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Background Saliva has garnered great interest as an alternative specimen type for molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data are limited on the relative performance of different molecular methods using saliva specimens and the relative sensitivity of saliva to nasopharyngeal (NP) swabs. Methods To address the gap in knowledge, we enrolled symptomatic healthcare personnel (n = 250) from Barnes-Jewish Hospital/Washington University Medical Center and patients presenting to the Emergency Department with clinical symptoms compatible with coronavirus disease 2019 (COVID-19; n = 292). We collected paired saliva specimens and NP swabs. The Lyra SARS-CoV-2 assay (Quidel) was evaluated on paired saliva and NP samples. Subsequently we compared the Simplexa COVID-19 Direct Kit (Diasorin) and a modified SalivaDirect (Yale) assay on a subset of positive and negative saliva specimens. Results The positive percent agreement (PPA) between saliva and NP samples using the Lyra SARS-CoV-2 assay was 63.2%. Saliva samples had higher SARS-CoV-2 cycle threshold values compared to NP swabs (P Conclusion These data demonstrate molecular assays have variability in performance for detection of SARS-CoV-2 in saliva.

The Lyra SARS-CoV-2 assay was the primary method for molecular testing performed at Barnes-Jewish Healthcare System in St. Louis, Missouri during the initial COVID-19 surge from mid-March to late-April 2020. We performed a retrospective analysis of 1,043 positive Lyra SARS-CoV-2 results during these 36 days to investigate associations between cycle threshold (CT) value and patient characteristics. Total RNA were extracted from NP or OP swabs using either the EasyMag or KingFisher automated extraction systems and quantified with RotorGene Q (Qiagen) or Applied Biosystems 7500 Fast Dx thermocyclers respectively. Notably, we found lower a significant median lower CT for samples tested on the KingFisher-ABI 7500 fastDX (KF/ABI) system compared to the EasyMag/RotorGene (EM/RGQ) platform. Since 77.5% of our tests were ran on the EM/RGQ pipeline we then perform additional analysis on these values and found that C T values in outpatient care settings compared to samples obtained in the emergency department or inpatient had significantly lower C T values. These collective findings suggests a difference in viral load amongst various patient populations.

Orthopedic patients are at risk for adverse postoperative cardiovascular outcomes. This pilot randomized controlled trial (RCT) of atorvastatin vs. placebo in orthopedic surgery patients was performed in order to assess: (1) the prevalence of perioperative myocardial injury; (2) the effect of atorvastatin on perioperative inflammation; and (3) the feasibility of performing a large RCT of statin therapy in orthopedic patients. Hip fracture (hip Fx) and total hip and knee replacement (THR and TKR) patients were randomized 1:1 to atorvastatin 40 mg daily vs. placebo, starting preoperatively and continuing until postoperative day (POD) 45. High-sensitivity cardiac troponin I (hs-cTnI), high-sensitivity C-reactive protein (hs-CRP), and interleukin-6 (IL-6) were measured preoperatively and on POD 2. Patients were monitored for adverse events until POD 90. Five hundred fifty-six patients were screened, 22 were recruited (4 hip Fx, 11 THR, 7 TKR), and 2 withdrew. Most (80%) had detectable hs-cTnI (> 1.1 pg/mL) preoperatively. Twenty percent had a perioperative rise in hs-cTnI (≥ 10 pg/mL), which was not blunted by atorvastatin. Hs-CRP rose in 19/20 patients, and IL-6 rose in all patients. However, atorvastatin did not blunt the rise in these inflammatory biomarkers. On POD 2, IL-6 and hs-cTnI levels correlated (ρ = 0.59, p = 0.02). Recruitment was limited by the high prevalence of statin use in the screened population and a high prevalence of exclusions among hip fracture patients. Perioperative myocardial injury and inflammation are common in orthopedic patients and do not appear to be reduced in those randomized to atorvastatin. NCT02197065

Importance The optimal international normalized ratio (INR) to prevent venous thromboembolism (VTE) in warfarin-treated patients with recent arthroplasty is unknown. Objective To determine the safety and efficacy of a target INR of 1.8 vs 2.5 for VTE prophylaxis after orthopedic surgery. Design, Setting, and Participants The randomized Genetic Informatics Trial (GIFT) of Warfarin to Prevent Deep Vein Thrombosis enrolled 1650 patients aged 65 years or older initiating warfarin for elective hip or knee arthroplasty at 6 US medical centers. Enrollment began in April 2011 and follow-up concluded in October 2016. Interventions In a 2 × 2 factorial design, participants were randomized to a target INR of 1.8 (n = 823) or 2.5 (n = 827) and to either genotype-guided or clinically guided warfarin dosing. For the first 11 days of therapy, open-label warfarin dosing was guided by a web application. Main Outcomes and Measures The primary outcome was the composite of VTE (within 60 days) or death (within 30 days). Participants underwent screening duplex ultrasound postoperatively. The hypothesis was that an INR target of 1.8 would be noninferior to an INR target of 2.5, using a noninferiority margin of 3% for the absolute risk of VTE. Secondary end points were bleeding and INR values of 4 or more. Results Among 1650 patients who were randomized (mean age, 72.1 years; 1049 women [63.6%]; 1502 white [91.0%]), 1597 (96.8%) received at least 1 dose of warfarin and were included in the primary analysis. The rate of the primary composite outcome of VTE or death was 5.1% (41 of 804) in the low-intensity-warfarin group (INR target, 1.8) vs 3.8% (30 of 793) in the standard-treatment-warfarin group (INR target, 2.5), for a difference of 1.3% (1-sided 95% CI, −∞ to 3.05%,P = .06 for noninferiority). Major bleeding occurred in 0.4% of patients in the low-intensity group and 0.9% of patients in the standard-intensity group, for a difference of −0.5% (95% CI, −1.6% to 0.4%). The INR values of 4 or more occurred in 4.5% of patients in the low-intensity group and 12.2% of the standard-intensity group, for a difference of −7.8% (95% CI, −10.5% to −5.1%). Conclusions and Relevance Among older patients undergoing hip or knee arthroplasty and receiving warfarin prophylaxis, an international normalized ratio goal of 1.8 compared with 2.5 did not meet the criterion for noninferiority for risk of the composite outcome of VTE or death. However, the trial may have been underpowered to meet this criterion and further research may be warranted. Trial Registration ClinicalTrials.gov Identifier:NCT01006733

Warfarin-dosing algorithms combine clinical factors and dosing history with the current international normalized ratio (INR) to estimate the therapeutic warfarin dose. Unfortunately, these approaches can result in an overdose if the INR is spuriously low. Our goal was to develop an alert mechanism based on prior INRs in addition to the current INR. Using data from the Genetics InFormatics Trial (GIFT) of Warfarin to Prevent DVT, we analyzed warfarin dose estimates for days 3 through 11 that were ≥10 % higher than an average of the previous two dose estimates. We fit a stepwise mixed model to current and prior dose estimates, and subsequently compared the root-mean-square-error (RMSE) in predicting the final therapeutic dose using the GIFT algorithm versus the mixed model. From 861 dosing records (obtain from 556 patients), 646 dosing records (75 %) were randomly selected for the derivation cohort and 215 dosing records (25 %) for the validation cohort. Using one prior dose estimate improved the accuracy of the warfarin dose estimate. Compared to a dose estimate based on current INR (GIFT algorithm), the mixed model reduced the RMSE in the derivation cohort by 0.0015 mg/day (RMSE 0.2079 vs. 0.2094; p = 0.039). In the validation cohort, the RMSE reduction was not significant. A mixed model of dose estimates based on the current and most recent INRs shows potential to improve the safety of warfarin dosing. Clinicians should be cautious about aggressively escalating the warfarin dose after an INR that is lower than expected.

