Precipitated opioid withdrawal in a patient started on olanzapine/samidorphan.

BACKGROUND: Olanzapine/Samidorphan (Lybalvi®) is a novel oral agent for the treatment of schizophrenia and bipolar I disorder. It was designed to reduce weight gain associated with olanzapine. Samidorphan is an analog of naltrexone, initially intended to treat substance use disorders by antagonizing mu, delta, and kappa opioid receptors. CASE REPORT: We present the case of a 36-year-old who took their first dose of olanzapine/samidorphan shortly before calling for emergency services. The patient took diphenhydramine and an epinephrine autoinjector for what they thought was an allergic reaction but continued to have symptoms. EMS reported involuntary muscle movements thought to be due to dystonia from olanzapine. In the ED, they experienced generalized muscle spasms lasting for several seconds and diaphoresis. Initially, the staff treated for a presumed dystonic reaction to olanzapine and administered diphenhydramine 25 mg IV, diazepam 2 mg IV, midazolam 5 mg IV, and benztropine 1 mg IV without improvement. It was later determined that the patient took 16 mg of buprenorphine SL daily. With this information, precipitated opioid withdrawal was felt to be the likely cause of symptoms. The patient received 16 mg of buprenorphine for an initial Clinical Opiate Withdrawal Scale (COWS) score of 11 with repeat COWS of 6. Why should an emergency physician be aware of this? Initiating olanzapine/samidorphan in the setting of chronic opioid therapy may result in precipitated opioid withdrawal. Additional SL buprenorphine may be a reasonable treatment modality.

Prolonged venom-induced consumptive coagulopathy following Mangshan pit viper (Protobothrops mangshanensis) envenomation despite Hemato Polyvalent antivenom administration.

This case report summarizes an envenomation by the Mangshan pit viper (Protobothrops mangshanensis), a rare, endangered, venomous snake endemic to Mount Mang of China, and the first reported use of Hemato Polyvalent antivenom (HPAV) for this species. The snakebite occurred in a United States zoo to a 46-year-old male zookeeper. He presented via emergency medical services to a tertiary center after sustaining a single P. mangshanensis bite to the abdomen and was transported with antivenom from the zoo. Within 2 hours of envenomation, he developed oozing of sanguineous fluid and ecchymosis at the puncture site, and about 4 hours post-bite, was treated with HPAV. His coagulation profile fluctuated with the following pertinent peak/nadir laboratory values and corresponding hospital day (HD): undetectable fibrinogen levels, d-dimer 8.89 mg/L and 7.43 mg/L, and INR 2.97 and 1.46 on HD zero and three, respectively. Other peak/nadir values included hemoglobin 9.7 g/dL and creatinine phosphokinase 2410 U/L on HD four and platelets 81 × 109/L on HD seven. The patient received a total of 30 vials of HPAV over 5 days and 1 unit of cryoprecipitate on HD six. Upon discharge on HD eight, laboratory studies were normalizing, except for platelets, and edema stabilized. This case describes an acute, recurrent, and prolonged venom-induced consumptive coagulopathy despite prompt administration and repeated doses of HPAV.

Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue.

BACKGROUND: Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. METHODS: Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. RESULTS: Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). CONCLUSIONS: This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.

Returns After Discharge From the Emergency Department Observation Unit: Who, What, When, and Why?

INTRODUCTION: The number of emergency department observation units (EDOU) and observation stays has continued to increase. Despite this, there is limited data on the characteristics of patients who return unexpectedly to the ED after EDOU discharge. METHODS: We identified the charts of all patients who were admitted to the EDOU of an academic medical center between January 2018-June 2020 and had a return to the ED within 14 days of discharge from the EDOU. Patients were excluded if they were admitted to the hospital from the EDOU, left against medical advice, or died in the EDOU. We manually extracted selected demographic factors, comorbidities, and healthcare utilization data from the charts. Physician reviewers identified return visits thought to be related to the index visit or potentially avoidable. RESULTS: During the study period, there were 176,471 ED visits, 4,179 admissions to the EDOU, and 333 return visits to the ED within 14 days from discharge from the EDOU, representing 9.4% of all patients discharged from the EDOU. We identified a higher rate of return for patients treated for asthma and lower rates of return for patients treated for chest pain or syncope than the overall return rate. Physician reviewers determined that 64.6% of unplanned returns were related to the index visit, and 4.5% were potentially avoidable. Of potentially avoidable visits, 53.3% occurred within 48 hours of discharge, supporting the use of this period as a potential quality metric. While there was no significant difference in the percentage of related return visits between males and females, there was a higher rate of potentially avoidable visits for male patients. CONCLUSION: This study adds to the limited body of literature on EDOU returns, finding an overall return rate of under 10%, with about two-thirds of returns determined to be related to the index visit and <5% considered to be potentially avoidable.

