Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

Effect of gabapentin on length of stay, opioid use, and pain scores in posterior spinal fusion for adolescent idiopathic scoliosis: a retrospective review across a multi-hospital system.

BACKGROUND: Gabapentin has been adopted in Enhanced Recovery After Surgery protocols as a means to reduce opioid consumption while maintaining adequate post-operative analgesia. The purpose of our study was to review and compare changes in length of stay, opioid use, and patient reported pain scores after the addition of gabapentin into five, distinct pain protocols for posterior spinal fusion in adolescent idiopathic scoliosis. METHODS: A retrospective review was completed using a database of electronic medical data from a single pediatric orthopedic healthcare system that was queried for patients with adolescent idiopathic scoliosis who underwent first-time posterior spinal fusion. Perioperative data including demographics, hospital length of stay, surgical details, opioid use, patient reported pain scores, and non-opioid analgesic use were collected. RESULTS: From December 2012 to February 2019, 682 hospitalizations for posterior spinal fusion in adolescent idiopathic scoliosis were identified with complete inpatient data; 49% were administered gabapentin. For the gabapentin cohort, the system saw no statistically significant effect on length of stay or pain averaged over POD#0-3. Opioid use was statistically lower averaged over POD#0-3. Individual sites saw variation on length of stay and opioid use compared to the system. CONCLUSION: In conclusion, system-wide data showed gabapentin containing protocols reduced opioid use while maintaining clinically equivalent analgesia. However, variations of individual site results make it difficult to conclude the degree to which gabapentin were responsible for this effect.

Structure-based design and discovery of potent and selective lysine-specific demethylase 1 (LSD1) inhibitors.

The histone demethylase LSD1 is a key enzyme in the epigenetic regulation of gene transcription. Here we present our efforts to discover small molecule reversible inhibitors of LSD1 as an attractive approach to treat hematologic malignancies and certain solid tumors. Using structure-based drug design, we designed and synthesized a novel series of heteroaromatic imidazole inhibitors that demonstrate potent inhibition of the demethylase activity and low nanomolar cell-based activity. This novel LSD1 inhibitor series was further optimized by attenuating the hERG inhibition and improving oral bioavailability.

Association of reactive oxygen species levels and radioresistance in cancer stem cells.

The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.

Characterizing antibiotic prophylaxis practices in pediatric deformity spinal surgery and impact on 30-day postoperative infection: an NSQIP pediatric database study.

PURPOSE: The aim of this study was to characterize antibiotic prophylaxis practices in pediatric patients who have received posterior arthrodesis for spinal deformity and understand how these practices impact 30-day postoperative infection rates. METHODS: This was a retrospective cohort study using the National Surgical Quality Improvement Program Pediatric database for year 2021. Patients 18 years of age or younger who received posterior arthrodesis for scoliosis or kyphosis correction were included. The outcome of interest was 30-day postoperative infection. Fisher's exact test and multivariable regression analysis were used to analyze the impact of intravenous antibiotic prophylaxis, intraoperative intravenous antibiotic redosing after 4 h, postoperative antibiotic prophylaxis, intraoperative topical antibiotics on 30-day postoperative infection, and various antibiotic prophylaxis regimens. RESULTS: A total of 6974 patients were included in this study. The 30-day infection rate was 2.9%. Presurgical intravenous antibiotic (11.5% vs. 2.7%, p = 0.005), postoperative antibiotic (5.7% vs. 2.4%, p < 0.01), and intraoperative topical antibiotic (4.0% vs. 2.7%, p = 0.019) were associated with significantly reduced infection rates. There was no significant difference in infection rates between patients that received cefazolin versus vancomycin versus clindamycin. The addition of Gram-negative coverage did not result in significant differences in infection rates. Multivariable regression analysis found postoperative intravenous antibiotics and intraoperative topical antibiotics to reduce infection rates. CONCLUSIONS: We found the use of presurgical intravenous antibiotics, postoperative intravenous antibiotics, and intraoperative topical antibiotics to significantly reduce infection rates. Results from this study can be applied to future research on implementation of standardized infection prevention protocols. LEVEL OF EVIDENCE: Level II.

The Effect of Adolescent Idiopathic Scoliosis on Natural Delivery and Epidural Use in Pregnant Females: A Matched Cohort Study.

