Selective MCL-1 inhibitor ABBV-467 is efficacious in tumor models but is associated with cardiac troponin increases in patients.

BACKGROUND: MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). METHODS: Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. RESULTS: Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. CONCLUSIONS: The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Apoptosis is a type of cell death that removes abnormal cells from the body. Cancer cells can have increased levels of MCL-1, a protein that helps cells survive and prevents apoptosis. ABBV-467 is a new drug that blocks the action of MCL-1 (an MCL-1 inhibitor) and could promote apoptosis. In animal models, ABBV-467 led to cancer cell death and delayed tumor growth. ABBV-467 was also studied in a clinical trial in 8 patients with multiple myeloma, a blood cancer. In 1 patient, ABBV-467 treatment prevented the cancer from getting any worse for 8 months. However, in 4 out of 8 patients ABBV-467 increased the levels of troponin, a protein associated with damage to the heart. This concerning side effect may impact the future development of MCL-1 inhibitors as anticancer drugs.

Reference intervals: past, present, and future.

Clinical laboratory test results alone are of little value in diagnosing, treating, and monitoring health conditions; as such, a clinically actionable cutoff or reference interval is required to provide context for result interpretation. Healthcare practitioners base their diagnoses, follow-up treatments, and subsequent testing on these reference points. However, they may not be aware of inherent limitations related to the definition and derivation of reference intervals. Laboratorians are responsible for providing the reference intervals they report with results. Yet, the establishment and verification of reference intervals using conventional direct methods are complicated by resource constraints or unique patient demographics. To facilitate standardized reference interval best practices, multiple global scientific societies are actively drafting guidelines and seeking funding to promote these initiatives. Numerous national and international multicenter collaborations demonstrate the ability to leverage combined resources to conduct large reference interval studies by direct methods. However, not all demographics are equally accessible. Novel indirect methods are attractive solutions that utilize computational methods to define reference distributions and reference intervals from mixed data sets of pathologic and non-pathologic patient test results. In an effort to make reference intervals more accurate and personalized, individual-based reference intervals are shown to be more useful than population-based reference intervals in detecting clinically significant analyte changes in a patient that might otherwise go unrecognized when using wider, population-based reference intervals. Additionally, continuous reference intervals can provide more accurate ranges as compared to age-based partitions for individuals that are near the ends of the age partition. The advantages and disadvantages of different reference interval approaches as well as the advancement of non-conventional reference interval studies are discussed in this review.

Estimation of Reference Intervals from Routine Data Using the refineR Algorithm-A Practical Guide.

BACKGROUND: Accurate reference intervals are essential for the interpretation of laboratory test results. Typically, they are determined by the central 95% range of test results from a predefined reference population. As these direct studies can face practical and ethical challenges, indirect methods using routine measurements offer an alternative approach. METHODS: We provide step-by-step guidance on how to apply an indirect method in practice using refineR, the most recently published indirect method, and showcase the application by evaluating real-world data of 12 prespecified analytes. Measurements were retrieved from ARUP Laboratories' data warehouse, and were obtained from routine patient testing on cobas c502 or e602 analyzers. Test results were prefiltered and cleaned and, if necessary, physiologically partitioned prior to estimating reference intervals using refineR. Estimated reference intervals were then compared to established intervals provided by the manufacturer. RESULTS: For most analytes, the reference intervals estimated by refineR were comparable to those provided by the manufacturer, shown by overlapping confidence intervals at both reference limits, or only the upper or lower limit. For thyroid-stimulating hormone, refineR estimated higher reference limits, while estimates for prealbumin were lower compared to the established reference interval. CONCLUSIONS: We applied the refineR algorithm to a variety of real-world data sets resulting in reference intervals similar to intervals previously established by direct methods. We further provide practical guidance and a code example on how to apply an indirect method in a real-world scenario facilitating their access and thus their use in laboratory settings.

Comparison of two chromogranin A assays and investigation of nonlinear specimens.

