), there were 132 diagnostic discrepancies (40.0%): 66 each for MM and NML (mean per case for both diagnoses, 6). In 9 instances (6.8%), the change was from positive (frozen) to negative (permanent) and in 43 (32.6%), from negative (frozen) to positive (permanent). There was poor agreement between frozen and permanent sections (kappa range per dermatopathologist, -0.1282 to 0.6615). If permanent histology is considered the "gold standard" for histologic evaluation, en face frozen sections are not suitable for accurate surgical margin assessment of melanocytic lesions.

Expression of desmin and smooth muscle myosin heavy chain in dermatofibromas.

BACKGROUND: The histopathologic features of dermatofibroma vary remarkably, and this diversity may occasionally cause problems in differentiating between benign and malignant mesenchymal lesions, including smooth muscle neoplasms. Immunohistochemical stains are sometimes necessary to clarify the histogenesis of a lesion. OBJECTIVE: To evaluate dermatofibromas for expression of desmin and smooth muscle myosin heavy chain (SM-MHC) antigens, which are commonly used as evidence of smooth muscle differentiation. METHODS: We studied 100 consecutive cases of dermatofibroma using hematoxylin-eosin-stained sections and immunoperoxidase staining with antibodies against desmin, SM-MHC, and smooth muscle actin. RESULTS: We found focal positivity for desmin in 9 cases, and in 2 of these cases, at least 10% of lesional cells showed strong expression. We found focal staining for SM-MHC in 10 cases, and in 2 of these cases, at least 10% of the lesional cells were positive. Regions positive for desmin and/or SM-MHC did not show definite histologic features of myogenous differentiation on hematoxylin-eosin-stained sections. All dermatofibromas expressing desmin and SM-MHC were also strongly positive for smooth muscle actin. CONCLUSIONS: About 10% of dermatofibromas show focal expression of desmin and SM-MHC, and this expression may be present in up to 10% to 15% of lesional cells. Thus, in dermal spindle cell lesions, focal expression of these muscle antigens, like that of smooth muscle actin, is not diagnostic of a smooth muscle tumor.

Squamous cell carcinoma in situ of the skin: history, presentation, biology and treatment.

Squamous cell carcinoma in situ (SCCIS) of the skin is a problem commonly dealt with by dermatologists. The classic presentation, originally described by Bowen, is easily recognized, but presentation on some anatomical surfaces may be associated with less than typical features. Major aetiological factors for this disease are UV light, human papillomavirus infection and immunosuppression. The natural course of SCCIS is usually prolonged, with treatment being appropriate, but not urgent. The choice of therapy requires consideration of the location of the lesion, and a desire for a high cure rate without causing loss of form, function or cosmesis. The immunomodulatory agent imiquimod has offered a significant advance for the topical treatment of SCCIS. Our improved understanding of the underlying biology of SCCIS permits us to make rational choices of treatment. In the future we may be able to determine which of these lesions may progress to invasive disease, and help us select the most effective therapy.

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.