Sample Dilution Research Articles

Abstract A014: A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction

Abstract Liquid biopsy continues to be a focus of the cancer diagnostics industry, primarily due to the non-invasive nature of sample collection, however the low levels of circulating tumor DNA (ctDNA) remain a challenge. Methylation has emerged as a promising biomarker for detecting ctDNA, yet detecting the cancerous signal in an excess of non-cancerous, unmethylated cfDNA in the background has been proven difficult. Harbinger Health has developed a novel method that depletes unmethylated background at the CpG-level from specific genomic regions of interest, resulting in an enrichment of the ctDNA fraction. We developed a background cfDNA depletion method that utilizes the endonuclease activity and the specificity of CRISPR/Cas12a to remove unmethylated DNA fragments at ctDNA methylation biomarker sites. First, we identified cancer-informative regions that showed consistent contiguous elevated methylation states in cancer compared to normal samples. We then designed guide RNAs that specifically bind and cut unmethylated CpG sites within the target regions. Cas depletion of bisulfite-converted DNA enriches cancer informative signal for bioanalysis. To test validity of our Cas complex designs, we created model samples that mimic fully unmethylated and fully methylated species. We spiked these cfDNA models into a background of genomic DNA from 5% to 0.01%. In triplicate, we examined cutting efficiency, multiplexing and specificity of Cas to unmethylated species of model DNA. Results were assessed using qPCR by comparing differences in Ct with and without Cas treatment. Additionally, we applied this depletion step during our library preparation process to show utility in methylation analysis of low tumor content liquid biopsy samples. Using both the model sample dilution series and bisulfite converted cfDNA samples, which represent a range of cancerous and background methylation signal, we deplete unmethylated targets using Cas prior to indexing PCR. This rendered unmethylated targets non-amplifiable, removing them from analysis. Results were assessed using both qPCR and sequencing data, such as methylated read counts over the target region. We demonstrated our analytical approach efficiently depletes non-cancer cfDNA by on average 9-fold, which increases the fraction of ctDNA signal and can be multiplexed. This background signal depletion driven by CRISPR is sensitive and specific, maintaining methylated DNA signal detection down to 0.01% at inputs of 10ng DNA. Our Cas-mediated depletion of background signal method has proven useful for enriching rare methylated fragments over background by improving the signal to noise ratio. This technique is novel by harnessing the specificity of CRISPR to target DNA in both a sequence and methylation state specific manner. The depletion of precise methylation states at the CpG-level make it suitable for use in multiple bioassays. Citation Format: Caitlin Gilley, Miguel Williams, Emily Neaga, Sarah Falotico, Kade Pettie, Anthony Shuber. A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A014.

UHPLC-QTOFMS Urine Drug Screening With Dilute-and-Shoot Sample Preparation and Vacuum-Insulated Probe-Heated Electrospray Ionization.

We developed a method for comprehensive urine drug screening by applying dilute-and-shoot extraction and vacuum-insulated probe-heated electrospray ionization with ultra-high performance liquid chromatography high-resolution quadrupole time-of-flight mass spectrometry (DS-UHPLC-VIP-HESI-QTOFMS). The method involved five-fold post-hydrolysis dilution of urine samples and chromatography on a C18 UHPLC column prior to QTOFMS analysis. The recently introduced VIP-HESI ion source was chosen due to its enhanced ionization efficiency and compatibility with UHPLC-QTOFMS. Extensive data was acquired in positive ion mode with a low collision energy (7 eV) and an elevated collision energy (30 eV), using the broadband collision-induced dissociation data acquisition scan mode that continuously generated high-resolution and accurate mass for parent and fragment qualifier ions, and parent ion isotopic patterns. Compound identification was performed against an in-house database with 1263 compound entries, using an automated post-run reverse target database search with preset identification criteria. Method validation with 56 different drugs showed acceptable results for the limit of identification (median 5 ng/mL), matrix effects (70-130%), repeatability of retention times (< 1%), mass accuracy (< 1 mDa), as well as for specificity and stability. As compared with an established UHPLC-QTOFMS method relying on solid-phase extraction and conventional electrospray ionization, DS-UHPLC-VIP-HESI-QTOFMS produced comparable results from authentic clinical urine samples for most drugs, but showed clearly improved detectability for pregabalin, gabapentin, and ritalinic acid. We anticipate that the new method will be a step forward for laboratories performing routine urine drug screening due to its fast turnaround time, reduced manual workload, cost efficiency, and broad substance coverage.

