A case of atypical IgA paraprotein interference on multiple chemistry assays: How to deal with it.

This case report discusses how paraproteins interfere with multiple chemistry analyses and protocols to overcome such obstacles. A serum specimen containing two monoclonal IgA (llight chain) paraproteins is subjected to a battery of tests on three wet chemistry platforms - AU5800, Cobas Pure, and Alinityci; the results were compared with those on a Vitros 350/ ECiQ dry chemistry platform. Paraprotein interference was found to affect the bilirubins, inorganic phosphate, and iron, whose repeat runs were also found to be irreproducible. Dilution with normal saline also failed to produce a satisfactory effect. Deproteinization by polyethylene glycol and dilution of the specimen with a normal serum specimen were observed to produce desirable results. Interference by IgA paraprotein on measurement of the bilirubin, phosphate, and iron in the wet chemistry system can be mitigated either by deproteinization or by dilution with normal serum.

A simple method to overcome paraproteinemic interferences in chemistry and immunoassays.

BACKGROUND: Interferences on chemistry and immunoassay results due to paraproteinemia may lead to erroneous diagnoses and treatment. Such interferences are difficult to recognize and even more difficult to deal with. This report describes 1 such case where multiple measurands were affected and how the interferant was overcome. CASE REPORT: Paraproteins present in an immunoglobulin (Ig)G-lambda multiple myeloma specimen interfered with results of total bilirubin, direct bilirubin, inorganic phosphate, iron, ferritin, and total thyroxine measured on 3 platforms: AU5800, Alinity ci, and cobas pure. Repeat testing upon dilution with normal saline or deproteinization by polyethylene glycol precipitation gave unsatisfactory results on some or all the affected measurands. Repeat testing after dilution of the interferant serum with a healthy serum corrected the anomalous results for all the affected measurands. CONCLUSION: Dilution of paraproteinemic serum with a healthy serum of known concentrations appears to be the most suitable method to negate the effects of paraproteinemic interferences.

Direct elemental analysis of plant oils by inductively coupled plasma mass spectrometry: Simple sample dilution combined with oxygen introduction into the plasma.

Assessment of trace metal concentrations in plant oils has been considered a crucial quality control marker for potential health risks, oil flavour, and oxidative stability. A straightforward inductively coupled plasma mass spectrometry (ICP-MS) methodology was developed and validated through introduction of argon:oxygen gas mixture into plasma, allowing for a direct elemental analysis of organic matrices. This approach offers the advantage of a simple one-step preparation of plant oil samples with negligible contamination risks. The complete solubilization of the oil matrix enables the determination of total metal content from a single test tube with low dilution factor of 5. The modified plasma conditions resulted in the development of a robust and accurate ICP-MS method providing limits of detection at sub ng·g-1 levels. The ICP-MS method allowed the determination of trace levels of Ba, Cd, Cu, Fe, Mn, Pb, Sn, V, and Zn in olive, sunflower and rapeseed oils.

Critical evaluation of strategies to achieve direct real-time PCR detection of swine pathogens in oral fluids.

Based on publications reporting improvements in real-time PCR (rtPCR) performance, we compared protocols based on heat treatment or dilution followed by direct rtPCR to standard extraction and amplification methods for the detection of porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), porcine epidemic diarrhea virus (PEDV), or Mycoplasma hyopneumoniae (MHP) in swine oral fluids (OFs). In part A, we subjected aliquots of positive OF samples to 1 of 4 protocols: protocol 1: heat (95°C × 30 min) followed by direct rtPCR; protocol 2: heat and cool (25°C × 20 min) followed by direct rtPCR; protocol 3: heat, cool, extraction, and rtPCR; protocol 4 (control): extraction and then rtPCR. In part B, positive OF samples were split into 3, diluted (D1 = 1:2 with Tris-borate-EDTA (TBE); D2 = 1:2 with negative OF; D3 = not diluted), and then tested by rtPCR using the best-performing protocol from part A (protocol 4). In part A, with occasional exceptions, heat treatment resulted in marked reduction in the detection of target and internal sample control (ISC) nucleic acids. In part B, sample dilution with TBE or OF produced no improvement in the detection of targets and ISCs. Thus, standard extraction and amplification methods provided superior detection of PRRSV, IAV, PEDV, and MHP nucleic acids in OFs.

