Digital process control of multi-step assays on centrifugal platforms using high-low-high rotational-pulse triggered valving.

Due to their capability for comprehensive sample-to-answer automation, the interest in centrifugal microfluidic systems has greatly increased in industry and academia over the last quarter century. The main applications of these "Lab-on-a-Disc" (LoaD) platforms are in decentralised bioanalytical point-of-use / point-of-care testing. Due to the unidirectional and omnipresent nature of the centrifugal force, advanced flow control is key to coordinate multi-step / multi-reagent assay formats on the LoaD. Formerly, flow control was often achieved by capillary burst valves which require gradual increments of the spin speed of the system-innate spindle motor. Recent advanced introduced a flow control scheme called 'rotational pulse actuated valves'. In these valves the sequence of valve actuation is determined by the architecture of the disc while actuation is triggered by freely programmable upward spike (i.e. Low-High-Low (LHL)) in the rotational frequency. This paradigm shift from conventional 'analogue' burst valves to 'digital' pulsing significantly increases the number of sequential while also improving the overall robustness of flow control. In this work, we expand on these LHL valves by introducing High-Low-High (HLH) pulse-actuated (PA) valving which are actuated by 'downward' spike in the disc spin-rate. These HLH valves are particularly useful for high spin-rate operations such as centrifugation of blood. We introduce two different HLH architectures and then combine the most promising with LHL valves to implement the time-dependent liquid handling protocol underlying a common liver function test panel.

Tailoring the work function of graphene via defects, nitrogen-doping and hydrogenation: A first principles study.

The effect of defects, nitrogen doping, and hydrogen saturation on the work function of graphene is investigated via first principle calculations. Whilst Stone-Wales defects have little effect, single and double vacancy defects increase the work function by decreasing charge density in theπ-electron system. Substitutional nitrogen doping in defect-free graphene significantly decreases the work function, because the nitrogen atoms donate electrons to theπ-electron system. In the presence of defects, these competing effects mean that higher nitrogen content is required to achieve similar reduction in work function as for crystalline graphene. Doping with pyridinic nitrogen atoms at vacancies slightly increases the work function, since pyridinic nitrogen does not contribute electrons to theπ-electron system. Meanwhile, hydrogen saturation of the pyridinic nitrogen atoms significantly reduces the work function, due to a shift from pyridinic to graphitic-type behavior. These findings clearly explain some of the experimental work functions obtained for carbon and nitrogen-doped carbon materials in the literature, and has implications in applications such as photocatalysis, photovoltaics, electrochemistry, and electron field emission.

Gray zone lymphoma: A case report and comprehensive review of literature.

Gray zone lymphoma is a very rare liquid malignancy that possesses intersecting features between primary mediastinal B-cell lymphoma and classic Hodgkin Lymphoma. In the case presented and accompanying literature review, we will discuss a patient with a chief complaint of shortness of breath and was found to have a mediastinal mass with biopsy consistent with mediastinal gray zone lymphoma. Herein, we explore the historical and recently updated diagnostic criteria of gray zone lymphoma from 2022 as well as the pathophysiology as it pertains to gene expression, while also reviewing the histological findings, epidemiology and treatment modalities.

Prostate cancer and microfluids.

Microfluidic systems aim to detect sample matter quickly with high sensitivity and resolution, on a small scale. With its increased use in medicine, the field is showing significant promise in prostate cancer diagnosis and management due, in part, to its ability to offer point-of-care testing. This review highlights some of the research that has been undertaken in respect of prostate cancer and microfluidics. Firstly, this review considers the diagnosis of prostate cancer through use of microfluidic systems and analyses the detection of prostate specific antigen, proteins, and circulating tumor cells to highlight the scope of current advancements. Secondly, this review analyses progressions in the understanding of prostate cancer physiology and considers techniques used to aid treatment of prostate cancer, such as the creation of a micro-environment. Finally, this review highlights potential future roles of microfluidics in assisting prostate cancer, such as in exosomal analysis. In conclusion, this review shows the vast scope and application of microfluidic systems and how these systems will ensure advancements to future prostate cancer management.

Characterisation of osteogenic and vascular responses of hMSCs to Ti-Co doped phosphate glass microspheres using a microfluidic perfusion platform.

