Patient Samples Research Articles

Isolating and Identification Of Bacteria Causing Urinary Tract Infections From Hospital Patients And Nursing Staff

The current study aimed to isolate and identification the bacteria that cause urinary tract infections from recumbent patients and nursing staff, using traditional and molecular methods. 700 Clinical samples were collected from Azadi Teaching Hospital and Kirkuk Teaching Hospital in Kirkuk city for the period from October 2023 to March 2024 from (500) recumbent patients and 200 nursing staff. 347 (49.6%) of the total samples showed positive results for bacterial growth when cultured on ideal culture media, including mannitol, blood, and MacConkey agars. 353(50.4%) of total samples were appear as negative results for bacterial growth. The findings showed the distribution of bacterial isolates obtained from urine samples of recumbent patients and nursing staff at Azadi Teaching Hospital. Escherichia coli (44.6% and 41.67%) was the most prevalent bacterial species, followed by (13.08% and 13.89%) K. pneumoniae, (11.54% and 11.11%) P. aeruginosa. while, the distribution of bacterial isolates obtained from urine samples of recumbent patients and nursing staff at Kirkuk Teaching Hospital. Escherichia coli (46.9% and 51.85%) was the predominant bacterial species, followed by (16.6% and 14.81%) K. pneumoniae and (9% and 11.11%) P. aeruginosa. for molecular study, 7(50.0%) out of a total of 14 E. coli isolates possessed the gyrA gene. On the other hand, 12(100.0%) isolates out of a total of 14 isolates of K. pneumoniae possess the gyrA gene, while 11(91.7%) isolate out of a total of 12 isolates of P. aeruginosa possesses the gyrA gene. It is concluded that the most common cause of urinary tract infections is Escherichia coli followed by K. pneumoniae and P. aeruginosa, and that one of the most important causes of antibiotic resistance is the presence of the gyrA gene, which causes bacterial resistance to fluoroquinolone.

Presumptive Identification of Clinically Significant Hemoglobin Variants Hb E, Hb S, Hb D in Hb A1c Capillary Electrophoresis.

The quantitation of glycated hemoglobin (Hb A1c) represents an average blood glucose level for a period of 2 to 3 months for diagnosing, monitoring, and managing diabetes mellitus. Unreliable results are reported when hemoglobin (Hb) variants are present in the sample. Patients are advised to use an alternate method due to the presence of the variant Hb and a reflex test to Hb electrophoresis to obtain precise information about the Hb variant. The present study utilizes x axis values from Hb A1c capillary electrophoresis (CE) to identify clinically significant Hb variants Hb E, S, and D. Patient samples (n = 60) that showed a variant peak in the Hb A1c test with an x axis value of 190 to 240 were selected for the study. The migration position of the Hb variant (x axis value) and variant percent of the Hb A1c test were compared with the x axis value and variant percent in the Hb electrophoresis test to presumptively identify the variants. The identity of the variants was confirmed using mass spectrometry (MS). Out of 60 samples, 20 samples were identified as Hb E (x axis 225-227), 20 samples were identified as Hb S (x axis 210-214), and 18 samples were identified as Hb D-Punjab (x axis 200-201). Two variants with an x axis value of 194 were identified as an α variant Hb Q India using MS. There is an overall negative shift of the x axis (-1 to -13 units) and a lower variant percent (-0.2% to -8.7%) in Hb A1c CE when compared with Hb electrophoresis. The present study highlights the significance of the x axis value and variant percent to identify clinically significant Hb variants in the Hb A1c CE test.

Does a Concise Patient-reported Outcome Measure Provide a Valid Measure of Physical Function for Cancer Patients After Lower Extremity Surgery?