STUDY DESIGN: Retrospective matched cohort study. OBJECTIVE: The aim of this study was to determine whether females with idiopathic scoliosis (IS), both with and without spine fusion, experience different rates of cesarean section (CS) and epidural anesthesia (EA) than females without scoliosis. SUMMARY OF BACKGROUND DATA: IS is a common spine condition with a higher prevalence in females. It is unclear whether females with scoliosis, treated nonoperatively or operatively, have different rates of cesarean delivery or EA. MATERIALS AND METHODS: Patients with IS who delivered in our integrated health care system during a 6-year period were identified (N = 1810). They were matched with a group without scoliosis who delivered during the same period (N = 1810). Rates and relative risk (RR) of CS and EA between cohorts and subgroups were calculated. RESULTS: The scoliosis cohort had significantly higher rates and RR of EA ( P = 0.002 and P = 0.004, respectively). Scoliosis patients treated nonoperatively had an 8% greater RR of EA ( P = 0.004) and had a significantly lower rate of CS (23.2% vs . 26%, P = 0.048) compared with the control group. Among only scoliosis patients, those treated with spine fusion had a 38% decreased RR of EA ( P < 0.001). Distal fusion level did not seem to influence the RR of EA or CS. CONCLUSIONS: Females with scoliosis were significantly more likely to receive EA at delivery compared with females without scoliosis. Rates and RR of cesarean delivery were not significantly lower among women with scoliosis, but females treated nonoperatively for scoliosis had a significantly lower CS rate than those without scoliosis. Females treated with spine fusion for scoliosis were far less likely to receive EA than both females without scoliosis and females with scoliosis treated nonoperatively.

Pre-Emptive Opioid-Sparing Medication Protocol Decreases Pain and Length of Hospital Stay in Children Undergoing Posterior Spinal Instrumented Fusion for Scoliosis.

Background: Poorly controlled post-operative pain following Posterior Spinal Instrumented Fusion (PSIF) for scoliosis may be associated with delayed ambulation and longer hospital stays. Multimodal analgesia use has been shown to provide superior analgesia with improved recovery and reduction of post-operative morbidity in other orthopedic subspecialties, but has not been described with pediatric patients undergoing spinal surgery. Objective: We describe a novel, pre-emptive, opioid-sparing pediatric pain medication protocol that is started two days prior to surgery, in accordance with first-order pharmacokinetics, and continued post-operatively until discharge with the goal of decreasing post-operative pain, improving early mobilization, and ultimately decreasing the patient's length of hospital stay. Methods: We retrospectively reviewed 116 PSIF cases from March 2014 to November 2017. Fifty-two patients received standard analgesia before August 2016, and 64 patients after August 2016 received the pre-emptive protocol consisting of a standardized combination of acetaminophen, celecoxib, and gabapentin two days prior to surgery and continued during their inpatient stay. Scheduled oxycodone and intravenous hydromorphone via patient controlled analgesia (PCA) were given to both groups equally during the post-operative hospital stay. We analyzed length of stay, total opioid consumption, and maximum pain scores per day from surgical to discharge date. Results: 116 patients were included: 64 patients in the pre-emptive group and 52 patients in the standard group. Length of hospital stay significantly differed, with means of 3.9 days in the pre-emptive group and 4.5 days in the standard analgesia group (p<0.05). Patients in the pre-emptive group recorded significantly lower maximal pain levels than those in the standard analgesia group on post-operative days #1 (4.9 vs. 5.8, p=0.0196), #3 (4.4 vs. 6.1, p=0.0006), and #4 (4.2 vs. 5.4, p=0.0393). Total post-operative morphine equivalents taken did not significantly differ between the two groups. Conclusion: This is a preliminary report demonstrating a significant decrease in maximal pain score and length of stay following PSIF on a cohort of patients receiving a novel pre-emptive opioid-sparing pain medication protocol based on first order pharmacokinetics. Future studies should investigate degree of mobilization and opioid consumption and maximal pain level after discharge from the hospital. Level of Evidence: III.

Recent advances in cancer stem cells.

