Implementation and assessment of an electronic clinical decision support tool to notify clinicians of critical hyperbilirubinemia in preterm neonates less than 35 weeks' gestation.

OBJECTIVES: Critical hyperbilirubinemia in preterm neonates, a condition requiring greater attention, is treated with phototherapy or exchange transfusion when bilirubin results exceed gestational age and age-specific medical decision levels (MDLs) to prevent bilirubin-induced neurologic damage. Conventional evaluation involves multiple manual steps and is poised to inconsistencies and delays. METHODS: We designed and implemented an electronic clinical decision support (CDS) tool to identify and alert neonatal intensive care unit clinicians of critical hyperbilirubinemia with a SmartZone alert. We evaluated the performance of our manual evaluation workflow, the accuracy of the electronic CDS tool, and the outcome of the electronic CDS tool to reduce the time to place orders for interventions. RESULTS: Among the 22 patients who met the criteria to have phototherapy ordered before implementing the electronic CDS tool, 20 (90%) had phototherapy ordered. Fourteen (70%) phototherapy orders were placed less than 24 hours, 4 phototherapy orders were placed 24 to 72 hours, and 2 orders were placed more than 72 hours after bilirubin results exceeded the corresponding MDLs. Among the 15 patients who met the criteria to have phototherapy ordered after implementing the electronic CDS tool, all (100%) received phototherapy orders, with 14 (93%) placed less than 24 hours and 1 order placed less than 48 hours. The electronic CDS tool identified all eligible patients correctly. The proportion of phototherapy ordered less than 24 hours increased from 70% to 93% after the implementation of the electronic CDS tool. CONCLUSIONS: The electronic CDS tool promoted more appropriate and timely intervention orders to manage critical hyperbilirubinemia in preterm neonates.

Over-the-counter and direct-to-consumer testing for Group A streptococcus.

Over-the-counter (OTC) and direct-to-consumer (DTC) tests have been gaining popularity due to their potential to provide accurate and quick diagnostic results without any test order from healthcare professionals, while providing patients the opportunity to actively engage in their own health management. Group A streptococcus is a common transmissible pathogen that leads to acute pharyngitis. Accurate and timely diagnosis of Group A streptococcus pharyngitis is critical to urge patients to seek professional healthcare, to support antibiotic stewardship, to reduce disease transmission, and to prevent rare but potentially life-threatening complications such as acute rheumatic fever, rheumatic heart disease, and poststreptococcal glomerulonephritis. This review provides an overview for OTC and DTC testing in general, discusses the clinical utilization of Group A streptococcus testing, analyzes the limitations and challenges of current Group A streptococcus testing methodologies if developed into OTC or DTC tests. Finally, this review provides an outlook for future developments that would further improve healthcare outcomes.

Development and Validation of a Liquid Chromatography Mass Spectrometry Method for Simultaneous Measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, Vitamin A, α-Tocopherol, and γ-Tocopherol.

OBJECTIVES: Fat-soluble vitamins are measured to identify deficiencies that may lead to rickets, osteomalacia, night blindness, and reversible motor and sensory neuropathies. We present a rapid liquid chromatography-mass spectrometry (LC-MS/MS) method that simultaneously measures 25-hydroxyvitamin D3 (25[OH]D3), epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol. METHODS: We mixed 100 µL serum with internal standard and extracted it by using supported liquid extraction plates. Reconstituted specimens were analyzed by LC-MS/MS with a 10-minute gradient. RESULTS: The method was linear, covering physiological levels with r2 > 0.99, and the total precision was less than 15% at all quality control levels. The lower limit of the measuring intervals for 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol were 4 ng/mL, 4 ng/mL, 4 ng/mL, 1 µg/dL, 0.2 µg/mL, and 0.2 µg/mL, respectively, with coefficient of variation of 20% or less. Recoveries were between 92% and 111% for National Institute of Standards and Technology reference materials and 81% and 122% for spike-recovery studies. Comparison studies for vitamin D total, vitamin A, and α-tocopherol demonstrated slopes between 1.04 and 1.11 and r2 between 0.94 and 0.96. Minimal matrix effect was observed for all analytes. CONCLUSIONS: We developed and validated a rapid LC-MS/MS method for the simultaneous measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol.

Parathyroid hormone.

