Principal Cells Research Articles

Thick Ascending Limb Specific Inactivation of Myh9 and Myh10 Myosin Motors Results in Progressive Kidney Disease and Drives Sex-specific Cellular Adaptation in the Distal Nephron and Collecting Duct.

Our previous work established a role for actin associated myosin motor proteins MYH9 and MYH10 in the trafficking of thick ascending limb (TAL) specific cargoes, uromodulin (UMOD) and Na+K+2Cl- cotransporter (NKCC2). Here, we have generated a TAL-specific Myh9&10 conditional knockout (Myh9&10 TAL-cKO) mouse model to determine the cell autonomous roles for MYH9&10 proteins in TAL cargo transport and to understand the consequence of TAL dysfunction in the adult kidney. Myh9&10 TAL-cKO mice develop progressive kidney disease with pathological tubular injury confirmed by histological changes, tubular injury markers, upregulation of ER stress/unfolded protein response pathway, and higher blood urea nitrogen and serum creatinine. However, male mice survive twice as long as female mice. We determined that the sexual dimorphism in morbidity is due to adaptation of the distal nephron and the collecting ducts in response to TAL dysfunction and significantly lower NKCC2 expression. We demonstrate that this triggers a compensatory mechanism involving sex-specific cellular adaptation within the distal tubules and collecting ducts to boost sodium reabsorption. While both sexes overcompensate by activating ENaC expression in the medullary collecting ducts resulting in hypernatremia, this is subdued in male Myh9&10 TAL-cKO mice as they initially promote higher sodium chloride cotransporter (NCC) expression within the distal nephron. Our results indicate that compromised TAL function results in maladaptation of medullary collecting duct cells, which acquire cortical-like properties, including ENaC expression. This work further confirms a cell autonomous role for myosin motor proteins MYH9&10 in the maintenance of NKCC2 expression in the TAL and uncover adaptive mechanisms of the distal nephron and the collecting duct segments in response to TAL dysfunction.

Restoring Compromised Cl- in D2 Neurons of a HD Mouse Model Rescues Motor Disability.

Huntington's disease (HD) is a progressive neurodegenerative disorder with no cure, characterized by significant neurodegeneration of striatal GABAergic medium spiny neurons (MSNs). Early stages of the disease are characterized by the loss of dopamine 2 receptor-expressing MSNs (D2 MSNs) followed by degeneration of dopamine 1 receptor-expressing MSNs (D1 MSNs), leading to aberrant basal ganglia signaling. While the early degeneration of D2 MSNs and impaired GABAergic transmission are well-documented, potassium chloride cotransporter 2 (KCC2), a key regulator of intracellular chloride (Cl-), and therefore GABAergic signaling, has not been characterized in D1 and D2 MSNs in HD. We aimed to investigate whether Cl- regulation was differentially altered in D1 and D2 MSNs and may contribute to the early degeneration of D2 MSNs in male and female symptomatic R6/2 mice. We used electrophysiology to record the reversal potential for GABAA receptors (EGABA), a read-out for the efficacy of Cl- regulation, in striatal D1 and D2 MSNs and their corresponding output structures. During the early symptomatic phase (P55-P65), Cl- impairments were observed in D2 MSNs in R6/2 mice, with no change in D1 MSNs. Cl- regulation was also dysfunctional in the globus pallidus externa, resulting in GABA-mediated excitation. When we overexpressed KCC2 in D2 MSNs using AAV-mediated delivery, we delayed the onset of motor impairments in R6/2 mice. We demonstrate that Cl- homeostasis is differentially altered in D1 and D2 MSNs and may contribute to the enhanced susceptibility of D2 MSNs during HD progression.Significance Statement Huntington's Disease is an inherited neurodegenerative disease caused by a repeat expansion in the Huntingtin gene and characterized by the sequential loss of dopamine 2 and dopamine 1 receptor-expressing medium spiny neurons (D2 and D1 MSNs) of the striatum. MSNs release GABA, which depends on proper Cl- regulation for inhibition. We asked whether Cl- homeostasis is differentially altered in D1 and D2 MSNs and their output structures, and whether this altered expression contributes to the pattern of degeneration between these two principal striatal cell types. Using electrophysiology, biochemistry, and fluorescence imaging, we determined that Cl- regulation was impaired in D2 MSNs in R6/2 mice, with no change in D1 MSNs. Cl- was also dysregulated in the globus pallidus externa resulting in excitatory GABA.

Activation of 5-HT7 receptors in the mouse dentate gyrus does not affect theta-burst-induced plasticity at the perforant path synapse.