Over the past 20 years, there has been a continuous shift in decentralizing clinical laboratory testing in the US. Historically, testing was conducted almost exclusively in centralized laboratories in hospitals. However, at present, approximately 53% of testing is performed in hospitals (including some near-patient testing in settings such as critical care and emergency rooms), 34% in commercial clinical laboratories (50% of which is done in Quest Diagnostics and LabCorp), and the remaining in physician office laboratories and other settings as well as directly by consumers using over-the-counter devices. These numbers vary depending on the state. For example, in New York State 368 million tests in chemistry were performed in 2014, 73% of which were processed in hospital laboratories and 23% in independent (commercial) laboratories (data courtesy of Dr. Robert Rej). There are many drivers for this triangulating dynamic shift, including cost reduction, convenience to patients and clinicians, quality improvement (when the frequency of performing a particular test in the hospital laboratory is low), and the use of novel technologies such as nanotechnology, microfluidics, electrical impedance, reflectance, and time-resolved fluorescence, which enabled the miniaturization of devices and the proliferation of point-of-care (POC)8 testing. Improvement in information technology has been instrumental in the process of decentralizing testing and providing clinicians with patient results; this is, however, not a trivial matter and the cost of the interface is not always justifiable. Regulatory requirements in terms of assuring competency of those performing testing, getting patient laboratory results into the medical records, billing appropriately, and providing patients with their test findings are only few of the challenges of decentralized testing. How is the testing dynamic shift in this $56 billion industry going to evolve over the next 5 and 10 years? What will be the impact of technology, reimbursement, and confidentiality in shaping this …

Background and importance Antiphospholipid syndrome (APS) is an autoimmune disorder associated with a hypercoagulable state and increased risk of intraoperative and postoperative thrombosis. Few neurosurgical studies have examined the management of these patients, though the standard of care in most other disciplines involves the use of anticoagulation therapy. However, this is associated with risks such as hemorrhage, thrombosis due to warfarin withdrawal, and is not compatible with operative intervention. Clinical presentation We report the cases of 2 antiphospholipid positive patients who were on anticoagulant therapy and underwent surgical bypasses and received perioperative management with plasmapheresis. The first was a 44-yr-old woman who presented with worsening vision, recurring headaches, and a known left internal carotid artery aneurysm that was unsuccessfully treated twice via extracranial to intracranial (ECIC) bypass at another institution. Preoperative tests at our institution revealed elevated beta 2 glycoprotein 1 IgA autoantibodies. The second case was a 24-yr-old woman with previously diagnosed APS, who presented for surgical evaluation of moyamoya disease after sustaining recurrent left hemispheric strokes. Both cases were managed with perioperative plasmapheresis to avoid the need for anticoagulation during the perioperative period, and both underwent successful ECIC bypass procedures without perioperative ischemic or hemorrhagic complications. Conclusion Management of neurosurgical patients with APS can be a precarious proposition. We describe the successful use of plasmapheresis and antiplatelet therapy to better manage patients undergoing neurosurgical procedures, specifically ECIC bypass, and feel this approach can be considered in future cases.

Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by the activation of the mononuclear phagocytic system. The diagnosis of HLH in adults is challenging not only because the majority of the reported data are from pediatric patients, but also because HLH occurs in many disease entities. This study reports the clinical and laboratory findings and prognostic factors of adult HLH in a large cohort managed at a single medical center from 2003 to 2014. Seventy-three patients met the HLH-2004 diagnostic criteria. The median age was 51 years (range, 18–82 years); 41 (56.2%) were male. Patients manifested fever, cytopenias, and elevated ferritin in >85% of cases. Likely causes of HLH were as follows: 30 (41.1%) infections, 21 (28.8%) malignancies, 5 (6.8%) attributed to autoimmune disorders, 1 (1.4%) primary immunodeficiency, 2 (2.7%) post solid organ transplantation, and 13 (17.8%) idiopathic. The median overall survival was 7.67 months. Patients with malignancy-associated HLH had a markedly worse survival compared with patients with non–malignancy-associated HLH (median overall survival 1.13 vs. 46.53 months, respectively; P 50,000 µg/L correlated with 30-day mortality. Survival after a diagnosis of HLH is dismal, especially among those with malignancy-associated HLH. The development of a registry for adults with HLH would improve our understanding of this syndrome, validate diagnostic criteria, and help develop effective treatment strategies. Am. J. Hematol. 90:220–224, 2015. © 2014 Wiley Periodicals, Inc.

Background The clinical utility of genotype-guided (pharmacogenetically based) dosing of warfarin has been tested only in small clinical trials or observational studies, with equivocal results. Methods We randomly assigned 1015 patients to receive doses of warfarin during the first 5 days of therapy that were determined according to a dosing algorithm that included both clinical variables and genotype data or to one that included clinical variables only. All patients and clinicians were unaware of the dose of warfarin during the first 4 weeks of therapy. The primary outcome was the percentage of time that the international normalized ratio (INR) was in the therapeutic range from day 4 or 5 through day 28 of therapy. Results At 4 weeks, the mean percentage of time in the therapeutic range was 45.2% in the genotype-guided group and 45.4% in the clinically guided group (adjusted mean difference, [genotype-guided group minus clinically guided group], −0.2; 95% confidence interval, −3.4 to 3.1; P=0.91). There ...

BACKGROUND: Blockade of platelet activation and aggregation can inhibit metastasis in preclinical models and is associated with cancer prevention. To test whether disruption of platelet function with clopidogrel and aspirin would decrease the number of circulating tumor cells (CTCs) in patients with metastatic breast cancer, a randomized phase II study was performed. METHODS: Patients with metastatic breast cancer who were not currently receiving cytotoxic chemotherapy were eligible. Patients were randomized to receive either clopidogrel and aspirin or to a control group receiving no treatment. Phlebotomy was performed at baseline, at 2 and 4 weeks, and monthly thereafter to obtain specimens to assess CTC, platelet aggregation, and thrombin activity. The primary end point was the proportion of patients with detectable CTCs at 1 month. RESULTS: Forty-eight patients were enrolled and 42 were evaluable at 1 month. Baseline CTC numbers were >/= 5 in 13% and >/= 1 in 65% of patients. Despite adequate platelet function inhibition in the treatment group, the proportion of patients with detectable CTCs was similar between the clopidogrel/aspirin and control groups at baseline (P = .21) and 4 weeks (P = .75), showing no treatment effect. Measured endogenous thrombin potential did not correlate with CTC number. No bleeding-related serious adverse events (SAEs) occurred. CONCLUSION: The baseline CTC numbers were lower than expected, decreasing the ability to detect an impact of platelet inhibition on CTCs. Clopidogrel and aspirin were well tolerated. Future studies evaluating the potential therapeutic role of antiplatelet therapy in breast cancer remain of interest, and they may be informed by these results.