Background: As a marker for functional and non-functional neuroendocrine tumors, serum chromogranin A (CgA) concentrations have shown value for detecting and monitoring disease. Here we describe a comparison between an established micro-titer plate assay (Cisbio CgA ELISA) and an analyzer-based assay (B·R·A·H·M·S CgA II KRYPTOR). Reference limits were established along with a performance evaluation of the KRYPTOR assay. Nonlinearity observed in approximately 0.03% of patients was also investigated. Methods: Samples were tested according to kit manufacturer's protocols. Reference limits were established for both assays testing the same cohort of healthy volunteers. Potential causes of nonlinearity investigated were HAMA, macromolecule effects and elevated serum creatinine. Results: KRYPTOR vs. Cisbio: slope=0.692, y-intercept=-40.0 (r2=0.967, n=186). Upper reference limits were 160 and 103 ng/mL for the Cisbio and KRYPTOR assays, respectively. Linearity: slope=1.012 (r2=0.998) with 95.0-105.5% recoveries. Precision: repeatability ≤2.4%, within-laboratory ≤3.1% (79 and 738 ng/mL). Limit of detection: 8 ng/mL. Strong nonlinear specimens (n=6) retested for HAMA interference generated differences (block-no block) ranging -3.2-4.2%. Polyethylene glycol precipitation recoveries ranged from 157 to >5714% for affected specimens versus 71-79% for normal specimens. Eight of 14 nonlinear specimens (57%) had elevated serum creatinine results (>1.20 mg/dL). Conclusions: The CgA II KRYPTOR assay performs acceptably for quantifying CgA in human serum. While adequate correlation is observed against the Cisbio ELISA, there is significant disagreement overall. Efforts to identify a cause of the nonlinearity observed in a small percentage of patients were inconclusive, but neither HAMA interference, macromolecule effects nor renal failure appear as major factors.

Clinical Utility and Analytical Aspects of Direct Measurements of Free Hormones Using Mass Spectrometry-Based Methods.

BACKGROUND: The free hormone (FH) hypothesis states that hormone action and the corresponding biological effects are mediated by the unbound (free) fraction of hormone in circulation. The in vivo relationship between protein-bound and FH is complex and dynamic. In most individuals, measurement of total hormone (TH) is usually adequate to reflect the hormone status; however, certain physiological conditions and/or medications can affect protein binding and alter FH concentration. In these cases, measurement of FH will provide a better measure of the bioactive hormone status than measurement of the TH. Measurement of FH presents many challenges, as the concentrations are very low and there are number of pitfalls, which may affect the measured concentrations. CONTENT: In this review, we discuss techniques used in the separation and direct quantitation of FH concentrations in biological samples using mass spectrometry for analysis. We also highlight clinical situations in which FH analysis is warranted and when mass spectrometry should be the preferred methodology over immunoassays. SUMMARY: Equilibrium dialysis, ultrafiltration, or size-exclusion separation coupled with liquid chromatography-tandem mass spectrometry provides a sensitive and specific method to measure FH concentrations. These direct methods are useful in iatrogenic or physiological states that alter hormone binding or metabolism.

Mitigation of Biotin Interference in Manual and Automated Immunoassays by Preconjugating Biotinylated Antibodies to the Streptavidin Surface as an Alternative to Biotin Depletion.

BACKGROUND: Streptavidin-to-biotin binding is one of the strongest noncovalent interactions in nature and incorporated into many immunoassays. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase results. To prevent biotin interference, we evaluated a method to preconjugate biotinylated antibodies to the assay's streptavidin solid surface before adding patient specimen and compared this technique to a biotin depletion protocol. METHODS: Biotin interference in 3 manual ELISAs and 2 automated immunoassays was established. Mitigation of biotin interference by preincubation was evaluated in each assay by adding biotinylated antibody to the streptavidin-coated surface before adding biotin- or PBS-spiked serum. Lastly, the preincubation method was compared to a biotin-depletion protocol to compare the effectiveness of mitigating biotin interference. RESULTS: In the presence of 400 µg/L biotin, analyte detection was reduced to 10% to 15% of total in the ELISA assays and to 15.2% in the automated sandwich (thyroglobulin) immunoassay. In the automated competitive (free thyroxine) immunoassay, biotin caused an increased detection of 551.6%. Preconjugation of the biotinylated capture antibody to the streptavidin surface in the ELISA assays resulted in 84% to 99% activity recovery, compared to 84% to 97% by a biotin depletion protocol. Similarly, automated sandwich and competitive immunoassays obtained 97.1% and 116.5% recovery by preconjugation, compared to 95.6% and 100.3% by the depletion method, respectively. CONCLUSION: This study demonstrates how assay redesign to include preconjugation of biotinylated capture antibody to streptavidin is an effective alternative to biotin-depletion methods to mitigate biotin interference.

Verifying Clinically Derived Reference Intervals for Daily Excretion Rates of Fractionated Metanephrines Using Modern Indirect Reference Interval Models.

