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Objectives We developed and participated in a 1-week laboratory medicine training presented from June 3, 2019, to June 7, 2019. Methods The training was a combination of daily morning lectures and case presentations as well as afternoon practical sessions in the clinical laboratory. The content was selected over months by local organizers and the visiting faculty and further modified on site to reflect local needs. Results Participants identified practice changes that could be realized in the short term but most faced significant barriers to implementation in the absence of structured and long-term follow-up. Conclusions In this report, we review insights learned from our experience and reflect on strategies for realistic, meaningful, and relevant contributions in the setting of laboratory medicine–oriented short-term programs.

Background The anion gap is primarily used in the diagnosis of acid-base disorders. We conducted a study to determine the anion gap reference interval in our patient population, investigated the workup of abnormal vs normal anion gaps, and examined the anion gap variation upon repeated testing. Methods A retrospective review was performed on 17137 adult and pediatric patients who presented to Yale-New Haven Hospital outpatient clinics, emergency department, or intensive care units between 2012 and 2017. Results We derived a new reference interval of 7 to 18 mmol/L with a median of 13 mmol/L in healthy adults with no significant differences owing to partitioning by sex or age. Based on the new reference interval, 5%, 23%, and 18% of healthy, emergency department, and intensive care unit adult patients, respectively, were misclassified as having high values with the previous interval of 6 to 16 mmol/L. However, there were no significant differences in the number of tests ordered in patients with anion gaps above and below the upper limit of the previous reference interval. The majority of increased anion gaps that were repeated normalized by 12 h. In a subgroup of healthy adult patients with annual testing, the median percent change in each patient's anion gap from 2015 to 2016 was approximately 13%. Conclusions The anion gap should be used with an appropriate reference interval to avoid misclassification. There may be a moderate degree of individuality that argues for comparing the anion gap with its baseline value in the same patient pending further studies that formally derive its biological variation.

OBJECTIVES To apply a simple method to validate testing for albumin, glucose, lactate dehydrogenase (LDH) and total protein (TP) in peritoneal, pleural, and cerebrospinal fluids (CSF) at a hospital in Liberia. METHODS Serum and body fluid specimens were mixed to create 100% serum and 25%, 50%, 75%, and 100% fluid tubes, which were tested on a Biotecnica BT3500. Differences less than 10% between calculated and measured concentrations were considered acceptable. RESULTS The means (confidence intervals) of the percent differences were: albumin/peritoneal 12.8 (6.0-19.7), albumin/pleural 2.8 (1.3-4.2), albumin/CSF 4.8 (2.2-7.5), glucose/peritoneal 4.0 (1.9-6.0), glucose/pleural 4.4 (3.1-5.7), glucose/CSF 2.9 (1.8-4.0), LDH/peritoneal 9.5 (6.3-12.7), LDH/pleural 9.5 (5.4-13.6), LDH/CSF 9.2 (5.2-13.3), TP/peritoneal 7.6 (3.8-11.4), TP/pleural 3.8 (1.5-6.2), and TP/CSF 4.5 (1.0-8.1). CONCLUSIONS All mean differences except for one were less than 10%, allowing for the adoption of clinical testing. The mixing study is a low-cost method for quality-assured testing that can be performed by resource-limited laboratories.

Objectives Thyroid dysfunction in pregnancy is associated with increased risk of adverse outcomes to mother and child. Trimester-specific reference intervals for thyroid function tests are not routinely provided by clinical laboratories. In this study, we present first- and second-trimester-specific reference intervals in a US population for thyroid-stimulating hormone (TSH), free thyroxine (FT4), total thyroxine (T4), and total triiodothyronine (T3) measured on Roche analyzers. Methods We used patient samples from first- and second-trimester prenatal screening. Samples were limited to singleton pregnancies and negative screening results for thyroid peroxidase and thyroglobulin antibodies. Analytes (TSH, FT4, T4, and T3) were measured on a Roche Modular e170 then verified on a Roche cobas e801. Results The reference intervals established on the e170 and verified on the e801 for the first trimester were 0.16 to 2.82 mIU/L for TSH, 12.0 to 18.5 pmol/L for FT4, 62.8 to 177.9 nmol/L for T4, and 1.5 to 3.4 nmol/L for T3. The reference intervals for the second trimester were 0.40 to 3.62 mIU/L for TSH, 10.2 to 16.6 pmol/L for FT4, 66.6 to 176.0 nmol/L for T4, and 1.56 to 3.6 nmol/L for T3. Conclusions This is the first report of trimester-specific reference intervals for thyroid function tests on Roche analyzers in the United States, and it is consistent with worldwide reports.