The study examined the effects of 5-HT7 receptor activation on GABAergic transmission within the dentate gyrus and plasticity at the glutamatergic perforant path input. Immunofluorescence imaging was performed using transverse hippocampal slices from transgenic mice expressing green fluorescent protein (GFP) under the Htr7 promoter. This was followed by whole-cell patch clamp electrophysiological recordings assessing the effects of pharmacologically activating 5-HT7 receptors on spontaneous inhibitory postsynaptic currents recorded from dentate granule cells and hilar mossy cells-two glutamatergic neuron types present in the dentate gyrus. Extracellular recordings of field excitatory postsynaptic potentials were then performed to assess whether 5-HT7 receptor activation influenced theta-burst stimulation-evoked plasticity of the perforant path synaptic input. It was found that parvalbumin and somatostatin interneurons in the dentate gyrus expressed GFP, which suggests they express 5-HT7 receptors. However, activation of 5-HT7 receptors had no effect on GABAergic transmission targeting mossy cells or granule cells. There was also no effect of 5-HT7 receptor activation on perforant path plasticity either with intact or blocked GABAA receptor signaling. The presence of 5-HT7 receptors in a subset of parvalbumin and somatostatin interneurons in the mouse dentate gyrus could mean that they are involved in the inhibitory control of dentate gyrus activity. However, this potential effect was not evident in slice recordings of inhibitory transmission targeting principal cells and did not affect perforant path plasticity. Further experiments are needed to fully elucidate the functional role of these receptors in the dentate gyrus.

COMPARISON OF YIELD AND QUALITY OF CIRCULATING CELL-FREE DNA FROM K2EDTA AND K3EDTA COLLECTIONS IN HEALTHY SUBJECTS

Background: Circulating cell-free DNA (cfDNA) is a biomarker for various clinical applications, including detecting and monitoring cancer. However, blood collection tubes can affect the yield and quality of cfDNA. Since specific cfDNA collection tubes are costly, K2EDTA and K3EDTA anticoagulant tubes are alternatives in routine clinical laboratories. Objectives: This study aimed to compare the efficiency of cfDNA extraction from plasma collected in K2EDTA and K3EDTA tubes and evaluate implementation for molecular diagnostics. Methods: Blood samples from 38 healthy subjects were collected in K2EDTA and K3EDTA tubes that were processed within 2 hours. The extracted cfDNA was measured and performed using SYBR Green-based qPCR for three endogenous reference genes (GAPDH, HPRT1, TFRC). The cfDNA yield and the amplification efficiency of these genes were compared between K2EDTA and K3EDTA tubes using the Mann-Whitney U test. Results: There were no significant differences in cfDNA concentration between K2EDTA and K3EDTA tubes (p=0.051). However, qPCR analysis revealed significantly higher copy numbers of TFRC and HPRT1 in K2EDTA tubes than in K3EDTA tubes (p<0.05). No significant difference was found for GAPDH. Conclusion: The results indicate that K2EDTA and K3EDTA tubes are an alternative option for cfDNA analysis if samples are processed quickly after a blood draw, which offers flexibility and cost savings in resource-limited areas.

Comparison of a two-step Tempus600 hub solution single-tube vs. container-based, one-step pneumatic transport system.

Efficient and timely transportation of clinical samples is pivotal to ensure accurate diagnoses and effective patient care. During the transportation process, preservation of sample integrity is crucial to avoid pre-analytical aberrations on laboratory results. Here, we present a comparative analysis between a two-step Tempus600 hub solution single-tube and a one-step, container-based pneumatic transport system (PTS) from Airco, for the in-house transportation of blood samples. Ten blood samples from healthy volunteers were split in 10 mL collection tubes filled at full or half capacity for transportation with the two PTS (about 250 m). To compare the impact of transportation, markers of hemolysis such as lactate dehydrogenase (LDH), potassium (K+), and the hemolysis index (HI), were determined. Additionally, differences in HI in routine samples and repeated transportation was investigated. To assess and compare the mechanistic impact profiles, we recorded the acceleration profiles of the two PTS using a shock data logger. Transportation using the Tempus600 hub solution resulted in 49 and 46 % higher HI with samples filled to total or half capacity, respectively. Routine samples transported with the Tempus600 hub solution showed a higher median HI by 23 and 33 %. Additionally, shock logger analysis showed an elevated amount of shocks (6.5 fold) and shock intensities (1.8 fold). The Tempus600 hub solution caused an increased number of unreportable LDH or K+ results based on the hemolysis index. However, it was only statistically significant for LDH (p<0.01 and p<0.08)- while the comparisons for K+ were not statistically significant (p<0.28 and p<0.56).

The biology of water homeostasis.