Item does not contain fulltext Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal, syndrome of excessive and ineffective activation of the immune system. The majority of the reported data on HLH is from pediatric patients and lacks specificity. This makes HLH diagnosis challenging especially in adults where HLH is triggered by many conditions and can resemble many disease entities. Elevated ferritin is one of the diagnostic criteria for HLH. We determined the conditions associated with elevated ferritin at our medical center to assess how specific ferritin is for predicting HLH. We retrospectively reviewed all ferritin results >10,000 mug/L in pediatric and adult patients. The most common condition associated with elevated ferritin was hematologic malignancy in adults (25.7%) and HLH in pediatric patients (48.9%). HLH was diagnosed in 14.2% of adults and 48.9% of children with ferritin >10,000 microg/L. Hyperferritinemia occurs in a variety of conditions and is not specific for adult or pediatric HLH. Common causes of elevated ferritin should be considered before entertaining the possibility of HLH, especially in adult patients.

Context.—Proper diagnosis and therapy of fibrinogen deficiency requires high-quality fibrinogen assays.Objective.—To assess the interlaboratory bias, precision, and grading of fibrinogen assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in coagulation.Design.—Two identical vials of normal plasma were sent to more than 3500 laboratories. Participants measured fibrinogen levels using local methods.Results.—Fifty different fibrinogen methods were evaluated. All-method bias was 8.3% (range of method-specific biases, 0.0%–27.0%) and all-method coefficient of variation was 7.7% (range of method-specific coefficients of variation, 0.7%–25.8%). After controlling for reagent/instrument type, mean fibrinogen levels were 11.6% higher for prothrombin time–based reagents compared to Clauss (P < .001), and coefficient of variation was 46% lower for mechanical endpoint instruments compared to photo-optical. Most testing events (97.4%) could be reliably graded as pass or fail using a target range of ±20% from the method mean (total pass rate, 98.8%). Total fail rate was 3.0-fold lower for mechanical instruments compared to photo-optical (0.5% versus 1.5%, P = .001). Nonetheless many photo-optical methods had very high precision and very low fail rates.Conclusions.—Fibrinogen assays showed highly variable methodology and performance characteristics. Bias, precision, and grading were affected by the type of reagent or instrument used.

The risk of venous thromboembolism (VTE) is higher after the total hip or knee replacement surgery than after almost any other surgical procedure; warfarin sodium is commonly prescribed to reduce this peri-operative risk. Warfarin has a narrow therapeutic window with high inter-individual dose variability and can cause hemorrhage. The genetics-informatics trial (GIFT) of warfarin to prevent deep vein thrombosis (DVT) is a 2 × 2 factorial-design, randomized controlled trial designed to compare the safety and effectiveness of warfarin-dosing strategies. GIFT will answer two questions: (1) does pharmacogenetic (PGx) dosing reduce the rate of adverse events in orthopedic patients; and (2) is a lower target international normalized ratio (INR) non-inferior to a higher target INR in orthopedic participants? The composite primary endpoint of the trial is symptomatic and asymptomatic VTE (identified on screening ultrasonography), major hemorrhage, INR ≥ 4, and death.

Introduction: Heparin-induced thrombocytopenia (HIT) is a complication of heparin exposure wherein antibodies (primarily IgG) specific for heparin/platelet-factor-4 (H/PF4) superstructures lead to platelet activation, consumptive thrombocytopenia, and acquired thrombophilia. The immune response is presumably elicited by exposure of a cryptic antigen when heparin and PF4 are bound. However, neither heparin nor PF4 is a foreign antigen in humans, raising the question whether loss of self-tolerance via aberrant antigen presentation by human leukocyte antigen (HLA) may contribute to the development of H/PF4 antibodies. We present evidence that development of high H/PF4 antibody levels is associated with specific HLA alleles, identification of which may elucidate mechanisms for antibody origin and principles for HIT risk stratification. Methods: We conducted a case-control study of 412 adult inpatients tested for enzyme-linked immunosorbent assay H/PF4 antibodies (Diagnostic Stago Asserachrom HPIA-ELISA) for HIT workup after having received intravenous unfractionated heparin. Positive cases (n=60) were individuals with assay optical density (OD) >1.00 while negative controls (n=352) had OD ≤1.00. DNA extracted from peripheral blood of cases and controls were sequenced at HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 loci (Illumina). High-resolution HLA typing results were analyzed for association with case/control status by Fisher's exact test. Results: Median (IQR) OD was 0.16 (0.08-0.34) for controls and 1.68 (1.23-2.31) for cases. The highest case OD was 3.00. Several HLA alleles were associated with case/control status (Figure 1). For class I loci, B*15:01 (odds ratio, OR: 3.94; 95%CI: 1.82-8.52), C*07:01 (2.43; 1.37-4.30), B*08:01 (2.05; 1.12-3.75), and B*49:01 (6.12; 1.21-31.08) were risk factors for developing H/PF4 antibodies, while B*07:02 (0.43; 0.20-0.94) was protective. For class II loci, DQB1*02:01 (2.50; 1.40-4.48), DRB1*03:01 (2.46; 1.37-4.40), DRB1*04:04 (2.65; 1.15-6.07), and DQB1*03:02 (1.88; 0.98-3.61) were risk factors. After adjusting for age, gender, race, and multiple comparison, B*15:01, C*07:01, DQB1*02:01, and DRB1*03:01 remained significant risk factors (p Discussion: We identified several HLA alleles associated with H/PF4 antibody formation, comprised almost entirely of well-known risk alleles for autoimmune disease. DQB1*02:01 (DQ2) and DQB1*03:02 (DQ8) are present in nearly all cases of celiac disease. DRB1*03:01 (DR3), linked with DQ2, is the single HLA allele most frequently associated with autoimmune diseases in the literature. B*08:01 (B8) is part of the "super B8" haplotype overlapping with DR3. B*15:01 (B15) has reported associations with type 1 diabetes, Grave's disease and Addison's disease. B*08:01, C*07:01, DQB1*02:01, and DRB1*03:01 appeared to constitute a distinct risk haplotype, separate from B*15:01 or DQB1*03:02. We also identified a protective allele, B*07:02, which has protective associations with ankylosing spondylitis. Our findings suggest individuals who develop high H/PF4 antibody levels following heparin exposure share HLA haplotypes with individuals who develop auto-antibodies in other contexts, perhaps via shared pathophysiology. A history of autoimmune disease may increase one's pre-test probability for HIT compared to the general population, and merits consideration when HIT is on the differential diagnosis. With additional outcomes research, such history might form a useful adjunct to the "4T" screen. Functional implications of HLA associations with HIT warrant further investigation. Figure 1. Human leukocyte antigen alleles associated with heparin/platelet-factor-4 antibody formation. Shown are histograms of optical density (OD) value, contingency-table counts for cases or controls having or missing an allele, odds ratios (OR) in base-2 logarithmic scale with 95% confidence interval, one-tailed Fisher's exact p-value, and known disease associations for each allele. Red: allele present, blue: allele absent, A: cases having allele, B: cases missing allele, C: controls having allele, D: controls missing allele. Disclosures No relevant conflicts of interest to declare.