Current functional assessment tools for orthopaedic oncology are long surveys that contribute to patients' survey fatigue and yet lack the ability to discern meaningful differences in a patient population that is often mobile but unable to perform strenuous activities. We sought to determine whether a shorter, novel tool based on existing, validated surveys could better capture differences in a sample of orthopaedic oncology patients. (1) Can a concise fixed-item functional tool derived from the 50 items in the Toronto Extremity Salvage Score for the lower extremity (TESS LE) and the Lower Extremity Functional Scale (LEFS) demonstrate similar responsiveness in terms of sensitivity and specificity? (2) What is the precision and accuracy of the concise tool compared with the TESS LE and LEFS? Functional outcome data were collected and maintained in a longitudinally maintained database at a single institution. Patients were included in the study if (1) they had undergone a tumor excision or a nononcologic orthopaedic procedure (for example, arthroplasty for osteoarthritis) for a bone or soft tissue tumor affecting lower extremity function, and (2) they had completed the LEFS, TESS LE, and Patient-Reported Outcomes Measurement Information System (PROMIS) global health tool on at least two clinic visits. Between September 2014 and April 2022, we treated 14,234 patients for primary bone or soft tissue sarcoma, metastatic disease to bone, or orthopaedic sequelae of chronic cancer care. Approximately 6% (854 of 14,234) were excluded due to the need of a language translator. Approximately 2% (278 of 13,380) refused or were unable to participate. Seventy-two percent (9433 of 13,102) of the patients had an operation on a lower extremity. Of these, 4% (339 of 9433) of the patients completed the TESS LE, LEFS, and Item 3 of the PROMIS global health tool on ≥ 2 clinic visits. Of the patients in the current study, 49% (167 of 339) were women, and 27% (93 of 339) had metastatic carcinoma. Twelve percent (41 of 339) of the patients died before the end of the study period. Spearman rank-order correlation, principal component analysis (PCA), and item response theory (IRT) modeling identified 14 highly discriminating items from the TESS LE and LEFS. Multiple linear stepwise regression (MLSR) was performed with the dependent variable being the summary score of the 14 items derived from the TESS LE and LEFS and standardized to a percentage of 100. The beta coefficient from the MLSR was used to derive a weight for each of the 14 items. Evaluation of the model with 10 to 17 variables was performed to ensure that the model with the 14 items met the most criteria for fit with the PCA, the receiver operating characteristic (ROC) curve, and the IRT modeling criteria. The responsiveness (sensitivity and specificity) of the change scores in the shortened 14-item survey, the 30-item TESS LE, and the 20-item LEFS as compared with the dichotomized changes in Item 3 of the PROMIS global health tool was evaluated using ROCs. The concordance (accuracy and precision) of the 14 items derived from the LEFS and TESS LE was evaluated. The responsiveness (sensitivity and specificity) of the shortened 14-item survey, the TESS LE, and the LEFS to the criterion target of the PROMIS global health tool (Item 3) was similar, with areas under the curve (AUCs) ranging from 0.62 to 0.65 for the ROC curves. The responsiveness of the 14-item survey to the TESS LE showed sensitivity of 96% and specificity of 90%, with an AUC of 0.98 (p < 0.001). The responsiveness of the 14 items to the LEFS showed sensitivity of 95% and specificity of 86%, with an AUC of 0.96. The validity of the 14 items to the TESS LE was measured by concordance, with a precision of 0.98 and an accuracy of 0.97. Concordance of the 14 items to the LEFS showed a precision of 0.98 and accuracy of 0.83. The concise 14 items derived from patient-reported responses in the TESS LE and LEFS outcome measures showed similar responsiveness (sensitivity and specificity) as the original TESS LE and LEFS for cancer patients after lower extremity orthopaedic surgery performed for oncologic and nononcologic indications. The concise 14 items have a similar ability to the TESS LE and LEFS to tell the clinician or patient how they are functioning compared with other patients. These 14 items are shorter than the combined 50 items of the TESS LE and LEFS while retaining the capacity to describe a broad range of lower extremity function for orthopaedic oncology patients. We have named the 14-item survey the Lower Extremity Oncology Functional Assessment Tool.Level of Evidence Level II, diagnostic study.

Immune triggers preceding neuralgic amyotrophy.

Infections and vaccinations have been identified as potential immunological triggers of neuralgic amyotrophy (NA), but the exact type and frequency of the preceding agents is unknown. This was a multicentre, prospective, observational, matched case-control study. NA was diagnosed by neuromuscular experts according to validated clinical criteria and electrodiagnostic studies. Clinical data and biological samples of NA patients were collected within 90 days from disease onset between June 2018 and December 2023. All NA patients were asked about prior infection and vaccination in the month before disease onset. Serological tests for hepatitis E virus, human immunodeficiency virus, severe acute respiratory syndrome coronavirus 2, Epstein-Barr virus, cytomegalovirus, parvovirus B19, varicella-zoster virus, Borrelia burgdorferi, Mycoplasma pneumoniae and Bartonella henselae were performed in a central laboratory. Each case was matched with a healthy control for age, sex, place of residence and time of blood collection. Fifty-seven patients and corresponding controls were included. The mean age was 45 years for both groups. NA onset was preceded by a symptomatic infectious trigger confirmed by microbiological tests in 15/57 (26.3%) patients. Coronavirus disease 2019 vaccination was considered a potential trigger in 7/57 (12.3%) subjects. An acute viral infection was associated with a bilateral involvement of the brachial plexus (p = 0.003, Cramèr's V = 0.43). Confirmed immune triggers (infection or vaccination) preceded disease onset in 22/57 (38.6%) NA cases. We suggest to test NA patients in the acute phase for intracellular antigens, especially in the case of concomitant bilateral involvement and hepatitis.

Resting-state functional connectivity in anxiety disorders: a multicenter fMRI study.

