C4d Detection Research Articles

Taylor dispersion analysis using capacitively coupled contactless conductivity detector

Taylor dispersion analysis (TDA) is a simple and absolute method to determine the hydrodynamic radius of solutes that respond to UV or fluorescence detections. To broaden the application range of TDA, it is necessary to develop new detection modes. This study aims to study capacitively coupled contactless conductivity detector (C4D) for the analysis of charged macromolecules. The detection sensitivities and hydrodynamic radii were compared for a C4D detector and a UV detector on positively or negatively charged polymers responding both to UV and C4D (poly-L-lysine and poly(acrylamide-co-2-acrylamido-1-methyl-propanesulfonate). The influence of the composition of the background electrolyte on the detection sensitivity has been studied and optimized for C4D detection. The influence of the molar mass and of the polymer chemical charge density on the C4D and UV sensitivities of detection have been investigated based on well-characterized copolymers samples of different molar masses and charge densities. The advantages and disadvantages compared to UV detection, as well as the range of applicability of C4D detection in TDA were identified. C4D detection can be an alternative method for sizing charged polymers of reasonable molar mass (typically below 105 g mol−1) that do not absorb in UV. A decline in the sensitivity of detection in C4D was observed for higher molar masses.

Complement C4d as a biomarker for systemic lupus erythematosus and lupus nephritis.

Background: Increasing studies in the last decade have led to the widespread understanding that C4d, a split product of complement component 4 (C4), is a potential biomarker for systemic lupus erythematosus (SLE) and lupus nephritis (LN).Purpose: The aim of this review is to summarize the highlights of studies investigating the use of C4d as a biomarker for diagnosing and monitoring SLE and LN patients.Data collection: we searched PubMed/Medline and Wanfang databases using the terms "C4d and systemic lupus erythematosus", "C4d and lupus nephritis", and "Complement C4d".Results: The deposition of C4d on circulating blood cells has been shown in several clinical studies to be a potential diagnostic marker that can be used to monitor patients with SLE. In addition, C4d deposits on circulating blood cells may be a helpful diagnostic marker for LN, one of the most severe complications of SLE. Meanwhile, studies utilizing renal biopsy specimens have indicated that C4d deposition in the renal peritubular capillaries of LN patients may predict more severe LN or a worse patient prognosis. Generally, a high plasma C4d level and a high plasma C4d/C4 ratio may also be promising indicators that can be used to monitor patients with SLE and LN.Conclusions: C4d detection may be a novel strategy for further clinical prediction and therapy.

Compact contactless conductometric, ultraviolet photometric and dual-detection cells for capillary electrophoresis via additive manufacturing

The growing demand for tailored detectors in capillary electrophoresis (CE), addressing tasks like field deployment or dual-detection analysis, emphasizes the necessity for compact detection cells. In this work, we propose cost-effective and user-friendly additive manufacturing (3D-printing) approaches to produce such miniaturized detection cells suitable for a range of CE applications. Firstly, capacitively-coupled contactless conductivity detection (C4D) cells of different sizes are fabricated by casting low-melting-point alloy into 3D-printed molds. Various designs of Faraday shields are integrated within the cells and compared. A mini-C4D cell (9.5×7.0×7.5 mm3) is produced, with limits of detection for alkaline cations ranging from 8-12 μM in a short–capillary based CE application. Secondly, ultraviolet photometric (UV-PD) detection cells are fabricated using 3D printing. These cells feature two narrow slits with a width of 60 μm, which are positioned along the path of incident and transmission light to facilitate collimation. A deep UV-LED (235 nm or 255 nm) is employed as the light source, and black resin is determined to be the optimal material for 3D printing the UV-PD cell, owing to its superior UV light absorption capabilities. The UV-PD cell is connected to the LED and photodetector through two optical fibers, making it easy to switch the light source and detector. The effective pathlength and stray light percentage for detecting on a 75 μm id capillary are 74 μm and 0.5 %, respectively. Thirdly, a dual-detection cell that combined C4D and UV-PD at a single detection point is proposed. The performance of direct detection by C4D and indirect detection by UV-PD is compared for detecting organic acids. The strategies for developing cost-effective compact detection cells facilitate the versatile integration of multiple detection methods in CE analysis.

