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Pharmacogenetic dosing improves the accuracy of warfarin dosing, but current pharmacogenetic dosing algorithms are less accurate in populations of African ancestry. The cytochrome P450 2C9*5 (CYP2C9*5) allele is found almost exclusively in populations of African ancestry, and in vitro studies suggest CYP2C9*5 is associated with reduced clearance of warfarin. The clinical relevance of this single-nucleotide variation (SNV) (formerly SNP) is uncertain. In this multicentered study of 2,298 patients (49% female, 35% Black) taking warfarin, we quantified the association between the CYP2C9*5 allele and warfarin requirements. The CYP2C9*5 SNV was present in 2.3% of Black and 0.07% of White patients. Without taking CYP2C9*5 into account, pharmacogenetic algorithms that include other SNVs overestimated the warfarin dose by 30% (95% confidence interval (19-40%), P 0.001), an average of 1.87 mg/day (SD 1.64) in heterozygotes (P 0.001). Noncarriers required a slightly (0.23 mg/day, SD 2.09) higher than predicted dose. Genotyping for CYP2C9*5 corrected the potential overdose and halved overall dosing error in heterozygotes. Patients carrying CYP2C9*5 require a clinically relevant reduction in warfarin dose. Given the potential to improve the accuracy and safety of warfarin dosing in populations of African ancestry, we have incorporated this SNV into a nonprofit website to assist warfarin initiation (www.WarfarinDosing.org).

Item does not contain fulltext INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of pathologic immune activation. Most studies on adult HLH have evaluated prognostic factors for overall survival; factors predicting early mortality have not been sufficiently investigated. METHODS: This was a collaborative study between Henry Ford Hospital and Barnes-Jewish Hospital. We identified all adult HLH patients with at least 2 ferritin levels within 30 days from admission. RESULTS: One-hundred twenty-four patients were identified. There were 77 males and 47 females; the median age at diagnosis was 48 years. Multivariate analysis showed that age (OR = 11.41; 95% CI:2.71-48.04; P = .001), hepatomegaly (OR = 15.68; 95% CI:3.24-75.96; P = .001), hyponatremia (OR = 5.94; 95% CI:1.76-20.1; P = .004), hypoalbuminemia (OR = 7.47; 95% CI:2.08-26.85; P = .002), and increasing ferritin levels (OR = 19.46; 95% CI:4.69-80.71; P

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.

INTRODUCTION The Sysmex XN-10 automated hematology analyzer (Sysmex Corporation) is routinely used in hematology laboratories to perform complete blood cell count with differential (CBC w/ diff). The sensitivity of this system for blast detection is unclear, since many prior studies evaluating the blast flagging capabilities of Sysmex XN series used the white precursor cell (WPC) channel, which is not cleared for use in the United States. METHODS We assessed the blast flagging capabilities of the Sysmex XN-10 compared with CellaVision (a cell image analyzer)-assisted visual hematology results. We evaluated the following flags: "blasts?/abnormal lymph?" and "immature granulocytes present" and compared differences in turnaround time between methods. RESULTS We collected data on 2239 CBC w/ diff Sysmex automated analyzer differential and CellaVision-assisted visual differential from the inpatient hematology-oncology population of a tertiary care medical center. Solely analyzing the first CBC/diff from each unique patient, both flags had a combined sensitivity of 100%, specificity of 50.2%, PPV of 21.7%, and NPV of 100%. The mean turnaround time for the automated differential was 19.5 minutes (SD 35.9 minutes) compared with 66.4 minutes for the CellaVision-assisted visual differential (SD 68.5 minutes; P

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.

Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome characterized by an uncontrolled hyper-inflammatory response. We assessed the transfusion requirements and predictors of 30-day mortality for adult HLH patients. We identified all adult patients with a diagnosis of HLH at a large academic hospital from October 2003 through February 2017. We extracted patients' clinical and laboratory data, including transfusion requirements, from their medical records. One-hundred sixteen patients were identified. Their median age was 48 years (range 18-82); 72(62%) were male. Median duration of hospital stay was 19 days (range 1-89 days). At 30 days from admission, 81(70%) patients were alive. Death was attributed to sepsis in 21 patients, lymphoma in six, bleeding in four, GVHD in one, liver failure in one, metastatic solid tumor in one, and unknown in one. Transfusion requirements at 30 days from admission were as follows: RBC, 86% of patients, median 6 units (range 1-58); platelets, 74% of patients, median 6 units (1-67); plasma, 40% of patients, median 4 units (1-56). Renal failure (OR = 4.39; P = 0.008) and hypofibrinogenemia (OR = 4.07; P = 0.009) correlated with 30-day mortality. The transfusion requirements for adult HLH patients were high. Our study indicated that renal insufficiency and hypofibrinogenemia are predictors of early death in adult HLH.