Using microspherical scaffolds as building blocks to repair bone defects of specific size and shape has been proposed as a tissue engineering strategy. Here, phosphate glass (PG) microcarriers doped with 5 mol % TiO2 and either 0 mol % CoO (CoO 0%) or 2 mol % CoO (CoO 2%) were investigated for their ability to support osteogenic and vascular responses of human mesenchymal stem cells (hMSCs). Together with standard culture techniques, cell-material interactions were studied using a novel perfusion microfluidic bioreactor that enabled cell culture on microspheres, along with automated processing and screening of culture variables. While titanium doping was found to support hMSCs expansion and differentiation, as well as endothelial cell-derived vessel formation, additional doping with cobalt did not improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled screening of culture parameters for cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing.

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay.

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Possible mechanisms of CO2 reduction by H2 via prebiotic vectorial electrochemistry.

Methanogens are putatively ancestral autotrophs that reduce CO2 with H2 to form biomass using a membrane-bound, proton-motive Fe(Ni)S protein called the energy-converting hydrogenase (Ech). At the origin of life, geologically sustained H+ gradients across inorganic barriers containing Fe(Ni)S minerals could theoretically have driven CO2 reduction by H2 through vectorial chemistry in a similar way to Ech. pH modulation of the redox potentials of H2, CO2 and Fe(Ni)S minerals could in principle enable an otherwise endergonic reaction. Here, we analyse whether vectorial electrochemistry can facilitate the reduction of CO2 by H2 under alkaline hydrothermal conditions using a microfluidic reactor. We present pilot data showing that steep pH gradients of approximately 5 pH units can be sustained over greater than 5 h across Fe(Ni)S barriers, with H+-flux across the barrier about two million-fold faster than OH--flux. This high flux produces a calculated 3-pH unit-gradient (equating to 180 mV) across single approximately 25-nm Fe(Ni)S nanocrystals, which is close to that required to reduce CO2. However, the poor solubility of H2 at atmospheric pressure limits CO2 reduction by H2, explaining why organic synthesis has so far proved elusive in our reactor. Higher H2 concentration will be needed in future to facilitate CO2 reduction through prebiotic vectorial electrochemistry.

Author Correction: Formation and purification of tailored liposomes for drug delivery using a module-based micro continuous-flow system.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices.

Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker. These methods were applied to immobilise transketolase (TK) in PMMA microfluidic devices. Binding efficiency studies showed that about 15% of the supplied TK was bound using the 1-step method and about 26% of the enzyme was bound by the 3-step method. However, the TK-catalysed reaction producing l-erythrulose performed in microfluidic devices showed that specific activity of TK in the device utilising the 1-step immobilisation method was approximately 30% higher than that of its counterpart. Reusability of the microfluidic device produced via the 1-step method was tested for three cycles of enzymatic reaction and at least 85% of the initial productivity was maintained. The device could be operated for up to 40 h in a continuous flow and on average 70% of the initial productivity was maintained. The simplified immobilisation method required fewer chemicals and less time for preparation of the immobilised microfluidic device compared to the 3-step method while achieving higher specific enzyme activity. The method represents a promising approach for the development of immobilised enzymatic microfluidic devices and could potentially be applied to combine protein purification with immobilisation.

Formation and purification of tailored liposomes for drug delivery using a module-based micro continuous-flow system.

Liposomes are lipid based bilayer vesicles that can encapsulate, deliver and release low-soluble drugs and small molecules to a specific target site in the body. They are currently exploited in several nanomedicine formulations. However, their development and application is still limited by expensive and time-consuming process development and production methods. Therefore, to exploit these systems more effectively and support the rapid translation of new liposomal nanomedicines from bench to bedside, new cost-effective and scalable production methods are needed. We present a continuous process flow system for the preparation, modification and purification of liposomes which offers lab-on-chip scale production. The system was evaluated for a range of small vesicles (below 300 nm) varying in lipid composition, size and charge; it offers effective and rapid nanomedicine purification with high lipid recovery (> 98%) combined with effective removal of non-entrapped drug (propofol >95% reduction of non-entrapped drug present) or protein (ovalbumin >90% reduction of OVA present) and organic solvent (ethanol >95% reduction) in less than 4 minutes. The key advantages of using this bench-top, rapid, process development tool are the flexible operating conditions, interchangeable membranes and scalable high-throughput yields, thereby offering simultaneous manufacturing and purification of nanoparticles with tailored surface attributes.