Water homeostasis is controlled by a brain-kidney axis that consists of central osmoreceptors, synthesis and secretion of arginine vasopressin (AVP) and AVP-responsive aquaporin-2 (AQP2) water channels in kidney collecting duct principal cells that facilitate water reabsorption. In addition to AVP, thirst represents a second line of defense to maintain water balance. Water balance disorders arise because of deficiency, resistance or inappropriate secretion of AVP or disturbances in thirst sensation (hypodipsia, polydipsia). People with water balance disorders are prone to develop hyponatremia or hypernatremia, which expose cells to osmotic stress and activate cell volume regulation mechanisms. This review covers several recent insights that have expanded our understanding of central osmoregulation, AQP2 regulation and cell volume regulation. This includes the role of with-no-lysine kinase 1 (WNK1) as a putative central osmolality sensor and, more generally, as an intracellular crowding sensor that coordinates the cell volume rescue response by activating sodium and potassium cotransporters. Furthermore, several new regulators of AQP2 have been identified, including for AVP-dependent AQP2 regulation (yes-associated protein, nuclear factor of activated T-cells, microRNAs) and AVP-independent AQP2 regulation (epidermal growth factor receptor, fluconazole, prostaglandin E2). It is also becoming increasingly clear that long-term cell volume adaptation to chronic hypotonicity through release of organic osmolytes comes at the expense of compromised organ function. This potentially explains the complications of chronic hyponatremia, including cognitive impairment, bone loss and vascular calcification. This review will illustrate why these new insights derived from basic science are also relevant for developing new approaches to treat water balance disorders.

Regulation of the gap junction interplay during postnatal development in the rat epididymis.

During postnatal development of the rat epididymis, a change in the expression of gap junction proteins, or connexins (Cxs), occurs, in which Gjb2 (Cx26) and Gja1 (Cx43) levels in the proximal epididymis are decreased, while Gjb1 (Cx32), Gjb4 (Cx30.3) and Gjb5 (Cx31.1) levels increase. The mechanism(s) responsible for the switch in Cx expression is unknown. The aim of this study is to identify the mechanisms responsible for the decrease in GJB2 protein levels and the increase in other Cxs during postnatal development. Results indicate that decreased Gjb2 expression for 48h does not alter the expression of other Cxs in RCE-1 principal cells, suggesting a lack of compensatory expression. Sequence analysis of both Gjb2 and Gjb1 promoters identified common multiple response elements to steroid hormones. Using RCE-1 cells, we observed that dexamethasone increased Gjb2 mRNA levels by twofold after 48h, while estradiol had no effect. Orchidectomy in rats resulted in a significant increase in GJB2 and decreased GJB1 in the caput and corpus epididymidis. Changes in Cxs protein levels were prevented by testosterone in orchidectomized rats. Similar results were observed in the prostate, another androgen-receptive organ. LNCaP cells, which are androgen-responsive, showed that exogenous dihydrotestosterone (DHT) decreased Gjb2 mRNA levels by approximately 50% concomitant with a 1.5-fold increase in Gjb1 levels. Using a GJB1 promoter construct we showed that DHT could induce transactivation of the luciferase transgene, while transactivation of two GJB2 promoters were unaltered. Results indicate that androgens and glucocorticoids regulate the expression of epididymal Cxs.

Case-Based Training on Best Practices in Effective Test Utilization for Clinical Laboratories: A Comparison of Two Educational Formats

Abstract Introduction/Objective While a survey of ASCP membership showed improvements being made in effective test utilization (ETU), there is an ongoing need for formal education on best practices. Case-based education drawn from real-world scenarios provides an opportunity to address this need, aimed at increasing the understanding of evidence- based guidelines to inform clinical test ordering and reduce overconsumption of supplies without compromising patient care. The poster presents two examples of case-based education presented in an innovative microlearning format and an eLearning format designed for clinical laboratory professionals. Methods/Case Report The activities presented 9 - 10 practical, real-world scenarios as educational cases along with assessment questions addressing a range of topics that included ETU for pancreatitis and cardiac injury, blood collection tube consumption and conservation, strategies for reducing repeat daily lab test ordering, and others. Each case was designed for learners to be able to review the content and answer the assessment questions in 5 minutes. In the microlearning activity, learners received the cases and questions via email on a scheduled frequency and completed them via mobile device. The questions were repeated up to 2 times to reinforce learning. Participants in the eLearning activity accessed the same cases and assessment questions via a different learning platform (with no staggered delivery). Results (if a Case Study enter NA) More than 2,500 learners have participated in one or both ETU activities, and feedback has been highly positive. The assessment results show proficiency gains for a range of laboratory personnel, including laboratory managers/supervisors, histotechnologists, medical laboratory scientists, laboratory technicians, pathologists, and other laboratory roles. Conclusion The results also show how case-based scenarios can help promote best practices in ETU, which may be particularly beneficial for laboratory decision-makers. In addition to showing increased understanding of ETU strategies, the results also pose lessons learned for designing similar types of case-based education.