OBJECTIVES: Biochemical testing of urinary metanephrines is useful in the diagnosis and monitoring of pheochromocytoma and paragangliomas. We investigated the feasibility of mixture decomposition (ie, indirect) methods in verifying clinically derived reference intervals for urinary deconjugated metanephrine metabolites. METHODS: Urinary 24-hour metanephrine and normetanephrine excretion results were extracted from our data warehouse and intervals were estimated by the modern variant of the Hoffmann method, maximum likelihood estimation (MLE), and gamma mixture model using R software. RESULTS: Hoffmann, MLE, and gamma mixture models provided metanephrine and normetanephrine intervals that closely matched those derived from clinical studies. However, three-component MLE and gamma models were required for normetanephrine in adult women because the Hoffmann method was not suitable. Some data transformations caused blending of the mixed distributions and subsequent widening of the reference interval estimation, emphasizing the importance of careful data transformation for Hoffmann and MLE analyses. Gamma mixture models gave overall good agreement without the need for data transformation. CONCLUSIONS: Indirect methods have utility in verifying reference intervals in 24-hour urine specimens collected by patients. We emphasize the benefits of applying multiple decomposition methods to corroborate findings and careful application of data transformation when using Gaussian-based models.

Arthroscopic Management of Synovial Chondromatosis of the Ankle Joint: A Systematic Review of the Literature.

BACKGROUND: Synovial chondromatosis (SC) of the ankle is a rare disorder in which metaplastic proliferation of synovia, tendon sheaths, and/or bursae leads to the formation of loose cartilaginous bodies within the joint space. While SC itself is a benign condition, its sequelae can be chronic and debilitating if left untreated, or if there is postoperative recurrence. Arthritic degeneration and malignant transformation to chondrosarcoma are among the more serious complications in the natural history of the disease; the latter occurs in approximately 5% of cases. We performed a systematic review of the literature on the arthroscopic management of ankle SC in order to better understand recurrence patterns and suggest an alternative approach to open arthrotomy. METHODS: Using predetermined inclusion and exclusion criteria, the PubMed and Embase databases were searched for relevant articles related to arthroscopic surgical management of patients with confirmed SC of the ankle. Article selection and data abstraction were performed in 3 steps by 3 independent reviewers. RESULTS: The initial search retrieved 116 articles, of which 15 were included. Those studies included 22 patients (14 to 63 years of age; 77% male) who were followed for a mean of 27 months (range, 8 weeks to 12 years) postoperatively. The cases represented a mix of primary and secondary SC etiologies, and almost entirely stage-III disease. Arthroscopic synovectomy with excision of loose bodies was a consistent feature of treatment, and bursectomy, debridement of osteochondral lesions or involved tendons, and osteophyte resection were performed as indicated. All but 1 case employed an anterior approach including anteromedial and anterolateral portals, and in several cases a 4-portal technique was used, which was subsequently proposed as a necessary approach for the definitive treatment of ankle SC. On the basis of the available data, complication and recurrence rates following arthroscopic management were very low. CONCLUSIONS: A growing pool of evidence is emerging to suggest favorable outcomes for arthroscopic treatment of SC of the ankle joint. However, more data on patient and surgical variables, as well as comparative studies with longer follow-up, are needed in order to draw definitive conclusions. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Cannabinol (CBN) Cross-Reacts with Two Urine Immunoassays Designed to Detect Tetrahydrocannabinol (THC) Metabolite.

BACKGROUND: The psychoactive component of cannabis, tetrahydrocannabinol (THC), is one of many cannabinoids present in the plant. Since cannabinoids have extensive structural similarity, it is important to be aware of potential cross-reactivity with immunoassays designed to detect THC metabolite. This is especially important as cannabinoid products are increasingly marketed as legal supplements. The objective of this study was to assess the cross-reactivity of 2 commercial immunoassays designed to detect THC metabolite with 4 cannabinoids: cannabidiol, cannabinol, cannabichromene, and cannabigerol. METHODS: Deidentified residual patient urine samples that tested negative for THC metabolite on initial testing were pooled and fortified with the above compounds to detect cross-reactivity. We next tested a range of CBN concentrations to determine what concentration of CBN was required to trigger a positive immunoassay result. Finally, we tested whether CBN has an additive effect with THC in the immunoassay by adding CBN to 21 samples weakly positive for THC by a mass spectrometry method but negative by the EMIT II Plus immunoassay. RESULTS: Both the EMIT II Plus assay and the Microgenics MultiGent assay demonstrated cross-reactivity with CBN. For the EMIT II Plus assay, about 5-fold more CBN than THC metabolite was required to produce an assay signal equivalent to the cutoff concentration, and CBN displayed an additive effect with THC metabolite. For the Microgenics assay, 20-fold more CBN than THC metabolite was required to cross the cutoff concentration. CONCLUSIONS: These data may help guide the need for confirmatory testing when results of THC metabolite testing by immunoassay are inconsistent with expectations.