Anxiety disorders (AD) are associated with altered connectivity in large-scale intrinsic brain networks. It remains uncertain how much these signatures overlap across different phenotypes due to a lack of well-powered cross-disorder comparisons. We used resting-state functional magnetic resonance imaging (rsfMRI) to investigate differences in functional connectivity (FC) in a cross-disorder sample of AD patients and healthy controls (HC). Before treatment, 439 patients from two German multicenter clinical trials at eight different sites fulfilling a primary diagnosis of panic disorder and/or agoraphobia (PD/AG, N = 154), social anxiety disorder (SAD, N = 95), or specific phobia (SP, N = 190) and 105 HC underwent an 8 min rsfMRI assessment. We performed categorical and dimensional regions of interest (ROI)-to-ROI analyses focusing on connectivity between regions of the defensive system and prefrontal regulation areas. AD patients showed increased connectivity between the insula and the thalamus compared to controls. This was mainly driven by PD/AG patients who showed increased (insula/hippocampus/amygdala-thalamus) and decreased (dorsomedial prefrontal cortex/periaqueductal gray-anterior cingulate cortex) positive connectivity between subcortical and cortical areas. In contrast, SAD patients showed decreased negative connectivity exclusively in cortical areas (insula-orbitofrontal cortex), whereas no differences were found in SP patients. State anxiety associated with the scanner environment did not explain the FC between these regions. Only PD/AG patients showed pronounced connectivity changes along a widespread subcortical-cortical network, including the midbrain. Dimensional analyses yielded no significant results. The results highlighting categorical differences between ADs at a systems neuroscience level are discussed within the context of personalized neuroscience-informed treatments. PROTECT-AD's registration at NIMH Protocol Registration System: 01EE1402A and German Register of Clinical Studies: DRKS00008743. SpiderVR's registration at ClinicalTrials.gov: NCT03208400.

Risk Factors for Gastric Cancer at RSUP Prof. Dr. I.G.N.G. Ngoerah Year 2018-2023

Gastric cancer is the fifth most common cancer and the fourth most common cause of death due to cancer in the world. The development of gastric cancer tends to be quite slow and takes years, so it is often found in the late stages. The cause of stomach cancer is not known for certain, but it is believed to be caused by many factors. This study aims to determine the risk factors for gastric cancer at RSUP Prof. Dr. I.G.N.G. Ngoerah was diagnosed in 2018-2023. This research was conducted using a retrospective descriptive method using secondary data in the form of medical records on 46 samples of gastric cancer patients who were controls or treated at RSUP Prof. Dr. I.G.N.G. Ngoerah was diagnosed in 2018-2023 The variables studied include the dependent variables, namely age, gender, peptic ulcers, blood type, obesity, alcohol consumption and smoking. The results of the analysis showed that gastric cancer patients at RSUP Prof. Dr. I.G.N.G. The majority are over 50 years old (82.6%), male (58.7%), have no peptic ulcers (89.1%), have blood type A (37%), underweight (50%), are not consume alcohol (97.8%), and do not smoke (97.8%).It can be concluded that the risk factors for gastric cancer patients at RSUP Prof. Dr. I.G.N.G. Ngoerah majority are over 50 years old, male, have no peptic ulcers, have blood type A, are underweight, do not consume alcohol and do not smoke. Keywords: Gastric Cancer, Risk Factors, RSUP Prof. Dr. I.G.N.G. Ngoerah

Understanding the journey towards rabies vaccination for travellers: Results of a cross-sectional survey with patients and providers in the US, Germany, Sweden, and Switzerland

BackgroundAlthough cases of rabies in international travellers are uncommon, they are a fatal risk which can be alleviated through vaccination prior to travel. As international travel recovers post-COVID, it is vital that travellers are made aware of the risk of rabies when travelling to endemic countries and supported to receive the vaccine when eligible. MethodsOnline surveys were conducted in the US, Germany, Sweden and Switzerland between November 2022–January 2023 with both patients and healthcare providers (HCPs). Eligibility criteria for patients included those eligible for rabies pre-exposure prophylaxis (PrEP) due to travel location and activity; HCPs had to be providers of travel vaccinations. In both surveys, questions were asked about discussion of rabies vaccination, decision of whether to administer a rabies vaccine, recommendation to get the rabies vaccine, and final decision to get a vaccine. ResultsThe final patient sample included n = 1557 patients who were eligible for rabies pre-exposure prophylaxis (US n = 504, Germany n = 353, Sweden n = 350, Switzerland n = 350) and n = 219 HCPs (US n = 75, Germany n = 75, Sweden n = 32, Switzerland n = 37). Although all patients in the sample were eligible for rabies vaccination, only 15 % felt they were at risk of getting rabies, and only 18 % received the rabies vaccine before their trip. HCPs reported discussing PrEP and/or PrEP and PEP with 30 % of patients presenting for travel vaccination advice, on average. ConclusionsAwareness and perception of rabies risk, and lack of consistent HCP discussion of the need for rabies PrEP may be major barriers to uptake of the vaccine for patients who are eligible to receive it.