Concise analysis of γ-hydroxybutyric acid in beverages and urine by capillary electrophoresis with capacitively coupled contactless conductivity detection using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid as background electrolyte

γ-Hydroxybutyric acid (GHB), a neurotransmitter or neuromodulator in the human central nervous system, is often abused in drug-facilitated sexual assaults due to its euphoric and sedative effects. While the analysis of GHB has received continuous attention, its inherent characteristics pose challenges. In the current study, capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) was built, and Good's buffers were evaluated as the background electrolytes for CE separation and C4D detection. On this basis, a simple and efficient CE-C4D method was developed for GHB analysis. Through theoretical discussion and experimental optimization, the separation of GHB and related positional isomers α-hydroxybutyric acid (AHB) and β-hydroxybutyric acid (BHB) was achieved within 4 min using 150 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as the running buffer. Under the optimized condition, the relative standard deviations of migration time and peak area were less than 1.1% and 4.5%, indicating good precision. The C4D signal of GHB showed a good linear relationship with GHB concentration in the range of 3-300 µM with a determination coefficient of 0.9997, and the detection limit was calculated to be 0.37 µM based on the signal-to-noise ratio of three. Furthermore, liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were comparatively studied for sample matrix purification. Combined with the optimized SPE procedure, the developed CE-C4D method has been successfully applied for the determination of exogenous GHB in spiked beverages and endogenous GHB in human urine.

Multimaterial 3D-printed contactless conductivity/laser-induced fluorescence dual-detection cell for capillary electrophoresis

利用多材料3D打印技术研制了用于毛细管电泳(CE)的二合一检测池,实现了电容耦合非接触电导(C4D)与共聚焦激光诱导荧光(LIF)两种检测方法在毛细管柱上同一位置同时检测。3D打印的检测池采用了导电的复合聚乳酸(PLA)材料制作C4D的屏蔽层,采用普通的绝缘PLA材料支撑C4D金属管电极并隔离屏蔽层。两根金属管电极通过“打印-暂停-打印”的方式嵌入到检测池中,两电极被2 mm厚的导电屏蔽层隔开,在屏蔽层中有一直径为1 mm的圆形通孔用于LIF检测。该检测池与带流通式进样接口的自组装CE系统联用,用于同时检测无机离子和异硫氰酸荧光素(FITC)标记的氨基酸。研究优化了C4D激励信号频率与电泳缓冲液浓度,选用的电泳缓冲溶液为10 mmol/L 3-吗啉丙烷-1-磺酸(MOPS)与10 mmol/L二(2-羟乙基)亚氨基三(羟甲基)甲烷(Bis-Tris)的混合溶液,选用C4D激励频率为77 kHz。二合一检测池应用于内径为25 μm的毛细管时,C 4D对Na+、K+和Li+的检出限分别为2.2、2.0和2.6 μmol/L; LIF对荧光素和FITC的检出限分别为7.6和1.7 nmol/L。两种检测方法的相对标准偏差在0.3%至4.5%之间(n=3),工作曲线的相关系数r 2≥0.9904。采用3D打印技术可以在实验室内实现复杂结构的制作,降低了制作的成本,且便于方法的推广和改进。

3D printed two-in-one on-capillary detector: Combining contactless conductometric and photometric detection for capillary electrophoresis