An accurate assessment of iron status in dialysis patients is important because both anemia and overtreatment with erythropoiesis-stimulating agents are associated with poor clinical outcomes. We have previously shown that both analytical and intra-individual (biological) variability in serum ferritin limits its utility as a proxy for iron stores in patients in this setting. As hepcidin is a direct regulator of iron status, its measurement might be useful for monitoring patients with iron dysregulation. We assessed short-term intra-individual variation of serum hepcidin in 28 patients with stable chronic kidney disease on hemodialysis. The intra-individual variability for serum hepcidin ranged from 9–79% during an initial 2-week to 12–85% over a 6-week period. The concentration of serum hepcidin was significantly correlated with serum C-reactive protein levels over the 6-week study period. Hence, significant intra-individual variability of hepcidin is likely dependent on short-term fluctuations in the inflammatory state. Thus, our results suggest that short-term measurement of serum hepcidin should not be used to guide clinical decisions regarding management of iron status in chronic hemodialysis patients.

Heparin-induced thrombocytopenia with thrombosis (HITT) is the most severe side effect of heparin administration. Patients with HITT may die or have permanent sequelae such as stroke or limb amputation. Contaminated heparin is associated with anaphylactic reactions and deaths by activating the contact system. It is also associated with high incidence of HIT via a yet unknown mechanism. This study showed that although oversulfated heparin byproduct induced thrombin activities in both normal and HIT patient plasmas through the contact system activation, authentic heparin induced thrombin activities only in HIT patient plasmas containing autoantibodies against protein/ heparin complex. These data suggest that the negatively charged immunoglobulin G (IgG)/platelet factor 4 (PF4)/heparin complex activate the contact system and produce thrombin in human plasma, and thrombin partially activates the platelets allowing subsequent platelet activation through IgG/Fc receptor II signaling. The newly discovered mechanism of heparin-induced thrombin activity could explain the increased incidence of HIT in patients exposed to contaminated heparin. Furthermore, the assays used in these studies would be valuable for HIT diagnosis, prevention, and treatment.

Warfarin demonstrates a wide interindividual variability in response that is mediated partly by variants in cytochrome P450 2C9 (CYP2C9) and vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1). It is not known whether variants in calumenin (CALU) (vitamin K reductase regulator) have an influence on warfarin dose requirements. We resequenced CALU regions in a discovery cohort of dose outliers: patients with high (>90th percentile, n = 55) or low ( G is associated with higher warfarin dose requirements, independent of known genetic and nongenetic predictors of warfarin dose in African Americans.

SummaryVia generation of vitamin K-dependent proteins, gamma-glutamyl carboxylase (GGCX) plays a critical role in the vitamin K cycle. Single nucleotide polymorphisms (SNPs) in GGCX, therefore, may affect dosing of the vitamin K antagonist, warfarin. In a multi-centered, cross-sectional study of 985 patients prescribed warfarin therapy, we genotyped for two GGCX SNPs (rs11676382 and rs12714145) and quantified their relationship to therapeutic dose. GGCX rs11676382 was a significant (p=0.03) predictor of residual dosing error and was associated with a 6.1% reduction in warfarin dose (95% CI: 0.6%-11.4%) per G allele. The prevalence was 14.1% in our predominantly (78%) Caucasian cohort, but the overall contribution to dosing accuracy was modest (partial R2 = 0.2%). GGCX rs12714145 was not a significant predictor of therapeutic dose (p = 0.26). GGCX rs11676382 is a statistically significant predictor of warfarin dose, but the clinical relevance is modest. Given the potentially low marginal cost of adding this SNP to existing geno-typing platforms, we have modified our non-profit website (www.WarfarinDosing.org) to accommodate knowledge of this variant.

Excessive bleeding after cardiac surgery can result in increased morbidity and mortality related to transfusion- and hypoperfusion-related injuries to critical organ systems. Our objective was to review mechanisms that result in bleeding after cardiac surgery as well as current and emerging interventions to reduce bleeding and transfusion. We discovered that of point-of-care (POC) tests of hemostatic function can facilitate the optimal management of excessive bleeding and reduce transfusion by facilitating administration of specific pharmacologic or transfusion-based therapy and by allowing physicians to better differentiate between microvascular bleeding and surgical bleeding. Emerging interventions like recombinant FVIIa have the potential to reduce bleeding and transfusion-related sequelae and may be life-saving; however, randomized, controlled trials are needed to confirm safety before they can be used as either first-line therapies for bleeding or bleeding prophylaxis. In conclusion, careful investigation of the role of new interventions is essential as the ability to reduce use of blood products, to decrease operative time and/or re-exploration rates has important implications for overall patient safety and health care costs.

Unexpectedly high rates of venous thromboembolic events (VTE) concurrent with the introduction of highly effective immune modulating drugs thalidomide and lenolidomide for treatment of multiple myeloma have focused attention on the incidence and underlying pathophysiology of VTE in patients with plasma cell dyscrasias, and on thromboprophylaxis approaches. While bleeding complications are relatively uncommon in patients with lymphoproliferative disorders, acquired von Willebrand syndrome, typically occurring in patients with monoclonal gammopathy of unknown significance, and acquired coagulopathies associated with primary amyloidosis can present with haemorrhagic complications and both are challenging to manage. This review highlights these important haemostasis-related complications of plasma cell dyscrasias and provides an overview of other uncommon bleeding and thrombotic events that can affect diagnostic and therapeutic management of clonal plasma cell disorders. Due to the infrequency of most of these haemostasis complications, available information is typically based on retrospective cases or series.

Laboratory assessment of fetal lung maturity assists obstetricians in estimating the risk of respiratory distress syndrome (RDS) when premature delivery of an infant is being considered. The status of fetal lung maturity is reflected in the concentrations of specific phospholipids and the number of surfactant-containing particles, or lamellar bodies, in amniotic fluid. Since lamellar body volumes are similar to platelets, automated hematology analyzers can provide rapid and precise lamellar body counts (LBCs). However, hematology analyzer manufacturers use different methods to enumerate platelets, leading to a lack of concordance between instrument brands when counting lamellar bodies. Laboratories performing LBCs should be aware of this important analytical variable when providing interpretations of LBC results.

Initiation of warfarin therapy is associated with bleeding owing to its narrow therapeutic window and unpredictable therapeutic dose. Pharmacogenetic-based dosing algorithms can improve accuracy of initial warfarin dosing but require rapid genotyping for cytochrome P-450 2C9 (CYP2C9) *2 and *3 single nucleotide polymorphisms (SNPs) and a vitamin K epoxide reductase (VKORC1) SNP. We evaluated 4 commercial systems: INFINITI analyzer (AutoGenomics, Carlsbad, CA), Invader assay (Third Wave Technologies, Madison, WI), Tag-It Mutation Detection assay (Luminex Molecular Diagnostics, formerly Tm Bioscience, Toronto, Canada), and Pyrosequencing (Biotage, Uppsala, Sweden). We genotyped 112 DNA samples and resolved any discrepancies with bidirectional sequencing. The INFINITI analyzer was 100% accurate for all SNPs and required 8 hours. Invader and Tag-It were 100% accurate for CYP2C9 SNPs, 99% accurate for VKORC1 -1639/3673 SNP, and required 3 hours and 8 hours, respectively. Pyrosequencing was 99% accurate for CYP2C9 *2, 100% accurate for CYP2C9 *3, and 100% accurate for VKORC1 and required 4 hours. Current commercial platforms provide accurate and rapid genotypes for pharmacogenetic dosing during initiation of warfarin therapy.