Development of graphitization method for low carbon aerosol filter samples with Automated Graphitization System AGE-3.

The wide applications of the radiocarbon (14C) approach in environmental, archeological, and geological research often necessitates the analysis of microgram-sized samples. The ability to measure low carbon samples is particularly relevant for aerosol particle filters, especially for samples from pristine environments. For this purpose, we investigated the sample dilution method for graphitization of low-carbon samples (20-200 µg C) with an Automated Graphitization System (AGE-3), and applied a mass balance equation for the calculation of 14C values. Materials with known 14C values (standards NIST-OXII and IAEA-C7) were diluted with 14C-free phthalic anhydride (PhA) until sufficient mass (500 µg C) for graphitization with the AGE-3 system was acquired. Reliable 14C values were obtained for samples with carbon amount in the range of 40-200 µg. Next, we adapted the dilution method for estimation of aerosol sample 14C values. Using it, we attained a precision of 0.71 ± 0.83 pMC for 14C measurements of aerosol samples containing 40-200 µg C. A shift of radiocarbon values to 5.07 pMC (average 3.08 ± 1.7 pMC) was observed for samples with low carbon content (<20 µg C). We determined that a precision of 2-3 pMC is acceptable for aerosol particle source apportionment studies. Using the sample dilution method, graphitization with AGE-3 of aerosol samples with carbon content >40 µg becomes a viable and efficient way of sample preparation for 14C analysis.

Importance of Observing the Progress Curve During Enzyme Assay in an Automated Clinical Chemistry Analyzer: A Case Study.

In the case presented here the reported result for total creatine phosphokinase led to the physician calling for report confirmation. The repeated test result was in keeping with the clinical picture and thus the previous erroneous result was amended. The incorrect result from auto analyzer was identified as failure to run the sample in dilution after instrumental flagging of possible substrate exhaustion evidenced by erroneous progress curve. A frequent reason for nonlinear progress curves is the presence of excess enzyme which can be easily misinterpreted as lower enzyme activity in a provided sample. Careful inspection of progress curve and predilution of sample in anticipated cases could avoid erroneous result.

Novel techniques to determine dilution ratios of raw wastewater and wastewater treatment plant effluent in the 5-day biochemical oxygen demand test.

The current 5-day biochemical oxygen demand (BOD5) test methods lack detailed information on sample dilution, which typically leads to an incorrect dilution ratio of samples and failed BOD5 testing. This study proposed an improved dilution technique that effectively determines the dilution ratios for raw wastewater and wastewater treatment plants (WWTPs) effluent. The numbers of raw wastewater and WWTPs effluent samples used in the BOD5 tests were 201 and 61, respectively. The experimental results demonstrated that both the raw wastewater and WWTPs effluent varied in BOD5 values with the sample dilution ratio, and these changes were more noticeable when the dissolved oxygen consumption ratio (DOCR) was less than 20% or when the dilution ratio was large. Assuming that the BOD5 value over the DOCR range of 40%-70% was true, the optimal DOCR range was 40%-90% for the raw wastewater and 40%-70% and 80%-90% for the WWTPs effluent, where the relative error of BOD5 values in these DOCR ranges was less than 10% depending on the dilution ratio. The correlation between the sample dilution ratio and the BOD5 value over the optimal DOCR range was considerably higher than that over the entire DOCR range. This was combined with the correlation equation between BOD5 and chemical oxygen demand to propose an equation that could determine more accurate sample dilution ratios for raw wastewater and WWTPs effluent compared to the conventional sample dilution methods for the BOD5 test.