Analytical performance of a point-of-care CBC hematology analyzer, including a 5-part differential: A prospective study to evaluate a microfluidic flow cytometry-based analyzer in waived settings.

A microfluidic flow cytometer-based point-of-care (POC) analyzer was validated against an in-laboratory hematology analyzer (Sysmex XN Automated Hematology System). Concordance on a full complete blood cell count (CBC) with 5-part differential, as performed by operators with no prior clinical laboratory experience, was evaluated. We prospectively collected 376 venous blood specimens (376) from individuals with self-reported medical conditions and from apparently healthy individuals. Forty-six additional remnant specimens were acquired to ensure coverage of analytic measuring ranges. Parallel testing was performed, with up to 7 hours between testing on the POC and Sysmex XN analyzers. Regression analysis resulted in r values of 0.998 to 0.932 for all parameters of a 5-part differential CBC other than basophils (0.709). The mean percentage bias from the reference method, inclusive of the upper and lower reporting limits, was less than 2% for parameters other than lymphocytes (-6.4%), monocytes (25.9%), eosinophils (12.2%), and basophils (-15%). Overall agreement on abnormal flagging was 93.3%. The Cito CBC microflow cytometer (CytoChip Inc) provides a CBC with a 5-part differential with accuracy, precision, and abnormal flagging equivalent to a moderate-complexity hematology analyzer. It has the key features required of a POC device that can be operated in a waived setting: minimum space requirements, rapid results, single-action measurement (no sample processing or dilution), ease of use, and minimal blood volume.

The Efficacy of Andexanet Alfa for the Reversal of Factor Xa Inhibitors Is Not Influenced by Hemodilution with Different Volume Expanders

Background: Andexanet alfa is a specific antidote for factor Xa (FXa) inhibitors. It is licensed to treat patients under FXa inhibitor therapy with life-threatening bleeding. Concomitantly, volume expanders are used to compensate for blood loss and maintain circulation. The competitive binding of andexanet to FXa inhibitors may be disrupted due to hemodilution, as shown by laboratory assays with high sample dilution. This study investigated the efficacy of andexanet for the reversal of FXa inhibitors under hemodilution. Methods: Blood from 10 healthy volunteers was anticoagulated with rivaroxaban and subsequently treated with four different volume expanders (Ringer’s solution, 4% gelatine, 5% and 20% human albumin (HA)) at two dilution levels (20% and 50%). After anticoagulation and hemodilution, andexanet was added according to the high-dose protocol. Blood samples were analyzed using a Russell’s viper venom (RVV) test on a Clot Pro® device, a thrombin generation assay, a fully automated coagulation analyzer and a chromogenic anti-FXa activity assay. Results: After anticoagulation, the median rivaroxaban concentration was 272 ng/mL (IQR 254–353). Anticoagulation with rivaroxaban caused a significant impairment of all coagulation parameters, which was further aggravated by hemodilution. After the administration of andexanet, coagulation parameters in anticoagulated samples were reversed to near baseline in all groups. Andexanet administration decreased the rivaroxaban plasma concentration in all groups to a median of &lt;10 ng/mL. In the anticoagulated, non-hemodiluted samples, anti-FXa activity was reduced by 98%. The anti-FXa activity in the anticoagulated, hemodiluted samples was reduced by approximately 96% in the 20% diluted samples and by about 93% in the 50% diluted samples. Conclusions: Our data indicate that FXa inhibitor reversal with andexanet is about 5% less effective with 50% hemodilution than in non-hemodiluted samples.