Therapeutic Drug Monitoring in Pregnant Patients.

During pregnancy, there are several physiological changes during each trimester that can affect the absorption, distribution, metabolism, and elimination of drugs. Although there is a potential need to understand the pharmacokinetics and pharmacodynamics of drugs in pregnant patients, therapeutic drug monitoring is not well established for various drug classes due to ethical and safety concerns regarding the neonate. Potential risks from in utero drug exposure to the fetus may impact growth and development and may cause malformations or teratogenesis. The clinician must consider the benefits of drug treatment for the pregnant mother versus the risk to the fetus, before prescribing medications during pregnancy. The objective of this review is to aid clinicians, pharmacists, and laboratorians in understanding the pharmacokinetic and pharmacodynamic changes during pregnancy, to provide drug class recommendations for monitoring therapy throughout pregnancy via therapeutic drug monitoring, and to highlight the recent directives of governing agencies on maternal and fetal health.

Sensitivity of C-reactive protein for the identification of patients with laboratory-confirmed bacterial infections in northern Tanzania.

OBJECTIVE: Identifying febrile patients requiring antibacterial treatment is challenging, particularly in low-resource settings. In South-East Asia, C-reactive protein (CRP) has been demonstrated to be highly sensitive and moderately specific in detecting bacterial infections and to safely reduce unnecessary antibacterial prescriptions in primary care. As evidence is scant in sub-Saharan Africa, we assessed the sensitivity of CRP in identifying serious bacterial infections in Tanzania. METHODS: Samples were obtained from inpatients and outpatients in a prospective febrile illness study at two hospitals in Moshi, Tanzania, 2011-2014. Bacterial bloodstream infections (BSI) were established by blood culture, and bacterial zoonotic infections were defined by ≥4 fold rise in antibody titre between acute and convalescent sera. The sensitivity of CRP in identifying bacterial infections was estimated using thresholds of 10, 20 and 40 mg/l. Specificity was not assessed because determining false-positive CRP results was limited by the lack of diagnostic testing to confirm non-bacterial aetiologies and because ascertaining true-negative cases was limited by the imperfect sensitivity of the diagnostic tests used to identify bacterial infections. RESULTS: Among 235 febrile outpatients and 569 febrile inpatients evaluated, 31 (3.9%) had a bacterial BSI and 61 (7.6%) had a bacterial zoonosis. Median (interquartile range) CRP values were 173 (80-315) mg/l in bacterial BSI, and 108 (31-208) mg/l in bacterial zoonoses. The sensitivity (95% confidence intervals) of CRP was 97% (83%-99%), 94% (79%-98%) and 90% (74%-97%) for identifying bacterial BSI, and 87% (76%-93%), 82% (71%-90%) and 72% (60%-82%) for bacterial zoonoses, using thresholds of 10, 20 and 40 mg/l, respectively. CONCLUSION: C-reactive protein was moderately sensitive for bacterial zoonoses and highly sensitive for identifying BSIs. Based on these results, operational studies are warranted to assess the safety and clinical utility of CRP for the management of non-malaria febrile illness at first-level health facilities in sub-Saharan Africa.