High variability in drug response and a narrow therapeutic index complicate warfarin therapy initiation. No existing algorithm provides recommendations on refining the initial warfarin dose based on genetic variables, clinical data, and international normalized ratio (INR) values. Our goal was to develop such an algorithm. We studied 92 patients undergoing primary or revision total hip or knee replacement. From each patient we collected a blood sample, clinical variables, current medications, and preoperative and postoperative laboratory values. We genotyped for polymorphisms in the cytochrome P450 (CYP) 2C9 and vitamin K epoxide reductase (VKORC1) genes. Using stepwise regression, we developed a model for refining the warfarin dose after the third warfarin dose. The algorithm explained four fifths of the variability in therapeutic dose (R2adj of 79%). Significant (P > .05) predictors were INR value after 3 doses (47% reduction per 0.25-unit rise), first warfarin dose (+7% per 1 mg), CYP2C9*3 and CYP2C9*2 genotype (−38% and −17% per allele), estimated blood loss (interacting with INR3), smoking status (+20% in current smokers), and VKORC1 (−11% per copy of haplotype A). If validated, this model should provide a safer, more effective process for initiating warfarin therapy.

Major, spontaneous bleeding is uncommon in patients with plasma cell dyscrasias despite frequent abnormal screening hemostasis tests. However, acquired von Willebrand deficiency and light-chain (AL) amyloidosis, and amyloidosis complicating multiple myeloma can present with serious hemorrhagic complications that are challenging to manage. While patients with monoclonal gammapathy of undetermined significance and multiple myeloma share an intrinsic increased risk of venous thromboembolic events (VTE), treatment with thalidomide and lenalidomide increases the incidence of VTE in certain multiple myeloma patient subsets. Our understanding of the complex interactions among malignant plasma cells, inflammatory and hemostasis pathways, and treatment modalities that combine to produce thrombotic complications is incomplete. Prospective, randomized trials are clearly needed to assist clinicians in providing optimal VTE prophylaxis to their patients with plasma cell dyscrasias.

Manual restraint and jugular venipuncture were used to obtain blood for hematology and coagulation tests for 18 captive Speke's gazelles (Gazella spekei). The hematocrit and hemoglobin values were slightly higher in Speke's gazelles than in domestic ruminants. The Speke's gazelles had a mean prothrombin time of 15.1 sec and a mean activated partial thromboplastin time of 24.2 sec. The pregnant female Speke's gazelles had shorter activated partial thromboplastin times than the males, but the difference was not significant. Ideally, prothrombin times and activated partial thromboplastin times would be compared to a healthy conspecific during a suspected bleeding crisis. Baseline prothrombin and activated partial thromboplastin times are presented here for Speke's gazelles because clotting times for exotic hoofstock are quite limited.

Background: This case conference reviews laboratory methods for assessing oxygenation status: arterial blood gases, pulse oximetry, and CO-oximetry. Caveats of these measurements are discussed in the context of two methemoglobinemia cases.Cases: Case 1 is a woman who presented with increased shortness of breath, productive cough, chest pain, nausea, fever, and cyanosis. CO-oximetry indicated a carboxyhemoglobin (COHb) fraction of 24.9%. She was unresponsive to O2 therapy, and no source of carbon monoxide could be noted. Case 2 is a man who presented with syncope, chest tightness, and signs of cyanosis. His arterial blood was dark brown, and CO-oximetry showed a methemoglobin (MetHb) fraction of 23%.Issues: Oxygen saturation (So2) can be measured by three approaches that are often used interchangeably, although the measured systems are quite different. Pulse oximetry is a noninvasive, spectrophotometric method to determine arterial oxygen saturation (SaO2). CO-oximetry is a more complex and reliable method that measures the concentration of hemoglobin derivatives in the blood from which various quantities such as hemoglobin derivative fractions, total hemoglobin, and saturation are calculated. Blood gas instruments calculate the estimated O2 saturation from empirical equations using pH and Po2 values. In most patients, the results from these methods will be virtually identical, but in cases of increased dyshemoglobin fractions, including methemoglobinemia, it is crucial that the distinctions and limitations of these methods be understood.Conclusions: So2 calculated from pH and Po2 should be interpreted with caution as the algorithms used assume normal O2 affinity, normal 2,3-diphosphoglycerate concentrations, and no dyshemoglobins or hemoglobinopathies. CO-oximeter reports should include the dyshemoglobin fractions in addition to the oxyhemoglobin fraction. In cases of increased MetHb fraction, pulse oximeter values trend toward 85%, underestimating the actual oxygen saturation. Hemoglobin M variants may yield normal MetHb and increased COHb or sulfhemoglobin fractions measured by CO-oximetry.

Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by the activation of the mononuclear phagocytic system. The diagnosis of HLH in adults is challenging not only because the majority of the reported data are from pediatric patients, but also because HLH occurs in many disease entities. This study reports the clinical and laboratory findings and prognostic factors of adult HLH in a large cohort managed at a single medical center from 2003 to 2014. Seventy-three patients met the HLH-2004 diagnostic criteria. The median age was 51 years (range, 18-82 years); 41 (56.2%) were male. Patients manifested fever, cytopenias, and elevated ferritin in85% of cases. Likely causes of HLH were as follows: 30 (41.1%) infections, 21 (28.8%) malignancies, 5 (6.8%) attributed to autoimmune disorders, 1 (1.4%) primary immunodeficiency, 2 (2.7%) post solid organ transplantation, and 13 (17.8%) idiopathic. The median overall survival was 7.67 months. Patients with malignancy-associated HLH had a markedly worse survival compared with patients with non-malignancy-associated HLH (median overall survival 1.13 vs. 46.53 months, respectively; P 0.0001). In a multivariable analysis, malignancy (hazard ratio = 12.22; 95% CI: 2.53-59.02; P = 0.002) correlated with poor survival. Ferritin50,000 µg/L correlated with 30-day mortality. Survival after a diagnosis of HLH is dismal, especially among those with malignancy-associated HLH. The development of a registry for adults with HLH would improve our understanding of this syndrome, validate diagnostic criteria, and help develop effective treatment strategies.