Kinetic Bacterial Endotoxin Testing Methods: Specifics of Analytical Conditions Exemplified by Medicinal Products from Different Classes

INTRODUCTION. Kinetic bacterial endotoxin (BE) testing methods based on amoebocyte lysate from the haemolymph of the horseshoe crab can quantify BEs in parenteral medicinal products across a broad range of concentrations. To develop a specific analytical procedure that is based on these methods and can be implemented into pharmaceutical quality control and standardisation, it is required to ascertain the conditions for its use, in particular, to determine the lowest product dilution that would neutralise interfering factors.AIM. This study aimed to develop an analytical procedure for the quantitative determination of BEs by the kinetic chromogenic method.MATERIALS AND METHODS. The study examined samples of medicinal products containing active substances from different classes, including a monoclonal antibody (mepolizumab) and a direct-acting anticoagulant (nadroparin). The BE limits for mepolizumab and nadroparin were ≤20 EU/mL and ≤95 EU/mL, respectively. The samples were tested at different dilutions, which did not exceed the maximum valid dilution. The measurements were performed using a BioТek EL×800 photocolourimeter and EndoScan-V software.RESULTS. The results for mepolizumab samples show that the kinetic chromogenic method can quantify BEs in mepolizumab medicinal products in the dilution range of 1:10 to 1:40. The study detected BEs in quantities of 0.0078–0.224 EU/mL within this dilution range. However, at a dilution ratio of 1:50 or higher, the method is suitable only for qualitative BE tests. For nadroparin medicinal products, the quantitative determination of BEs should start at a dilution of 1:1000 (the study demonstrated no influence of interfering factors at this dilution).CONCLUSIONS. The authors developed analytical procedures for the determination of BEs in mepolizumab and nadroparin medicinal products by the kinetic chromogenic method (method D). When developing analytical procedures for the quantification of BEs in individual medicinal products, the developer should determine the lowest sample dilution that would neutralise interfering factors. Subsequently, the dilution should be specified in the documentation for the analytical procedure. This approach can enhance assay accuracy and save time and resources in routine testing.

New latex agglutination assay for the determination of lactoferrin in human milk

BackgroundLactoferrin (LF) in human milk has various biological properties and contributes to the prevention of preterm birth complications. Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used methods to measure LF in human milk, but this method is time-consuming and laborious. In Japanese human milk banks, the concentration of LF in donor human milk (DHM) is measured routinely. Here, we reported a rapid, simple, and accurate method for determining LF in human milk using a new reagent based on a latex agglutination assay.MethodsWe obtained 208 human milk pools from 148 mothers, and samples were collected before and after Holder pasteurization. Milk samples were diluted 100- or 200-fold and LF concentrations were measured by a latex agglutination assay using an automated analyzer. The reagent was validated in terms of repeatability, linearity, detection limit, recovery, and comparison with ELISA.ResultsThe coefficient of variation (CV) for intra-assay precision ranged from 0.6 to 5.0% in human milk with high, medium, and low LF concentrations. The linearity was also tested by serial sample dilution and was confirmed up to 16 µg/mL with a detection limit of 0.2 µg/mL. The recovery rates in a spiked recovery test were ranged from 90 to 120% at high, medium, and low concentrations of LF. Furthermore, a strong correlation was observed between LF levels determined by the latex agglutination assay and ELISA (r = 0.978, p < 0.001, n = 255). The regression equation was y = 0.991x + 0.545 (r2 = 0.974, p < 0.001). Compared with ELISA, the latex agglutination assay reduces the measurement time by 160 min and the cost by 55%.ConclusionsThe latex agglutination assay used to determine LF in human milk is rapid, simple, and accurate enough to be used routinely. Its use may contribute to the quick and easy provision of appropriate DHM to preterm infants.

Determining diagnostic sensitivity loss limits for sample pooling in duplex rtPCR surveillance testing: Theileria orientalis and Anaplasma marginale.