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

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by excessive activation of the immune system, resulting in overproduction of inflammatory cytokines. Patients usually present with high fever, cytopenias, hyperferritinemia, and hepatosplenomegaly, and their disease process ranges from mild to fatal multiorgan failure. HLH is a heterogeneous group of disorders that can be triggered by infections, neoplasms, or autoimmune diseases. The HLH diagnosis can be difficult to confidently confirm in critically ill patients while waiting for pathology or reference laboratory results to return, delaying the diagnosis with significantly worsened outcomes. The current HLH-2004 diagnostic guidelines were originally developed for pediatric cases and were not validated to diagnose secondary HLH, whether in children or adults. In addition, some laboratory findings that are common among HLH patients such as hypoalbuminemia and elevated liver enzymes are not represented in the HLH-2004 guidelines. Even more challenging for clinicians is that many of the diagnostic features of this syndrome are nonspecific. For example, the clinical presentation of HLH can meet the diagnostic criteria of systemic inflammatory response syndrome, viral infections, or neoplastic diseases. It is necessary to revisit the diagnostic criteria for HLH by validating the clinical and laboratory findings in large prospective HLH prospective clinical trials or by establishing registries. This will improve our understanding of HLH, help validate and develop newer, more specific, and more rapidly obtainable diagnostic criteria, and, eventually, result in earlier therapy with more consistent monitoring of the response.

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

Background Ehrlichiosis is an acute febrile tick-borne disease which can rarely be a trigger for secondary hemophagocytic lymphohistiocytosis (HLH). Methods We reviewed our experience with Ehrlichia infections at a tertiary-care academic medical center. Results Over 10 years, 157 cases of ehrlichiosis were identified. Ten patients (6.4%) had infection with E. ewingii, 7(4.5%) of whom were transplant patients as compared to 3(1.9%) non-transplant patients (p = .035). Transplant patients were more likely to have leukopenia and elevated creatinine compared to immunocompetent patients; length of hospital stay and early mortality were not different between the two groups. Ten patients met the HLH-2004 diagnosis criteria, which could be an underestimation of HLH occurrence as most patients were not completely evaluated for these criteria. We calculated the H-Score to find the probability of HLH; 25 patients scored high making the occurrence rate of HLH at least 16%. Ehrlichia-induced HLH patients (N = 25) had more anemia, thrombocytopenia, elevated creatinine and AST. Moreover, they had a significantly longer hospital stay (median 9 days) compared to patients without HLH (median 4 days) (p = .006). Conclusions Ehrlichia-induced HLH is a potential serious complication with relatively high occurrence rate; patients manifest severe disease with end-organ damage requiring longer hospital stay.

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.

Zoonotic infections can result in life-threatening complications that can manifest with hemophagocytic lymphohistiocytosis (HLH)/cytokine storm syndrome (CSS). Zoonotic bacterial infections constitute the largest group of reported cases associated with HLH among other zoonotic infections. There is a growing list of zoonotic bacterial infections associated with HLH/CSS and these include Brucella spp., Rickettsia spp., Ehrlichia, Coxiella burnetii, and Mycobacterium spp.

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.