Context.—D-dimer is widely used for exclusion, or as an aid in diagnosis, of venous thromboembolism (VTE); however, the D-dimer assay methods available from manufacturers and the laboratory application of those methods vary widely. Objectives.—To describe the current laboratory practice regarding the assay and reporting of D-dimer. Design.—Laboratories' D-dimer proficiency testing data were analyzed and laboratory practices regarding the performance and reporting of D-dimer were surveyed. Results.—Initial grading of D-dimer proficiency testing demonstrated high variability within and among methods. This variability continued to be present for several years after attempts to intervene. The number of laboratories using D-dimer to exclude VTE grew from 1500 in 2004 to more than 3500 in 2012. Survey and proficiency testing data demonstrated that 33% of laboratories changed the type or magnitude of units from that recommended by the manufacturer, a practice associated with as much as a 20-fold increase in the failure of proficiency testing. Many laboratories used a threshold for the exclusion of VTE that is higher than that recommended by the manufacturer. Many laboratories continue to use qualitative assays with insufficient sensitivity for exclusion of VTE. Conclusions.—There is considerable variability both within and among quantitative methods used to assay D-dimer by laboratories. Laboratory practice continues to vary widely regarding the type and magnitude of units reported and the setting of the threshold for the exclusion of VTE. Although improved, the variability continues despite initial efforts to intervene.

Warfarin's monopoly on oral anticoagulation therapy, as well as that of other vitamin K antagonists derived from coumarin, is over. Two new classes of drugs, oral direct thrombin inhibitors (DTI)2 and oral direct factor Xa inhibitors (DFXaI), are steadily obtaining regulatory approval for prevention of thromboembolic events in patients who have atrial fibrillation, undergo hip or knee replacement surgery, or require treatment of pulmonary emboli and deep vein thromboses. The results from an expanding list of large, well-conducted randomized clinical trials confirm the efficacy and safety of DTI and DFXaI drugs as noninferior or superior to standard prophylactic or therapeutic anticoagulation therapies (1). Presently, the US Food and Drug Administration (FDA) and regulatory agencies in other countries have approved one DTI (dabigatran etexilate, Pradaxa®) and two DFXaI (rivaroxaban, Xarelto®; apixaban, Eliquis®) for one or more indications, and clinical trials that were recently completed or are underway will likely bring more drugs in these two classes to market with approval for additional indications (2). The common mechanism of action of these small molecules is reversible blockage of the active enzyme site of thrombin or factor Xa. Thrombin converts fibrinogen to fibrin, and factor Xa converts the zymogen prothrombin to thrombin. Inhibition of either coagulation factor reduces the rate of fibrin clot formation. Unlike warfarin or heparin, DTI and DFXaI activities are not dependent on decreased synthesis of coagulation factors or acceleration of the effects of antithrombin, an endogenous inhibitor of thrombin and factor Xa. DTI and DFXaI have many advantages compared with coumarins, including rapid onset of anticoagulation, no dietary restrictions, few drug–drug interactions, and more predictable pharmacokinetics. As a result, they are prescribed as fixed doses without adjustments on the …

Summary The introduction of several oral direct anticoagulants within the past 2–3 years has dramatically changed clinical practice and has also impacted on utilization and interpretation of coagulation laboratory testing. This article reviews the effects of the oral thrombin inhibitor, dabigatran, and the oral factor Xa inhibitors, rivaroxaban and apixaban, on screening and diagnostic coagulation tests, and describes methods for measuring the their anticoagulant activity in plasma. Currently, there are evidence gaps regarding the role of laboratory testing for surveillance and management of adverse events associated with these new anticoagulants which do not require routine therapeutic drug monitoring. This is a rapidly changing field, and coagulation laboratory experts have a major role in ensuring patients receive appropriate testing and accurate interpretations of results.

After a decade of clinical investigation, pharmacogenetic-guided initial dosing of warfarin is at a crossroads. Genotypes for two single nucleotide polymorphisms (SNPs) in the cytochrome P 450 2C9 gene, affecting warfarin metabolism, and one SNP in vitamin K reductase complex 1 gene, affecting warfarin sensitivity, account for approximately 30% of therapeutic warfarin dosing variability in whites and Asians. Incorporating this genetic information, along with patient's age, body size, and other clinical information improves the accuracy of initial warfarin dosing. Currently, there is insufficient evidence to support the clinical benefits and cost effectiveness of routine warfarin pharmacogenetics. Results from ongoing international randomized clinical trials should provide clarity about the place of warfarin pharmacogenetics in personalized medicine.

Context.—Hereditary and acquired deficiencies of antithrombin (AT), protein C (PC), and protein S (PS) are risk factors for venous thromboembolism. Proper diagnosis requires high-quality assays for these proteins. Objective.—To determine the accuracy and interlaboratory precision of AT, PC, and PS assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in thrombophilia and to grade the performance of laboratories. Design.—Standardized normal plasma with assigned analyte values was sent in 2 separate challenges to participating laboratories. Participants measured AT, PC, and PS levels using local methods. Results.—When compared with the assigned values for the international standard, the order of assay accuracy from highest to lowest was AT activity, PC antigen, AT antigen, total PS antigen, PC activity, PS activity, and free PS antigen (range of assay bias, 2.6%–8.8%). The order of assay precision from highest to lowest was PC activity, AT activity, AT antigen, total PS antigen, PS activity, free PS antigen, and PC antigen (range of assay coefficient of variation, 6.1%–20.0%). Most testing events (87.8%) could be graded as pass or fail using a target range of ±3 standard deviations from the method-specific mean. The pass rate was 98.2% for all AT, PC, and PS testing events combined. Conclusions.—Accuracy and precision were higher for AT assays and lower for PC and PS assays. It was feasible to grade individual laboratory performance.

A 32-year-old woman presented with an acute anterior-lateral myocardial infarction 7 days postpartum. Coronary angiography revealed an occlusive left main coronary artery dissection. After coronary artery bypass surgery the patient's cardiac function improved and stabilized. Thrombocytopenia and a femoral artery thrombosis after 9 days of heparin exposure marked the development of heparin-induced thrombocytopenia and thrombosis that was successfully managed with argatroban, a direct thrombin inhibitor anticoagulant.

Well-characterized genes that affect warfarin metabolism (cytochrome P450 (CYP) 2C9) and sensitivity (vitamin K epoxide reductase complex 1 (VKORC1)) explain one-third of the variability in therapeutic dose before the international normalized ratio (INR) is measured. To determine genotypic relevance after INR becomes available, we derived clinical and pharmacogenetic refinement algorithms on the basis of INR values (on day 4 or 5 of therapy), clinical factors, and genotype. After adjusting for INR, CYP2C9 and VKORC1 genotypes remained significant predictors (P < 0.001) of warfarin dose. The clinical algorithm had an R(2) of 48% (median absolute error (MAE): 7.0 mg/week) and the pharmacogenetic algorithm had an R(2) of 63% (MAE: 5.5 mg/week) in the derivation set (N = 969). In independent validation sets, the R(2) was 26-43% with the clinical algorithm and 42-58% when genotype was added (P = 0.002). After several days of therapy, a pharmacogenetic algorithm estimates the therapeutic warfarin dose more accurately than one using clinical factors and INR response alone.

