An aphid symbiont confers protection against a specialized RNA virus, another increases vulnerability to the same pathogen.

Insects often harbour heritable symbionts that provide defence against specialized natural enemies, yet little is known about symbiont protection when hosts face simultaneous threats. In pea aphids (Acyrthosiphon pisum), the facultative endosymbiont Hamiltonella defensa confers protection against the parasitoid, Aphidius ervi, and Regiella insecticola protects against aphid-specific fungal pathogens, including Pandora neoaphidis. Here, we investigated whether these two common aphid symbionts protect against a specialized virus A. pisum virus (APV), and whether their antifungal and antiparasitoid services are impacted by APV infection. We found that APV imposed large fitness costs on symbiont-free aphids and these costs were elevated in aphids also housing H. defensa. In contrast, APV titres were significantly reduced and costs to APV infection were largely eliminated in aphids with R. insecticola. To our knowledge, R. insecticola is the first aphid symbiont shown to protect against a viral pathogen, and only the second arthropod symbiont reported to do so. In contrast, APV infection did not impact the protective services of either R. insecticola or H. defensa. To better understand APV biology, we produced five genomes and examined transmission routes. We found that moderate rates of vertical transmission, combined with horizontal transfer through food plants, were the major route of APV spread, although lateral transfer by parasitoids also occurred. Transmission was unaffected by facultative symbionts. In summary, the presence and species identity of facultative symbionts resulted in highly divergent outcomes for aphids infected with APV, while not impacting defensive services that target other enemies. These findings add to the diverse phenotypes conferred by aphid symbionts, and to the growing body of work highlighting extensive variation in symbiont-mediated interactions.

An In Silico Exploration of the Factors That Affect the Precision of the Bethesda Assay.

OBJECTIVES: Despite more than 40 years of experience performing the Bethesda assay (BA), poor intra- and interlaboratory precision remains the biggest laboratory challenge to date. METHODS: The BA procedure was modeled using stochastic simulation techniques to determine the precision of the BA up to dilutions of 1:4,096, to estimate the minimum significant relative change at various inhibitor titers, and to understand the laboratory procedural variables that could significantly affect the performance of the BA at high dilutions. RESULTS: Selecting the lowest dilution tube with a residual activity closest to 25% for calculating the reported Bethesda titer (BT), using a factor activity assay with a coefficient of variation less than or equal to 7.5% in the range of 15% to 50% factor activity level, performing the factor activity measurement in replicates, and minimizing pipette volumetric error resulted in the lowest imprecision in the reported BT. The factor neutralization kinetics of the inhibitor appear to have little impact on the precision of the assay if the incubation time is greater than 90 minutes. CONCLUSIONS: This in silico model will assist future laboratory efforts in standardizing the quantification of specific coagulation factor inhibitors and improving the precision of the reported results.

Hemostatic prophylaxis and colonoscopy outcomes for patients with bleeding disorders: A retrospective cohort study and review of the literature.

INTRODUCTION: Hemostatic prophylaxis (HP) is recommended for patients with bleeding disorders (PWBD) before invasive procedures. However, evidence-based guidelines are needed to determine optimal HP strategies. AIM: To determine outcomes of HP for PWBD undergoing colonoscopy. METHODS: We undertook a retrospective cohort study of HP and outcomes of colonoscopy procedures performed between 9 November 1993 and 13 February 2018 for PWBD who received care in the Mayo Clinic Comprehensive Hemophilia Treatment Center. RESULTS: During the study period, 73 PWBD (58 with milder phenotypes: haemophilia, von Willebrand disease [subtypes 1 and 2; II, VII and XI deficiency]) underwent 141 procedures. Preprocedural HP was given to 61%, and interventions were performed in 47%. Of the 39% without preprocedural HP, postprocedural HP was given for 11%. One major (0.7%; 6 days postprocedure despite HP) and 10 minor (7%) bleeding complications occurred, which tended to be in patients with severe disease and/or after excision of larger polyps. There was no significant difference in the rate of bleeding complications with or without preprocedural HP (8.1% vs 5.5%, respectively; P = .74, Fisher's exact test). CONCLUSION: The low bleeding rates in our cohort suggest that preprocedure HP may be withheld for patients with mild bleeding disorders who undergo colonoscopy with a low likelihood of requiring an intervention or who require only low-risk intervention. This strategy may be best used in experienced centres, provided optimal local hemostasis measures are undertaken and postprocedural HP is rapidly available if high-risk intervention is required. Further studies are needed to determine optimal evidence-based HP strategies for PWBD undergoing colonoscopy.