Selective inhibition of HDAC class IIA as therapeutic intervention for KMT2A-rearranged acute lymphoblastic leukemia

KMT2A-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards histone deacetylase (HDAC) inhibition. Most broad-spectrum HDAC inhibitors simultaneously target multiple human HDAC isoforms. Consequently, they often induce toxicity and especially in combination with other therapeutic agents. Therefore, more specifically targeting HDAC isoforms may represent a safer therapeutic strategy. Here we show that shRNA-mediated knock-down of the class IIA HDAC isoforms HDAC4, HDAC5, and HDAC7 results in apoptosis induction and cell cycle arrest in KMT2A-rearranged ALL cells. In concordance, the HDAC4/5 selective small molecule inhibitor LMK-235 effectively eradicates KMT2A-rearranged ALL cell lines as well as primary patient samples in vitro. However, using a xenograft mouse model of KMT2A-rearranged ALL we found that the maximum achievable dose of LMK-235 was insufficient to induce anti-leukemic effects in vivo. Similar results were obtained for the specific class IIA HDAC inhibitors MC1568 and TMP195. Finally, LMK-235 appeared to exert minimal anti-leukemic effects in vivo in combination with the BCL-2 inhibitor venetoclax, but not enough to prolong survival in treated mice. In conclusion, class IIA HDAC isoforms represent attractive therapeutic target in KMT2A-rearranged ALL, although clinical applications require the development of more stable and efficient specific HDAC inhibitors.

Expedient measurement of total protein in human serum and plasma via the biuret method using fiber optic probe for patient samples and certified reference materials.

The biuret method is currently recognized as a reference measurement procedure for serum/plasma total protein by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). However, as the reaction involved in this method is highly time-dependent, to ensure identical measurement conditions for calibrator and samples for high accuracy, a fast and simple measurement procedure is critical to ensure the precision and trueness of this method. We measured serum/plasma total protein using a Cary 60 spectrophotometer coupled with a fiber optic probe, which was faster and simpler than the conventional cuvette method. The biuret method utilizing alkaline solutions of copper sulfate and potassium sodium tartrate was added to the sample and calibrator (NIST SRM 927e) incubated for 1h before measurement. A panel of samples consisting of pooled human serum, single donor serum, and certified reference materials (CRMs) from three sources were measured for method validation. Sixteen native patient samples were measured using the newly developed biuret method and compared against clinical analyzers. Additionally, the results of three cycles of a local External Quality Assessment (EQA) Programme submitted by participating clinical laboratories were compared against the biuret method. Our biuret method using fiber optic probe demonstrated good precision with within-day relative standard deviation (RSD) of 0.04 to 0.23% and between-day RSD of 0.58%. The deviations between the obtained values and the certified values for all three CRMs ranged from -0.38 to 1.60%, indicating good method trueness. The routine methods using clinical analyzers were also found to agree well with the developed biuret method using fiber optic probe for EQA samples and native patient samples. The biuret method using a fiber optic probe represented a convenient and reliable way of measuring serum total protein. It also demonstrated excellent precision and trueness using CRMs and patient samples, which made the method a simpler candidate reference method for serum protein measurement.

Association between length of hospital stay before and after surgery and nutritional risk according to NRE-2017 - A secondary analysis of a cohort study.

to evaluate the predictive ability of the Nutritional Risk Emergency - 2017 (NRE) to predict prolonged length of stay, ICU admission intra-mortality and readmission, severe postoperative complications. a prospective cohort was conducted with surgical patients admitted in a public tertiary hospital. The NRE-2017 tool was applied for detecting malnutrition risk in hospitalized patients. Surgical complications were assessed by Clavien-Dindo. Patients were followed during hospitalization to identify length of stay as well as stay after surgery in the Intensive Care Unit (ICU). Regression analysis was performed to assess the association between risk of malnutrition and clinical outcomes. we included 162 elective surgery patients; 79 patients were identified with nutritional risk using the NRE-2017 (≥ 1.5) tool and 83 without nutritional risk. Patients with nutritional risk were at higher risk of prolonged hospitalization [18 (10-36) days vs. 13 (7-23 days); p: 0.006] and ICU hospitalization [6 (2-14 days vs. 3.5 (1-7 days; p: 0.020]. There was an association between surgical complications and nutritional risk independently, but the significance was lost when adjusting the analysis. There was no association with mortality and readmission in this sample of patients. the NRE-2017 tool was associated with hospital stay in those patients at nutritional risk, however there was no association with mortality and readmission.

Clinical, developmental and serotonemia phenotyping of a sample of 70 Italian patients with Phelan-McDermid Syndrome