In this work, for the first time, a 3D printed two-in-one on-capillary detector, combining contactless conductometric detection (C4D) and photometric detection (PD), is fabricated for capillary electrophoresis (CE). The C4D Faraday shield (FS) is printed using electrically conductive composite polylactic acid (PLA) to minimize the stray capacitance. Non-conductive PLA is also used to print the insulator of FS to prevent the electrical conduction with two stainless steel electrodes. A novel collimator, consisting of two partially aligned pinholes, is printed by conductive material to collimate the light-emitting diode beam. The C4D detection has a signal-to-noise ratio of 1092 ± 2 for 200 μM potassium on a 25 μm id capillary. The PD detection shows excellent linearity with stray light down to 8% and an effective path length at 73% of a 75 μm id capillary. The analytical performance is demonstrated by CE separation and detection of cations. PD shows limits of detection (LODs) of 1.3, 0.9, and 1.7 μM for cobalt, copper and zinc, which are complexed with 4-(2-Pyridylazo) resorcinol, while C4D shows LODs of 1.2, 1.4, 21 and 2.6 μM for potassium, sodium, cobalt and zinc, respectively.

Capillary electrophoresis with dual C4D/UV detection for simultaneously determining major metal cations and whey proteins in milk.

A reliable and simple CE method with dual C4D and UV detection modes for simultaneous determination of major metal cations and whey proteins in milk samples was developed. Sample pretreatment comprised dilution, acidification to pH 4.55 with 10 mM AcOH and centrifugation. The complete separation of metal cations K+, Ca2+, Na+, and Mg2+ and whey proteins α-Lac, and β-Lg could be achieved respectively within 10 min and 20 min in a simple BGE composed of 1.0 M AcOH, 12 mM l-His and 2 mM 18-crown-6 with pH 2.74 at a voltage/current of +15 kV/12.5 μA. The samples were injected hydrodynamically by a pressure of 50 mbar for 5 s, the excitation voltage and excitation frequency of the C4D detector were 80 V and 1000 kHz, respectively and the detection wavelength of UV detection was set at 200 nm. In cation analysis, the range of the detection limit was 0.05-0.10 mg L-1 for C4D detection and 0.10-0.50 mg L-1 for UV detection, respectively, and the relative standard deviations (RSD%, n = 5) of intraday and interday analysis were 0.37-0.55% and 0.46-0.79% for the relative migration time, and 2.51-4.12% and 3.65-4.91% for the peak area, respectively. In whey protein analysis, the detection limits of β-Lg and α-Lac analysis were 5 mg L-1 and 3 mg L-1, respectively and the relative standard deviations (RSD%, n = 5) of intraday and interday analysis were 0.29-0.31% and 0.43-0.48% for the migration time and 2.89-3.25% and 3.29-4.18% for the peak area, respectively. The content of four major metal cations and two whey proteins in various types of milk samples was obtained. The results indicated that the content of metal cations varied little in milk samples of different brands and prices, while the content of whey proteins, as thermosensitive active proteins, varied greatly among different heat-treated milk samples.

Automated detection algorithm for C4d immunostaining showed comparable diagnostic performance to pathologists in renal allograft biopsy.

A deep learning-based image analysis could improve diagnostic accuracy and efficiency in pathology work. Recently, we proposed a deep learning-based detection algorithm for C4d immunostaining in renal allografts. The objective of this study is to assess the diagnostic performance of the algorithm by comparing pathologists' diagnoses and analyzing the associations of the algorithm with clinical data. C4d immunostaining slides of renal allografts were obtained from two different institutions (100 slides from the Asan Medical Center and 86 slides from the Seoul National University Hospital) and scanned using two different slide scanners. Three pathologists and the algorithm independently evaluated each slide according to the Banff 2017 criteria. Subsequently, they jointly reviewed the results for consensus scoring. The result of the algorithm was compared with that of each pathologist and the consensus diagnosis. Clinicopathological associations of the results of the algorithm with allograft survival, histologic evidence of microvascular inflammation, and serologic results for donor-specific antibodies were also analyzed. As a result, the reproducibility between the pathologists was fair to moderate (kappa 0.36-0.54), which is comparable to that between the algorithm and each pathologist (kappa 0.34-0.51). The C4d scores predicted by the algorithm achieved substantial concordance with the consensus diagnosis (kappa = 0.61), and they were significantly associated with remarkable microvascular inflammation (P = 0.001), higher detection rate of donor-specific antibody (P = 0.003), and shorter graft survival (P < 0.001). In conclusion, the deep learning-based C4d detection algorithm showed a diagnostic performance similar to that of the pathologists.