BACKGROUND: Extracorporeal photopheresis (ECP) is commonly used to treat patients with graft-versushost disease (GVHD) and lung transplant rejection (LTR) in our institution. The quantitative relationship between the number of white blood cells treated during ECP and the cell count in peripheral blood is unclear. STUDY DESIGN AND METHODS: Patients with GVHD and LTR receiving ECP with either UVAR XTS or CELLEX (Therakos) were prospectively recruited for this study. A complete cell count with differential was performed on preprocedural peripheral blood and samples from the collected buffy coats. Correlation analysis and linear regression were performed between cell counts in peripheral blood and buffy coat. Collection efficiency was compared between UVAR XTS and CELLEX. RESULTS: In all 52 patients, lymphocyte counts in buffy coat and peripheral blood showed strong correlation (r values were 0.85 and 0.983 for UVAR XTS and CELLEX, respectively; p < 0.001) with slopes of 2 and 5.1 for UVAR XTS and CELLEX, respectively (p < 0.001). The quantitative relationship remained robust in patients stratified by diagnoses. Monocytes also showed consistent correlation and linearity, but not neutrophils or combined white blood cells, red blood cells, or platelets. CELLEX enriched approximately twice as many lymphocytes and monocytes than UVAR XTS per procedure (p < 0.001). CONCLUSION: The preprocedural peripheral lymphocyte count can predict the number of lymphocytes within the buffy coat collected during ECP, which may justify the use of peripheral lymphocyte count as a surrogate for the cell dose treated per procedure. Peripheral monocyte counts may serve as an alternative. CELLEX is more efficient in collecting lymphocytes and monocytes than UVAR XTS under conditions tested. E xtracorporeal photopheresis (ECP) is a treatment modality that entails leukapheresis, mixing of the collected buffy coat and 8-methoxypsoralen (8-MOP) ex vivo, irradiation byultravioletA(UVA)light,andreinfusionoftreatedwhite blood cells (WBCs) back to the patient. UVA light activates 8-MOP, which crosslinks the DNA strands by forming covalent bonds with the pyrimidine bases. ECP was initially applied in the treatment of cutaneous T-cell lymphoma (CTCL) and a loss of viability was observed in lymphocytes. 1

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.

Background Extracorporeal photopheresis (ECP) is an established treatment for graft-versus-host disease (GVHD). Various haematocrit thresholds have been used to trigger red blood cells transfusion prior to ECP. Moderate-to-severe GVHD is frequently complicated by anaemia; the safety and collection efficiency with a lower haematocrit for ECP is unknown. Methods We prospectively enrolled 26 consecutive adult GVHD patients with haematocrits between 25% and 28·9% who received ECP on the CELLEX system. Preprocedural transfusion was withheld. We monitored the adverse events and transfusions avoided. A complete blood cell count with differential was performed on preprocedural peripheral blood and buffy coat collected. Lymphocyte fold enrichment (LFE) was compared between this cohort and two historical control groups with haematocrits of 29% or higher. Results Red Blood Cells transfusion was avoided in the lower-haematocrit cohort without adverse events. The median LFE was 4·5 (95%CI, 3·1–5·7) in the lower-haematocrit cohort and 5·2 (95%CI, 4·1–6·5) in the higher-haematocrit CELLEX-treated control group. The median difference was 0·7 (95%CI, −0·3 to 2·0, P = 0·14). It could not be established that the lower-haematocrit cohort was non-inferior to the higher-haematocrit control group with a prespecified non-inferiority margin of 1·3. However, LFE was significantly higher in the lower-haematocrit cohort than the higher-haematocrit UVAR XTS-treated control group (P < 0·01). Conclusion Buffy coat can be collected for ECP using CELLEX in GVHD patients with a haematocrit of 25% or higher, with a collection efficiency superior to that in patients with higher haematocrits but treated using UVAR XTS. No increase in adverse events was observed at these lower haematocrits.

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.