BackgroundPhelan-McDermid syndrome (PMS) is caused by monoallelic loss or inactivation at the SHANK3 gene, located in human chr 22q13.33, and is often associated with Autism Spectrum Disorder (ASD).ObjectivesTo assess the clinical and developmental phenotype in a novel sample of PMS patients, including for the first time auxometric trajectories and serotonin blood levels.Methods70 Italian PMS patients were clinically characterized by parental report, direct medical observation, and a thorough medical and psychodiagnostic protocol. Serotonin levels were measured in platelet-rich plasma by HPLC.ResultsOur sample includes 59 (84.3%) cases with chr. 22q13 terminal deletion, 5 (7.1%) disruptive SHANK3 mutations, and 6 (8.6%) ring chromosome 22. Intellectual disability was present in 69 (98.6%) cases, motor coordination disorder in 65 (92.9%), ASD in 20 (28.6%), and lifetime bipolar disorder in 12 (17.1%). Prenatal and postnatal complications were frequent (22.9%-48.6%). Expressive and receptive language were absent in 49 (70.0%) and 19 (27.1%) cases, respectively. Decreased pain sensitivity was reported in 56 (80.0%), hyperactivity in 49 (80.3%), abnormal sleep in 45 (64.3%), congenital dysmorphisms in 35 (58.3%), chronic stool abnormalities and especially constipation in 29 (41.4%). Parents reported noticing behavioral abnormalities during early childhood immediately after an infective episode in 34 (48.6%) patients. Brain MRI anomalies were observed in 53 (79.1%), EEG abnormalities in 16 (23.5%), kidney and upper urinary tract malformations in 18 (28.1%). Two novel phenotypes emerged: (a) a subgroup of 12/44 (27.3%) PMS patients displays smaller head size at enrollment (mean age 11.8 yrs) compared to their first year of neonatal life, documenting a deceleration of head growth (p < 0.001); (b) serotonin blood levels are significantly lower in 21 PMS patients compared to their 21 unaffected siblings (P < 0.05), and to 432 idiopathic ASD cases (p < 0.001).ConclusionsWe replicate and extend the description of many phenotypic characteristics present in PMS, and report two novel features: (1) growth trajectories are variable and head growth appears to slow down during childhood in some PMS patients; (2) serotonin blood levels are decreased in PMS, and not increased as frequently occurs in ASD. Further investigations of these novel features are under way.

Adrenal Vein Sampling for Primary Aldosteronism: Recommendations From the Australian and New Zealand Working Group.

Adrenal vein sampling (AVS) is the current recommended procedure for identifying unilateral subtypes of primary aldosteronism (PA), which are amenable to surgery with the potential for cure. AVS is a technically challenging procedure usually undertaken by interventional radiologists at tertiary centres. However, there are numerous variations in AVS protocols relating to patient preparation, sampling techniques and interpretation which may impact the success of AVS and patient care. To reduce practice variations, improve the success rates of AVS and optimise patient outcomes, we established an Australian and New Zealand AVS Working Group and developed evidence-based expert consensus recommendations for the preparation, performance and interpretation of AVS. These recommendations can be used by all healthcare professionals in a multidisciplinary team who look after the diagnosis and management of PA.

Traditional Indigenous-Amazonian Therapy Involving Ceremonial Tobacco Drinking as Medicine: A Transdisciplinary Multi-Epistemic Observational Study.

Although the tobacco plant has been employed as a medicinal and sacred herb by Indigenous cultures across the Americas, its usage drastically changed after the 15th-century colonial arrival; its large-scale commodification and global marketing once brought to Europe lead to hedonic and addictive uses harmful to health. As a consequence, tobacco smoking is now one of the largest public health problems worldwide. However, in the Peruvian Amazon, a region of origin of tobacco species, Indigenous healers still know how to use the plant for therapeutic purposes. Due to a general disregard of Indigenous knowledge and stigma, these uses have however not so far been clinically investigated. We hence conducted for the first time a clinical field study assessing a sample of patients treated by a traditional healer specialized in tobacco in the Peruvian Amazon (observational design, pilot study, N = 27). The study was conducted within a transdisciplinary and multi-epistemic medical frame, in close partnership with an Amazonian healer. We used validated self-report scales to quantitatively assess mental health variables before and after the weeklong treatment, and mixed-methods to report experienced effects. Paired-samples t-tests comparing pre- and post-treatment scores revealed significant reductions in anxiety, depression, perceived stress, and general symptom indicators. Experienced effects included initial physical discomfort, followed by psychologically or existentially/spiritually significant insights. Our findings point to a sophisticated therapeutic approach based on Indigenous knowledge of tobacco applications, which should be further investigated. The study also contributes to the burgeoning scientific field on therapeutic uses of contentious psychoactive plants.

Massively parallel reporter assays identify enhancer elements in oesophageal Adenocarcinoma.

Cancer is a disease underpinned by aberrant gene expression. Enhancers are regulatory elements that play a major role in transcriptional control and changes in active enhancer function are likely critical in the pathogenesis of oesophageal adenocarcinoma (OAC). Here, we utilise STARR-seq to profile the genome-wide enhancer landscape in OAC and identify hundreds of high-confidence enhancer elements. These regions are enriched in enhancer-associated chromatin marks, are actively transcribed and exhibit high levels of associated gene activity in OAC cells. These characteristics are maintained in human patient samples, demonstrating their disease relevance. This relevance is further underlined by their responsiveness to oncogenic ERBB2 inhibition and increased activity compared to the pre-cancerous Barrett's state. Mechanistically, these enhancers are linked to the core OAC transcriptional network and in particular KLF5 binding is associated with high level activity, providing further support for a role of this transcription factor in defining the OAC transcriptome. Our results therefore uncover a set of enhancer elements with physiological significance, that widen our understanding of the molecular alterations in OAC and point to mechanisms through which response to targeted therapy may occur.