On-Demand Seizures Facilitate Rapid Screening of Therapeutics for Epilepsy.

Animal models of epilepsy are critical in drug development and therapeutic testing, but dominant methods for pharmaceutical evaluation face a tradeoff between higher throughput and etiological relevance. For example, in temporal lobe epilepsy, a type of epilepsy where seizures originate from limbic structures like the hippocampus, the main screening models are either based on acutely induced seizures in wild type, naïve animals or spontaneous seizures in chronically epileptic animals. Both types have their disadvantages - the acute convulsant or kindling induced seizures do not account for the myriad neuropathological changes in the diseased, epileptic brains, and spontaneous behavioral seizures are sparse in the chronically epileptic models, making it time-intensive to sufficiently power experiments. In this study, we took a mechanistic approach to precipitate seizures "on demand" in chronically epileptic mice. We briefly synchronized principal cells in the CA1 region of the diseased hippocampus to reliably induce stereotyped on-demand behavioral seizures. These induced seizures resembled naturally occurring spontaneous seizures in the epileptic animals and could be stopped by commonly prescribed anti-seizure medications such as levetiracetam and diazepam. Furthermore, we showed that seizures induced in chronically epileptic animals differed from those in naïve animals, highlighting the importance of evaluating therapeutics in the diseased circuit. Taken together, we envision our model to advance the speed at which both pharmacological and closed loop interventions for temporal lobe epilepsy are evaluated.

Proteomic analysis of CD29+ Müller cells reveals metabolic reprogramming in rabbit myopia model

The prevalence of myopia is rapidly increasing, significantly impacting the quality of life of affected individuals. Prior research by our group revealed reactive gliosis in Müller cells within myopic retina, prompting further investigation of their role in myopia, which remains unclear. In this study, we analyzed protein expression changes in CD29+ Müller cells isolated from a form deprivation-induced rabbit model of myopia using magnetic activated cell sorting to investigate the role of these cells in myopia. As the principal glial cells in the retina, Müller cells exhibited significant alterations in the components of metabolic pathways, particularly glycolysis and angiogenesis, including the upregulation of glycolytic enzymes, such as lactate dehydrogenase A and pyruvate kinase, implicated in the adaptation to increased metabolic demands under myopic stress. Additionally, a decrease in the expression of proteins associated with oxygen transport suggested enhanced vulnerability to oxidative stress. These findings highlight the proactive role of CD29+ Müller cells in modifying the retinal environment in response to myopic stress and provide valuable insights into mechanisms that could help mitigate myopia progression.

Hierarchical Classification of Factors Associated With Noninvasive Prenatal Testing Failures and Its Impact on Pregnancy Outcomes

Abstract Objective To conduct a hierarchical classification analysis of the nonreportable results of noninvasive prenatal testing in an attempt to reduce failure rates and provide pregnant women with accurate information to alleviate their anxiety. Methods In this study, 30,039 singleton pregnancies who underwent noninvasive prenatal testing in a single center from May 2019 to April 2022 were collected, and 811 samples with initial noninvasive prenatal testing failure were retrospectively analyzed. Grouping was based on the reasons for initial test failure; tracking the noninvasive prenatal testing results and prenatal diagnosis results (if any) of the “z-scores in the gray area” group and analyzing the possible influencing factors of the “low fetal fraction” group in the pre-experimental and experimental period by using one-way analysis of variance, Mann-Whitney U test, and χ2 test; and tracking the pregnancy outcomes of the test failures samples to analyze the risk of perinatal complications and adverse pregnancy outcomes of the different types of test failures. Results None of the samples' initial nonresults because of z-scores in the gray area were ultimately found to have chromosomal aneuploidy. However, pregnancy complications (P = 0.018) and a high likelihood of adverse pregnancy outcomes (P = 0.048) may still occur. Maternal gestational age (P &lt; 0.001), body mass index (P &lt; 0.001), library concentration (P &lt; 0.001), and fetal gender (P &lt; 0.001) were considered to be the associated factors for the initial low fetal fraction results. This may be associated with pregnancy complications (P &lt; 0.001) and a high likelihood of adverse pregnancy outcomes (P = 0.034). The body mass index (P = 0.015) and time between draws (P = 0.001) were associated with the second test’s success. The incidence of low fetal fraction samples was more frequent with blood collection tubes of the G type than with the K type (P &lt; 0.001). Conclusion Initial nonresults because of z-scores in the gray area did not imply an increased risk of aneuploidy, but vigilance is needed for an increased risk of pregnancy complications and adverse pregnancy outcomes. Because of the low fetal fraction, the initial absence of results may be related to the assay method, as well as the effect of blood collection tubes and the need to be alert to the risk of pregnancy complications and adverse pregnancy outcomes.