Objectives Despite extensive research on procalcitonin (PCT)-guided therapy in lower respiratory tract infections, the association between PCT and bacterial pneumonia remains unclear. Methods We evaluated retrospectively the performance of PCT in patients presenting with lower respiratory tract infection symptoms and grouped by seven diagnoses. All patients had microbial testing, chest imaging, and CBC counts within 1 day of PCT testing. Results Median PCT level in patients diagnosed with bacterial pneumonia was significantly higher than in patients diagnosed with other sources of infections or those not diagnosed with infections. Median PCT levels were not different among patients grouped by type or quantity of pathogen detected. They were significantly higher in patients with higher pathogenicity scores for isolated bacteria, those with abnormal WBC count, and those with chest imaging consistent with bacterial pneumonia. A diagnostic workup that included imaging, WBC count, and Gram stain had an area under the receiver operating characteristic curve of 0.748, and the addition of PCT increased it to 0.778. Conclusions PCT was higher in patients diagnosed with bacterial pneumonia. Less clear is its diagnostic ability to detect bacterial pneumonia over and above imaging and laboratory data routinely available to clinicians.

Loss of glutamine synthetase (GS) in hippocampal astrocytes has been implicated in the causation of human mesial temporal lobe epilepsy (MTLE). However, the mechanism by which the deficiency in GS leads to epilepsy is incompletely understood. Here we ask how hippocampal GS inhibition affects seizure phenotype and neuronal activation during epilepsy development (epileptogenesis). Epileptogenesis was induced by infusing the irreversible GS blocker methionine sulfoximine (MSO) unilaterally into the hippocampal formation of rats. We then used continuous video-intracranial electroencephalogram (EEG) monitoring and c-Fos immunohistochemistry to determine the type of seizures and spatial distribution of neuronal activation early (1-5days postinfusion) and late (16-43days postinfusion) in epileptogenesis. Early in epileptogenesis, seizures were preferentially mild (stage 1-2), activating neurons in the entorhinal-hippocampal area, the basolateral amygdala, the piriform cortex, the midline thalamus, and the anterior olfactory area. Late in epileptogenesis, the seizures were generally more severe (stages 4-5) with neuronal activation extending to the neocortex, the bed nucleus of the stria terminalis, the mediodorsal thalamu\s, and the central nucleus of the amygdala. Our findings demonstrate that inhibition of GS focally in the hippocampal formation triggers a process of epileptogenesis characterized by gradual worsening of seizure severity and involvement of progressively larger neuronal populations over a period of several weeks. Knowledge about the underlying mechanism of epileptogenesis is important because such knowledge may result in more specific and efficacious treatments of MTLE by moving away from large and poorly specific surgical resections to highly targeted surgical or pharmacological interventions of the epileptogenic process.

BackgroundWaste persists in healthcare and negatively impacts patients. Clinicians have direct control over test ordering and ongoing international efforts to improve test utilisation have identified multifaceted approaches as critical to the success of interventions. Prior to 2015, Yale New Haven Health lacked a coherent strategy for laboratory test utilisation management.MethodsIn 2015, a system-wide laboratory formulary committee was formed at Yale New Haven Health to manage multiple interventions designed to improve test utilisation. We report here on specific interventions conducted between 2015 and 2017 including reduction of (1) obsolete or misused testing, (2) duplicate orders, and (3) daily routine lab testing. These interventions were driven by a combination of modifications to computerised physician order entry, test utilisation dashboards and physician education. Measurements included test order volume, blood savings and cost savings.ResultsTesting for a number of obsolete/misused analytes was eliminated or significantly decreased depending on alert rule at order entry. Hard stops significantly decreased duplicate testing and educational sessions significantly decreased daily orders of routine labs and increased blood savings but the impact waned over time for select groups. In total, we realised approximately $100 000 of cost savings during the study period.ConclusionThrough a multifaceted approach to utilisation management, we show significant reductions in low-value clinical testing that have led to modest but significant savings in both costs and patients’ blood.