OBJECTIF: Identifier les patients fébriles nécessitant un traitement antibactérien est un défi, en particulier dans les milieux à faibles ressources. En Asie du Sud-Est, il a été démontré que la protéine C-réactive (CRP) est très sensible et modérément spécifique dans la détection des infections bactériennes et qu'elle réduit en toute sécurité les prescriptions antibactériennes inutiles dans les soins primaires. Comme les données sont rares en Afrique subsaharienne (ASS), nous avons évalué la sensibilité de la CRP dans l'identification des infections bactériennes sévères en Tanzanie. MÉTHODES: Des échantillons ont été obtenus auprès de patients hospitalisés et ambulatoires dans une étude prospective sur les maladies fébriles dans deux hôpitaux à Moshi, en Tanzanie de 2011 à 2014. Les infections bactériennes du sang (IBS) ont été identifiées par la culture du sang et les infections bactériennes zoonotiques ont été définies par une élevation ≥ 4 fois le titre des anticorps entre les sérums en aiguë et en convalescence. La sensibilité de la CRP dans l'identification des infections bactériennes a été estimée en utilisant des seuils de 10, 20 et 40 mg/L. La spécificité n'a pas été évaluée parce que la détermination des résultats faux positifs de la CRP était limitée par le manque de tests de diagnostic pour confirmer les étiologies non bactériennes et parce que la confirmation des vrais cas négatifs était limitée par la sensibilité imparfaite des tests de diagnostic utilisés pour identifier les infections bactériennes. RÉSULTATS: Sur 235 patients ambulatoires fébriles et 569 patients hospitalisés fébriles évalués, 31 (3.9%) avaient une IBS et 61 (7.6%) avaient une zoonose bactérienne. Les valeurs médianes (intervalle interquartile) de la CRP étaient de 173 (80-315) mg/L dans les IBS et de 108 (31-208) mg/L dans les zoonoses bactériennes. La sensibilité (intervalles de confiance à 95%) de la CRP était de 97% (83-99%), 94% (79-98%), 90% (74-97%) pour identifier les IBS et 87% (76-93% ), 82% (71-90%), 72% (60-82%) pour les zoonoses bactériennes, en utilisant des seuils de 10, 20 et 40 mg/L respectivement. CONCLUSION: La CRP était modérément sensible pour les zoonoses bactériennes et hautement sensible pour l'identification des IBS. Sur la base de ces résultats, des études opérationnelles sont justifiées pour évaluer la sécurité et l'utilité clinique de la CRP pour la prise en charge des maladies fébriles non paludiques dans les établissements de santé de premier niveau en ASS.

Ferritin in serum and urine: A pilot study.

Serum ferritin reflects total body iron stores, thus a low serum ferritin is used as a parameter of iron deficiency. In healthy adults in Japan, urine ferritin levels were about 5% of serum ferritin levels, with a correlation coefficient of 0.79. It is not known whether a low urine ferritin could serve as a non-invasive screen for iron deficiency. If so, this might be useful for neonates and young children, avoiding phlebotomy to screen for iron deficiency. However, for urinary ferritin screening to be feasible, ferritin must be measurable in the urine and correlate with serum ferritin. Testing should also clarify whether the iron content of ferritin in serum and urine are similar. In this pilot feasibility study we measured ferritin in paired serum and urine samples of healthy adult males, healthy term neonates, growing preterm neonates, and children who had very high serum ferritin levels from liver disorders or iron overload. We detected ferritin in every urine sample, and found a correlation with paired serum ferritin (Spearman correlation coefficient 0.78 of log10-transformed values). These findings suggest merit in further studying urinary ferritin in select populations, as a potential non-invasive screen to assess iron stores.

Short-term oral gavage administration of adenine induces a model of fibrotic kidney disease in rats.

INTRODUCTION: The adenine model of kidney disease typically involves dietary delivery of adenine over several weeks. This model can be variable in its disease progression and can result in significant mortality. In the current study, the amount of adenine delivered to rats was controlled by utilizing oral gavage administration over a short period in an attempt to induce robust renal pathology while addressing variability and viability of the animals. METHODS: Adenine (150 or 200 mg/kg) was administered via oral gavage for 10 consecutive days, and assessed over a total of 20 days. RESULTS: Both adenine dose groups manifested pathophysiological features of kidney disease such as proteinuria, elevated serum creatinine and BUN, and tubulointerstitial fibrosis. The animals also displayed a decline in glomerular filtration rate. Renal mRNA expression of genes associated with injury, inflammation, and fibrosis (i.e., Col1a1, Acta2, Serpine1, Timp1, Fn-Eda, Tgfb1, Ccl2, Nlrp3, Aqp1 and Ccnd1) were elevated as were urinary biomarkers that have translational utility (i.e., clusterin, KIM-1, MCP-1, OPN, NGAL, B2M, calbindin, and cystatin C). All disease endpoints were more pronounced in the 200 mg/kg group, however, while measures of tissue fibrosis were sustained, there was partial recovery by day 20 in functional readouts. No mortality was observed in either dose group. DISCUSSION: Short-term delivery of adenine via precise gavage delivery induced a robust model with hallmarks of fibrotic kidney disease, had limited variance between animals, and no animal morbidity within the 20 days studied. This model represents a methodical alternative to long-term dietary dosing of adenine.