Parathyroid hormone is an essential regulator of extracellular calcium and phosphate. PTH enhances calcium reabsorption while inhibiting phosphate reabsorption in the kidneys, increases the synthesis of 1,25-dihydroxyvitamin D, which then increases gastrointestinal absorption of calcium, and increases bone resorption to increase calcium and phosphate. Parathyroid disease can be an isolated endocrine disorder or part of a complex syndrome. Genetic mutations can account for diseases of parathyroid gland formulation, dysregulation of parathyroid hormone synthesis or secretion, and destruction of the parathyroid glands. Over the years, a number of different options are available for the treatment of different types of parathyroid disease. Therapeutic options include surgical removal of hypersecreting parathyroid tissue, administration of parathyroid hormone, vitamin D, activated vitamin D, calcium, phosphate binders, calcium-sensing receptor, and vitamin D receptor activators to name a few. The accurate assessment of parathyroid hormone also provides essential biochemical information to properly diagnose parathyroid disease. Currently available immunoassays may overestimate or underestimate bioactive parathyroid hormone because of interferences from truncated parathyroid hormone fragments, phosphorylation of parathyroid hormone, and oxidation of amino acids of parathyroid hormone.

Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume.

OBJECTIVES: Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Citrated whole blood (200 µL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. RESULTS: This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. CONCLUSIONS: Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

Smith-Lemli-Opitz Syndrome in a newborn infant with developmental abnormalities and low endogenous cholesterol.

BACKGROUND: Patients with Smith-Lemli-Opitz Syndrome (SLOS) have defective endogenous cholesterol synthesis, and present with decreased cholesterol levels and multiple developmental dysmorphologies. CASE DESCRIPTION: A newborn infant with normal XY karyotype and normal microarray was born with multiple developmental defects and ambiguous genitalia. The patient was diagnosed with SLOS, following biochemical genetic analysis of serum 7-DHC concentrations. The clinical course of the patient was further complicated by the comorbidities associated with SLOS and the bacterial infections. CONCLUSION: We provide a detailed biochemical profile of the SLOS patient. The report can help us further understand the pathological impacts of cholesterol synthesis deficiency and provide relevant clinical management with outcome of this rare genetic disorder.

Reengineering Critical Laboratory Testing for Timely Chemotherapeutic Management.

BACKGROUND: Delivery of cytotoxic therapy is a complex multifaceted process that involves harmonized collaboration between all systems involved. Optimizing laboratory turnaround time (TAT) ensures timely delivery of chemotherapy, which potentially translates into improved patient outcomes and satisfaction. In this study, we aimed to reduce the laboratory TAT for key laboratory tests to optimize the timely administration of chemotherapy. METHODS: TAT data for complete blood count (CBC) and comprehensive metabolic panel (CMP) included specimen collection to receipt (Col-Rcv), specimen receipt to result release (Rcv-Res), and the overall TAT from specimen collection to result release (Col-Res). Work flows were reconfigured to transport CBC specimens directly to the hematology laboratory after collection and to treat all CMP samples from chemotherapy clinics as urgent [i.e., shortest turnaround time (STAT)]. From the CMP, total bilirubin and creatinine-the 2 key analytes for liver and renal toxicity assessment before chemotherapy drug administration-were analyzed on ABL 800 whole blood analyzers to further improve the laboratory TAT. RESULTS: CBC showed a significant reduction in the median (Col-Res) TAT to 16 min (P < 0.0001). For CMP, by processing all specimens as STAT samples, the median (Col-Res) TAT was reduced from 74 min to 54 min (P < 0.0001), and it was further reduced to 9 min (P < 0.0001) for total bilirubin and creatinine. CONCLUSION: Careful work flow analysis and reengineering of preanalytical and analytical process for key laboratory tests significantly reduced median overall TAT to <20 min, which helped facilitate more timely delivery of chemotherapy, without necessitating the construction of a satellite laboratory.

Validation of an Extensive CYP2D6 Assay Panel Based on Invader and TaqMan Copy Number Assays.

BACKGROUND: CYP2D6 is involved in the oxidative metabolism of approximately 20% of all clinically used medications. Genotyping cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6), is a challenge because of the high complexity of the locus. METHODS: Twenty-nine CYP2D6 sequence variants were genotyped in 50 deidentified patient samples and 29 Coriell DNAs by Invader assay, and results were compared with Infiniti assay and Sanger sequencing. To determine CYP2D6 copy number, 3 TaqMan real-time hydrolysis probes were used and results were compared with long-range PCR. Discrimination of the duplicated alleles was done on 17 DNA samples with 3 copies of CYP2D6 by long-range PCR followed by Invader genotyping and single nucleotide extension for the comparison. RESULTS: Complete concordance was observed for all samples between platforms except for 2 samples due to the lack of the *45 allele in the Infiniti panel. Reproducibility with the Invader assay and TaqMan copy number was 100%. Analytical sensitivity using DNA with 2 copies was determined to be 10 ng DNA for the Invader assay and 1 ng/µL DNA for the TaqMan assay, respectively. Complete concordance and reproducibility were observed for duplicated allele discrimination with the exception of 1 sample, determined to be *29/*43X2 by the Invader test and *1X2/*29 by the Infiniti method, which did not test for *43. CONCLUSIONS: This validation study showed that Invader and TaqMan assay combined panel provides an attractive, valid, highly accurate, and reproducible approach for CYP2D6 genotyping for clinical implementation.