A combinatorial culture strategy to develop pseudomyxoma peritonei organoid models.

Few preclinical models of pseudomyxoma peritonei (PMP) have been developed, probably due to the tumor's low incidence and its peculiar characteristics of slow growth. Therefore, there is a need to develop more refined PMP models that better reflect its characteristics. The aim of the study is to develop a culture strategy to generate organoid models derived from PMP patient samples. We followed a strategy based on combinatorial culture conditions that include the different factors essential for PMP growth and that mimic the microenvironment present in the patients. We cultured PMP samples in the presence of the various factors produced by the niche environment of PMP. We obtained 12 PMP organoid models, each of which grows under specific culture conditions. PMP-derived organoids show long-term expansion capacity and reproduce the genetic landscape and histological phenotype of the tumor of origin. The organoids we developed faithfully reproduce the key features of PMP disease and will allow us to understand the biology of PMP. With them, we will be able to identify key regulatory networks that support PMP progression, providing a platform for multilevel preclinical testing, identify novel diagnostic biomarkers, and generate novel targets for patient treatments.

B-310 Freezing and thawing serum without mixing prior to analysis causes false low Thyroglobulin results

Abstract Background Thyroglobulin (Tg), a glycoprotein that is synthesized in follicular cells of the thyroid gland, is the precursor of Thyroxin (T4) and Triiodothyronine (T3). Tg is a biomarker used for many clinical conditions, including monitoring for recurrence in patients with differentiated thyroid carcinoma. Typically, Tg levels drop to very low or undetectable levels after surgical excision and/or radioiodine ablation of the thyroid, with post-operative levels greater than 1-2 ng/ml indicating potential disease recurrence. Discordant Tg results in a patient sample were identified during a quality check of Tg results. This study investigates the root of cause of the discrepant Tg results. Given that: 1) Tg analyses were performed twice a week at our laboratory due to low daily test volume and patients’ samples received on an unanalytical day were stored at -20°C until analysis; 2) Tg analyses were performed by various medical technologists; 3) sample mixation prior to analysis was not routine practice for our automated laboratory, we hypothesized that the discrepancy was due to inconsistent sample handling, e.g., mixation vs unmixation prior to analysis. Methods Analysis of the daily QC and PT results in the period of the incident occurence ruled out causes in analytical phase. We split 52 de-identified patient serum samples into two equal aliquots and subsequently stored both aliquots at -20°C for 48 hours. Both aliquots of each sample were thawed at room temperature for 1 hour. One of the aliquots was analyzed for Tg without mixing, and the other was analyzed after mixing using a Vortexer for 4 seconds at 250 xg. To investigate if Tg concentrations form a gradient from the top to the bottom of a red-top vacutainer tube, five patient samples with Tg at 2.59, 2.79, 2.92, 52.37 and 144.12 ng/mL were thawed without mixing following storage at -20°C for 48 hours. Five layer (600 μl/layer) aliquots were carefully and sequentially taken from top to bottom for each specimen, and the Tg concentration in the aliquot of each layer was determined using the Beckman Coulter Access-2 Immunoassay. Results Of the 52 patients samples, 14 Tg results were excluded from statistical analysis due to exceeding the assay dynamic range (2 above and 12 below the upper and low limits, respectively). Analysis of the remaining 38 patient samples showed the mean of Tg concentrations was 22.8% (SD 18%) higher in mixed samples than the un-mixed samples. Regression analysis showed an R2 = 0.9976 (Mixed Tg = 1.1523 x Un-mixed + 0.4174, range 0.1 - 275.7 ng/mL, intercept 95%CI -0.721 to 1.556, slop 95%CI 1.1331 to 1.1715, p-value &amp;lt;0.001). Results of Tg gradient analysis showed significant higher Tg values in the 2nd, 3rd, 4th and 5th than the 1st layer by 26% (SD 11%), 63% (26%), 112% (57%), and 160% (76%), respectively. Conclusions False Tg values were found in samples without mixing before analysis after thawing from frozen state, due to formation of Tg concentration gradient in test tube. For obtaining reliable results, samples must be mixed before analysis when thawed from frozen storage.

A-085 Comparing CO2 stability in patient samples and proficiency testing material

