Presenting Characteristics, Treatment Patterns, and Outcomes among Patients with Venous Thromboembolism during Hospitalization for COVID-19.

Venous thromboembolism (VTE) is common in patients with coronavirus disease-2019 (COVID-19). However, limited data exist on patient characteristics, treatments, and outcomes. To describe the clinical characteristics, treatment patterns, and short-term outcomes of patients diagnosed with VTE during hospitalization for COVID-19. This is a prospective multinational study of patients with incident VTE during the course of hospitalization for COVID-19. Data were obtained from the Registro Informatizado de la Enfermedad TromboEmbólica (RIETE) registry. All-cause mortality, VTE recurrences, and major bleeding during the first 10 days were separately investigated for patients in hospital wards versus those in intensive care units (ICUs). As of May 03, 2020, a total number of 455 patients were diagnosed with VTE (83% pulmonary embolism, 17% isolated deep vein thrombosis) during their hospital stay; 71% were male, the median age was 65 (interquartile range, 55-74) years. Most patients (68%) were hospitalized in medical wards, and 145 in ICUs. Three hundred and seventeen (88%; 95% confidence interval [CI]: 84-91%) patients were receiving thromboprophylaxis at the time of VTE diagnosis. Most patients (88%) received therapeutic low-molecular-weight heparin, and 15 (3.6%) received reperfusion therapies. Among 420 patients with complete 10-day follow-up, 51 (12%; 95% CI: 9.3-15%) died, no patient recurred, and 12 (2.9%; 95% CI: 1.6-4.8%) experienced major bleeding. The 10-day mortality rate was 9.1% (95% CI: 6.1-13%) among patients in hospital wards and 19% (95% CI: 13-26%) among those in ICUs. This study provides characteristics and early outcomes of patients diagnosed with acute VTE during hospitalization for COVID-19. Additional studies are needed to identify the optimal strategies to prevent VTE and to mitigate adverse outcomes associated.

Venous thromboembolism characteristics and outcomes among RIETE patients tested and untested for inherited thrombophilia.

ABSTRACT: Inherited thrombophilia (IT) workup is commonly pursued in patients with venous thromboembolism (VTE). Recent American Society of Hematology guidelines recommend a selective approach to IT testing, nevertheless, evidence on whether thrombophilia testing can actually improve patient-important outcomes through tailored management is limited. Data from the large, prospective Registro Informatizado de Enfermedad TromboEmbólica (RIETE) registry were analyzed to compare VTE risk factors, management, and outcomes between patients who were tested for IT and untested patients, during anticoagulant treatment and after its discontinuation. Among 103 818 patients enrolled in RIETE, 21 089 (20.3%) were tested for IT, 8422 (8.1%) tested positive, and 82 729 (79.7%) were not tested. IT testing was more frequent in patients with VTE provoked by minor risk factors and less common in those with major risk factors such as surgery or active cancer. Choices of anticoagulant treatment did not differ based on IT testing results. Untested patients exhibited inferior outcomes across all VTE categories, with higher rates of VTE recurrence, major bleeding, mortality, and notably, cancer-related mortality. After treatment discontinuation, IT-negative patients with surgically provoked VTE showed lower recurrence rates. For immobilization-related VTE as well as in estrogen-related VTE, no significant differences in recurrence rates were observed between IT-negative and IT-positive patients. However, IT-negative patients with pregnancy or postpartum-related VTE had significantly lower recurrence rates. Patients with unprovoked VTE, particularly those testing positive for IT, had high recurrence rates after treatment. These findings underscore the complex role of IT testing in managing VTE, supporting personalized treatment strategies that consider VTE risk factors and comorbidities. The trial was registered at www.clinicaltrials.gov as #NCT02832245.

Enoxaparin for Long-Term Therapy of Venous Thromboembolism in Patients with Cancer and Renal Insufficiency.