Contactless conductivity detector from printed circuit board for paper-based analytical systems

This work presents a capacitively coupled contactless conductivity detector (C4D) etched out from a printed circuit board (PCB) as potential sensor for paper-based analytical systems. Two lines of any desirable pattern forming 35-μm thick planar copper electrodes were produced on a PCB plate (40 mm × 60 mm) by photolithography. The final PCB plate was covered with polypropylene film to serve as the insulating layer for the C4D detector. The film also protected the copper electrodes from corrosion. Electrodes made in this planar geometry make the PCB-C4D suitable as sensor for flat devices such as paper-based analytical devices. For this work, plain paper strips were employed as sample reservoir and as fluidic channel without hydrophobic pattern. A dried paper strip was first placed over the sensor, followed by dispensing a fixed volume of the liquid sample onto the paper. Entrapment of the liquid sample in the paper strip leads to reproducible size and position of the detection zone of the sample liquid for the capacitive coupling effect. High precision was obtained with %RSD ≤1% (n = 18) for standard solutions of KCl. Soil suspensions could be analyzed without prior filtration by placing a drop onto the paper strip extending away from the detector zone. The paper strip filtered out soil particles at the surface of the paper. Therefore, only soil filtrate moved towards the detection zone by lateral flow. The C4D detection using paper strip showed high tolerance to soil suspension with turbidity up to 6657 NTU, offering direct analysis of soil salinity. Cleaning with moist tissue paper between samples is adequate even for dirty samples such as soil suspension. We also monitored conductivity of acid-base reaction in the microfluidic paper channels, which was later applied to the quantification of bicarbonate in water and in antacid tablet (“Soda Mint Tablet”).

Simultaneous determination of three sulfanilamide artificial sweeteners in foodstuffs by capillary electrophoresis coupled with contactless conductivity detection based on porous aromatic frameworks enhanced solid phase extraction

In this paper, a simple and easy method of solid phase extraction (SPE) followed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) is evaluated as a novel approach for the simultaneous determination of acesulfame-K (ACE), sodium saccharin (SAC), and sodium cyclamate (CYC) in foodstuffs without derivatization. To reduce the complex matrix interference resulting from the constituents of samples and enriched targets, porous aromatic frameworks (PAFs) enhanced SPE, a suitable sample pretreatment procedure, was introduced. Several factors affecting extraction efficiency and electrophoretic separation were investigated. Additionally, the interaction mechanisms between the host (PAF-6) and guests (ACE, SAC, and CYC) were studied. Under the optimum conditions, three sulfanilamide artificial sweeteners were baseline separated within 8 min, exhibiting a linear calibration over two orders of magnitude (R2 &gt; 0.995). The limits of detection (LOD) and quantification (LOQ) were considered better than those usually obtained by CE with ultraviolet and C4D detection. The proposed SPE–CE–C4D method has been successfully applied to analyse beverage samples and candied fruits with recoveries in the range of 78.89%–92.00%.

C4d detection and histological patterns in the diagnosis ofantibody-mediated rejection after lung transplantation: a single-centre study.