Abstract Background Total carbon dioxide (tCO2) measurements are important in the assessment of acid-base disorders. The collection and storage conditions of specimens are important preanalytical factors that can influence the accuracy of tCO2 measurements. Proficiency testing (PT) materials are used to assess the accuracy and reliability of a laboratory’s test performance. Unsuccessful performance on PT challenges can be an indicator of procedural issues. However, the stability of tCO2 in PT material compared to physiologic specimens is unknown and no guidance is provided to laboratories on how to handle PT samples for tCO2 measurement. This study aims to compare tCO2 stability in PT material with that of plasma samples when exposed to ambient air at room temperature. Methods Using enzymatic endpoint measurement on Vitros 4600 Chemistry System, tCO2 was measured in PT material (n = 5) and patient samples (n = 6) at baseline and after exposure to air for 15, 30, 60, 90, and 120 minutes. 500μL of each sample was aliquoted into EZ-nest tube adapters and baseline measurements were immediately analyzed. Average percent difference from baseline was calculated and &amp;gt;-10% bias was considered acceptable. Results The average percent difference from baseline in PT samples exceeded -10% as early as 30 minutes; whereas -3% bias was observed in patient samples at 60 minutes. Results are summarized in the table below. Conclusions The assessment of tCO2 stability ensures the reliability of laboratory measurements. Our results indicate tCO2 in PT material is not stable when exposed to air beyond 15 minutes, however tCO2 in most patient samples remained stable beyond 120 minutes. Understanding the stability limits of tCO2 in PT material allows us to devise workflows that prevent sample degradation during PT events. By establishing best practices for maintaining tCO2 integrity in PT material, laboratories can enhance the accuracy of proficiency testing results.

A-111 “Activation” Of macro-ast by pyridoxal phosphate supplementation

Abstract Background Type 1 macro-AST is a high molecular weight AST (Aspartate Aminotransferase) formed between an AST monomer and immunoglobulins in plasma, which can cause elevated AST values. It is believed that reduced clearance or excretion of macro-AST can cause elevated AST results. Here we report a case of macro-AST that produced high AST values only by the AST assay that includes pyridoxal phosphate (PLP), not by the one without PLP. Case description A 54 year-old male patient diagnosed with disseminated Bartonellosis was monitored with ‘liver function” tests on a monthly basis, with AST varying between 24 to 73 U/L (Reference interval, 13-47 U/L). The patient’s AST value suddenly jumped to 378 U/L when the laboratory switched to an AST assay supplemented with PLP (named ASTPM) on Roche Cobas c702 analyzer (c702). When the same patient sample was tested using the previous AST assay which does not include PLP, a result of 14 U/L was obtained. Macro-AST was suspected, as all other “liver function” tests were within reference interval. Methods Protein-A and Protein-G were used to remove immunoglobulins from the patient sample and another sample with matching AST as a control, respectively. Three hundred microliters of washed Protein-A or Protein-G Sepharose bead slurry was mixed with 300 microliters of each sample, respectively. Each mixture was incubated at 37oC for 3 hours on a rocker. After centrifugation, the supernatant was transferred to a new tube. AST, ASTPM, and all other tests were performed on c702, and results corrected for dilution. The recovery of enzyme activity or immunoglobulin concentration after the removal of immunoglobulins was calculated, respectively. Results In the original sample, the ratio of ASTPM to AST was 28 (389 U/L vs. 14 U/L) for the patient, and 1.1 (304 U/L vs. 275 U/L) for the control. After the complete removal of immunoglobulin G (IgG), the recovery of ASTPM for the patient was 3% and for the control was 74%. The recovery of ALTPM (supplemented with PLP) was 89% for the patient and 85% for the control, showing the expected loss of enzymatic activity by Protein-A or Protein-G treatment. Conclusions The significant reduction of ASTPM activity (recovery of 3%) after removal of IgG in the patient sample proved that the AST enzyme was associated with IgG. The slight increase of ASTPM activity compared to AST (ratio of 1.1) in the control sample was an expected effect of supplementing PLP in the AST assay, however, the huge increase of ASTPM activity (ASTPM/AST ratio of 28) in the patient sample suggests that PLP “activated” the macro-AST and produced high AST result. This is a concerning effect of supplementing PLP in AST assays because it can cause spurious AST elevations in cases of macro-AST. As more laboratories begin to employ the PLP supplemented aminotransferase assays, it is important to acknowledge the interactions between PLP and macro-AST and the role of the laboratory to identify macro-AST in order to avoid unnecessary, costly, and time-consuming testing and procedures for these patients.

B-268 Detection of xylazine in fentanyl-positive urine drug screen specimens using a quantitative LCMS/MS method