Evaluation of clean-up procedures and sample dilution in multi-residue pesticide analysis of spices and herbs by UPLC-MS/MS.

The matrix effect in complex spices and herbs presents a great challenge to the simultaneous, rapid, quantitative analysis of multiple pesticides by mass spectrometry. The aim of this work was to develop and validate an effective UPLC-MS/MS multi-residue method for the analysis of pesticide residues to reduce matrix effects in four spices and one herb and infer which component caused the matrix effect in black pepper. In this paper, we highlight the importance of reducing matrix effects. SPE and dilution factors combined with d-SPE were two approaches to reduce matrix effects compared with d-SPE, and dilution (5-fold) combined with d-SPE was more effective than SPE in reducing matrix effects. With the validated d-SPE method combined with dilution (5-fold), 22 pesticides showed satisfactory recovery (77 to 114%) and RSDs (1.4%-19.1%) at three spiked concentrations in dried chilli pepper, star anise, mint, and cinnamon skin, but not in black pepper. Compared to the other three spices and one herb, black pepper appeared to have a stronger matrix effect. D-SPE method combined with dilution (5-fold) was not suitable for black pepper and required more efficient purification methods and higher dilution factors. With the validated SPE method combined with 10-fold dilution, 19 pesticides showed satisfactory recovery (71% to 113%) and RSDs (2.1%-18.2%) at three spiked concentrations in black pepper. We ascertained that piperine was the component contributing most to the matrix effect in black pepper. The method was used to monitor 15 market samples.

Rapid LC-MS/MS Determination of Hesperidin in Fermented Tea Prepared from Unripe Satsuma Mandarin (Citrus unshiu) Fruits and Third-crop Green Tea (Camellia sinensis) Leaves.

For improving quality control in the fermented tea production process and advancing the corresponding food labeling with function claims, a rapid and robust hesperidin analysis method using LC-MS/MS with the sample dilution approach was developed by following internationally accepted criteria of the Association of Official Analytical Chemists (AOAC). The linear correlation coefficient (r2) of the regression line was 0.9997 in the concentration range of 0.025 - 2.5 mg/L. The matrix effect evaluated using regression line slope values was negligible. The recovery rate of 100.7% indicated improved trueness. The performance of the newly developed method in determining the hesperidin content of fermented tea samples did not significantly vary from that of a well-established, conventional method. The HorRat values of intra- and inter-laboratory reproducibility studies were both within the acceptable range, indicating sufficient accuracy of the newly developed method according to the AOAC criteria.

Simultaneous multielement analysis by ICP-MS with simple whole blood sample dilution and its application to uremic patients undergoing long-term hemodialysis.

Few studies were reported on trace elements' alterations in uremic patients undergoing long-term (>24 months) hemodialysis (HD), especially by using the whole blood as the biological fluid for the measuring purpose. Our objective was to develop an improved micro-sampling inductively coupled plasma-mass spectrometry (ICP-MS) method to determine the levels of Ca, Mg, Cu, Zn, Fe, Mn, Se and Pb in uremic patients receiving long-term HD. A ICP-MS method with a modified whole blood sample preparation procedure with small volumes was established and applied for the simultaneous quantification of the various elements in uremic patients undergoing long-term HD. 124 eligible uremic patients receiving long-term HD (75 males and 49 females) and 77 healthy subjects (54 males and 23 females) were recruited and Ca, Mg, Cu, Zn, Fe, Mn, Se and Pb levels were further determined. Our results revealed that uremic patients with HD had significantly higher blood levels of Ca, Mg, Zn and Pb and lower Cu, Fe, Se and Mn concentrations than healthy controls. In conclusion, a reproducible and reliable ICP-MS method using minimal whole blood sample volume (50 µL) with a simple dilution-based preparation procedure was successfully improved, validated and applied. Uremic patients undergoing long-term HD might be at increased risk of some essential trace elements deficiency (especially for Cu, Fe and Se) or toxic trace element excess (Pb) in respect to healthy subjects. Monitoring of blood levels and supplementation of some trace elements may be indicated in uremic patients undergoing long-term HD.

Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system.

Protein purification processes in basic research using ÄKTA™ liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS™ autosampler and a modular sample in-line dilution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion. The chromatographic setup enabled purification of a large array of cytosolic and membrane proteins from small-scale expression cultures produced in insect cells necessary to develop and optimize isotope-labeling strategies for nuclear magnetic resonance spectroscopy applications, resulting in a reduction in experiment time of about 20% per run for both cytosolic and membrane protein purification schemes. However, when queuing multiple samples the throughput increased by 66% and 75%, respectively. In addition, a novel system configuration is presented, where two column valves can be operated independently. This allows for the design of purification loops to increase purity of the target protein.

Mismatch between sample diluent and eluent: Maintaining integrity of gradient peaks using in silico approaches.

The mismatch of elution strength between the sample diluent and the eluent causes undesirable peak deformations for large sample volumes in gradient liquid chromatography. The solution to that problem consists in diluting the sample solution in a weak solvent. But the minimum dilution factor has to be determined by the user given some performance objectives. In silico approaches are applied in this work to find such adequate dilution factors. Two calculations methods are proposed for the prediction of peak distortions. The first comprehensive method is based on solving numerically the mass balance equations for all the analytes and the strong solvent. An excellent agreement between the experimental and the calculated gradient chromatograms is observed (sample diluent: acetonitrile/water, 50/50, v/v; injection volume: 15 µL; linear gradient: 5%-95% acetonitrile during 3 min) for five compounds (acetanilide, coumarin, benzoin, bi-naphthol, and dibutylphthalate) injected into a 2.1 × 50 mm column packed with 1.7 µm XBridge-C18 particles. This first method happens to be highly time-consuming and impractical for common users. Experimental work and calculation times are then minimized by applying a second method based on the basics of retention and dispersion of injected pulses. Despite being less accurate than the first method, the agreement between the experimental and calculated peak width remains physically meaningful allowing the experimenter to rapidly guess the required sample dilution factor for any combination of injected volume and strong solvent concentration in the sample solution.

High-throughput gas chromatography-mass spectrometry analysis of pesticide residues in spices by using the enhanced matrix removal-lipid and the sample dilution approach.

Spices are known as difficult matrices that contain variable amounts of fats, piperine or many other matrix constituents. The increasing sensitivity of new GC-MS/MS platforms opens new approaches to analyze pesticide residue in complex matrices such as spices, by sample dilution. The aim of this work is to develop and validate an effective multiresidue method for the analysis of pesticide residues in spices by GC-MS/MS. In this paper, we highlight the importance of reducing matrix interferences generated from co-extractive components of spices. Moreover, we emphasize the concern of obtaining clean extracts requiring less instrument maintenance. By evaluating the total ion chromatograms (TIC) on GC-Orbitrap-MS of different extracts using various sorbents, QuEChERS citrate using EMR-Lipid sorbent resulted in the cleanest extract among Z-Sep, Primary secondary amine (PSA), Oasis® Prime HLB, and Supelclean™ Ultra cartridges that consist of a top bed of PSA, C18 and Grashsphere™ 2031 and a bottom bed of Z-Sep. Later, the analyses were performed on a GC-QQQ-MS/MS, applying a 15 min runtime method covering 205 compounds. The samples were diluted 25 times before the injection bearing in mind that the instrumental LOQs reached (iLOQ) were 2 ng g-1 and the method LOQs (mLOQ) were 50 ng g-1. Good recoveries between 70 and 120% with RSDs lower than 20% were observed for 90% of the compounds in black pepper and for 83% of the compounds in cayenne pepper. To demonstrate the applicability of the proposed method, 50 real dried and non-dried spice samples were analyzed. The most detected pesticides were metalaxyl, chlorpyrifos, tebuconazole, ethion, and chinomethionate.