Clinical characteristics and outcomes using dexmedetomidine in nonintubated patients: A poison center observational study.

PURPOSE: Dexmedetomidine is a central α2 agonist commonly used on intubated patients. It is increasingly being used off-label in nonintubated agitated patients. We sought to determine the overall clinical course, adverse effects, and need for subsequent mechanical ventilation in toxicology patients after treatment with dexmedetomidine. METHODS: This was a retrospective cohort study conducted by chart review of electronic records from the Virginia Poison Control Center from January 1, 2019 to February 4, 2022. Inclusion criteria consisted of all poison center cases where dexmedetomidine was used. The primary outcome was the presence or absence of clinical improvement following dexmedetomidine use. Secondary outcomes included adverse effects, subsequent intubation, or death. RESULTS: During this study period, there were 220 cases in which dexmedetomidine was used to treat agitation. After exclusions, 70 cases were analyzed. The categories included antimuscarinic (n = 19), polysubstance (n = 16), sedative withdrawal (n = 10), unknown agitation (n = 7), sympathomimetic (n = 5), baclofen withdrawal (n = 3), unknown ingestion (n = 3), sedative/hypnotic (n = 2), antipsychotic (n = 2), hallucinogenic (n = 2), and opioid withdrawal (n = 1). Clinical improvement occurred in 62 of 70 patients (89%). There were no deaths. A total of 4 patients were intubated after starting dexmedetomidine, 2 for refractory agitation and 2 for hypoxia after aspiration. CONCLUSION: Global clinical improvement was observed in the agitated toxicology patients administered dexmedetomidine. There was one case of intubation secondary to oversedation. Dexmedetomidine could be a useful adjunctive treatment for agitated toxicologic patients but should be studied further before routinely used.

Retained bullets and lead toxicity: a systematic review.

INTRODUCTION: Lead toxicity secondary to retained bullet(s) (RB) after a penetrating gunshot wound is a rare but likely underdiagnosed condition, given the substantial number of firearm injuries in the United States. There is currently no consensus on the indications for surveillance, chelation, or surgical intervention. OBJECTIVE: The purpose of our review is to summarize the literature on systemic lead toxicity secondary to RBs to help guide clinicians in the management of these patients. METHODOLOGY: The primary literature search was conducted in Medline (PubMed), EMBASE, Cochrane, and CENTRAL using the following MESH terms: "chelation" and "lead poisoning" or "lead toxicity" or "lead" and "bullet" or "missile" or "gunshot", or "bullet". RESULTS: The search identified 1,082 articles. After exclusions, a total of 142 articles were included in our final review, the majority of which were case reports. Several factors appear to increase the risk of developing lead toxicity including the location of the RB, the presence of a fracture or recent trauma, number of fragments, hypermetabolic states, and bullet retention duration. Particularly, RBs located within a body fluid compartment like an intra-articular space appear to be at a substantially higher risk of developing lead toxicity. Even though patients with lead toxicity from RBs will have similar symptoms to patients with lead toxicity from other sources, the diagnosis of lead poisoning may occur months or years after a gunshot wound. Symptomatic patients with high blood lead levels (BLLs) tended to improve with a combination of chelation and surgical removal of RBs. CONCLUSIONS: We suggest surveillance with serial BLLs should be performed. Patients with intra-articular RBs appear to be at increased risk of lead toxicity and if possible, early surgical removal of the RBs is warranted, especially given that signs of toxicity are vague, and patients may not have access to follow-up. Long-term chelation should not be used as a surgical alternative and management should be multidisciplinary.

The addition of FAIMS increases targeted proteomics sensitivity from FFPE tumor biopsies.

Mass spectrometry-based targeted proteomics allows objective protein quantitation of clinical biomarkers from a single section of formalin-fixed, paraffin-embedded (FFPE) tumor tissue biopsies. We combined high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) to increase assay sensitivity. The modular nature of the FAIMS source allowed direct comparison of the performance of FAIMS-PRM to PRM. Limits of quantitation were determined by spiking synthetic peptides into a human spleen matrix. In addition, 20 clinical samples were analyzed using FAIMS-PRM and the quantitation of HER2 was compared with that obtained with the Ventana immunohistochemistry assay. FAIMS-PRM improved the overall signal-to-noise ratio over that from PRM and increased assay sensitivity in FFPE tissue analysis for four (HER2, EGFR, cMET, and KRAS) of five proteins of clinical interest. FAIMS-PRM enabled sensitive quantitation of basal HER2 expression in breast cancer samples classified as HER2 negative by immunohistochemistry. Furthermore, we determined the degree of FAIMS-dependent background reduction and showed that this correlated with an improved lower limit of quantitation with FAIMS. FAIMS-PRM is anticipated to benefit clinical trials in which multiple biomarker questions must be addressed and the availability of tumor biopsy samples is limited.