Importance Low-density lipoprotein cholesterol (LDL-C), a key cardiovascular disease marker, is often estimated by the Friedewald or Martin equation, but calculating LDL-C is less accurate in patients with a low LDL-C level or hypertriglyceridemia (triglyceride [TG] levels ≥400 mg/dL). Objective To design a more accurate LDL-C equation for patients with a low LDL-C level and/or hypertriglyceridemia. Design, Setting, and Participants Data on LDL-C levels and other lipid measures from 8656 patients seen at the National Institutes of Health Clinical Center between January 1, 1976, and June 2, 1999, were analyzed by the β-quantification reference method (18 715 LDL-C test results) and were randomly divided into equally sized training and validation data sets. Using TG and non–high-density lipoprotein cholesterol as independent variables, multiple least squares regression was used to develop an equation for very low-density lipoprotein cholesterol, which was then used in a second equation for LDL-C. Equations were tested against the internal validation data set and multiple external data sets of either β-quantification LDL-C results (n = 28 891) or direct LDL-C test results (n = 252 888). Statistical analysis was performed from August 7, 2018, to July 18, 2019. Main Outcomes and Measures Concordance between calculated and measured LDL-C levels by β-quantification, as assessed by various measures of test accuracy (correlation coefficient [R2], root mean square error [RMSE], mean absolute difference [MAD]), and percentage of patients misclassified at LDL-C treatment thresholds of 70, 100, and 190 mg/dL. Results Compared with β-quantification, the new equation was more accurate than other LDL-C equations (slope, 0.964; RMSE = 15.2 mg/dL;R2 = 0.9648; vs Friedewald equation: slope, 1.056; RMSE = 32 mg/dL;R2 = 0.8808; vs Martin equation: slope, 0.945; RMSE = 25.7 mg/dL;R2 = 0.9022), particularly for patients with hypertriglyceridemia (MAD = 24.9 mg/dL; vs Friedewald equation: MAD = 56.4 mg/dL; vs Martin equation: MAD = 44.8 mg/dL). The new equation calculates the LDL-C level in patients with TG levels up to 800 mg/dL as accurately as the Friedewald equation does for TG levels less than 400 mg/dL and was associated with 35% fewer misclassifications when patients with hypertriglyceridemia (TG levels, 400-800 mg/dL) were categorized into different LDL-C treatment groups. Conclusions and Relevance The new equation can be readily implemented by clinical laboratories with no additional costs compared with the standard lipid panel. It will allow for more accurate calculation of LDL-C level in patients with low LDL-C levels and/or hypertriglyceridemia (TG levels, ≤800 mg/dL) and thus should improve the use of LDL-C level in cardiovascular disease risk management.

Background Procalcitonin (PCT) rises early upon bacterial infection and has a long-half life, making it useful in the diagnosis of infections and antibiotic stewardship. We sought to determine the utility of PCT in diagnosing pneumonia (PNU) in the patient population presenting to our hospital during real-world clinical practice. Methods PCT results from patients in 2015 were reviewed retrospectively. Patients were eligible for inclusion if all of following criteria were met: PCT in 2015 and a lower respiratory tract culture, respiratory virus testing, chest x-ray, and white blood cell (WBC) count within 1 day of the PCT. Patients who opted out of research and those with mycobacterial infections and culture-positive infections of body sites other than urine and lower respiratory tract were excluded from further analysis. Results A total of 400 patients remained with 413 eligible PCT results across the ED (109), ICU (134), and inpatient (167) and outpatient (3) areas with a mean (SD) age of 66.2 (18.1). PCT was higher in patients with multiple pathogens reported on their respiratory cultures, direct fluorescence antibody (DFA), or PCR-based tests (mean ± SD = 2.43 ± 0.74 ng/mL; N = 32), than those with no pathogens reported (3.25 ± 1.13 ng/mL; N = 224; P < .05). Patients were grouped for the presence or absence of clinically defined PNU, according to a modification of the Centers for Disease Control (CDC) PNU1 criteria incorporating (1) chest x-ray results, (2) altered WBC number/altered mental status/fever, and (3) respiratory/breathing signs. PCT was higher in patients with clinically defined PNU, and the high PCTs were consistent with positive chest x-rays (criterion 1), and positive criterion 2 but not criterion 3. Incorporation of an elevated PCT >0.1 ng/mL into the PNU score slightly improved the area under the ROC curve (AUC) for the algorithm’s detection of PNU against the final clinical diagnosis (0.73 without PCT vs 0.76 with PCT). Furthermore, higher PCT was associated with higher 30-day and 1-year mortality. Conclusion PCT results were largely consistent with other markers of PNU such as imaging and CDC criterion 2, which suggests that PCT can be useful in evaluating for the presence of PNU. However, the PCT may not add additional information to assist in decision making above the already commonly ordered tests.