BACKGROUND: The optimal therapy of venous thromboembolism (VTE) in cancer patients with renal insufficiency (RI) is unknown. Current guidelines recommend to use low-molecular-weight heparin over direct oral anticoagulants to treat VTE in cancer patients at high risk of bleeding. METHODS: We used the Registro Informatizado Enfermedad Tromboemb00F3lica (RIETE) registry to compare the 6-month incidence rates of (1) VTE recurrences versus major bleeding and (2) fatal pulmonary embolism (PE) versus fatal bleeding in three subgroups (those with mild, moderate, or severe RI) of cancer patients receiving enoxaparin monotherapy. RESULTS: From January 2009 through June 2022, 2,844 patients with RI received enoxaparin for ≥6 months: 1,432 (50%) had mild RI, 1,168 (41%) moderate RI, and 244 (8.6%) had severe RI. Overall, 68, 62, and 12%, respectively, received the recommended doses. Among patients with mild RI, the rates of VTE recurrences versus major bleeding (4.6 vs. 5.4%) and fatal PE versus fatal bleeding (1.3 vs. 1.2%) were similar. Among patients with moderate RI, VTE recurrences were half as common as major bleeding (3.1 vs. 6.3%), but fatal PE and fatal bleeding were close (1.8 vs. 1.2%). Among patients with severe RI, VTE recurrences were threefold less common than major bleeding (4.1 vs. 13%), but fatal PE was threefold more frequent than fatal bleeding (2.5 vs. 0.8%). During the first 10 days, fatal PE was fivefold more common than fatal bleeding (2.1 vs. 0.4%). CONCLUSION: Among cancer patients with severe RI, fatal PE was fivefold more common than fatal bleeding. The recommended doses of enoxaparin in these patients should be revisited.

Cancer-Associated Thrombosis: Trends in Clinical Features, Treatment, and Outcomes From 2001 to 2020.

Background: Despite advances in cancer and venous thromboembolism (VTE) management, the epidemiology of cancer-associated thrombosis management over time remains unclear. Objectives: We analyzed data from the RIETE (Registro Informatizado de la Enfermedad Trombo Embólica) registry spanning 2001 to 2020 to investigate temporal trends in clinical characteristics and treatments for cancer-associated thrombosis. Methods: Using multivariable survival regression, we examined temporal trends in risk-adjusted rates of symptomatic VTE recurrences, major bleeding, and death within 30 days after incident VTE. Results: Among the 17,271 patients with cancer-associated thrombosis, there was a progressive increase in patients presenting with pulmonary embolism (from 44% in 2001-2005 to 55% in 2016-2020; P < 0.001 for trend), lung (from 12.7% to 18.1%; P < 0.001) or pancreatic cancer (from 3.8% to 5.6%; P = 0.003), and utilization of immunotherapy (from 0% to 7.4%; P < 0.001). Conversely, there was a decline in patients with prostate cancer (from 11.7% to 6.6%; P < 0.001) or carcinoma of unknown origin (from 3.5% to 0.7%; P < 0.001). At the 30-day follow-up, a reduction was observed in the proportion of patients experiencing symptomatic VTE recurrences (from 3.1% to 1.1%; P < 0.001), major bleeding (from 3.1% to 2.2%; P = 0.004), and death (from 11.9% to 8.4%; P < 0.001). Multivariable analyses revealed a decreased risk over time for VTE recurrence (adjusted subdistribution HR [asHR]: 0.94 per year; 95% CI: 0.92-0.98), major bleeding (asHR: 0.98; 95% CI: 0.96-0.99), and death (aHR: 0.97; 95% CI: 0.96-0.98). Conclusions: In this multicenter study of cancer patients with VTE, there was a decline in thrombotic, hemorrhagic, and fatal events from 2001 to 2020. (Registro Informatizado de la Enfermedad Trombo Embólica [RIETE]; NCT02832245).

Predictors of recurrence of cancer-associated venous thromboembolism after discontinuation of anticoagulant therapy: a multicenter cohort study.