Summary. Background: CYP2C9 and VKORC1 genotypes predict therapeutic warfarin dose at initiation of therapy; however, the predictive ability of genetic information after a week or longer is unknown. Experts have hypothesized that genotype becomes irrelevant once international normalized ratio (INR) values are available because INR response reflects warfarin sensitivity. Methods: We genotyped the participants in the Prevention of Recurrent Venous Thromboembolism (PREVENT) trial, who had idiopathic venous thromboemboli and began low-intensity warfarin (therapeutic INR 1.5–2.0) using a standard dosing protocol. To develop pharmacogenetic models, we quantified the effect of genotypes, clinical factors, previous doses and INR on therapeutic warfarin dose in the 223 PREVENT participants who were randomized to warfarin and achieved stable therapeutic INRs. Results: A pharmacogenetic model using data from day 0 (before therapy initiation) explained 54% of the variability in therapeutic dose (R2). The R2 increased to 68% at day 7, 75% at day 14, and 77% at day 21, because of increasing contributions from prior doses and INR response. Although CYP2C9 and VKORC1 genotypes were significant independent predictors of therapeutic dose at each weekly interval, the magnitude of their predictive ability diminished over time: partial R2 of genotype was 43% at day 0, 12% at day 7, 4% at day 14, and 1% at day 21. Conclusion: Over the first weeks of warfarin therapy, INR and prior dose become increasingly predictive of therapeutic dose, and genotype becomes less relevant. However, at day 7, genotype remains clinically relevant, accounting for 12% of therapeutic dose variability.

Background: The application of pharmacogenetic results requires demonstrable correlations between a test result and an indicated specific course of action. We developed a computational decision-support tool that combines patient-specific genotype and phenotype information to provide strategic dosage guidance. This tool, through estimating quantitative and temporal parameters associated with the metabolism- and concentration-dependent response to warfarin, provides the necessary patient-specific context for interpreting international normalized ratio (INR) measurements. Methods: We analyzed clinical information, plasma S-warfarin concentration, and CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) and VKORC1 (vitamin K epoxide reductase complex, subunit 1) genotypes for 137 patients with stable INRs. Plasma S-warfarin concentrations were evaluated by VKORC1 genotype (−1639G>A). The steady-state plasma S-warfarin concentration was calculated with CYP2C9 genotype–based clearance rates and compared with actual measurements. Results: The plasma S-warfarin concentration required to yield the target INR response is significantly (P < 0.05) associated with VKORC1 −1639G>A genotype (GG, 0.68 mg/L; AG, 0.48 mg/L; AA, 0.27 mg/L). Modeling of the plasma S-warfarin concentration according to CYP2C9 genotype predicted 58% of the variation in measured S-warfarin concentration: Measured [S-warfarin] = 0.67(Estimated [S-warfarin]) + 0.16 mg/L. Conclusions: The target interval of plasma S-warfarin concentration required to yield a therapeutic INR can be predicted from the VKORC1 genotype (pharmacodynamics), and the progressive changes in S-warfarin concentration after repeated daily dosing can be predicted from the CYP2C9 genotype (pharmacokinetics). Combining the application of multivariate equations for estimating the maintenance dose with genotype-guided pharmacokinetics/pharmacodynamics modeling provides a powerful tool for maximizing the value of CYP2C9 and VKORC1 test results for ongoing application to patient care.

Summary. Background: Warfarin is commonly prescribed for prophylaxis and treatment of thromboembolism after orthopedic surgery. During warfarin initiation, out-of-range International Normalized Ratio (INR) values and adverse events are common. Methods: In orthopedic patients beginning warfarin therapy, we developed and prospectively validated pharmacogenetic and clinical dose refinement algorithms to revise the estimated therapeutic dose after 4 days of therapy. Results: The pharmacogenetic algorithm used the cytochrome P450 (CYP) 2C9 genotype, smoking status, peri-operative blood loss, liver disease, INR values and dose history to predict the therapeutic dose. The R2 was 82% in a derivation cohort (n = 86) and 70% when used prospectively (n = 146). The R2 of the clinical algorithm that used INR values and dose history to predict the therapeutic dose was 57% in a derivation cohort (n = 178) and 48% in a prospective validation cohort (n = 146). In 1 month of prospective follow-up, the percent time spent in the therapeutic range was 7% higher (95% CI: 2.7–11.7) in the pharmacogenetic cohort. The risk of a laboratory or clinical adverse event was also significantly reduced in the pharmacogenetic cohort (Hazard Ratio 0.54; 95% CI: 0.30–0.97). Conclusions: Warfarin dose adjustments that incorporate genotype and clinical variables available after four warfarin doses are accurate. In this non-randomized, prospective study, pharmacogenetic dose refinements were associated with more time spent in the therapeutic range and fewer laboratory or clinical adverse events. To facilitate gene-guided warfarin dosing we created a non-profit website, http://www.WarfarinDosing.org.

A 47-year-old African American woman was evaluated for a prolonged prothrombin time (PT) result obtained before she underwent right total hip arthroplasty. The patient had no history of gastrointestinal or intracranial bleeding, epistaxis, or hemarthrosis. However, she reported a tendency toward easy limb bruising and menorrhagia, which required iron supplementation. She had a negative family history of abnormal bleeding. Initial laboratory studies included findings within reference intervals for complete blood cell count and activated partial thromboplastin time (aPTT) (30.8 s, reference interval 23–36 s), prolonged PT (20.3 s, reference interval 11.0–15.0 s), and International Normalized Ratio (INR) (1.78, reference interval 0.9–1.2). No preanalytical artifacts were identified, and the result of a repeat PT was also prolonged. ### laboratory evaluation of prolonged results for screening coagulation tests PT and aPTT are commonly requested screening tests. In vivo, the initiation of coagulation depends on tissue factor–mediated factor VII (FVII) activation, and sustained thrombin generation requires activation of factors XI, IX, VIII, X, and V. For the interpretation of PT and aPTT results, however, coagulation factor activation culminating in a fibrin clot can be organized into intrinsic, extrinsic, and common pathways (Fig. 1⇓ ). An isolated result showing aPTT prolongation suggests a deficiency or inhibitor of one or more of the intrinsic pathway clotting factors (prekallikrein, high molecular weight kininogen, factors XII, XI, IX, and VIII). An isolated PT prolongation suggests a deficiency or inhibition of the extrinsic pathway (FVII), but mild factor X, V, and II deficiencies are also possible causes. Prolongation of both aPTT and PT suggests a deficiency or inhibition of the common pathway coagulation factors (factor X, V, and II), or a qualitative or quantitative fibrinogen defect. Figure 1. Coagulation factor activation culminating in the generation of a fibrin clot. HMWK, high molecular weight kininogen; PK, prekallikrein; TF, tissue factor. When evaluating an unexpected prolonged aPTT and/or PT result, the …

Initiation of warfarin therapy using trial-and-error dosing is problematic. Our goal was to develop and validate a pharmacogenetic algorithm. In the derivation cohort of 1,015 participants, the independent predictors of therapeutic dose were: VKORC1 polymorphism -1639/3673 G>A (-28% per allele), body surface area (BSA) (+11% per 0.25 m(2)), CYP2C9(*)3 (-33% per allele), CYP2C9(*)2 (-19% per allele), age (-7% per decade), target international normalized ratio (INR) (+11% per 0.5 unit increase), amiodarone use (-22%), smoker status (+10%), race (-9%), and current thrombosis (+7%). This pharmacogenetic equation explained 53-54% of the variability in the warfarin dose in the derivation and validation (N= 292) cohorts. For comparison, a clinical equation explained only 17-22% of the dose variability (P < 0.001). In the validation cohort, we prospectively used the pharmacogenetic-dosing algorithm in patients initiating warfarin therapy, two of whom had a major hemorrhage. To facilitate use of these pharmacogenetic and clinical algorithms, we developed a nonprofit website, http://www.WarfarinDosing.org.