Event-triggered logical flow control for comprehensive process integration of multi-step assays on centrifugal microfluidic platforms.

The centrifugal "lab-on-a-disc" concept has proven to have great potential for process integration of bioanalytical assays, in particular where ease-of-use, ruggedness, portability, fast turn-around time and cost efficiency are of paramount importance. Yet, as all liquids residing on the disc are exposed to the same centrifugal field, an inherent challenge of these systems remains the automation of multi-step, multi-liquid sample processing and subsequent detection. In order to orchestrate the underlying bioanalytical protocols, an ample palette of rotationally and externally actuated valving schemes has been developed. While excelling with the level of flow control, externally actuated valves require interaction with peripheral instrumentation, thus compromising the conceptual simplicity of the centrifugal platform. In turn, for rotationally controlled schemes, such as common capillary burst valves, typical manufacturing tolerances tend to limit the number of consecutive laboratory unit operations (LUOs) that can be automated on a single disc. In this paper, a major advancement on recently established dissolvable film (DF) valving is presented; for the very first time, a liquid handling sequence can be controlled in response to completion of preceding liquid transfer event, i.e. completely independent of external stimulus or changes in speed of disc rotation. The basic, event-triggered valve configuration is further adapted to leverage conditional, large-scale process integration. First, we demonstrate a fluidic network on a disc encompassing 10 discrete valving steps including logical relationships such as an AND-conditional as well as serial and parallel flow control. Then we present a disc which is capable of implementing common laboratory unit operations such as metering and selective routing of flows. Finally, as a pilot study, these functions are integrated on a single disc to automate a common, multi-step lab protocol for the extraction of total RNA from mammalian cell homogenate.

Mimicking insect communication: release and detection of pheromone, biosynthesized by an alcohol acetyl transferase immobilized in a microreactor.

Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis, is achieved in a novel microfluidic system designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an "artificial gland", i.e., a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behavior as 3 virgin females in most of the parameters considered.

Trends in prostatic adenocarcinoma tumor volume by visual estimation in prostatectomy specimens.

We retrospectively reviewed 1792 consecutive radical prostatectomies (RP) from 2003 to 2006 at a single institution to establish tumor volume reference values, to determine current trends in visually estimated prostate adenocarcinoma tumor volume, and to characterize cases with no residual cancer on RP. Tumor volumes were recorded and subsequently stratified as very low, 0-1%; low, 1.1-10%; intermediate, 10.1-20%; high, 20.1-50%; and very high, >50%, with incidences of 11.7%, 52.1%, 21.5%, 13.2%, and 1.5%, respectively. The incidence of very low volume tumors increased within the time period (p=0.04). Seminal vesicle involvement was detected in 5.0% of cases and lymph node metastasis occurred in 1.4%. Volume categories statistically correlated with seminal vesicle invasion (p=0) and lymph nodes metastases (p=0). Eleven cases of no residual cancer (0.6%) were identified with a non-statically significant increase during the study (p=0.07). The rising incidence of very low volume tumors should be considered by clinicians when discussing treatment options with patients. A discrete tumor volume should be provided for RP specimens as it may be an important prognostic factor.

Blastic plasmacytoid dendritic cell neoplasm with leukemic presentation, lacking cutaneous involvement: Case series and literature review.

The diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN) can be challenging, owing to the absence of traditional lineage-specific markers, but is facilitated by CD4/CD56 co-expression and frequent skin involvement. Herein, we present our collective experiences with three BPDCN cases lacking cutaneous presentation and the inherent diagnostic pitfalls. Taken in context of similar historical cases, we suggest that BPDCN with "leukemic" presentation (L-BPDCN) otherwise presents no major distinguishing features and is at least as aggressive as its cutaneous-involved BPDCN counterpart.

Pheromone synthesis in a biomicroreactor coated with anti-adsorption polyelectrolyte multilayer.