BACKGROUND: Data on recurrence after the end of anticoagulation for a first event of cancer-associated venous thromboembolism (VTE) are scarce. OBJECTIVES: Our aim was to assess predictors of VTE recurrence during a 1-year follow-up period. METHODS: This study is an analysis of RIETE, an international, multicenter, prospective cohort study of patients diagnosed with VTE. Patients had to have active cancer at the time of VTE and to have withdrawn from anticoagulation after 3 months of full treatment. Analyses were performed using Fine and Gray models, with death as a competing risk, and multiple imputation of missing data was performed by chained equations. RESULTS: Among 14 318 patients with cancer-associated VTE, 3414 had undergone time-limited anticoagulation for at least 3 months. The cumulative incidence function for recurrent VTE was 10.2% (95% CI, 9.1-11.5) at 1 year. Chronic kidney disease (a subhazard ratio [sHR] of 1.08 for 10-mL/min decrease in glomerular filtration rate; 95% CI, 1.02-1.14); cancer of the lung, brain, stomach, esophagus, liver, or ovary (sHR, 3.56; 95% CI, 1.07-11.80; compared with cancer of the oropharynx, larynx, or melanoma); cancer of the pancreas, the biliary tract, or of unknown origin (sHR, 6.86; 95% CI, 1.89-24.85); inferior vena cava filter (sHR, 3.16; 95% CI, 1.75-5.71); postthrombotic syndrome (sHR, 2.09; 95% CI, 1.06-4.15); and residual pulmonary thrombotic obstruction (sHR, 2.58; 95% CI, 1.38-4.82) were predictive of recurrence. Surgery during the 2 months before VTE was predictive of absence of recurrence (sHR, 0.60; 95% CI, 0.40-0.92). CONCLUSION: One year after anticoagulant cessation for cancer-associated VTE, approximately 10% of patients experienced recurrence. Discontinuing anticoagulant therapy seems safe, mainly in surgery-associated VTE.

Short versus long course therapy in the treatment of febrile urinary tract infections in men based on serum PSA values.

BACKGROUND: Febrile urinary tract infections (fUTI) in men are frequently complicated with subclinical prostatic involvement, measured by a transient increase in serum prostate-specific-antigen (sPSA). The aim of this study was to evaluate recurrence rates in a 6-month follow-up period of 2-week versus 4-week antibiotic treatment in men with fUTI, based on prostatic involvement. Clinical and microbiological cure rates at the end-of-therapy (EoT) were also assessed. METHODS: Open label, not-controlled, prospective study. Consecutive men diagnosed of fUTI were included. Duration of therapy was 2 weeks for patients with a sPSA level <5mg/L (short duration therapy, SDT) or 4 weeks for PSA >5 mg/L (long duration therapy, LDT). RESULTS: Ninety-one patients were included; 19 (20%) received SDT. Median age was 56.9 years (range 23-88). Bacteremia was present in 9.8% of patients (Escherichia coli was isolated in 91%). Both groups had similar demographic, clinical characteristics and laboratory findings. Median PSA levels were 2.3 mg/L in the SDT group vs 23.4 mg/L in the LDT group. In the 6-month visit, 26% of patients had achieved complete follow-up. Nonsignificant differences between groups were found neither in recurrence rates after 6 months (9% in SDT vs 10% in LDT) nor in clinical or microbiological cure rates at EoT (100% in SDT vs 95% in LDT and 95% in SDT vs 93% in LDT respectively). CONCLUSIONS: One fifth of men with fUTI did not present apparent prostatic involvement. A 2-week regimen seems adequate in terms of clinical, microbiological cure and recurrence rates for those patients without PSA elevation.

Bleeding risk in hospitalized patients with COVID-19 receiving intermediate- or therapeutic doses of thromboprophylaxis.

INTRODUCTION: Some local protocols suggest using intermediate or therapeutic doses of anticoagulants for thromboprophylaxis in hospitalized patients with coronavirus disease 2019 (COVID-19). However, the incidence of bleeding, predictors of major bleeding, or the association between bleeding and mortality remain largely unknown. METHODS: We performed a cohort study of patients hospitalized for COVID-19 that received intermediate or therapeutic doses of anticoagulants from March 25 to July 22, 2020, to identify those at increased risk for major bleeding. We used bivariate and multivariable logistic regression to explore the risk factors associated with major bleeding. RESULTS: During the study period, 1965 patients were enrolled. Of them, 1347 (69%) received intermediate- and 618 (31%) therapeutic-dose anticoagulation, with a median duration of 12 days in both groups. During the hospital stay, 112 patients (5.7%) developed major bleeding and 132 (6.7%) had non-major bleeding. The 30-day all-cause mortality rate for major bleeding was 45% (95% confidence interval [CI]: 36%-54%) and for non-major bleeding 32% (95% CI: 24%-40%). Multivariable analysis showed increased risk for in-hospital major bleeding associated with D-dimer levels >10 times the upper normal range (hazard ratio [HR], 2.23; 95% CI, 1.38-3.59), ferritin levels >500 ng/ml (HR, 2.01; 95% CI, 1.02-3.95), critical illness (HR, 1.91; 95% CI, 1.14-3.18), and therapeutic-intensity anticoagulation (HR, 1.43; 95% CI, 1.01-1.97). CONCLUSIONS: Among patients hospitalized with COVID-19 receiving intermediate- or therapeutic-intensity anticoagulation, a major bleeding event occurred in 5.7%. Use of therapeutic-intensity anticoagulation, critical illness, and elevated D-dimer or ferritin levels at admission were associated with increased risk for major bleeding.