The influence of microchannel structure on double emulsion droplets formation

Microfluidics for the preparation of double emulsion droplets for a wide range of applications in medicine, agronomy, and materials science. The requirements for droplet preparation vary in different fields, and these needs can be met by altering fluid parameters, microchannel geometries, and other means. Therefore, this paper explores the influence of microfluidic device geometry on double-emulsion droplet molding using a VOF approach. The influence of variations in the length between the inject tube of the inner phase and inlet of the collection tube, the outlet width, the angle between phases, and the outlet angle on the double-emulsion droplet formation distance as well as on the area and other parameters were emphasized. The results show that increasing the length between the inner phase injection tube and inlet of the collection tube helps to improve the stability of droplet formation; the change of the outlet width has less effect on droplet formation; and the change of the angle between the phases tends to lead to the shift of the droplet formation mode, which has a greater effect on droplet formation. This study can provide a reference for optimizing the microchannel structure.

Utility of Reticulocyte Hemoglobin Equivalent in Screening for Iron Deficiency in Pregnancy.

Ferritin, commonly used for diagnosing iron deficiency (ID) in pregnancy, is limited by high cost and false elevations during inflammation. Reticulocyte hemoglobin equivalent (Ret-He), an alternative marker for ID, is unaffected by inflammation and analyzed on the same collection tube as the standard complete blood count (CBC). We aimed to determine the accuracy of Ret-He in detecting ID in pregnancy compared to ferritin in a U.S. This prospective cohort study enrolled 200 pregnant participants, recruited in any trimester if a CBC was drawn as part of routine prenatal care. For those who agreed to participate, Ret-He and ferritin were collected concurrently with the CBC. ID was defined as ferritin level below 30 ng/mL. Patients were classified into three groups based on hemoglobin and ferritin results to determine the severity of ID: no ID, ID alone, and ID anemia (IDA). Four participants with anemia but normal ferritin were excluded. Receiver operating curve analysis, including the area under the curve (AUC), was performed to assess the accuracy of Ret-He in detecting ID. A one-way ANOVA (analysis of variance) with post-hoc analysis was used to compare differences in Ret-He between the three groups of ID severity. The prevalence of ID in our cohort was 82% (161/196). The AUC for Ret-He was 0.65 (95% confidence interval: 0.55-0.75), indicating suboptimal discrimination between patients with and without ID. Ret-He was significantly different among the three groups (p < 0.001). In post-hoc analysis, Ret-He was significantly lower in the IDA group compared to the ID group (p < 0.001) but there was only a trend of lower Ret-He in the ID group compared to the non-ID group (p = 0.38). Ret-He has low accuracy in diagnosing ID in pregnancy. It may be useful in detecting severe ID resulting in anemia but not a mild iron-deficient state resulting in ID only. · The prevalence of ID in our cohort was 82%.. · Ret-He has low accuracy in diagnosing ID in pregnancy.. · Ferritin is preferable when readily available..

Focal Adhesion Kinase (FAK) inhibition induces membrane accumulation of aquaporin2 (AQP2) through endocytosis inhibition and actin depolymerization in renal epithelial cells.

Cellular trafficking of the water channel aquaporin 2 (AQP2) is regulated by the actin cytoskeleton in collecting duct principal cells (PC) to maintain proper water balance in animals. Critical actin depolymerization/polymerization events are involved in both constitutive AQP2 recycling, and the pathway stimulated by vasopressin receptor signaling. Focal adhesion kinase (FAK) plays an important role in modulating the actin cytoskeleton through inhibiting small GTPases, and multiple studies have shown the involvement of FAK in insulin and cholesterol trafficking through actin regulation. To understand whether FAK contributes to water reabsorption by the kidney, we performed a series of in vitro experiments to examine the involvement of FAK and its signaling in mediating AQP2 trafficking in cultured renal epithelial cells. Our data showed that FAK inhibition by specific inhibitors caused membrane accumulation of AQP2 in AQP2expressing LLCPK1 cells by immunofluorescence staining. AQP2 membrane accumulation induced by FAK inhibition is associated with significantly reduced endocytosis of AQP2 via the clathrin-mediated endocytosis pathway. Moreover, AQP2 membrane accumulation induced by FAK inhibition also occurred in cells expressing the constitutive dephosphorylation mutant of AQP2, S256A. This was confirmed by immunoblotting using a specific antibody against phospho-serine 256 AQP2, supporting a phosphorylation independent mechanism. Finally, we demonstrated that inhibition of FAK caused reduced RhoA signaling and promoted F-actin depolymerization. In conclusion, our study identifies FAK signaling as a pathway that could provide a novel therapeutical avenue for AQP2 trafficking regulation in water balance disorders.

Disruption of mitochondrial electron transport impairs urinary concentration via AMPK-dependent suppression of aquaporin-2.