Introduction This randomized, exploratory study compared the incidence of heparin-dependent antibodies associated with subcutaneous (SC) desirudin or heparin given for deep-vein thrombosis prophylaxis following cardiac and thoracic surgery. Materials and Methods Adult patients scheduled for elective cardiac or thoracic surgery received desirudin 15 mg SC twice daily or unfractionated heparin 5000 units SC thrice daily. Duration of thrombosis prophylaxis was determined by the treating physician. Primary outcome measure was the incidence of new antibody formation directed against platelet factor 4 (PF4)/heparin complex. Secondary outcomes included bleeding and thrombotic complications. Blood was tested for anti-PF4/heparin antibodies at baseline, after surgery prior to study drug administration, postdrug day (PDD) 2, PDD 7, and at 1 month. Doppler studies were done before discharge. Results Of 120 patients, 61 received desirudin, 59 received heparin. New PF4/heparin antibodies occurred in 10.2% and 13.6% of desirudin- and heparin-treated patients, respectively. Among desirudin patients with no heparin exposure, none (0/36) developed PF4/heparin antibodies versus 17.1% with heparin exposure. Incidence of deep venous thrombosis was 4.9% and 3.4% in the desirudin and heparin groups, respectively. Two heparin-group patients developed pulmonary embolism. Two patients per group had bleeding events; no patients required re-exploration for bleeding complications. Median chest tube output was similar with desirudin (900 mL) and heparin (692 mL) as was blood transfusion requirements of more than 2 units (5/61, desirudin; 2/59 heparin). Conclusions The incidence of thrombotic events was low in both groups. There were no safety concerns, and desirudin was not associated with anti-PF4/heparin antibodies.

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.

Acquired autoantibodies targeting coagulation factors are fortunately rare. However, unfamiliarity with them can lead to misinterpretation of clinical presentations and screening laboratory tests, erroneous or delayed diagnoses, and inappropriate management decisions which may result in severe morbidity or mortality. Using acquired neutralizing antibodies to Factor VIII (acquired hemophilia A) as a model for clinical presentation, laboratory diagnosis, management of bleeding, and immunosuppression treatment, this chapter reviews our current knowledge of acquired inhibitors of the coagulation system.

Vitamin K is a cofactor for human glutamic acid carboxylase enzymes in multiple tissues and organs. The dominant vitamin K activity is gamma-carboxylation of selective glutamic acid residues on coagulation factors X, IX, VII, and II. Humans predominantly rely on green vegetable dietary sources of vitamin K rather than synthesis of vitamin K by intestinal bacteria. Vitamin K deficiency bleeding in newborns is uncommon, potentially devastating, and preventable with vitamin K prophylaxis. Other causes of vitamin K deficiency include prolonged anorexia, malabsorption, and rapid depletion without adequate intake during acute medical and surgical events. The significance and mechanisms of antibiotic-associated vitamin K deficiency remain controversial and incompletely investigated. Management options for vitamin K deficiency include oral and parenteral vitamin K and coagulation factor replacement. Ingestion of “superwarfarin” rodenticides produces a severe and prolonged coagulopathy with potential for major bleeding complications.

Disseminated intravascular coagulation (DIC) is a clinicopathological syndrome in which the consequences of systemic activation of coagulation by a disease, illness, or trauma can produce microvascular thrombi, organ dysfunction, consumption of platelets, and coagulation factors. Depending upon the severity and tempo of the initiating event, DIC may be asymptomatic or cause severe thrombotic or hemorrhagic complications including death. The primary driver of DIC in most conditions is tissue factor pathway activation of thrombin, although many cellular and complex activation pathways are also involved. Conditions in which bleeding is the dominant complication of DIC will be a focus of this chapter.

Warfarin sodium is commonly prescribed for the prophylaxis and treatment of venous thromboembolism. Dosing algorithms have not been widely adopted because they require a fixed initial warfarin dose (eg, 5 mg) and are not tailored to other factors that may affect the international normalized ratio (INR).To develop an algorithm that could predict a therapeutic warfarin dose based on drug interactions, INR response after the initial warfarin doses, and other clinical factors.We used stepwise regression to quantify the relationship between these factors in patients beginning prophylactic warfarin therapy immediately prior to joint replacement. In the derivation cohort (n = 271), we separately modeled the therapeutic dose after 2 and 3 initial doses. We prospectively validated these 2 models in an independent cohort (n = 105).About half of the therapeutic dose variability was predictable after 3 days of therapy: R2 was 53% in the derivation cohort and 42% in the validation cohort. INR response after 3 warfarin doses (INR3) inversely correlated with therapeutic dose (p0.001). Intraoperative blood loss transiently, but significantly, elevated the postoperative INR values. Other significant (p0.03) predictors were the first and second warfarin doses (+7% and +6%, respectively, per 1 mg), and statin use (-15.0%). The model derived after 2 warfarin doses explained 32% of the variability in therapeutic dose.We developed and validated algorithms that estimate therapeutic warfarin doses based on clinical factors and INR response available after 2-3 days of warfarin therapy. The algorithms are implemented online at www.WarfarinDosing.org.