To prepare a biosynthetic module in an infochemical communication project, we designed a silicon/glass microreactor with anti-adsorption polyelectrolyte multilayer coating and immobilized alcohol acetyl transferase (atf), one of the key biosynthetic enzymes of the pheromone of Spodoptera littoralis, on agarose beads inside. The system reproduces the last step of the biosynthesis in which the precursor diene alcohol (Z,E)-9,11-tetradecadienol is transformed into the major component (Z,E)-9,11-tetradecadienyl acetate. The scope of this study was to analyze and implement a multilayer, anti-adsorption coating based on layer-by-layer deposition of polyethylenimine/dextransulfate sodium salt (PEI/DSS). The multilayers were composed of two PEI with molecular weights 750 and 1.2 kDa at pH 9.2 or 6.0. Growth, morphology, and stability of the layers were analyzed by ellipsometry and atomic force microscopy (AFM). The anti-adsorption functionality of the multilayer inside the microreactor was validated. The activity of His(6)-(atf) was measured by gas chromatography coupled to mass spectrometer (GC-MS).

Acute promyelocytic leukemia at time of relapse commonly demonstrates cytogenetic evidence of clonal evolution and variability in blast immunophenotypic features.

Despite the success of the current therapy for patients with acute promyelocytic leukemia (APL), relapse occurs in up to 30% of patients. The characteristics of relapsed APL are not well described. We evaluated a group of APL cases at relapse and compared the clinicopathologic, immunophenotypic, molecular, and cytogenetic findings with those at initial diagnosis. From a group of 207 patients with APL, in 38 patients morphologic evidence of relapse developed. In 30 patients relapse was isolated to bone marrow, and 8 had extramedullary disease. Blasts were morphologically stable in 37 patients. Changes in the immunophenotypic profile were common, the most frequent being gain of CD34, HLA-DR, or CD33 and attenuation or loss of CD13. Cytogenetic changes were common at relapse. The size of the PML-RARalpha fusion transcript was invariable. We conclude that changes in the immunophenotype and cytogenetic evidence of clonal evolution are common in APL at the time of relapse.

Rapid and reliable confirmation of acute promyelocytic leukemia by immunofluorescence staining with an antipromyelocytic leukemia antibody: the M. D. Anderson Cancer Center experience of 349 patients.

BACKGROUND: The authors evaluated the utility of immunofluorescence staining with an antipromyelocytic leukemia (anti-PML) antibody for patients with a suspected diagnosis of new or relapsed acute promyelocytic leukemia (APL) and correlated the findings with the results of other established diagnostic modalities. METHODS: Bone marrow (BM) and/or peripheral blood (PB) smears from 349 patients in whom the diagnosis of APL was considered were assessed with the anti-PML antibody using immunofluorescence. The study group included 199 patients with confirmed APL and 150 with other conditions. The results of conventional cytogenetics, reverse transcription polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) performed on these patients were correlated with the PML results. RESULTS: Among patients with confirmed APL, anti-PML antibody was positive in 182 of 184 BM and 32 of 33 PB smears. Conventional cytogenetics demonstrated t(15;17)(q22;q12) in 166 of 182 (91%) patients; 10 had a normal karyotype, 4 had insufficient mitoses to grow in culture, 1 was inconclusive, and 1 was 48, XX, +8, +8. Anti-PML staining was positive in 9 of 10 with a normal karyotype and in all 4 cases with insufficient mitoses. RT-PCR and FISH were positive for PML-retinoic acid receptor-alpha in 169 of 172 (98%) and 90 of 94 (96%) cases, respectively. Among the patients without APL, 148 of 150 (98.6%) were negative with anti-PML antibody. The sensitivity and specificity of the test were 98.9% and 98.7%, respectively. CONCLUSIONS: PML immunofluorescence staining is a rapid (<4 hours turnaround time) and reliable frontline diagnostic approach that can facilitate initiation of targeted therapy, particularly in clinical settings where cytogenetic and molecular testing are not readily available.

Expression of glypican 3 in ovarian and extragonadal germ cell tumors.