To expand surveillance testing capacity through sample pooling, a thorough understanding is needed of how sample dilution through pooling affects the sensitivity of candidate assays. We validated a robust and representative framework for assessing the dilution effect of sample pooling using duplex rtPCR surveillance of Theileria orientalis and Anaplasma marginale, both of which are causative agents of severe anemia in cattle and a serious threat to the cattle industry in Virginia and many other states. We used 200 known-positive samples with Ct values representative of typical surveillance results in a series of pools in which we re-tested each sample individually, followed by each sample diluted in equal volumes with negative samples to make pools of 2, 4, 6, 8, and 10 total samples. We compared the Ct values of the individual positives with the Ct values of each pool size to determine if Ct values increase past the limit of detection in the 45-cycle assay. We observed a maximum of 2% sensitivity loss (no more than 2 of 100 samples returned a false-negative result) for both T. orientalis and A. marginale during the pooling series, with lower-than-expected average Ct increase and sensitivity loss. We conclude that pooling up to 10 samples would be acceptable for regional surveillance of T. orientalis and A. marginale using our rtPCR assay. The described strategy is applicable to validate pooling for a wide range of single and duplex rtPCR assays, which could expand efficient disease surveillance.

Development of microflow ultra high performance liquid chromatography-mass spectrometry metabolomic assays for analysis of mammalian biofluids

Introduction and objectivesThe application of untargeted metabolomics assays using ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) to study metabolism in biological systems including humans is rapidly increasing. In some of these studies there is a requirement to collect and analyse low sample volumes of biofluids (e.g. tear fluid) or low cell and tissue mass samples (e.g. tissue needle biopsies). The application of microflow, capillary or nano liquid chromatography (≤ 1.0 mm column internal diameter (i.d.)) theoretically should accomplish a higher assay sensitivity compared to analytical liquid chromatography (2.1–5.0 mm column internal diameter). To date, there has been limited research into microflow UHPLC-MS assays that can be applied to study samples of low volume or mass.MethodsThis paper presents three complementary UHPLC-MS assays (aqueous C18 reversed-phase, lipidomics C18 reversed-phase and Hydrophilic Interaction Liquid Chromatography (HILIC)) applying 1.0 mm internal diameter columns for untargeted metabolomics. Human plasma and urine samples were applied for the method development, with porcine plasma, urine and tear fluid used for method assessment. Data were collected and compared for columns of the same length, stationary phase and stationary phase particle size but with two different column internal diameters (2.1 mm and 1.0 mm).Results and conclusionsAll three assays showed an increase in peak areas and peak widths when applying the 1.0 mm i.d. assays. HILIC assays provide an advantage at lower sample dilutions whereas for reversed phase (RP) assays there was no benefit added. This can be seen in the validation study where a much higher number of compounds were detected in the HILIC assay. RP assays were still appropriate for small volume samples with hundreds of compounds being detected. In summary, the 1.0 mm i.d. column assays are applicable for small volume samples where dilution is required during sample preparation.

Dual on-the-move electrochemical immunoassays for the simultaneous determination of amyloid-β (1−42) and Tau in Alzheimer's patient samples

Here, we report catalytic micromotors (MM)-based electrochemical immunoassays for the on-the-move dual and simultaneous determination of Amyloid-β (Aβ-42) and Tau protein (Tau) (MMAβ-42-MMTau) as relevant Alzheimer’s disease biomarkers in brain tissue, cerebrospinal fluid, and plasma diagnosed samples. Combining the binding capacity of the antibody's functionalized polypyrrole (PPy) layer of MM with the self-propulsion from the PtNPs layer thanks to the decomposition of hydrogen peroxide, the approach yielded excellent detection limits (LODAβ-42=0.04 ng/mL, LODTau= 0.4 pg/mL) using low sample volumes (30 µL) and short analysis times (15 min) to detect both biomarkers. Quantitative analysis by MMAβ-42-MMTau was carried out without any clinical sample dilution (linear ranges are between 0.1 and 5 ng/mL for Aβ-42 and from 1 to 106 pg/mL in the case of Tau), highlighting the versatility of the approach to quantify Aβ-42 and Tau levels at different dynamic ranges. MMAβ-42-MMTau showed superior analytical capabilities to the single molecule counting technology (SMCx) during quantitative analysis in all sample classes tested, reporting a difference in quantitative levels for both biomarkers between healthy and diseased individuals and an increase in the levels with disease progression, except in plasma samples where no relationship between biomarker levels and disease progression was found.