Systemic toxicity from subcutaneous brimonidine injection successfully treated with naloxone.

Brimonidine is a topical ophthalmic alpha-2 adrenergic agonist solution used to treat glaucoma. The toxidrome includes drowsiness, lethargy, hypotension, bradycardia, and respiratory depression when ingested in infants. We report a case of intentional subcutaneous injection of brimonidine in an elderly patient resulting in hypotension and CNS depression that responded to naloxone. A 73-year-old female with a past medical history significant for glaucoma, hypertension, and indwelling pacemaker presented to the emergency department after injecting her brimonidine tartrate ophthalmic solution subcutaneously (SQ). The patient was not taking any antihypertensive medications or opioids. Initial presentation consisted of lethargy, a paced rhythm of 60 bpm, and blood pressure of 91/24 mmHg with a MAP of 46. Due to central nervous system depression, 3 mg of intranasal naloxone was administered. The patient was treated with intravenous fluids and escalating doses of naloxone and required a continuous infusion. Mental status and vital signs subsequently improved. The patient was admitted to the ICU and naloxone was subsequently weaned over 12 h. Systemic central alpha-2 adrenergic agonist toxicity resulted from SQ brimonidine injection. Central alpha-2 adrenergic agonist overdoses present as sympatholytic effects including CNS depression, bradycardia, hypotension, and may mimic the opioid toxidrome. Brimonidine SQ injection has not previously been reported and this case has similar findings to other central alpha-2 adrenergic agonist poisonings. Naloxone has previously shown variable reversal of CNS depression in central alpha-2 overdose. In this case, high-dose naloxone was useful for reversing CNS depression and hemodynamic instability.

Social support and outcomes in older adults with lung cancer.

BACKGROUND: Insufficient social support is associated with increased mortality among older adults. Lung cancer is primarily a disease of older adults and is the leading cause of all cancer deaths. We assessed the association of social support with outcomes among older adults with lung cancer. MATERIALS AND METHODS: Adults age 65 and older with lung cancer with a completed geriatric assessment (GA) were assessed. Emotional social support (ES) and tangible (material, instrumental) support (TS) measures and patient characteristics were obtained from the GA. The electronic health record was used to extract clinical variables. Simple linear regression models evaluated the association between social support scales with patient and clinical factors. RESULTS: 79 adults were assessed. White race was positively associated with ES score (p=.04), while higher BMI (p=.03), depression (p=.03) and anxiety (p=.02) were associated with worse ES. Higher BMI was associated with higher/better TS score (p=.02) while living alone was associated with lower/worse TS score (p=.03). Completion of platinum-based doublet chemotherapy with immunotherapy as planned was associated with higher ES scores (p=.02) and higher TS scores (p=.02). Disease progression was associated with lower ES scores (p=.03). CONCLUSION: Social support may influence clinical outcomes in older adults with lung cancer. As lung cancer often portends to poor prognosis, social support may be an important prognostic indicator.

Global Extraction from Parallel Reaction Monitoring to Quantify Background Peptides for Improved Normalization and Quality Control in Targeted Proteomics.

Mass spectrometry-based targeted proteomics employs heavy isotope-labeled proteins or peptides as standards to improve accuracy and precision. The input sample amount is often determined by the total quantity of endogenous proteins or peptides, as defined by spectrophotometric assays, before the heavy-isotope standards are spiked into the samples. Errors in spectrophotometric measurements, which may be due to low sensitivity or chemical or biological interference, have a direct impact on the quantitative mass spectrometry results. Currently used targeted proteomics workflows cannot identify or correct deviations that arise from differences in the input sample amount. We have developed a workflow, global extraction from parallel reaction monitoring (PRM), to identify and quantify thousands of background peptides that are inherently acquired by PRM experiments. These background peptides were used to identify differences in the input sample amount and to reduce this variance by intensity-based, post-acquisition normalization. This approach was then applied to a xenograft study to improve the quantification of human proteins in the presence of mouse tissue contamination. In addition, these background peptides also provided a direct source of quality control metrics related to sample handling and preparation.

The addition of a lipid-rich dietary supplement eliminates seizure-like activity and paralysis in the drosophila bang sensitive mutants.