Urinary concentration is an energy-dependent process that minimizes body water loss by increasing aquaporin-2 (AQP2) expression in collecting duct (CD) principal cells. To investigate the role of mitochondrial (mt) ATP production in renal water clearance, we disrupted mt electron transport in CD cells by targeting ubiquinone (Q) binding protein QPC (UQCRQ), a subunit of mt complex III essential for oxidative phosphorylation. QPC-deficient mice produced less concentrated urine than controls, both at baseline and after type 2 vasopressin receptor stimulation with desmopressin. Impaired urinary concentration in QPC-deficient mice was associated with reduced total AQP2 protein levels in CD tubules, while AQP2 phosphorylation and membrane trafficking remained unaffected. In cultured inner medullary CD cells treated with mt complex III inhibitor antimycin A, the reduction in AQP2 abundance was associated with activation of 5' adenosine monophosphate-activated protein kinase (AMPK) and was reversed by treatment with AMPK inhibitor SBI-0206965. In summary, our studies demonstrated that the physiological regulation of AQP2 abundance in principal CD cells was dependent on mt electron transport. Furthermore, our data suggested that oxidative phosphorylation in CD cells was dispensable for maintaining water homeostasis under baseline conditions, but necessary for maximal stimulation of AQP2 expression and urinary concentration.

B-013 Improving the Workflow of Blood and Coagulation Tests in clinical laboratory through the Utilization of Laboratory Automation Track Systems

Abstract Background The Laboratory Automation Track System (TLA) is an automated system designed for executing highly repetitive tasks within a laboratory, commonly applied in biochemical and immunological testing systems. It aims to reduce human errors in specimen handling, enhance the overall process quality, and provide better Turnaround Time (TAT) from specimen collection to result reporting. For the first time in our laboratory, we have integrated the blood and coagulation systems with TLA. Consequently, we seek to evaluate the differences in the testing workflow and reporting efficiency of the blood and coagulation systems after being connected to TLA. Methods Abbott GLP systems Track was the TLA system for connecting with blood and coagulation systems. Two Sysmex XN-9100 fully automatic hematology systems and two Sysmex CS-5100 fully automatic coagulation analyzers were developed for blood and coagulation system, respectively. TUNYEN Laboratory Information System was established for Laboratory information system (LIS). Results Through the analysis of quality indicators and integration with intelligent information systems, improvements have been made, including the reduction of blood collection tube usage, efficient manpower utilization management, tracking of specimen progress and achievement of timely reporting rates.(1)Approximately 2,915 blood collection test tubes are saved every month, with a converted cost of 0.15 USD per tube, and a total saving of 437 USD. And reduce the carbon emissions of 32 kgCO2e, comply with the spirit of ESG sustainable development.(2)The TLA no longer requires staff to collect and deliver specimens as in the past,reducing staff and enhancing work efficiency.One of manpower was saved and new inspection items were developed.(3)Tracking of specimen progress:The laboratory has set up a specimen tracking system to monitor the specimen delivery location and provide timely response to the clinical status.The one-hour TAT for blood and coagulation increased from 82.0% and 76.9% before improvement to 91.8% and 93.3% after improvement respectively. Conclusions The implementation of Laboratory Automation Track Systems (TLA) leads to a reduction in medical errors and specimen sample volume, while also enhancing accuracy and precision, thereby improving the safety of laboratory staff. Although the introduction of TLA may require an initial investment, it brings the benefits of automation, offering ease and efficiency to laboratory processes.

A-070 Evaluation of Plain and Serum-Separator Blood Collection Tubes for the Measurement of Steroid Hormones by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Abstract Background Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is the preferred method for quantifying steroids. Commonly measured steroids 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and dehydroepiandrosterone (DHEA). Although serum separator blood collection tubes may not be recommended but their frequent use is attributed to the ease of serum separation. Moreover, the serum separator tube may be the only available option for analysis, without the need for additional sample collection. Under such conditions, from certain serum separator tubes, we observed occasional interference in LC-MS/MS steroid method. We pursued to evaluate the performance of blood collection tubes without and those with serum separators, for the quantification of specified steroids by LC-MS/MS. Methods Blood samples were collected from 10 volunteers using both plain and serum separator blood collection tubes procured from two major suppliers (Greiner and BD). The specified steroids were measured by LC-MS/MS method. The procedure involved addition of labeled internal standards to the samples and extraction of steroids in methyl tert-butyl ether. Subsequently, the extract dried under a stream of nitrogen, and the residue was reconstituted in the mobile phase. LC-MS/MS analysis involved multiple reaction monitoring (MRM) for the specified steroids and their internal standards. Results The figure shows a sample chromatogram from a subject with blood samples collected in a plain (A) and serum separator tubes from Greiner (B) and BD (C1 and C2, a zoomed of C1). In this sample, from the plain tube, the concentrations of 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone and DHEA were 27, 55, 690, 99 and 283 ng/mL respectively. No significant interference was observed in Greiner serum separator tube. Whereas BD serum separator tube exhibited several erroneous peaks making the quantitation of several steroids challenging or inaccurate. Conclusions In the described LC-MS/MS method, BD serum-separator tubes exhibited erroneous peaks. These peaks were not seen in Greiner serum-separator tubes.