Recent developments and persisting challenges in the prevention and treatment of venous thromboembolism in patients with hematological malignancies.

Venous thromboembolism (VTE) is a common complication of cancer. Its treatment is challenging because of the high risk for both VTE recurrence and bleeding. Evidence is particularly scarce for patients with hematological malignancies. This review aims to summarize new developments in anticoagulation for the prevention and treatment of VTE in patients with active cancer, largely derived from the formal introduction of direct anticoagulants (DOACs) in this population. We then offer our recommendations for the thromboprophylaxis and treatment of VTE in patients with hematological disorders (mature lymphoid disorders, plasma cell disorders, myeloproliferative neoplasms, myelodysplastic syndrome and acute leukemia/stem cell transplant). We conclude by emphasizing the lack of high-quality evidence in a majority of these settings, caution about the use of DOACs in clinical situations where evidence is lacking and, finally, note the importance of involving patients in the decision-making process.

Evaluation of the SAMe-TT2R2 score to predict the quality of anticoagulation control in patients with venous thromboembolism treated with vitamin K antagonists: Findings from the RIETE registry.

BACKGROUND: The time in therapeutic range (TTR) of patients with venous thromboembolism (VTE) treated with vitamin K antagonists (VKA) is usually below recommended, leading to higher frequency of vascular events, bleeding and mortality. The SAMe-TT2R2 prediction score discriminates those patients with high or low probability of obtaining poor INR control and its use is recommended in patients with atrial fibrillation. Its usefulness has been evaluated in patients with VTE, with conflicting results. METHOD: We included consecutive patients enrolled in Registro Informatizado Enfermedad TromboEmbolica (RIETE), a prospective multicenter VTE registry, treated with VKA for >90 days and a minimum of 3 INR determinations. We analyzed the relationship between the SAMe-TT2R2 score and TTR, determined by the Rosendaal method and by the percentage of INR determinations (after excluding the first month). A ROC curve was calculated considering a cut-off point of TTR ≥65% for good anticoagulation control. RESULTS: 3893 patients were included and classified in high (1411 patients) or low (2482 patients) probability of obtaining poor INR control according to the total score obtained (0-1 points versus 2 points, respectively). TTR, calculated by direct method and Rosendaal method, was 51.2 (±23.4) and 55.4 (±25.9) in the high probability group; and 54.4 (±23.0) and 58.2 (±25.6) in the low probability group, respectively (p < 0.001 for both comparisons). The outcomes were similar between groups. The predictive capacity of the SAMe-TT2R2 score showed an area under the ROC curve of 0.54 (CI 95% 0.52-0.56) and 0.53 (CI 95% 0.51-0.55). CONCLUSIONS: In patients with VTE treated with VKA, the SAMe-TT2R2 score discriminated those patients with high probability of obtaining poor INR control, but with a low predictive capacity. Further studies are required to assess the usefulness of the score in clinical decision-making.

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.

Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL.

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.

Performance evaluation of three platforms with ultrasensitive ligand-binding assay potential.

AIM: We evaluated three immunoassay-based technologies and their biomarker kits, by determining precision, parallelism and detectability of analytes of interest. MATERIALS & METHODS: We compared ultrasensitive assays for three biomarkers: interleukins IL-6, IL-13 and IL-17A using kits obtained from Roche (IMPACT platform - proprietary platform), Singulex (Erenna®) and Quanterix (Simoa™). We defined the true LLOD as the LLOQs, and provided disease-specific parallelism results and detectability levels for endogenous analyte, which were good across platforms, though they varied from analyte to analyte. CONCLUSION: We highlight a simplified approach employed for evaluating ultrasensitive kits and provide an overview of the methodologies used to compare available assays. All three platforms are able to detect very low-level analytes. We recommend all three platforms for detection of very low-level analytes.