Antibody-mediated rejection (AMR) is an emerging and challenging issue in transplantation. Endothelial deposition of C4d and microvascular inflammation (MI) are reliable markers of AMR in renal and cardiac transplantation, but remain controversial in the lung. Our aim was to assess C4d immunohistochemistry and histological patterns for the diagnosis of lung AMR. We reviewed 158 transbronchial biopsies (TBBs) (n=85 clinically indicated, and n=73 surveillance TBBs) from 48 recipients, blinded to clinical and serological data. C4d was scored as 0, 1+ (<10%), 2+ (10-50%) or 3+ (>50%). TBBs were reassessed for MI and acute lung injury (ALI). Donor-specific antibodies (DSAs), acute clinical graft dysfunction and chronic lung allograft graft dysfunction (CLAD) were recorded. C4d3+, C4d2+, C4d1+ and C4d0 occurred respectively in four (2.5%), six (3.8%), 28 (17.7%) and 120 (75.9%) TBBs. MI and ALI were rare but more frequent in C4d1-3+ TBBs than in the absence of C4d. C4d2+ was frequently observed with concomitant infection. Among the surveillance TBBs, only two (2.7%) showed MI. Neither ALI nor C4d3+ was diagnosed on surveillance TBBs. No significant association was found between histopathological findings and DSAs. All four patients with C4d3+ could retrospectively be diagnosed with AMR and developed CLAD. Although rare, diffuse C4d deposition appears to be a strong indication of acute clinical AMR in lung transplant patients, whereas intermediate C4d2+ requires more investigations. In stable patients, histopathology and C4d may lack the sensitivity to diagnose subclinical AMR. This emphasises the need for a multidisciplinary evaluation of each suspected AMR case, and the need for complementary diagnostic tools.

Bacterial Concentration Detection using a PCB-based Contactless Conductivity Sensor.

Capacitively coupled contactless conductivity detection (C4D) is an improved approach to avoid the problems of labor-intensive, time-consuming and insufficient accuracy of plate count as well as the high-cost apparatus of flow cytometry (FCM) in bacterial counting. This article describes a novel electrode-integrated printed-circuit-board (PCB)-based C4D device, which supports the simple and safe exchange of capillaries and improves the sensitivity and repeatability of the contactless detection. Furthermore, no syringe pump is needed in the detection, it reduces the system size, and, more importantly, avoids the effect on the bacteria due to high pressure. The recovered bacteria after C4D detection at excitation of 25 Vpp and 60–120 kHz were analyzed by flow cytometry, and a survival rate higher than 96% was given. It was verified that C4D detection did not influence the bacterial viability. Moreover, bacteria concentrations from 106 cells/mL to 108 cells/mL were measured in a linear range, and relative standard deviation (RSD) is below 0.2%. In addition, the effects on bacteria and C4D from background solutions were discussed. In contrast to common methods used in most laboratories, this method may provide a simple solution to in situ detection of bacterial cultures.

Clinical impact of complement (C1q, C3d) binding De Novo donor-specific HLA antibody in kidney transplant recipients.

IntroductionComplement binding activity of donor-specific HLA antibodies (DSA) has been suggested as a new tool to stratify immunologic risk in kidney transplantation (KT). The objective of this study was to evaluate the clinical implication of C1q/C3d binding activity of de novo DSA (dnDSA) in KT recipients.Material and methodsA total of 161 pretransplant DSA-negative recipients were monitored for dnDSA at the time of biopsy. C1q/C3d binding activities of dnDSA were assessed using C1qScreen assay (One lambda, USA) and Lifecodes C3d detection assay (Immucor, USA), respectively. Clinical outcomes including biopsy-proven antibody mediated rejection (AMR), C4d detection and post-biopsy graft survival were investigated.ResultsDe-novo DSAs were detected in fifty-four (33.5%) patients (HLA class I only, n = 19; class II only, n = 29; both class I and II, n = 6). Of them, complement binding activities were detected in 26 (48.1%) patients, including 17 C1q+ and 24 C3d+ patients. Both C1q and C3d positivity were associated with increased mean fluorescence intensity values of dnDSA. Complement binding activity of dnDSA enhanced the incidence of AMR (25.0% in C1q-C3d-, 36.4% in C1q+/C3d- or C1q-/C3d+, and 60.0% in C1q+/C3d+ patients) (P <0.001). The incidence of AMR was not different between patients with C1q+ and those with C3d+ dnDSA (64.7%, 11/17 versus 45.8%, 11/24, P = 0.238). In comparison between C1q and C3d assay according to HLA specificity, C1q+ HLA class I ± II dnDSA was the best predictor for AMR (odds ratio: 27.2). C1q+/C3d+ dnDSA was associated with more C4d deposition in allograft tissue and inferior post-biopsy graft survival. Clinical outcomes were not significantly different between C1q+ and C3d+ dnDSA-positive patients.ConclusionDetection of complement binding activity using both C1q and C3d assays can be a further prognostic marker for predicting AMR and allograft outcome in dnDSA+ kidney transplant patients.