High-Sensitivity Micro LC-MS/MS Assay for Serum Estradiol without Derivatization.

BACKGROUND: There are considerable demands to accurately measure estradiol (E2) at low concentrations (<20 pg/mL) in postmenopausal women, men, pediatric patients, and patients receiving breast cancer treatment. Most current high-sensitivity LC-MS/MS E2 methods require large sample volumes and involve complex sample preparations with dansyl chloride derivatization. Our study aims to develop a high-sensitivity, underivatized method using micro LC-MS/MS to reliably measure E2 concentrations below 5 pg/mL by the use of low sample volume. METHODS: A total of 290 µL of sample was mixed with internal standard (IS), E2-d4, and extracted with a mixture of hexane/ethyl acetate (90/10) (v/v). After extraction, sample was separated by Eksigent Ekspert™ micro LC 200 system with a flow rate of 35 µL/min in a total run time of 3.5 min and detected by SCIEX QTRAP 6500 mass spectrometer in a negative mode using transitions: 271/145 (quantifier) and 271/143 (qualifier). In this method, it was crucial to use HPLC columns with stability at a pH >10. RESULTS: The validation study demonstrated broad linear ranges (3.0-820.0 pg/mL) with r2 > 0.999. Total precision was below 15% at all QC levels, and limit of quantification (LOQ) was 3.0 pg/mL. Our method showed good correlation with E2 RIA (r2 = 0.96, bias = -1.0 pg/mL) and modest correlation with E2 Roche Cobas automated immunoassay (r2 = 0.86, bias = 6.0 pg/mL). CONCLUSIONS: In conclusion, we developed and validated a routinely applicable micro LC-MS/MS method without derivatization for E2 in blood samples with an LOQ of 3.0 pg/mL.

Clinical evaluation of the QMS® Tacrolimus immunoassay.

BACKGROUND: Tacrolimus, a widely used immunosuppressant, inhibits T-lymphocyte signal transduction and cytokine upregulation. We evaluated and compared the performance of a newly developed tacrolimus immunoassay method to LC-MS/MS. METHOD: Analytical performance was assessed using quality control materials and whole blood patient samples. Interferences studies were performed using pooled whole-blood samples spiked with each interferent, respectively. Comparison studies were conducted using 145 de-identified whole blood samples collected after routine tacrolimus analysis by LC-MS/MS. RESULTS: CVs were between 3.9 and 8.1% and the method was linear (r(2)=0.99) up to 30.0 ng/ml. Calibration was stable ≤12 days and LOQ was 0.7 ng/ml (14.4% CV). Bilirubin (≤48 mg/dl), hemoglobin (≤345 mg/dl), and triglycerides (<2800 mg/dl) showed no significant interference. Comparison (Passing-Bablok regression) for all samples showed a proportional bias of 17%. Comparisons of liver and kidney transplant patients showed slope biases of 22% and 31%, respectively, whereas other remaining transplant patients (stem cell, heart, lung, and islet) showed a slope bias of 0.98. CONCLUSIONS: Overall, the QMS Tacrolimus Immunoassay showed good analytical performance. Comparison studies showed a proportional bias of 17%, which can be attributed to the significant number of liver and kidney transplant patients present in this study (121/145).

Reducing the risk of hyperammonemia from transfusion of stored red blood cells.

Ammonia concentration increases in red cell units (RBCs) during storage. We measured absolute amounts of ammonia (AA) per unit serially in stored RBCs and before and after removal of the supernatant by volume reduction (VR) or washing. Ammonia increased 6.4-fold in untreated units over 31 days. VR decreased AA 3.7-fold, whereas washing decreased it 38-fold (p<0.0001). At least for certain patients, e.g., infants receiving large volume transfusions and patients in liver failure, it may be advisable to use RBCs as fresh as possible and to limit infusion (by VR or washing) of ammonia in the supernatant.

The mechanism of peptidyl transfer catalysis by the ribosome.

The ribosome catalyzes two fundamental biological reactions: peptidyl transfer, the formation of a peptide bond during protein synthesis, and peptidyl hydrolysis, the release of the complete protein from the peptidyl tRNA upon completion of translation. The ribosome is able to utilize and distinguish the two different nucleophiles for each reaction, the α-amine of the incoming aminoacyl tRNA versus the water molecule. The correct binding of substrates induces structural rearrangements of ribosomal active-site residues and the substrates themselves, resulting in an orientation suitable for catalysis. In addition, active-site residues appear to provide further assistance by ordering active-site water molecules and providing an electrostatic environment via a hydrogen network that stabilizes the reaction intermediates and possibly shuttles protons. Major questions remain concerning the timing, components, and mechanism of the proton transfer steps. This review summarizes the recent progress in structural, biochemical, and computational advances and presents the current mechanistic models for these two reactions.