Enhancing sensitivity in fluorescence measurements across an ultra-wide concentration range through optimizing combined-segments strategy

Fluorescence measurement technology is crucial in analytical instrumentation. However, when applied in fields such as environmental science and medical research for quantification purposes, it requires prior estimation of sample concentration and sample dilution to ensure measurement precision. This requirement presents challenge for its broader application in online fluorescence sensing. This study addresses the challenge of achieving high-precision measurements across an ultra-wide concentration range, which is critical for applications ranging from environmental monitoring to clinical diagnostics. The binary segmentation method can initially achieve the goal of ultra-wide range fluorescence measurement, but it fails to maintain sensitivity and precision across the entire range, with relative errors exceeding ±15%. Therefore, we propose an optimizing combined-segments strategy, which examines the effects of adjusting the fluorescence reception position, range, and region interval parameters of the fluorescence reception probes on measurement sensitivity limits. This strategy aims to find the optimal measurement sensitivity limit (lmopt) and the best combined-segments configuration based on more than one quantitative relationship curves. Experimental results demonstrate that within a concentration range 20 times broader than the linear range, it can effectively maintain relative errors within ±5%. This enhances the applicability of fluorescence sensing techniques in various practical settings and advances online and direct fluorescence measurement technologies across an ultra-wide concentration range.

Distance-based analytical device integrated with carbon nanomaterials for sarcosine quantification in human samples.

Astraightforward distance-based paper analytical device (dPAD) was developedfor monitoring sarcosine levels in human samples for the rapid diagnosis and prognosis of prostate cancer and related symptoms. This assay eliminates the need for the expensive horseradish peroxidase (HRP) enzyme by utilizing carbon nanodots (CDs) as a peroxidase-like nanozyme. The proposed dPAD sensor consists of a sample zone pre-deposited with sarcosine oxidase (SOx) and CDs, and a detection zone containing 3,3',5,5'-tetramethylbenzidine (TMB). When a solution containing sarcosine is added to the sample zone, hydroxyl radicals (•OH) are produced through SOx oxidation and subsequent peroxidase catalysis by the CDs. The formed •OH radicals immediately flow to the detection zone via capillary force, where they oxidize TMB, resulting in a visible colour change from colourless to blue. Sarcosine quantification is effortlessly accomplished by measuring the distance of the blue colour in the detection zone. The developed dPAD offers a linear working range between 12.5 and 35.0nmol L-1 (R2 = 0.9959) and a detection limit (LOD) of 10.0nmol L-1. This covers the clinical range for urinary sarcosine determination, thereby suggesting no additional sample preparation or dilution is needed. The sensor shows high precision with the highest relative standard deviation (RSD) of 4.58% and demonstrates excellent selectivity with no observed interferences. Furthermore, recovery studies in human control urine samples ranged from 98.67 to 101.50%, with the highest RSD of 2.03%. Correspondingly, our dPAD method showed a great match with the performance of a commercial ELISA method for detecting sarcosine in human control serum. The sensor is more cost-effective, user-friendly, and accessible than previous methods. Overall, the proposed method represents a promising analytical tool for sarcosine quantification. The concept is also applicable for broader analytical applications in detecting other biomolecules.

Bacterial Binding to Polydopamine-Coated Magnetic Nanoparticles.

In medical infections such as blood sepsis and in food quality control, fast and accurate bacteria analysis is required. Using magnetic nanoparticles (MNPs) for bacterial capture and concentration is very promising for rapid analysis. When MNPs are functionalized with the proper surface chemistry, they have the ability to bind to bacteria and aid in the removal and concentration of bacteria from a sample for further analysis. This study introduces a novel approach for bacterial concentration using polydopamine (pDA), a highly adhesive polymer often purported to create antibacterial and antibiofouling coatings on medical devices. Although pDA has been generally studied for its ability to coat surfaces and reduce biofilm growth, we have found that when coated on magnetic nanoclusters (MNCs), more specifically iron oxide nanoclusters, it effectively binds to and can remove from suspension some types of bacteria. This study investigated the binding of pDA-coated MNCs (pDA-MNCs) to various Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and several E. coli strains. MNCs were successfully coated with pDA, and these functionalized MNCs bound a wide variety of bacterial strains. The efficiency of removing bacteria from a suspension can range from 0.99 for S. aureus to 0.01 for an E. coli strain. Such strong capture and differential capture have important applications in collecting bacteria from dilute samples found in medical diagnostics, food and water quality monitoring, and other industries.