Noninvasive quantification of intrarenal allograft C4d deposition with targeted ultrasound imaging.

Antibody-mediated rejection (AMR) has emerged as a major cause of renal allograft dysfunction. C4d, a specific marker for AMR diagnosis, was strongly recommended for routine surveillance; however, currently, C4d detection is dependent upon tissue biopsy, which is invasive and provides only local semi-quantitative data. Targeted ultrasound imaging has been used extensively for noninvasive and real-time molecular detection with advantages of high specificity and sensitivity. In this study, we designed C4d-targeted microbubbles (MBC4d ) using a streptavidin-biotin conjugated method and detected C4d deposition in vivo in a rat model of AMR by enhanced ultrasound imaging. This noninvasive procedure allowed successful acquisition of the first qualitative image of C4d deposition in a wide renal allograft section, which reflected real-time C4d distribution in grafts. Moreover, we introduced normal intensity difference for quantitative analysis, which exhibited a nearly linear correlation with the grade of C4d deposition according to pathologic analysis. In addition, this approach showed no influence on survival rates and pathologic features in the microbubble injection groups, thereby demonstrating its safety. These findings demonstrated a simple, noninvasive, quantitative, and safe evaluation method for C4d, with the utility of this approach potentially preventing patients from having to undergo an invasive biopsy.

Do C1q-Positive Donor Specific Antibodies have Prognostic Effect in Patients with Resistant Antibody-Mediated Rejection Treated with Bortezomib? Single-Centre Experience

Introduction and Aims Donor-specific antibodies (DSAs) examination is a crucial part of antibody-mediated rejection (AMR) diagnosis. C1q-positive (+) DSAs are associated with poor graft survival. We analyzed results of patients (pts) treated for acute resistant AMR by a bortezomib-based regimen with retrospectively performed C1q assay. The aim of this work was to analyze the treatment effect on DSA levels and potential effect of C1q+ DSAs on graft survival. Methods We retrospectively analyzed documentation of 772 pts who underwent renal transplantation (Tx) between 1/2012-6/2015. Novel therapeutic approach to resistant acute AMR in kidney transplant recipients was applied in 23 pts (3%) based on administration of bortezomib (B) [1 cycle of 4 doses of B (1.3 mg/m2)], small doses of i.v. steroids, plasmapheresis (PP) and a dose of rituximab (375mg/m2). This protocol was administered after conventional treatment had failed. Resistant AMR was defined as persisting deterioration or non-function of renal allograft in pts with histological verification of AMR, positive C4d staining and detection of DSA receiving standard antirejection treatment with PP + IVIG. C1q assay was performed 3 times – before Tx, at the time of diagnosis and after AMR treatment. Pts were followed for minimum of 24 months. Results Therapy of resistant acute AMR was administered to 23 pts after kidney Tx with median peak PRA 52%, actual PRA 36%, mean HLA mismatch in HLA-A 1.2 ± 0.4, HLA-B 1.7 ± 0.5, HLA-DR 1.3 ± 0, with median of 5.8 years on dialysis. 3 pts underwent 1st kidney Tx, while 20 pts reTx. All pts received induction therapy with antithymocyte globulin (n=22) or basiliximab (n=1), and maintenance immunosuppression with tacrolimus, mycophenolate mofetil/enteric-coated mycophenolate sodium and corticosteroids. Diagnosis of resistant acute AMR was made on 14th POD (7- 60 days). Using bortezomib regimen in treating resistant acute AMR led to decrease in DSA quantity in HLA especially in class I (p=0.005), class II (p = 0.015), but not in DQ (p= 0.2). No significant improvement of renal function was observed during the follow-up. The pts whose levels of serum creatinine increased more than 25% of baseline level in 6 months after administration of protocol with B, are progressors (n=11). The progressors graft survival was 57% in 2 years. The treatment protocol was not effective in decreasing C1q+ DSAs class I (p=0.25), class II (p=0.69), DQ (p=0.58). We detected C1q+ in 11 out of 11 progressors, compared with 3 out of 12 non-progressors (p<0.001). C1q+ DSAs correlated with C4d+ in graft biopsies at the time of AMR diagnosis (p=0.0012). Conclusions Bortezomib-based regimen was effective against class I and II DSAs, but it did not affect DQ DSAs. Pts with any C1q+ DSAs were more likely to have progressive graft dysfunction compared to those with C1q-negative DSAs. C1q positivity is a promising prognostic marker in pts with resistant AMR.