The theory of cancer stem cells states that a subset of cancer cells within a tumor has the ability to self-renew and differentiate. Only those cells within a tumor that have these two properties are called cancer stem cells. This concept was first demonstrated in the study of leukemia where only cells with specific surface antigen profiles were able to cause leukemia when engrafted into immunodeficient mice. In recent years solid tumors were studied utilizing similar techniques in mice. Human tumors where evidence of cancer stem cells has been published include tumors of the breast, brain, pancreas, head and neck, and colon. If this difference in tumorigenicity of cancer cells also occurs in patients, then the ability to enrich for cancer stem cells lays an important groundwork for future studies where mechanisms involved in cancer stem cells can now be investigated.

Intramedullary nailing of pediatric femoral shaft fracture.

Intramedullary nail fixation of pediatric long bone fracture, particularly femoral shaft fracture, has revolutionized the care and outcome of these complex injuries. Nailing is associated with a high rate of union and a low rate of complications. Improved understanding of proximal femoral vascularity has led to changes in nail insertion methodology. Multiple fixation devices are available; selection is based on fracture type, patient age, skeletal maturity, and body mass index. A thorough knowledge of anatomy and biomechanics is required to achieve optimal results without negatively affecting skeletal development.

Microdose protocol stereoradiography has similar reliability to standard low-dose protocol during concurrent Sanders skeletal maturity staging.

PURPOSE: Decreasing radiation exposure is important for scoliosis patients who require serial imaging. Microdose protocol stereoradiography is now increasingly utilized. Previous studies have reported similar reliability of concurrent Sanders skeletal maturity staging based on standard low-dose stereoradiography and standard hand radiographs. The purpose of our study was to investigate the reliability and radiation exposure of concurrent Sanders staging using microdose protocol compared to a standard protocol for adolescent idiopathic scoliosis. We hypothesized that surgeon-performed Sanders staging would have similar reliability when comparing microdose and standard-dose imaging protocols. METHODS: A randomized survey of 30 hand images using standard protocol spinal stereoradiography and an equal number from microdose protocol were distributed to six experienced pediatric orthopaedic spine surgeons. Images were graded by each surgeon according to the Sanders skeletal maturity grading system. Items were again randomized and graded after a 2-week interval. Fleiss' weighted kappa for inter and intraobserver reliability was calculated and an unpaired t test was used to test for significance. RESULTS: Interobserver reliability for all modalities was in the strong to almost perfect agreement (average weighted κ > 0.8) range. For the microdose protocol, κ was 0.82 and 0.84 for each separate round of grading. Standard low-dose protocol κ was 0.83 and 0.79. Intraobserver κ was 0.86 for microdose and 0.82 for standard. Average radiation for microdose was significantly less radiation (82.6%) than standard stereoradiography (0.3 ± 0.1 mGy vs. 1.9 ± 0.4 mGy, p < 0.001). CONCLUSIONS: Sanders staging reliability of a well-positioned hand during scoliosis stereoradiography was similarly excellent for both microdose and standard low-dose protocol. Microdose protocol used less radiation while still preserving the reliability of Sanders staging.

Risk of early complication following anterior vertebral body tethering for idiopathic scoliosis.