Dynamics of optical properties of sequentially diluted lucigenin aqueous solutions according to luminescence data.

The article discusses optical properties (luminescence) of diluted (24 dilution factor) lucigenin (Lc) aqueous solutions. Six series of Lc aqueous solutions, with 50 samples in each series, were studied. The series were diluted on different days within random schedules, following a unified procedure: the first sample in all the series was the Lc (C Lc = 8.2 × 10-7mol/L) stock solution, while the rest of the samples were obtained by successive dilution with the ratio of 24. For the first three samples, the Lc luminescence intensity decrease appropriately complied with the exponential function model (the dilution ratio: none for the stock solution, for the second and the third, 24 and 24 × 24 = 242, respectively). Starting from the fourth sample for statistical processing of luminescence data, the seven largest values were selected from the built rank distribution of emission intensity values. This method helps eliminate the influence of "random large bounces" when calculating the correlation coefficient. Up to the 50th studied sample, a challenging linear gradual decrease in the intensity of recorded photometric values was noted (correlation coefficients for all series being close to -0.9). Similar analysis of six reference series of pure water "dilution" samples did not exhibit any correlation between the highest emission values in the studied wavelength range (specific for Lc bandwidth, 480-505nm) and the sample's dilution number. It can be assumed that photometric values, recorded in the series of Lc sequentially diluted aqueous solutions after substance (Lc) elimination (theoretically expected after the 13th sample within the used experimental setup), could be attributed to the gradual destruction of long-lived aqueous structures formed in the process of hydration of Lc molecules during its dissolution.

Who Keeps Changing My Results? - A Case of Heterophile Antibody Interference

Abstract Introduction/Objective Heterophile antibodies are known to cause interference in immunoassays leading to misleading results causing diagnostic confusion and unnecessary testing and/or treatment. Many clinically important analytes are tested using antibody-based assays, as such, these are prone to interference. Methods/Case Report We describe a case of a 73 year old male who was referred with history of hypocalcemia and his previous lab tests were suggestive for idiopathic hypoparathyroidism, Hashimoto’s disease and Addison’s disease raising physician concern for a possible Type 1 autoimmune endocrinopathy. One of his results (cortisol) was outside the analytical measurement range and hence the sample was diluted and tested again, but the result was much less than what was expected, prompting an investigation. Investigation for an interfering substance included serial dilution and analysis of the same sample for the same analytes by different methods (different immunoassay, mass spectrometry or both). Based on non-parallelism in serial dilution results, discordant results with other methods, and loss of interference after dilution it was determined that the most likely cause was a heterophile antibody. Results (if a Case Study enter NA) NA Conclusion Awareness of the possibility that interference by heterophile antibodies can lead to erroneous laboratory results is important to prevent unnecessary testing and inappropriate diagnosis/management.

Virtual Instruments for Peak-Overlapping Studies to Determine Low- and High-Concentration Components with Ion Chromatography: Potassium and Sodium.

We developed the LabVIEW-based virtual instruments (VIs) to bridge a gap in commercial software and to enable systematic peak-overlapping studies to recognise the concentration levels enabling reliable simultaneous determination of major and minor constituents in samples with wide concentration proportions. The VIs were applied to a case study of the ion chromatographic determination of potassium as minor and sodium as a major ion with an IonPac CS12A column and 50 μL injection loop. Two successive studies based on multilevel two-factorial response surface experimental designs, (1) a model peak-overlapping study based on single-ion injections, and (2) an accuracy and precision study, provided guidelines for real sample analyses. By adjusting sample dilutions so that the sodium mass concentration was set to 340 mg/L, the simultaneous determination of potassium in the presence of sodium was possible in samples with sodium over potassium concentration ratios between 14 and 341. The relative expanded uncertainty associated with potassium ion determination was between 0.52 and 4.4%, and the relative bias was between -3.8 and 1.9%. We analysed Ringer's physiologic solutions, standard sea, trisodium citrate anticoagulant, and buffered citrate anticoagulant solutions. We confirmed that the VI-supported peak-overlapping studies contributed to the quality of results by enabling the evidence-based choices of concentration levels adjusted by a dilution.