Application of C4d Immunohistochemistry on Routinely Processed Tissue Sections for the Diagnosis of Autoimmune Bullous Dermatoses.

The diagnosis of autoimmune bullous dermatoses relies greatly on direct immunofluorescence (DIF) examination performed on frozen tissue sections, showing deposits of immunoglobulins and/or C3 on specific cutaneous structures. However, frozen material is not always available for DIF; therefore, alternative techniques are needed in the diagnostic procedure. We therefore tested the usefulness of C4d immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections for the diagnosis of bullous pemphigoid (BP) and pemphigus (P). A retrospective immunohistochemical study was performed on biopsies of BP (n: 29) and P (n: 22, including 19 Pemphigus vulgaris and 3 paraneoplastic), submitted for routine histological examination and compared with DIF on the corresponding frozen sections. Twenty-five BP biopsies (86.2%) showed C4d deposits seen as a linear labeling along the dermal-epidermal junction and on the membrane of basal keratinocytes. Seventeen P biopsies (77.2%) showed C4d deposits in a classical "intercellular" pattern, predominating on the lower epidermal layers. The sensitivity, specificity, positive predictive value, and negative predictive value reached 86%, 98%, 96%, and 92% in BP, respectively and 77%, 98%, 94%, and 92% in P, respectively. Furthermore, in the cases where serological tests were available, the sensitivity of C4d detection was higher than that of enzyme-linked immunosorbent assay/indirect immunofluorescence in both BP (87% vs. 67%) and P (82% vs. 54.5%). We conclude that DIF on frozen sections still remains the gold standard for the immunopathological diagnosis of BP and P; however, in the absence of frozen material, C4d immunohistochemistry performed on routinely processed biopsy material can be of considerable help in confirming the diagnosis.

Deciphering complement interference in anti-human leukocyte antigen antibody detection with flow beads assays.

Anti-human leukocyte antigen (HLA) antibody detection in solid-phase flow beads assays can be quenched by complement activation, but the precise mechanism of this interference is not fully elucidated yet. Using the Luminex flow beads screening assay for detection of anti-HLA antibodies, we analyzed the binding of high concentrations of the pan class I anti-HLA monoclonal antibody W6/32 in neat normal, ethylenediaminetetraacetic acid-treated normal and complement factors C1q, C4/C3, C2, C3, factor B or C5-depleted human sera, using anti-mouse immunoglobulin G as the detection antibody. Complement activation and binding to beads were revealed using anti-human C1q, C4d, and C3d antibodies. To translate our findings to the human setting, we used the class I and class II HLA single-antigen flow beads assays and sera from four patients with high titers of antibodies. Detection of W6/32 did not suffer any interference with C1q and C4/C3-depleted sera. A partial quenching was observed with C2, C3, and factor B-depleted sera, but was more pronounced with the factor B-depleted serum. W6/32 was undetectable in presence of C5-depleted serum. The binding of activation products derived from C3 principally, and also from C4, impaired immunoglobulin G and C1q detection. Accordingly, C4d detection was hindered by deposition of activated C3. Similar findings were obtained with patients' sera. Binding of C4 and C3 activation products is the main responsible for complement interference in flow beads assays. A complete quenching requires complement activation through C3 cleavage and its amplification by the alternative pathway.