PURPOSE: The purpose of this study was to determine peri-operative morbidity associated with anterior vertebral body tethering (aVBT) for idiopathic scoliosis. METHOD: Of 175 patients treated with aVBT, 120 patients had 2 year follow up and were included in this study. Prospectively collected clinical and radiographic data was analyzed retrospectively. RESULTS: Pre-operatively, the mean patient age was 12.6 year (8.2-15.7 year), Risser 0-3, with main thoracic scoliosis 51.2° (40-70°). Immediately post-operative, scoliosis improved to 26.9° (6-53°; p < 0.05), at 1-year post-operative was 23.0° (- 11 to 50°; p < 0.01 vs immediate post-op) and at 2-year post-operative was 27.5° (- 5 to 52; p = 0.64 vs immediate post-op). Pre-operative T5-T12 kyphosis was 16.0° (- 23 to 52°), post-operative was 16.9° (- 7 to 44°), at 1-year was 17.5° (- 14 to 61°) and at 2-year was 17.0° (- 10 to 50°; p = 0.72 vs pre-op). All patients underwent thoracoscopic approach, EBL 200 ml (20-900 ml), surgical time 215.3 min (111-472 min), anesthesia time 303.5 min (207-480 min), ICU stay of 0.2 day (0-2 days), and post-operative hospital stay 4.5 days (2-9 days). During the in-hospital peri-operative period, there were no unplanned return to the operating room (UPROR) and there was a 0.8% rate of complication: one pneumothorax requiring reinsertion of chest tube. By 90 days post-operative, there was no UPROR and a 5% rate of complication. Five additional patients developed complications after discharge: one CSF leak treated with blood patch injection in the clinic and resolved, two pleural effusions requiring chest tubes, one superficial wound infection and one pneumonia treated with outpatient antibiotics. By 1-year post-op, there was a 1.7% rate of UPROR and 8.3% rate of complication. Four additional patients developed complications beyond 90 days: two upper limb paresthesia required outpatient medical management, one CSF leak which initially treated blood patch injection in the clinic initially which then required UPROR, and one compensatory lumbar curve add on that was treated with extension of the tether. By 2-years post-op, there was a 6.7% rate of UPROR and 15.8% rate of complication. 9 additional complications developed after 1 year. One curve progression, one keloid scar, one right leg weakness, 4 cable failures and 2 curve overcorrections. CONCLUSION: This large, multicenter series of aVBT demonstrated a 15.8% complication rate and a 6.7% UPROR rate at 2 year post-operatively. This early study during the learning curve of aVBT found higher rates of CSF leaks and overall complications than would be expected for PSFI at 1 year post-operatively and a higher rate of overall complications and of UPROR than would be expected for PSFI at 2 year post-operatively. As is common with new procedures, the complication rate may fall with further experience.

A history of bracing for idiopathic scoliosis in North America.

The care of the patient with scoliosis has a history extending back over two millennia with cast and brace treatment being a relatively recent endeavor, the modern era comprising just over half a century. Much of the previous literature provides a modest overview with emphasis on the history of the operative management. To better understand the current concepts of brace treatment of scoliosis, an appreciation of the history of bracing would be helpful. As such, we review the history of the treatment of scoliosis with an emphasis on modern brace treatment, primarily from a North American perspective. Our review utilizes consideration of historical texts as well as current treatises on the history of scoliosis and includes discussion of brace development with their proponents' rationale for why they work along with an appraisal of their clinical outcomes. We provide an overview of the current standards of care and the braces typically employed toward that standard including: the Milwaukee brace, the Wilmington brace, the Boston brace, the Charleston brace, the Providence brace and the SpineCor brace. Finally, we discuss future trends including improvements in methods of determining the critical period of peak growth velocity in children with scoliosis, the exciting promise of gene markers for progressive scoliosis and "internal bracing" options.

Discovery of CC-90011: A Potent and Selective Reversible Inhibitor of Lysine Specific Demethylase 1 (LSD1).

Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure-activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).

The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: a possible molecular basis for the site-specific cancer risk modification.

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

An inhibitory metabolite leads to dose- and time-dependent pharmacokinetics of (R)-N-{1-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxy-phenyl)-acetamide (AMG 487) in human subjects after multiple dosing.

(R)-N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]-pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxyphenyl)-acetamide (AMG 487) is a potent and selective orally bioavailable chemokine (C-X-C motif) receptor 3 (CXCR3) antagonist that displays dose- and time-dependent pharmacokinetics in human subjects after multiple oral dosing. Although AMG 487 exhibited linear pharmacokinetics on both days 1 and 7 at the 25-mg dose, dose- and time-dependent kinetics were evident at the two higher doses. Nonlinear kinetics were more pronounced after multiple dosing. Area under the plasma concentration-time curve from 0 to 24 h [AUC((0-24 h))] increased 96-fold with a 10-fold increase in dose on day 7 compared with a 28-fold increase in AUC((0-24 h)) on day 1. These changes were correlated with time- and dose-dependent decreases in the metabolite to parent plasma concentrations, suggesting that these changes result from a decrease in the oral clearance (CL) of AMG 487 (e.g., intestinal/hepatic first-pass metabolism and systemic CL). The biotransformation of AMG 487 is dependent on CYP3A and results in the formation of two primary metabolites, a pyridyl N-oxide AMG 487 (M1) and an O-deethylated AMG 487 (M2). One of these metabolites, M2, undergoes further metabolism by CYP3A. M2 has also been demonstrated to inhibit CYP3A in a competitive (K(i)=0.75 microM) manner as well as via mechanism-based inhibition (unbound K(I)=1.4 microM, k(inact)=0.041 min(-1)). Data from this study implicate M2-mediated CYP3A mechanism-based inhibition as the proximal cause for the time-dependent pharmacokinetics of AMG 487. However, the sequential metabolism of M2, nonlinear AMG 487 pharmacokinetics, and the inability to accurately determine the role of intestinal AMG 487 metabolism complicates the correlation between M2 plasma concentrations and the time-dependent AMG 487 pharmacokinetic changes.