Specific Immune Response to Phospholipase B-Like 2 Protein, a Host Cell Impurity in Lebrikizumab Clinical Material.

Host cell proteins are manufacturing process-related impurities that may co-purify with the product despite extensive efforts to optimize the purification process. The risks associated with these impurities can vary and may be patient and/or therapeutic dependent. Therefore, it is critical to monitor and control the levels of these impurities in products and their potential impact on safety and efficacy. Lebrikizumab is a humanized immunoglobulin G4 monoclonal antibody (mAb) that binds specifically to soluble interleukin 13. This mAb is currently in phase III clinical development for the treatment of asthma. Following initial phase III studies, the material used in lebrikizumab clinical trials was found to have a process-related impurity identified as Chinese hamster ovary phospholipase B-like 2 (PLBL2) which co-purified with lebrikizumab. The immunogenic potential of PLBL2 and its potential impact on the immunogenicity of lebrikizumab in clinical studies were therefore evaluated. Data from the clinical studies demonstrated that ∼90% of subjects developed a specific and measurable immune response to PLBL2. Given the high incidence of antibodies to PLBL2 as well as the comparable safety profile observed between placebo- and drug-treated subjects, no correlation between safety events and anti-PLBL2 antibodies could be made. Additionally, no impact on the incidence of anti-lebrikizumab antibodies was observed, suggesting the lack of an adjuvant effect from PLBL2. Interim analysis from ongoing phase III studies using material with substantially reduced levels of PLBL2 with patients having had longer exposure shows significantly less and dose-dependent frequency of immune responses to PLBL2.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Strategies to compare clinical antitherapeutic antibody data when changing assay platforms: a case study.

Zhihua Julia Qiu has over 20 years post PhD experience in academic institutes, pharmaceutical industry and biotechnology startup settings; focused on novel therapeutics discovery and development and diagnostic tools. She is currently a Scientist in the Bioanalytical Sciences department at Genentech; responsible for developing, evaluating and implementing Bioanalytical strategy to support protein therapeutics development. That includes assay development and validation to evaluate PK, antitherapeutic antibodies as well as biomarkers in both nonclinical and clinical studies for Immunology and Oncology indications. In addition, she has led the evaluation of multiple novel technology platforms and transitioning assay platform to enable continuous support for the development of protein therapeutics and antibody-drug conjugates.

Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper.

Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

Índices de riesgo de recurrencia en la enfermedad tromboembólica venosa no provocada / Predicting risk indices of recurrence in the unprovoked venous thromboembolic disease

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Development of a novel homogenous electrochemiluminescence assay for quantitation of ranibizumab in human serum.

A solution-phase electrochemiluminescence assay (ECLA) was developed to quantify ranibizumab in serum from patients treated with this biotherapeutic for neovascular age-related macular degeneration. Ranibizumab, a recombinant humanized Fab ("fragment, antigen binding"), binds with high affinity and specificity to vascular endothelial growth factor A (VEGF-A), inhibiting its activity. Fab molecules contain the amino acid sequence that binds antigen and are composed of one constant and one variable domain from each heavy and light chain of the antibody. High assay sensitivity was required to enable pharmacokinetic (PK) evaluation of ranibizumab-dosed patients in clinical trials. Our assay's lower limit of quantitation is 300 pg/mL ranibizumab in neat serum, achieving a 67-fold improvement in sensitivity relative to a conventional ELISA-based PK method. In this assay, ruthenium-labeled affinity-purified rabbit anti-ranibizumab antibodies and biotinylated rhVEGF are added to serum samples. During overnight incubation, these two labeled molecules bind to ranibizumab, and the resulting immune complex is then captured by streptavidin-coated paramagnetic beads and analyzed for electrochemiluminescence. The ranibizumab PK ECLA has a reporting range of 300-24,000 pg/mL, based on accuracy and precision parameters. It showed high precision for both intra- and inter-assay analyses. Recovery of ranibizumab from 10 individual donors averaged between 100% and 119% of nominal concentration. There was no cross-reactivity observed in the assay to other recombinant humanized antibodies (whole molecules or monoclonal antibody fragments) or human IgG. To our knowledge, this report represents the first description of development and validation of an ECLA-based PK assay for a recombinant humanized Fab therapeutic agent.