A-122 Evaluation of the Performance of the BD MiniDraw™ Capillary Blood Collection System Under Various Transport Conditions

Abstract Background Specimen transport is a vital component of the preanalytical phase. External factors such as temperature fluctuations, vibrations, shock, and prolonged transport times may negatively impact specimen integrity and compromise test results. A study was performed to evaluate the effects of temperature extremes and simulated transportation on specimen quality in the BD MiniDraw™ Capillary Blood Collection System with BD MiniDraw™ H&amp;H and SST™ Capillary Blood Collection Tubes (BD MiniDraw™ H&amp;H, BD MiniDraw™ SST™). The BD MiniDraw™ Capillary Blood Collection System enables capillary blood collection by trained healthcare workers in non-traditional settings (i.e., retail pharmacies and clinics). Methods Sixteen adult participants were enrolled from the on-site Associate Sample Collection Program. Venous blood was collected via venipuncture into reference comparator and no-additive tubes. Venous blood from no-additive tubes was then pooled into a secondary container for aliquoting into BD MiniDraw™ SST™ and MiniDraw™ H&amp;H tubes. Thirty tubes per tube type were separated into 6 groups with storage and simulated transport conditions conducted in accordance with ASTM D7386-16 and ASTM D4169-22, as applicable: Group 1a: No transport, 2-8°C storage uncontrolled humidity, 49-52-hour storage; Group 1b: Air/road transport, 2-8°C storage uncontrolled humidity, 49-52-hour storage; Group 2a: No transport, 20-24°C storage, uncontrolled humidity, 5-7-hour storage; Group 2b: Road transport, 20-24°C storage, uncontrolled humidity, 5-7-hour storage; Group 3a: Road transport, 38-40°C storage, 90% relative humidity, 5-7-hour storage; Group 3b: Air/road transport, 38-40°C storage, 90% relative humidity, 49-52-hour storage Following clotting, centrifugation, and storage for the BD MiniDraw™ SST™ tubes, testing was performed for selected chemistry analytes (Albumin, Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALKP), Blood Urea Nitrogen, Chloride, Calcium, Cholesterol, Creatinine, High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Sodium, Total Bilirubin (TBIL), Total Protein, Triglycerides) on the Siemens Atellica® Solution. Testing was performed for hemoglobin/hematocrit on the Sysmex XN-1000 in the BD MiniDraw™ H&amp;H Tubes. Visual quality (barrier formation, hemolysis) and tube integrity (physical damage, blood leakage, cap removal, tube fit into adaptor) were also evaluated. Mean biases and confidence intervals within the specific analyte clinical acceptance limit for each group comparison was considered clinically equivalent. Results Clinical equivalence was demonstrated for all analytes for each group comparison except ALKP, ALT, HDL, LDL, TBIL and hematocrit for the extreme transport/storage conditions for Group 3b versus Group 3a. Complete barrier formation with acceptable visual hemolysis was noted in all specimens post transport. Tube integrity was maintained post transport as well. Conclusions Analytes were not impacted across a wide range of temperature and transport conditions. However, exposing samples to extreme high temperature, high humidity conditions may affect analyte performance. This reiterates the importance of monitoring conditions to ensure accurate test results. No impact to visual observations of sample quality and tube physical integrity was noted at different storage and transportation extremes.

B-242 Improved Patient Satisfaction With Novel Capillary Blood Collection Devices as an Alternative to Venipuncture for Applications in Clinical Proteomic Assays