Isolation and molecular characterization of cancer stem cells in MMTV-Wnt-1 murine breast tumors.

In human breast cancers, a phenotypically distinct minority population of tumorigenic (TG) cancer cells (sometimes referred to as cancer stem cells) drives tumor growth when transplanted into immunodeficient mice. Our objective was to identify a mouse model of breast cancer stem cells that could have relevance to the study of human breast cancer. To do so, we used breast tumors of the mouse mammary tumor virus (MMTV)-Wnt-1 mice. MMTV-Wnt-1 breast tumors were harvested, dissociated into single-cell suspensions, and sorted by flow cytometry on Thy1, CD24, and CD45. Sorted cells were then injected into recipient background FVB/NJ female syngeneic mice. In six of seven tumors examined, Thy1+CD24+ cancer cells, which constituted approximately 1%-4% of tumor cells, were highly enriched for cells capable of regenerating new tumors compared with cells of the tumor that did not fit this profile ("not-Thy1+CD24+"). Resultant tumors had a phenotypic diversity similar to that of the original tumor and behaved in a similar manner when passaged. Microarray analysis comparing Thy1+CD24+ tumor cells to not-Thy1+CD24+ cells identified a list of differentially expressed genes. Orthologs of these differentially expressed genes predicted survival of human breast cancer patients from two different study groups. These studies suggest that there is a cancer stem cell compartment in the MMTV-Wnt-1 murine breast tumor and that there is a clinical utility of this model for the study of cancer stem cells.

A Five-year Review of the Designated Leadership Positions of Pediatric Orthopaedic Society of North America: Where Do Women Stand?

Despite the growing number of women entering medical school, female representation among orthopedic surgery is the lowest compared with all areas of medicine. In 2014, 47.7% of students entering medical school were women, but only 13.7% of orthopedic residents were women. Pediatric orthopedics have been successful in enrolling women compared with other orthopedic subspecialties. This is an investigation of female representation among the Pediatric Orthopaedic Society of North America membership roster, providing insight into the effect on the increased gender diversity in the membership of an organization and its correlation with leadership positions at different levels within the organization.

A fully automated plasma protein precipitation sample preparation method for LC-MS/MS bioanalysis.

This report describes the development and validation of a robust robotic system that fully integrates all peripheral devices needed for the automated preparation of plasma samples by protein precipitation. The liquid handling system consisted of a Tecan Freedom EVO 200 liquid handling platform equipped with an 8-channel liquid handling arm, two robotic plate-handling arms, and two plate shakers. Important additional components integrated into the platform were a robotic temperature-controlled centrifuge, a plate sealer, and a plate seal piercing station. These enabled unattended operation starting from a stock solution of the test compound, a set of test plasma samples and associated reagents. The stock solution of the test compound was used to prepare plasma calibration and quality control samples. Once calibration and quality control samples were prepared, precipitation of plasma proteins was achieved by addition of three volumes of acetonitrile. Integration of the peripheral devices allowed automated sequential completion of the centrifugation, plate sealing, piercing and supernatant transferral steps. The method produced a sealed, injection-ready 96-well plate of plasma extracts. Accuracy and precision of the automated system were satisfactory for the intended use: intra-day and the inter-day precision were excellent (C.V.<5%), while the intra-day and inter-day accuracies were acceptable (relative error<8%). The flexibility of the platform was sufficient to accommodate pharmacokinetic studies of different numbers of animals and time points. To the best of our knowledge, this represents the first complete automation of the protein precipitation method for plasma sample analysis.