Warfarin is an effective, commonly prescribed anticoagulant used to treat and prevent thrombotic events. Because of historically high rates of drug-associated adverse events, warfarin remains underprescribed. Further, interindividual variability in therapeutic dose mandates frequent monitoring until target anticoagulation is achieved. Genetic polymorphisms involved in warfarin metabolism and sensitivity have been implicated in variability of dose. Here, we describe a novel variant that influences warfarin requirements. To identify additional genetic variants that contribute to warfarin requirements, screening of DNA variants in additional genes that code for drug-metabolizing enzymes and drug transport proteins was undertaken using the Affymetrix drug-metabolizing enzymes and transporters panel. A DNA variant (rs2108622; V433M) in cytochrome P450 4F2 (CYP4F2) was associated with warfarin dose in 3 independent white cohorts of patients stabilized on warfarin representing diverse geographic regions in the United States and accounted for a difference in warfarin dose of approximately 1 mg/day between CC and TT subjects. Genetic variation of CYP4F2 was associated with a clinically relevant effect on warfarin requirement.

Introduction: Extracorporeal photopheresis (ECP) is an effective therapy for patients with acute or chronic graft-versus-host disease (GVHD) who have significant skin involvement. During ECP, buffy coat is collected from the peripheral blood (PBL), mixed with 8-methoxypsoralen (8-MOP), and exposed to ultraviolet-A (UVA). Photo-activated 8-MOP crosslinks DNA in nucleated cells, which are infused back to the patient to induce an immune modulatory effect. The exact mechanism of ECP and the optimal cellular dose to treat are unknown. We previously demonstrated a linear relationship between lymphocyte counts in the PBL and the buffy coat, with the collection efficiency affected primarily by the type of ECP machine used (UVAR or CELLEX; Therakos, West Chester, PA). It is therefore feasible to track the cellular dose of ECP by measuring the lymphocyte count in the PBL prior to each procedure. Another outstanding question is whether ECP can be successfully performed at a decreased hematocrit, which is a common finding among patients with GVHD. Due to concerns over the collection efficiency and patient safety, our protocol recommends transfusion of red cells prior to ECP when a patient's hematocrit is in the range of 28-30%. However, there is no evidence from human subjects supporting the relevance of this specific threshold. In this study, we compared the collection efficiency during ECP in GVHD patients with hematocrits between 24% and 29% and those with hematocrits of 29% or higher. Methods: Patients with GVHD and hematocrits between 24% and 29% were prospectively recruited for this study which was approved by the institutional review board (n=26). The hematocrit of 24% approximated the red cell transfusion threshold for our transplant patient population. One ECP procedure was performed for each patient using the CELLEX with 1500 mL of whole blood processed. PBL was drawn within 24 hours prior to the procedure for a complete cell count with automated differentials. At the end of cell collection but prior to 8-MOP treatment, 1 mL of well-mixed buffy coat was drawn for the complete cell count. Spearman correlation coefficient was calculated for the counts of each nucleated cell type, including neutrophils, lymphocytes and monocytes, in the PBL versus the buffy coat. The collection efficiency, or fold enrichment, was evaluated using the ratio of lymphocyte count in the buffy coat to that in the PBL. The ratio was then compared among the current cohort (lower-hct-CELLEX group) and two historical control groups with hematocrits of 29% or higher who were treated with either UVAR (higher-hct-UVAR group n=6) or CELLEX (higher-hct-CELLEX group, n=16). Mann-Whitney test was performed with a double-sided p value of 0.05. Data visualization and statistical analysis were performed using Prism 6. Results: Consistent with our previous findings in patients with higher hematocrits, there was a significant correlation between lymphocyte counts in the PBL and the buffy coat in the lower-hematocrit-CELLEX group (r=0.66, p=0.0002) (Figure 1A). A significant correlation was also observed for monocytes (r=0.46, p=0.02) but not neutrophils (r=0.26, p=0.20). The distribution of lymphocyte counts was fit into a linear regression model with a slope of 4.22 (95% CI: 3.06-5.37, p < 0.0001). The fold enrichment for lymphocytes was higher in the lower-hct-CELLEX group than in the higher-hct-UVAR group (medians: 4.94 versus 1.66, p=0.008). The fold enrichment for lymphocytes was not significantly different between the lower-hct-CELLEX group and the higher-hct-CELLEX group (medians: 4.94 versus 5.22, p=0.27) (Figure 1B). According to previous protocols, approximately 1-2 units of red blood cells would have been transfused for each patient in the lower-hct-CELLEX group prior to each procedure. These transfusions were completely avoided without any adverse events observed throughout the study. Conclusions: The fold enrichment of lymphocytes during ECP using the CELLEX is comparable between GVHD patients with hematocrits between 24% and 29% and patients with hematocrits of 29% or higher. Unnecessary red blood cell transfusion may be avoided without significant increase in adverse events. The efficacy and safety of this practice warrant further investigation in a larger cohort. Disclosures No relevant conflicts of interest to declare.

Tremendous improvements have been made in the outcomes of patients with acute venous thromboembolic events (VTE) since Murray et al. successfully administered heparin to postsurgery patients in 1937 (1). Advances include accurate methods for diagnosis of deep vein thromboses (DVT) and pulmonary emboli (PE); effective regimens for initial and long-term anticoagulation; and wide acceptance of a system for standardization of thromboplastin sensitivities to the anticoagulant effects of warfarin (International Normalized Ratio; INR). In this issue of Clinical Chemistry , van den Besselaar et al. (2) address an unresolved aspect of anticoagulation therapy: standardization of activated partial thromboplastin time (APTT) reagents for monitoring heparin therapy. Heparin is a glycosaminoglycan extracted from mast cells of porcine intestinal mucosa or bovine lung. It is composed of long chains of alternating d-glucuronic acid and N -acetyl-d-glucosamine sugar residues, which undergo a series of chemical modifications, primarily sulfation, leading to unique pentasaccharide sequences that serve as a binding site for antithrombin III (AT III) (3). The anticoagulant effect of heparin is mediated through this interaction, which markedly accelerates the rate of AT III inhibition of thrombin (factor IIa) and factor Xa. Heparin polysaccharides are heterogeneous in length and anticoagulation activity and range in mass from 5000 to 30 000 kDa. Commercial preparations are calibrated in USP units, 1 unit being defined as the quantity that prevents 1.0 mL of citrated sheep plasma from clotting for 1 h after the addition of 0.2 mL of 10 g/L CaCl2 (3). Low-molecular-weight heparins (LMWH) are produced from unfractionated heparin to yield smaller polysaccharides with average molecular masses of 4000–5000 kDa. These shorter molecules lose the ability to accelerate AT III inhibition of thrombin but retain the ability to catalyze factor Xa inhibition. Decreased in vivo protein binding improves LMWH bioavailability and leads to …