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction.

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/µL), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/µL and 10,000 copies/µL, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

The bronchoalveolar lavage dilution conundrum: an updated view on a long-standing problem.

Bronchoalveolar lavage (BAL) is used by researchers to study molecular interactions within healthy and diseased human lungs. However, the utility of BAL fluid measurements may be limited by difficulties accounting for dilution of the epithelial lining fluid (ELF) sampled and inconsistent collection techniques. The use of endogenous markers to estimate ELF dilution has been proposed as a potential method to normalize acellular molecule measurements in BAL fluid, but these markers are also imperfect and prone to inaccuracy. The focus of this report is to review factors that affect the interpretation of acellular molecule measurements in lung ELF and present original data comparing the performance of several BAL dilution markers during health and in a human endobronchial endotoxin challenge model of acute inflammation. Our findings suggest that incomplete ELF and lavage fluid mixing, flux of markers across the alveolar barrier, and lung inflammation are all possible factors that can affect marker performance. Accounting for these factors, we show that commonly used markers including urea, total protein, albumin, and immunoglobulin M are likely unreliable BAL dilution markers. In contrast, surfactant protein D appears to be less affected by these factors and may be a more accurate and biologically plausible marker to improve the reproducibility of acellular BAL component measurements across individuals during health and inflammatory states.NEW & NOTEWORTHY In this report, mathematical prediction models and real-world measurements are used to compare the performance of molecular markers of dilution in bronchoalveolar lavage fluid samples. Effects of acute inflammation within individual subjects are highlighted. These findings inform recommendations for normalizing measurements across bronchoalveolar lavage samples and highlight the need for additional markers to improve the rigor of translational studies utilizing bronchoalveolar lavage measurements.

Liquid Handling Technologies: A Study through Major Discoveries and Advancements

Liquid handling is an essential process in laboratories involving automatically transferring accurate amounts of liquids for various task completions such as sample preparation, pipetting, dilution, and mixing. Automated liquid handling systems (ALHS) help to minimize the errors, complications, and difficulties that are faced with manual methods of liquid handling by efficiently enhancing the accuracy, precision, and reproducibility of experimental workflows and have transformed life sciences aiding research in various fields. Processes like Nuclear Magnetic Resonance (NMR) spectrometry, mass spectrometry, and acoustic droplet ejection with ALHS showcase tremendous potential. Outdated manual machinery is being replaced rapidly with automated tools. We have observed that these laboratory setups are sophisticated and costly. It is challenging to access such technologies, especially in poor or developing countries. At the nanoscale level, handling and evaporation of biomaterials becomes a great issue. Multitudinous devices can combat the cost issue along with utilization of single equipment without the enhancement of add-on modules. Liquid handling devices enhance the quality of experiments contributing to efficient dealing of circumstances like COVID-19. They aid in genomics and proteomics and play a pivotal role in liquid manipulation, drug screening, quantifications, forensic studies and many more. Liquid handling devices with robotic equipment can perform multiple tasks and can assist in a wide range of fields. We anticipate that in the coming years, our laboratories will be furnished with advanced, robotic technologies and cause a revolution in the scientific scenery.

12568 “Unusual Presentation Of Pituitary Macroadenoma: Co-secretion Of GH And Prolactin With Prozone Phenomenon"