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.

Hemophagocytic lymphohistiocytosis (HLH) is a rare potentially fatal syndrome characterized by an uncontrolled hyperinflammatory response. The secondary form of HLH is usually triggered by a causative agent. Ehrlichia chaffeensis is a rare trigger of secondary HLH. We present a case series of five adolescents and adults diagnosed with Ehrlichia-induced HLH and we discuss their clinical and laboratory findings. We also review the literature for similar cases. Between October 2003 and June 2014, we identified 76 cases of HLH in adolescents and adults, 5 of which were induced by Ehrlichia. All 5 patients had fever, cytopenias, hypertriglyceridemia, and high ferritin. Hyperferritinemia was striking with a median admission ferritin of 47,290 μg/L (range: 2,863-85,517). In addition to the positive Ehrlichia PCR testing on peripheral blood of all patients, two patients with neurologic symptoms tested positive for E. chaffeensis in CSF specimens. Early treatment with doxycycline was effective. After a median follow up of 7.3 months, all patients were alive and none had recurrence of HLH. Clinicians should consider E. chaffeensis as a potential trigger for HLH especially in areas with tick activity. Prompt diagnosis and treatment with doxycycline are required for a better outcome.

The U.S. College of American Pathologists (CAP) has conducted a focused study of the proficiency testing for von Willebrand disease (vWD) analysis from 2003 to 2005. This report summarizes the findings regarding the accuracy and precision of the various assays at different analyte levels, as well as the influence of the reference material used to construct the assay standard curve. The results show that testing of von Willebrand factor (vWF):antigen (vWF:Ag) and ristocetin cofactor activity (vWF:RCo) is reasonably accurate, with all-method mean values falling within 3.2 and 5.6%, respectively, of the International Society on Thrombosis and Haemostasis Secondary Coagulation Standard (lot 2) assigned values. vWF:Ag measurements are reasonably precise (all-method coefficients of variation [CVs] = 10.7 to 15.1%), even at lower levels of vWF. The highest precision was observed for immunoturbidometric assays (CVs, 6.3 to 9.7%). vWF:RCo measurements are less precise (all-method CVs, 23.3 to 30.9%). The reference materials used in the standard curves for immunoturbidometric vWF:Ag assays appear to have accurately assigned vWF values for the majority of commercial suppliers.

Documentation of preanalytical conditions that could result in inaccurate results and misdiagnoses of patients is important. It has recently been reported that a significant loss of factor VIII (FVIII) and von Willebrand factor (vWF) activity occurs when citrated whole blood is stored on ice. We tested the hypothesis that the cold-dependent loss of FVIII and vWF activity is due to the formation of cryoprecipitate and is reversed by rewarming the citrated whole blood before centrifugation and removal of plasma. We collected venous blood from 10 healthy subjects into 3.2% sodium citrate glass tubes. One tube was immediately centrifuged and the plasma was stored at -20 degrees C. Following storage in an ice bath (4 degrees C) for 3.5 hours, 1 tube was immediately centrifuged and processed while the second tube was placed in a 37 degrees C water bath for 5 minutes then centrifuged and processed. Subsequently, plasma samples were quickly thawed at 37 degrees C and the following tests were performed: prothrombin time (PT), partial thrombin time (aPTT), FVIII activity, vWF antigen (vWF:Ag), and vWF activity (vWF:Act). Means for each analyte from the 2 tubes stored at 4 degrees C for 3.5 hours with or without rewarming were compared to baseline tube using the Student t test. Compared to the baseline tube results, PT and aPTT showed no significant changes in either of the tubes stored at 4 degrees C for 3.5 hours. However, FVIII, vWF:Ag, and vWF:Act were significantly lower in the tubes stored at 4 degrees C for 3.5 hours, but no differences were detected between baseline and rewarmed tube results. In conclusion, prolonged storage of citrated whole blood at 4 degrees C causes a clinically significant reduction of FVIII and vWF activities. The losses are completely reversed by rewarming the tube prior to processing. This is consistent with a reversible cryoprecipitation of vWF and FVIII rather than a cold-activated enzymatic degradation.