OBJECTIVE: To investigate the effect that a diet supplemented with KetoCal 4:1, a commercially available dietary formula consisting of a 4:1 ratio of fats to carbohydrates plus proteins, had on the seizure-like activity (SLA) and paralysis normally exhibited by the Drosophila Bang-sensitive (BS) paralytic mutants following mechanical shock. METHODS: Given that dietary changes are known to reduce seizures in humans and animal models, three BS mutants, easily-shocked (eas), bang-senseless (parabss), and technical knockout (tko), were fed a standard cornmeal/yeast/sugar diet supplemented with 10% KetoCal 4:1 (KetoCal-sup diet). Newly eclosed BS flies were fed this diet for 3-7 days and the effect this had on SLA, paralysis, locomotor activity, triglyceride levels, and glucose levels was examined. RESULTS: All three genotypes displayed significant reductions in SLA and BS sensitivity following mechanical shock. After only 3 days on the diet, 95% of tko flies no longer exhibited SLA or paralysis, and near complete suppression of the BS phenotype was seen by day 7. In the case of eas, there was a 78% reduction of SLA after 3 days on the diet and SLA was completely suppressed by day 7. The parabss flies showed a similar but less robust reduction of SLA on the diet as there was only a 68% reduction of SLA and paralysis following 7 days on the diet. The diet did not suppress activity globally as tko flies had increased basal locomotor activity on the diet while the parabss and eas flies showed no significant change in basal activity. The KetoCal-sup diet did not significantly alter the triglyceride levels or the total glucose levels in the BS mutants. In addition, the SLA and BS suppression was maintained even when the BS mutants were transitioned back to a standard fly diet. CONCLUSIONS: The SLA and paralysis associated with the Drosophila BS phenotype can be effectively suppressed by transient exposure to a KetoCal-sup diet. This suppression was not dependent upon long term maintenance of the diet and it was not associated with alterations in total glucose or triglyceride levels in these flies.

Isolation of clinically relevant concentrations of bone marrow mesenchymal stem cells without centrifugation.

BACKGROUND: This study examined the quality of bone marrow aspirates extracted using a novel, FDA cleared method to optimally target cells from the inner cortical iliac bone surface without the need for centrifugation. This method employs small draws from a single puncture that promote only lateral flow from multiple sites (SSLM method). The study utilized the Marrow Cellutions bone marrow aspiration system (MC system) which is based on the SSLM method and compared the MC system directly to bone marrow concentrates (BMAC) generated by centrifugation of aspirates harvested with a standard aspiration needle. METHODS: Three direct comparisons were conducted evaluating the SSLM draws and BMACs derived from the same patient from contralateral iliac crests. The levels of TNCs/mL, CD34+ cells/mL, CD117+ cells/mL, and CFU-f/mL were compared between the various bone marrow preparations. The cellular content of a series of SSLM draws was also analyzed to determine the total nucleated cell (TNC) count and the concentration of mesenchymal stem/progenitor cells as measured by colony forming unit fibroblasts (CFU-f). RESULTS: In direct comparisons with BMAC systems, SSLM draws yielded significantly higher CFU-f concentrations and comparable concentrations of CD34+ and CD117+ cells. In addition, the average quantity of TNCs/mL in a series of 30 patients utilizing the SSLM draw was 35.2 × 106 ± 17.1 × 106 and the average number of CFU-f/mL was 2885 ± 1716. There were small but significant correlations between the TNCs/mL and the CFU-fs/mL using the SSLM method as well as between the age of the patient and the CFU-fs/mL. CONCLUSIONS: The MC Device, using the SSLM draw technique, produced concentrations of CFU-fs, CD34+ cells and CD117+ cells that were comparable or greater to BMACs derived from the same patient. Given the rapid speed and simplicity of the MC Device, we believe this novel system possesses significant practical advantages to other currently available centrifugation based systems.

Targeted data-independent acquisition for mass spectrometric detection of RAS mutations in formalin-fixed, paraffin-embedded tumor biopsies.

Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2. When the assay was applied to tumor samples with known KRAS or NRAS mutations (G12A, G12D, G12V, and G13D), RAS-mutant and wild-type peptides were successfully detected in 11 of 13 biopsy samples. Mutation statuses obtained by DIA were concordant with those obtained by DNA sequencing, and yields of mutant peptide (mutant peptide/[mutant + wild-type peptides]) exhibited linear correlation with yields of RAS-mutant mRNA. When applied to biopsy samples with failed DNA testing results, the DIA assay identified an additional RAS-mutated sample. SIGNIFICANCE: Proteomic detection of RAS mutations by DIA in tumor biopsies can provide solid evidence of mutant RAS protein regardless of the mutation types and sites in exon 2. This robust method could rescue samples that fail genomic testing due to insufficient tumor tissue or lack of sequenceable DNA. It can be used to explore the relationship between protein expression level of mutant RAS and therapeutic outcome.

Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography/electrospray ionization multiple reaction monitoring mass spectrometry (LC/ESI-MRM-MS) are more commonly used in clinical research. Both technologies have certain disadvantages, namely, the nonspecificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-matrix-assisted laser desorption/ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05-2.0 fmol/µg of total lysate protein and with coefficients of variation < 15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis-only 25 µg of total protein-and are therefore suitable for analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 h.

Multiple Hip Intra-Articular Steroid Injections Increase Risk of Periprosthetic Joint Infection Compared With Single Injections.

BACKGROUND: Patients with hip osteoarthritis often temporize their symptoms with multiple intra-articular steroid hip injections (IASHIs) before undergoing total hip arthroplasty (THA). Although there is recent evidence to suggest that IASHI can lead to an increased risk of future periprosthetic joint infection (PJI), the potential increase in risk of PJI after multiple IASHIs compared with single IASHI remains largely unknown. The aim of the study was to evaluate whether multiple IASHIs are associated with increased risk of PJI compared with single IASHI in THA patients. METHODS: We evaluated 2 cohorts of patients consisting of 106 patients who received 2 or more IASHI in the year before THA and a matched group of 350 patients who received one IASHI in the 12 months before THA. RESULTS: The single and multiply-injected patient cohorts had an infection rate of 2.0% and 6.6% (7/350 and 7/106), respectively (P = .04, odds ratio 3.30) and average follow-up of 28.9 and 24.2 months. The 2 cohorts did not differ with regard to age, gender, American Society of Anesthesiologist score, presence of diabetes mellitus, or body mass index. CONCLUSION: In comparison with patients with single IASHI, multiple IASHIs are associated with an increased risk of PJI significantly higher than the elevated risk reported with single injection before THA. The present study findings would be clinically useful in counseling patients who are considering temporizing their symptoms with multiple IASHIs before undergoing THA.

Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach.

Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.

Increased Depth and Breadth of Plasma Protein Quantitation via Two-Dimensional Liquid Chromatography/Multiple Reaction Monitoring-Mass Spectrometry with Labeled Peptide Standards.

Absolute quantitative strategies are emerging as a powerful and preferable means of deriving concentrations in biological samples for systems biology applications. Method development is driven by the need to establish new-and validate current-protein biomarkers of high-to-low abundance for clinical utility. In this chapter, we describe a methodology involving two-dimensional (2D) reversed-phase liquid chromatography (RPLC), operated under alkaline and acidic pH conditions, combined with multiple reaction monitoring (MRM)-mass spectrometry (MS) (also called selected reaction monitoring (SRM)-MS) and a complex mixture of stable isotope-labeled standard (SIS) peptides, to quantify a broad and diverse panel of 253 proteins in human blood plasma. The quantitation range spans 8 orders of magnitude-from 15 mg/mL (for vitamin D-binding protein) to 450 pg/mL (for protein S100-B)-and includes 31 low-abundance proteins (defined as being <10 ng/mL) of potential disease relevance. The method is designed to assess candidates at the discovery and/or verification phases of the biomarker pipeline and can be adapted to examine smaller or alternate panels of proteins for higher sample throughput. Also detailed here is the application of our recently developed software tool-Qualis-SIS-for protein quantitation (via regression analysis of standard curves) and quality assessment of the resulting data. Overall, this chapter provides the blueprint for the replication of this quantitative proteomic method by proteomic scientists of all skill levels.

Effect of Posterior Tibial Slope on Flexion and Anterior-Posterior Tibial Translation in Posterior Cruciate-Retaining Total Knee Arthroplasty.

Reduced posterior tibial slope (PTS) and posterior tibiofemoral translation (PTFT) in posterior cruciate-retaining (PCR) total knee arthroplasty (TKA) may result in suboptimal flexion. We evaluated the relationship between PTS, PTFT, and total knee flexion after PCR TKA in a cadaveric model. We performed a balanced PCR TKA using 9 transfemoral cadaver specimens and changed postoperative PTS in 1° increments. We measured maximal flexion and relative PTFT at maximal flexion. We determined significant changes in flexion and PTFT as a function of PTS. Findings showed an average increase in flexion of 2.3° and average PTFT increase of 1mm per degree of PTS increase when increasing PTS from 1° to 4° (P<.05). Small initial increases in PTS appear to significantly increase knee flexion and PTFT.