Abstract Disclosure: F. Lopez Maldonado: None. A. Rodriguez Gonzalez: None. A. Mayorga Leon: None. E. Leon Milan: None. Background: Plurihormonal pituitary adenomas (10-15% cases) are linked to acromegaly, especially in young patients. ∼8% are associated with MEN1. They produce growth hormone (GH), prolactin (PRL), and sometimes thyroid-stimulating hormone (TSH), causing clinical effects from GH and excess PRL. Most are large and invasive in ∼50% of cases. Case Report: A 37-year-old male with no significant medical history, exhibited monthly right temporal headaches, ipsilateral eye pain, nausea, and vomiting, that responded to analgesics and antibiotics. Two years later he presented increased headaches, fainting, and decreased vision in the right eye. Over the next four years, his headaches worsened, leading to complete vision loss in the right eye and compromised vision in the left eye. Evaluation by Internal Medicine revealed bradylalia, recent memory changes, a prominent supraorbital arch, pale optic disc with centered vessels in the right eye. Additionally, macroglossia, prognathism, and acromegaly-like changes in extremities were noted. Campimetry revealed left temporal hemianopsia. Hormonal profile: LH 0.04 mIU/mL (1.24 - 8.6 mIU/mL), FSH 0.21 mIU/mL (1.27 - 19.2 mIU/mL), TSH 4.78 mIU/mL (1.24 - 19.2 mIU/mL), Total T4 2.04 mcg/dL (4.5 - 10.9 mcg/dL), PRL 1.1 ng/mL (2.8 - 13.13 ng/mL) (at dilution 1:100) &amp;gt;20,000 ng/mL, Cortisol 0.903 mcg/dL (3.7 - 19.4 mcg/dL), IGF-1 1001 ng/mL (41 - 246 ng/mL), GH 12 ng/mL (0.03 - 2.47 ng/mL), Total Testosterone 0.13 ng/mL (1.6 - 8.7 ng/mL) MRI report: Invasive macroadenoma measuring 49x43x48 mm with partial inclusion of the right cavernous sinus and complete inclusion of the left cavernous sinus, bilateral carotid involvement with left predominance, involvement over the optic chiasm, pituitary stalk, floor of the sella turcica with migration towards the sphenoidal sinus. Diagnosis: Co-secreting PRL and GH pituitary macroadenoma, Secondary Hypogonadotropic Hypogonadism, Secondary Hypothyroidism, Secondary Adrenal Insufficiency. Treatment: Prednisone 5 mg PO QD, Cabergoline 0.5 mg PO QD 5/7 (MWFSS), Levothyroxine 100 mcg PO QD, Sostenon 240 mg/mL IM every 3 weeks. One week post-treatment initiation, the patient reported improvement.Follow-up labs showed normalized hormone levels. MRI revealed a 37x34x28 mm pituitary macroadenoma with bleeding and cystic changes. Surgery was deferred due to the favorable response to medical treatment. An annual MRI was performed, which showed a decrease in tumor size. By the seventh year, the tumor had reduced by 98% under the same treatment. Conclusion: Macroprolactinomas pose diagnostic challenges due to the prozone effect, requiring sample dilution for accurate diagnosis. Reflecting on the unexpected, this tumor co-secretes GH, which is uncommon; however, it responded effectively to medical treatment. Presentation: 6/3/2024

A-136 Icteric Interference is not Resolved with Sample Dilution in Serum Creatinine and Total Protein Assays

Abstract Background Icterus is a common endogenous interference that can cause falsely low results for serum creatinine and total protein. It can be defined by elevated unconjugated (indirect) and conjugated (direct) bilirubin levels and is most commonly associated with liver dysfunction. Recent literature suggests that dilutions can be a reliable method to reduce bilirubin interference. The purpose of this study was to determine whether the vendor’s on-board sample dilution can eliminate icteric interference with creatinine and total protein. Methods Creatinine (enzymatic) and total protein interference studies were performed on an automated chemistry analyzer (Roche Diagnostics, cobas c702). Low, mid, and high sample pools for each analyte were divided into four equal volumes. To achieve an icterus index (II) of approximately 40 (approximately 40 mg/dL of bilirubin), one sample pool was spiked with unconjugated bilirubin, and one pool was spiked with conjugated bilirubin. An equal amount of diluent was added into each control pool and remained at an II of approximately 0. The icteric and control samples were measured for conjugated and unconjugated bilirubin, serum indices, and the target analyte. Using the on-board dilution function, the icteric creatinine and total protein pools were diluted using a 1:4 and 1:3 dilution, respectively. All samples were analyzed in triplicate. Percent variance was found by calculating the difference between the average dilution result and control result and then dividing by the average control result. Results Conclusions Only modest alleviation of icteric interference was noted with dilution. The average bias was 10% for creatinine and 24% with total protein, after dilution. The on-board sample dilution is not effective in eliminating icteric interference when measuring both creatinine and total protein.