Reversible LSD1 inhibition with HCI-2509 induces the p53 gene expression signature and disrupts the MYCN signature in high-risk neuroblastoma cells.

Lysine-Specific Demethylase 1 (LSD1) over-expression correlates with poorly differentiated neuroblastoma and predicts poor outcome despite multimodal therapy. We have studied the efficacy of reversible and specific LSD1 inhibition with HCI-2509 in neuroblastoma cell lines and particularly the effect of HCI-2509 on the transcriptomic profile in MYCN amplified NGP cells. Cell survival assays show that HCI-2509 is cytotoxic to poorly differentiated neuroblastoma cell lines in low micromole or lower doses. Transcriptional profiling of NGP cells treated with HCI-2509 shows a significant effect on p53, cell cycle, MYCN and hypoxia pathway gene sets. HCI-2509 results in increased histone methyl marks and p53 levels along with cell cycle arrest in the G2/M phase and inhibition of colony formation of NGP cells. Our findings indicate that LSD1 inhibition with HCI-2509 has a multi-target effect in neuroblastoma cell lines, mediated in part via p53. MYCN-amplified neuroblastoma cells have a targeted benefit as HCI-2509 downregulates the MYCN upregulated gene set.

Evaluation of a new venous catheter blood draw device and its impact on specimen hemolysis rates.

OBJECTIVES: Blood collections from peripheral intravenous catheters offer several benefits to patients, including reduced needle punctures and patient discomfort, but they risk reducing the quality of blood specimens analyzed by the laboratory. In an effort to balance analytical quality of test results with patient-centered care initiatives, a needle-less blood collection device called PIVO™ was evaluated at two institutions. The primary objective of this study was to assess the ability of the PIVO™ device to provide high-quality blood specimens for laboratory testing compared to current blood collection methods. METHODS: Blood specimens drawn using the PIVO™ device were prospectively flagged. A retrospective review was performed comparing the degree and rate of hemolysis for PIVO™ blood collections to both concurrent and historical hemolysis rates for other collection methods. RESULTS: Approximately 7600 PIVO™ blood draws were performed across the two institutions. The hemolysis rates of samples collected with PIVO™ were evaluated using 2380 flagged collections, containing approximately 1200 test orders requiring hemolysis index measurements. The hemolysis rate of PIVO™-flagged samples (1.8%) was statistically superior to the venipuncture and central line blood collection methods (3.3%), reducing the risk of hemolysis during a venous blood draw by 39%. CONCLUSIONS: PIVO™ collections facilitated improvement in the rate and degree of sample hemolysis when compared to venipuncture and central line blood collections. These findings suggest that PIVO™ is capable of delivering samples that are superior to current blood collection methods in terms of hemolysis rate as well as reducing the number of invasive venipunctures required for laboratory testing.

Targeting Anti-TGF-ß Therapy to Fibrotic Kidneys with a Dual Specificity Antibody Approach.

Targeted delivery of a therapeutic agent to a site of pathology to ameliorate disease while limiting exposure at undesired tissues is an aspirational treatment scenario. Targeting diseased kidneys for pharmacologic treatment has had limited success. We designed an approach to target an extracellular matrix protein, the fibronectin extra domain A isoform (FnEDA), which is relatively restricted in distribution to sites of tissue injury. In a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis, injury induced significant upregulation of FnEDA in the obstructed kidney. Using dual variable domain Ig (DVD-Ig) technology, we constructed a molecule with a moiety to target FnEDA and a second moiety to neutralize TGF-ß After systemic injection of the bispecific TGF-ß + FnEDA DVD-Ig or an FnEDA mAb, chemiluminescent detection and imaging with whole-body single-photon emission computed tomography (SPECT) revealed significantly higher levels of each molecule in the obstructed kidney than in the nonobstructed kidney, the ipsilateral kidney of sham animals, and other tissues. In comparison, a systemically administered TGF-ß mAb accumulated at lower concentrations in the obstructed kidney and exhibited a more diffuse whole-body distribution. Systemic administration of the bispecific DVD-Ig or the TGF-ß mAb (1-10 mg/kg) but not the FnEDA mAb attenuated the injury-induced collagen deposition detected by immunohistochemistry and elevation in Col1a1, FnEDA, and TIMP1 mRNA expression in the obstructed kidney. Overall, systemic delivery of a bispecific molecule targeting an extracellular matrix protein and delivering a TGF-ß mAb resulted in a relatively focal uptake in the fibrotic kidney and reduced renal fibrosis.