Mass Spectrometric and Contactless Conductivity Detection Approaches in the Determination of Muscle Relaxants by Capillary Electrophoresis

Novel and rapid capillary electrophoresis-coupled tandem mass spectrometry (CE-MS/MS) and capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) methods have been developed for the separation and determination of three neuromuscular blocking agents: pancuronium, vecuronium, and rocuronium. In both cases, the separation was conducted in background electrolytes based on acidic acetate-ammonium buffers to avoid possible decomposition of the analytes that are known to be unstable in alkaline media. Baseline resolution of the analytes was achieved in the presence of modified γ-cyclodextrin by CE with C4D detection. The two detection techniques were compared with regard to analytical figures of merit including linear dynamic range, limit of detection, limit of quantification, precision, and accuracy. The calibration curves showed good linearity for both detection methods examined (characterized by r2 ≥ 0.9908). The LODs of the CE-MS/MS and the CE-C4D methods differed at least by two orders of magnitude considering all analytes. The differences in precision and accuracy of these methods were evaluated and discussed. The assays of pancuronium, vecuronium, and rocuronium in commercial injection solutions by CE-MS/MS and CE-C4D were performed and the results compared.

Rapid and sensitive analysis of three polyphenols in tobacco by CE using homemade C4D with a mini detection cell

A rapid, sensitive, and practical CE with C(4) D detection was developed for the analysis of three polyphenols (rutin, scopoletin, and chlorogenic acid) in tobacco samples. The constructed mini detection cell (12 mm × 10 mm × 10 mm) of C(4) D featured with small inner cell volume (∼2 nL), smaller noise (<0.9 mV), repeatability, high strength and durableness. Three polyphenols were ultrasonically extracted with methanol-water (70:30, v/v) solution following SPE cleanup. The CE method was optimized with the running buffer of 150 mmol L(-1) 2-amino-2-methyl-1-propanol (pH 11.2), and the applied separation voltage of +20 kV over a capillary of 50 μm id × 375 μm od × 50 cm (38 cm to the C(4) D window, 41.5 cm to the UV detector window), which gave a baseline separation of three polyphenols within ca. 6 min. The method provided the limits of quantification (S/N = 10) at about 0.08-0.15 μg g(-1) for three polyphenols, whereas the overall recoveries ranged from 82% to 88%. The proposed method has been successfully applied to measure three polyphenols in the actual tobacco samples, and their contents were calculated and evaluated.

Separation of selected transition metals by capillary chelation ion chromatography using acetyl-iminodiacetic acid modified capillary polymer monoliths

Capillary housed laurylmethacrylate-co-ethylene dimethacrylate (LMA-co-EDMA) polymer monoliths were fabricated, functionalised with varying amounts of vinyl azlactone, followed by immobilisation of iminodiacetic acid (IDA), forming a range of acetyl-iminodiacetic acid (AIDA) functionalised monoliths, applied to the chelation ion chromatographic separation of selected transition and heavy metals. A number of monoliths of varying length and ligand density were prepared, resulting in increased cation retention and chromatographic resolution on those displaying the highest capacity. Ligand density and related column capacity were confirmed visually using scanning capacitively coupled contactless conductivity detection (sC4D) techniques. Column temperature studies to determine retention mechanism and the effect of temperature on the retention of Mn(II), Cd(II) and Cu(II) was investigated, showing an increase in retention with increased temperature for Cd(II) and Cu(II), whilst a decrease in retention was obtained for Mn(II). Isocratic capillary chelation ion chromatographic separations of Mn(II), Cd(II) and Cu(II) were obtained, with dual peak detection demonstrated using combined on-column C4D detection and UV–Visible detection following the post-capillary column reaction of the eluted metals with 4-(2-pyridylazo) resorcinol (PAR).