Abstract Background Fentanyl, a synthetic opioid used clinically for severe pain management, sedation, and as a perioperative analgesic, has been at the forefront of fatal drug-involved overdoses for almost a decade. Xylazine, an α-2 adrenergic receptor agonist, is an increasingly common adulterant in illicitly manufactured fentanyl products and analogs. Given its class, xylazine is non-responsive to naloxone, an opioid receptor antagonist used to reverse opioid overdose. Importantly, the U.S. Drug Enforcement Administration reported xylazine in 23% of seized fentanyl powders and 7% of fentanyl pills; additionally, a recent study by Kariisa et. al. identified an increase of xylazine presence in fatal fentanyl-involved overdoses (2.9% in January 2019 to 10.9% in June 2022) across twenty states.1 Collectively, these trends indicate a need for increased surveillance of xylazine in overdose-presenting patients to inform appropriate clinical responses. In this study, we developed a sensitive LC-MS/MS method for xylazine quantitation and used it to determine xylazine presence in our fentanyl positive patients. Methods An LC-MS/MS method for quantitation of xylazine from urine samples was developed using a Waters Xevo TQ-XS and reversed-phase CORTECS C8 2.7 µm column. MRMs and optimal MS tuning parameters were generated for xylazine and xylazine-d6 via IntelliStart, with transition ions of 221.18→90.04 (Quan) and 221.18→136.31 (Qual) selected for xylazine quantitation against a 227.23→89.98 xylazine-d6 transition as an internal standard. An LC method was developed using water (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) and methanol (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) gradient for a 4-minute runtime. Calibrators were prepared by addition of the pure drug to water, while QCs were prepared using healthy volunteer urine that was confirmed negative in a routine urine drug screen (UDS). Calibrators were value-assigned using a reference laboratory. A simple processing method consisting of the addition of 200 µL methanol containing 25 ng/mL xylazine-d6 to 50 µL of standards, QCs, or urine was used for sample preparation. Remnant fentanyl-positive patient samples (determined by ARK Fentanyl II Immunoassay - Abbott Architect C16000 analyzer) submitted for routine UDS evaluation were collected for xylazine analysis. Results Using custom xylazine standards, the method yielded a linear range from 2 ng/mL to 500 ng/mL with an average R2 of 0.9995 across runs (n = 12). The LLoQ for this assay was 2 ng/mL with a CV of 18.1%. The intra-assay CVs were &amp;lt; 5.0% and inter-assay CVs were &amp;lt; 15.0%. Of preliminary patient samples tested (n = 25), xylazine was detected in 32% of fentanyl-positive samples, ranging in concentration from 2.8 ng/mL to 192.8 ng/mL with an average of 64.8 ng/mL. Conclusions Here we report a simple LC-MS/MS method for quantitation of xylazine in remnant fentanyl-positive UDS specimens by LC-MS/MS. Preliminary testing revealed a xylazine positivity rate of 32% in fentanyl-positive patients. Uncomplicated sample work-up makes this method applicable to xylazine surveillance in all UDS-positive patient samples in high-throughput clinical laboratories. Reference: 1. Kariisa M, et. al. “Illicitly Manufactured Fentanyl-Involved Overdose Deaths with Detected Xylazine.” MMWR Morb Mortal Wkly Rep 2023;72:721-727.

A-115 Iron assay improvement: The need for thiourea addition in Ferene/Ferrozine reagent cartridge

Abstract Background Iron assay is often ordered along with ferritin, transferrin, and iron binding capacity in patients suspected of having iron deficiency anemia or iron overload. A ferene/Ferrozine chromogen-based iron assay is more common in College of American Pathology accredited laboratories across U.S. Recently, a product recall/field safety notice was issued by a major manufacturer for several lots of ferene based iron reagent due to unacceptable positive bias in patient iron results. However, the reason(s) underlying this positive bias was not provided. In this report, we investigated the possible causes for the recall of specific lots of iron assay reagents. Methods The performance of 2 lots of recalled iron test kits was compared against 2 good lots (Sentinel Diagnostics) on the Alinity c analyzer utilizing approved calibrator lots, quality control (QC) materials (Bio-Rad Laboratories), and excess/leftover de-identified patient samples. Spectral scan (in the visible wavelength) of good and recalled lots of reagents, R1 and R2 was performed on the Tecan microplate reader. Trace elements in R1 and R2 reagents were estimated with Quantitative Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Spiking experiments in calibrator, discarded patient samples, and reagents were undertaken to further elucidate the cause and prevention of positive bias seen with recalled reagent lots. Graphs and statistical analysis were generated using Graphpad prism 10.1. Results Iron levels in QC materials and de-identified patient samples (n=42) tested with 2 recalled lot of reagents on Alinity c analyzer showed a positive bias of 15-36% compared to unimpacted lots of reagents. Calibrator and blank absorbance of recalled lots was much higher than the unimpacted lots. Percentage change in the basal absorbance of recalled vs unimpacted R2 reagent measured from 500 to 600 nm in 25 nm increments and corrected to 700 nm ranged from 150 to 250%. No such changes were noted with both R1 reagents. Interestingly, ICP-MS heavy metal panel analysis revealed that R2 reagent of the recalled lots had unanticipated presence of Cu (∼22 υg/dL) and lead (7.5 υg/L), which was not present in R1 reagents. Increased concentrations of Cu spiking (100, 200, and 400 υγ/dL) in R2 reagent from recalled lots discernibly increased the Cal Factor values as well as patient iron results relative to unspiked reagent. Further, the presence of Cu over time deteriorated the accuracy of R2 reagent reflecting on very high rise in Cal Factor and patient iron values. Interestingly, TU addition to R2 reagent from unimpacted lot prevented the Cu-induced spurious rise in Cal Factor and iron measurements in the patient samples. Further, TU exhibited sustained preventive effect on Cu-induced iron measurement changes over time. Conclusions Based on our investigation, we conclude that copper contamination of R2 reagent is the possible cause for the positive bias in the recalled iron assay reagent lots. We recommend the addition of TU to Ferene containing R2 reagent pack as a simple and effective step to prevent Cu-induced false elevation in iron values.