Cerebral function and viability are critically dependent on efficient delivery of oxygen and glucose through the microvasculature. Here, we studied individual microvessels in the intact brain using high-resolution confocal imaging and long-term time-lapse two-photon microscopy across the lifetime of a mouse. In the first postnatal month, we found large-scale sprouting but to our surprise the majority of sprouts underwent pruning and only a small fraction became perfused capillaries. After the first month, microvessel formation and elimination decreased and the net number of vessels stabilized. Although vascular stability was the hallmark of the adult brain, some vessel formation and elimination continued throughout life. In young adult mice, vessel formation was markedly increased after exposure to hypoxia; however, upon return to normoxia, no vessel elimination was observed, suggesting that new vessels constitute a long-term adaptive response to metabolic challenges. This plasticity was markedly reduced in older adults and aging where hypoxia-induced angiogenesis was absent. Our study describes, for the first time in vivo patterns of cerebral microvascular remodeling throughout life. Disruption of the observed balance between baseline turnover and vascular stability may underlie a variety of developmental and age-related degenerative neurological disorders.

Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. HTLV-I associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by multiple organ invasion. We have shown that ATL-derived cells induce angiogenesis and communicate with endothelial cells through gap junctions. This results in induction of endothelial-derived metalloproteinases, sub-endothelial basement membrane degradation followed by endothelial cell retraction allowing neoplastic lymphocytes extravasation. In this study we have used the specific tyrosine kinase inhibitor of VEGF receptors PTK787, functional blocking antibodies against VEGF and the gap junction communication inhibitor18-a-G to dissect the respective role of VEGF signaling pathway and cell-to-cell communication in endothelial cell retraction and tumor cell extravasation. PTK787 has mild growth inhibitory and cytotoxic effects on both endothelial and tumor cells at a concentration of 50μM and significantly inhibited cell proliferation at 100μM. Hence, 20μM PTK787 which did not exhibit any cytotoxic or antiproliferative effects on both cell types was used in experiments that last 24 hours or longer (in vitro tube formation assay and invasion assay). Endothelial cells monolayers incubated with either ATL derived cells or their cell-free conditioned medium for 48 hours induced in vitro tube formation in Matrigel angiogenesis assay. This was inhibited by both anti-VEGF antibody and 18-a-G and to a greater extent and in a dose dependent manner by PTK787. Importantly, PTK787, and to a lesser extent anti-VEGF antibody and 18-alpha-G significantly attenuated the ability of ATL-derived cells to traverse endothelial cell monolayers in double compartment invasion assays. Signaling cascade that is activated upon VEGF stimulation of endothelial cells was investigated in confluent endothelial cells cocultured with ATL derived cells or cell-free conditioned medium in the absence or presence of PTK787 or anti-VEGF antibody, using phospho-specific antibodies against FAK Tyr 861, FAK Tyr 397, Src, ERK, PLCgamma, and Akt/PKB, known to be signal mediators in the Ras-Raf-MEKK-ERK signaling cascade. The addition of ATL derived cells or cell-free conditioned medium resulted in a transient activation of the above signal mediators with a peak increase at 15 minutes and gradual return to baseline after 60 minutes. This activation was down regulated in the presence of anti-VEGF antibody and to a greater extent in the presence of PTK787. This data establishes a novel role of VEGF signaling in tumor cell invasion and supports the potential use of PTK787 in the treatment of ATL and other hematological malignancies with angiogenic and invasive properties.