Multi-function microfluidic platform for sensor integration.

The limited availability of metabolite-specific sensors for continuous sampling and monitoring is one of the main bottlenecks contributing to failures in bioprocess development. Furthermore, only a limited number of approaches exist to connect currently available measurement systems with high throughput reactor units. This is especially relevant in the biocatalyst screening and characterization stage of process development. In this work, a strategy for sensor integration in microfluidic platforms is demonstrated, to address the need for rapid, cost-effective and high-throughput screening in bioprocesses. This platform is compatible with different sensor formats by enabling their replacement and was built in order to be highly flexible and thus suitable for a wide range of applications. Moreover, this re-usable platform can easily be connected to analytical equipment, such as HPLC, laboratory scale reactors or other microfluidic chips through the use of standardized fittings. In addition, the developed platform includes a two-sensor system interspersed with a mixing channel, which allows the detection of samples that might be outside the first sensor's range of detection, through dilution of the sample solution up to 10 times. In order to highlight the features of the proposed platform, inline monitoring of glucose levels is presented and discussed. Glucose was chosen due to its importance in biotechnology as a relevant substrate. The platform demonstrated continuous measurement of substrate solutions for up to 12 h. Furthermore, the influence of the fluid velocity on substrate diffusion was observed, indicating the need for in-flow calibration to achieve a good quantitative output.

Reducing blood volume requirements for clinical pathology testing in toxicologic studies-points to consider.

In preclinical safety assessment, blood volume requirements for various endpoints pose a major challenge. The goal of this working group was to review current practices for clinical pathology (CP) testing in preclinical toxicologic studies, and to discuss advantages and disadvantages of methods for reducing blood volume requirements. An industry-wide survey was conducted to gather information on CP instrumentation and blood collection practices for hematology, clinical biochemistry, and coagulation evaluation in laboratory animals involved in preclinical studies. Based on the survey results and collective experience of the authors, the working group proposes the following "points to consider" for CP testing: (1) For most commercial analyzers, 0.5 mL and 0.8 mL of whole blood are sufficient for hematology and biochemistry evaluation, respectively. (2) Small analyzers with low volume requirements and low throughput have limited utility in preclinical studies. (3) Sample pooling or dilution is inappropriate for many CP methods. (4) Appropriate collection sites should be determined based on blood volume requirements and technical expertise. (5) Microsampling does not provide sufficient volume given current analyzer and quality assurance requirements. (6) Study design considerations include: the use of older/larger animals (rodents), collection of CP samples before toxicokinetic samples, use of separate subsets of mice for hematology and clinical biochemistry testing, use of a priority list for clinical biochemistry, and when possible, eliminating coagulation testing.

Is dilution important: Factitious Total Creatine Kinase in case of Rhabdomyolysis?

Factitious test reports may result in incorrect diagnosis and incorrect management. Such incorrect diagnosis can be prevented by a vigilant biochemist. We report a case of Rhabdomyolysis presenting with extremely low total Creatine Kinase (CK) levels which was factitious. Running the sample in dilution resulted in a very high value of total CK which could have been missed if the sample was not run in dilution and the diagnosis of Rhabdomyolysis could have been missed.

Impact of asymmetrical flow field-flow fractionation on protein aggregates stability.