Germ cell tumors (GCTs), rare malignancies that occur in a wide range of locations and display variable histologic patterns, may pose diagnostic challenges. Glypican 3 (GPC3), a membrane-bound heparan sulfate proteoglycan, has been shown to be a novel diagnostic marker in testicular GCT. However, GPC3 expression in ovarian and extragonadal GCT has not been reported. We evaluated GPC3 immunoreactivity in GCTs from 63 patients (57 children and 6 adults), including 14 ovarian and 20 extragonadal primary GCTs and 8 metastases along with 21 primary testicular GCTs for comparison. All 33 yolk sac tumors (YSTs) and both choriocarcinomas were immunoreactive for GPC3. In contrast, a minority of immature (4/10) and mature (4/35) teratomas were positive. No positivity was seen in 6 embryonal carcinomas or 5 germinomas. GPC3 is differentially expressed in ovarian and extragonadal GCTs, with expression predominantly observed in YSTs and choriocarcinoma.

Topoisomerase II alpha expression in testicular germ cell tumors.

OBJECTIVES: Inhibitors of topoisomerase II alpha (TopoIIalpha), an enzyme with a crucial role in DNA maintenance, are included in the chemotherapy protocols for testicular germ cell tumors (GCTs). Despite the success of current chemotherapy regimens, a significant number of patients experience relapse. We analyzed TopoIIalpha expression in primary and metastatic testicular GCTs because this enzyme is a target for some antineoplastic agents. METHODS: Primary GCT specimens from 109 patients, including 57 seminomas and 52 mixed GCTs (41 embryonal carcinomas, 23 yolk sac tumors, 19 seminomas, 8 choriocarcinomas, 17 teratomas with immature elements, and 16 teratomas with mature elements), were obtained from our archives. The metastatic lesions from 11 of the patients with mixed GCTs included seven teratomas with mature components, five embryonal carcinomas, one yolk sac tumor, one choriocarcinoma, and one teratoma with immature components. Representative sections were subjected to immunohistochemistry with monoclonal antibody against TopoIIalpha, and the nuclear staining findings were evaluated. RESULTS: Most embryonal carcinoma (100%), yolk sac tumor (95%), seminoma (88%), and choriocarcinoma (62%) components of the GCTs were TopoIIalpha immunoreactive. None of the teratoma specimens with mature elements expressed TopoIIalpha. CONCLUSIONS: The results of our study have shown that TopoIIalpha is expressed in most seminomas, embryonal carcinomas, yolk sac tumors, and choriocarcinomas, suggesting a possible mechanism of sensitivity of these components to TopoIIalpha inhibitors. Teratomas with mature and immature elements expressed low levels of TopoIIalpha, which might contribute to their chemoresistance. These findings imply that the variable chemoresponsiveness of testicular GCTs could have an underlying molecular basis.

Glypican 3: a novel marker in testicular germ cell tumors.

Glypican 3 (GPC3), a membrane-bound heparin sulfate proteoglycan, may play a role in promoting embryonic cell growth and differentiation. GPC3 is mutated in Simpson-Golabi-Behmel syndrome, characterized by tissue overgrowth and an increased risk of embryonal malignancies. Recently, GPC3 was reported to be one of the over-expressed genes in testicular yolk sac tumors by gene expression microarray analysis. However, the presence of the GPC3 protein in germ cell tumors has never been investigated. The purpose of the study was to investigate the GPC3 expression in various histologic components of testicular germ cell tumors using immunohistochemistry and to assess its possible utility as a diagnostic marker. Tumors from 71 patients were examined, including components of 42 seminomas, 37 embryonal carcinomas, 24 yolk sac tumors, 20 teratomas with mature elements, 16 teratomas with immature elements, and 7 choriocarcinomas. Cytoplasmic and membranous immunoreactivity was semiquantitatively evaluated. All yolk sac tumor (24/24) and choriocarcinoma (7/7) components were immunoreactive for GPC3, whereas only 38% of teratomas with immature elements and 8% of embryonal carcinomas expressed GPC3. There was no immunoreactivity in benign testicular tissue, intratubular germ cell neoplasia, seminomas (0/42), or teratomas with mature elements (0/20). We conclude that the oncofetal protein GPC3 is a novel immunohistochemical marker in testicular germ cell tumors with differential expression in defined histologic subtypes. Our findings suggest a possible role of GPC3 in tumor cell differentiation. Furthermore, GPC immunostaining may be useful in the pathologic diagnosis of nonseminomatous germ cell tumors, particularly yolk sac tumor, and choriocarcinoma.