Abstract Background Two new clinical diagnostic tests are available that aid in the identification of lung nodules as likely malignant or likely benign. One test is an ELISA that measures the levels of seven autoantibodies to tumor-associated antigens that aids in the identification of patients with likely malignant lung nodules who may benefit from timely intervention. The other is an LC-MS/MS-based (MRM) test that measures the ratio of two proteins (LG3BP and C163A) combined with several clinical and radiological factors to aid in the identification of patients with likely benign lung nodules who may benefit from additional CT surveillance. Since commercialization, we have found that test access may be improved by providing alternatives to traditional venipuncture. Therefore, we conducted evaluation studies with two novel non-fingerstick capillary blood collection devices. These devices puncture the skin by utilizing either microneedles (Device A) or a lancet (Device B) for capillary blood collection. Methods Blood specimens were collected from consented donors using one of the two capillary blood collection devices and by venipuncture into K2EDTA blood collection tubes. Feedback was also solicited from the healthcare professionals and donors via a standard questionnaire. The whole blood specimens were shipped to the testing laboratory where the samples were processed according to the respective clinical assay workflows. Feasibility testing for the ELISA-based assay was conducted with both devices using normal healthy donor specimens (105 donors for Device A and 43 for Device B). Clinical evaluation was executed utilizing only Device B (77 donors with lung cancer and 13 with an indeterminate lung nodule). Feasibility testing for the LC-MS/MS-based assay was conducted with both devices using healthy donor specimens (103 donors for Device A and 41 for Device B), while clinical evaluation utilized only Device B (80 donors with lung cancer and 30 with an indeterminate lung nodule). Results Both capillary blood collection devices revealed good correlations to venous blood draws for the ELISA-based test during feasibility. The two devices also showed good correlations for the LC-MS/MS-based test, with the results from Device A requiring a correction factor. Due to technical and operational challenges throughout feasibility, Device B became the preferred device to be evaluated for the clinical study. Good correlations for both diagnostic tests were observed from the clinical study. During clinical evaluation, healthcare professionals and donors were asked to rate the ease-of-use, pain, and speed of collection when using Device B. Most healthcare professionals (108/126) rated the device as easy to use. Ninety-five percent of the donors reported minimal pain while the remainder reported moderate pain. Ninety-one percent of the donors felt that the time required to collect blood using Device B was acceptable. Sixty percent of the donors preferred the capillary collection device (Device B), 17% preferred venipuncture, while 23% reported no preference for blood collection. Conclusions The novel capillary blood collection devices demonstrated high correlations for two clinical diagnostic tests. Feedback collected from healthcare professionals and donors revealed that the majority preferred the capillary blood collection device as compared to traditional venipuncture for blood collection.

B-040 Direct Aspiration of Cryogenic Matrix Tubes on the Siemens Atellica System With Epic-Beaker Integration of External Barcode IDs for Analysis Small Volume Biobanked Specimens

Abstract Background Most core chemistry and immunochemistry clinical analyzers are designed for direct sampling of primary blood tubes with volumes ranging from 3.5-10 mL. This limits analysis of low volume pediatric specimens and biobanked aliquoted samples from clinical trials without manual technologist intervention. This work outlines a microsampling and data transmission scheme for analysis of 500 uL aliquots of biobanked serum collected for the Nutrition for Precision Health study powered by the NIH All of Us Research Program without relabeling secondary aliquot tubes after receipt. Methods Commercial plastic clip bearings sourced from IGUS (LCM-08-02) were used to stabilize the matrix tube in a standard 5 mL Sarstedt aliquot tube. 96-well format Matrix Tubes (ThermoFisher 3719-11) with pre-printed 10-digit side barcodes were programmed in the Atellica software as a custom false bottom tube with specifications height 75 mm, diameter 12 mm, bottom offset 41 mm and were loaded using a dedicated false bottom tray. Manifest files (.csv) containing the sample ID, clinical trials participant ID, collection date/time, age and sex were ingested into Epic via an Incoming Orders HL7 interface. Non-human patient records were generated using the participant ID as an external patient ID and patient name and calculating the Date of Birth based on the age in years given in the manifest file. Test orders were created based on assigned testing for the sample ID in the manifest. The sample ID corresponding the preprinted Matrix Tube barcode ID was inserted into the Epic container identifier field. Results Test orders were automatically transmitted to the Atellica Data Manger (ADM) allowing specimens to be run on the Atellica System (CH and IM modules) and results transmitted to Epic utilizing the established Data Innovations (DI) interface and processes used for patient samples without the need for Epic generated labels. Successful scanning and recognition of Matrix Tube barcodes was 100%. Complete aspiration of a Comprehensive Metabolic Panel, Lipid Panel, Iron, TIBC, hsCRP, insulin, and H, I, L indices occurred for 50% of samples tested without intervention. Approximately 250 uL of the 500 uL aliquot remained after aspiration of all tests (247.7 uL total volume) from the Matrix Tube. No probe crashes were observed despite the narrower 8 mm diameter of the Matrix Tubes versus the standard 12- or 13-mm diameter of primary blood collection tubes. Conclusions This Epic-DI-ADM data workflow and microsampling adaptation for Matrix Tube aspiration allows greater container type flexibility for the receipt and analysis of external samples delivered in non-primary blood tubes for clinical laboratories. Future work involving adjustment of the false bottom tube offset depth may reduce unusable sample volume allowing for a higher percentage of complete aspiration without intervention. This work is significant because it suggests existing clinical analyzers are easily adaptable to microsampling smaller volume tubes without significant effort.