The impact of asymmetrical flow field-flow fractionation (AF4) on protein aggregate species is investigated with the aid of multiangle light scattering (MALS) and dynamic light scattering (DLS). The experimental parameters probed in this study include aggregate stability in different carrier liquids, shear stress (related to sample injection), sample concentration (during AF4 focusing), and sample dilution (during separation). Two anti-streptavidin (anti-SA) IgG1 samples composed of low and high molar mass (M) aggregates are subjected to different AF4 conditions. Aggregates suspended and separated in phosphate buffer are observed to dissociate almost entirely to monomer. However, aggregates in citric acid buffer are partially stable with dissociation to 25% and 5% monomer for the low and high M samples, respectively. These results demonstrate that different carrier liquids change the aggregate stability and low M aggregates can behave differently than their larger counterparts. Increasing the duration of the AF4 focusing step showed no significant changes in the percent monomer, percent aggregates, or the average Ms in either sample. Syringe-induced shear related to sample injection resulted in an increase in hydrodynamic diameter (dh) as measured by batch mode DLS. Finally, calculations showed that dilution during AF4 separation is significantly lower than in size exclusion chromatography with dilution occurring mainly at the AF4 channel outlet and not during the separation. This has important ramifications when analyzing aggregates that rapidly dissociate (<∼2s) upon dilution as the size calculated by AF4 theory may be more accurate than that measured by online DLS. Experimentally, the dhs determined by online DLS generally agreed with AF4 theory except for the more well retained larger aggregates for which DLS showed smaller sizes. These results highlight the importance of using AF4 retention theory to understand the impacts of dilution on analytes.

Improved liquid-liquid extraction with inter-well volume replacement dilution workflow and its application to quantify BMS-927711 in rat dried blood spots by UHPLC-MS/MS.

An UHPLC-MS/MS method was developed and validated to quantify BMS-927711, a drug candidate to treat migraine, in rat dried blood spots (DBS). The DBS samples were extracted using an improved liquid-liquid extraction (LLE) strategy involving in the sonication of DBS punches in 20% MeOH aqueous solution containing the internal standard, [(13)C2, D4]-BMS-927711, and then with a 100mM NH4OAc buffer solution, followed by an automated LLE with EtOAc-hexane (70:30, v/v). The presence of 20% MeOH as an organic modifier in the elution solution significantly improved the analyte elution efficiency and assay performance. A novel inter-well volume replacement dilution workflow was introduced for DBS sample dilution before LLE step. This was a simple two-step process, firstly a small portion of the DBS blank solution was discarded, and then the same volume of a concentrated DBS sample solution was spiked into the leftover blank solution to achieve a desired dilution. Chromatographic separation was achieved on an Acuity UPLC(®) BEH C18 column (2.1mm×50mm, 1.7µm) and the analyte was detected by selected reaction monitoring (SRM) with positive electrospray ionization on an AB Sciex Triple Quad 5500 mass spectrometer. The standard curve was linear from 5.00 to 5000ng/mL with assay precision ≤4.9% CV, and assay accuracy within ±3.1%Dev of the nominal values. Accurate sample dilution was achieved by using inter-well volume replacement with a precision of ≤4.2% CV and an accuracy of ±3.3% for dilution QC at 50,000ng/mL with 100-fold dilution (n=18). This robust UHPLC-MS/MS assay has been successfully applied to the non-clinical studies in rats. By using inter-well volume replacement workflow, accurate dilution was demonstrated using only one DBS blank sample for a typical dilution of <50-fold, and using only two blank DBS samples for a dilution of up to 625-fold. Moreover, this new workflow makes it easier to automate DBS sample dilution.

Evaluation of matrix effects in the analysis of volatile organic compounds in whole blood with solid-phase microextraction.

The complexity and matrix variability of biological samples requires an accurate evaluation of matrix effects. The dilution of the biological sample is the simplest way to reduce or avoid the matrix effect. In the present study, a set of volatile organic compounds with different volatilities was used to assess the effect of the dilution of blood samples on the extraction efficiency by headspace solid-phase microextraction. It was found that there was a significant matrix effect but that this effect differs significantly depending on the volatility of the compound. A 1:2 (blood/water) dilution was enough to allow quantitative recoveries of those compounds with boiling points <100°C. For compounds with boiling points between 100 and 150°C, the matrix effect was stronger and a 1:5 dilution was required. The dilution of blood samples proved to be inefficient for quantitative recovery of compounds with boiling points >150°C. A 1:5 dilution of the sample allowed detection limits in the range of nanogram per liter to be obtained. This was sufficient to detect the main volatile compounds present in blood and contamination after exposure.