The Mayo Clinic Experience With Psychogenic Purpura (Gardner-Diamond Syndrome).

BACKGROUND: The objective of this study was to describe presentation, natural history, management and long-term outcomes of patients with psychogenic purpura (PP), also known as Gardner-Diamond Syndrome. METHODS: In this retrospective study, records of patients with a diagnosis of PP seen at Mayo Clinic, Rochester from 1976 to 2016 were reviewed. Available literature regarding PP was also comparatively reviewed. RESULTS: Seventy-six patients with a diagnosis of PP were identified and 54/76 (71%) experienced a prodromal sensation. The Condensed MCMDM-1 bleeding score, excluding cutaneous manifestations, was <3 in 91% of patients. Laboratory tests of primary and secondary hemostasis were normal. Fifty-four percent of patients had an underlying psychiatric diagnosis. Management approaches included psychological counseling and psychiatry evaluation in 44 patients. Pharmacologic treatment for 30 patients included psychotropic agents, antihistamines, hormonal medications and anti-inflammatory agents. At a median follow-up of 5years (range 1-34),13/28 (46.4%) experienced recurrent ecchymoses and 6 continued to seek hematology follow-up at Mayo Clinic, Rochester. Our data was similar to the aggregate data from case reports in the literature. CONCLUSIONS: For patients with unexplained recurrent ecchymosis a diagnosis of PP should be considered. Diagnosis is one of exclusion and initial evaluation should include documenting a bleeding score and obtaining laboratory tests assessing primary and secondary hemostasis. The relatively low bleeding scores together with laboratory assessments support that PP is primarily a dermal rather than a systemic bleeding diathesis. In our cohort, addressing psychological stressors was the most effective treatment; however pharmacologic therapy can be used for refractory disease.

Clinical and laboratory diagnosis of autoimmune factor V inhibitors: A single institutional experience.

BACKGROUND: Coagulation factor V inhibitors (FV-i) may occur in patients with congenital FV deficiency or previously hemostatically normal patients (autoimmune (AI)-FV-i). Most of the published literature is confined to case reports. OBJECTIVE: Describe clinical and laboratory features of AI-FV-i identified through the Special Coagulation Laboratory at Mayo Clinic, Rochester, Minnesota. METHODS: In this retrospective study individuals with FV-i screens performed from January 1999 to February 2017 were identified through the special coagulation laboratory database. Clinical presentation, management, and outcomes were collected for our institutional patients while detailed laboratory data was collected for all tested patients. RESULTS: Of patients with FV-i managed at our institution, 2/8 (25%) patients experienced no bleeding. There was no correlation between inhibitor titers and/or FV activity (FV:C) levels and clinical bleeding. Hemostatic management included fresh frozen plasma, platelet transfusion, activated prothrombin complex concentrates, and recombinant factor VIIa. Only 2 patients received immunomodulatory treatment. FV-i mixing studies with normal pooled plasma (n = 26) demonstrated inhibition on immediate mix but progressive inhibition after 1 h of incubation could not be demonstrated. 71% of platelet neutralization procedures were falsely positive while 59% of DRVVT assays were indeterminate. CONCLUSION: FV-i demonstrates immediate inhibition on mixing studies; however our limited data does not support a time dependent inhibition. Our clinical cohort confirms the variable clinical phenotype for individuals with FV-i and supports the notion that management of FV-i should be guided by clinical symptoms and not FV:C or FV-i titer.

Diagnostic laboratory standardization and validation of platelet transmission electron microscopy.

Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2-4.0. Mean DG/plt was associated with IPF (p = .01, R2 = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (p > .17, R2 < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.

Lack of grading agreement among international hemostasis external quality assessment programs.

: Laboratory quality programs rely on internal quality control and external quality assessment (EQA). EQA programs provide unknown specimens for the laboratory to test. The laboratory's result is compared with other (peer) laboratories performing the same test. EQA programs assign target values using a variety of methods statistical tools and performance assessment of 'pass' or 'fail' is made. EQA provider members of the international organization, external quality assurance in thrombosis and hemostasis, took part in a study to compare outcome of performance analysis using the same data set of laboratory results. Eleven EQA organizations using eight different analytical approaches participated. Data for a normal and prolonged activated partial thromboplastin time (aPTT) and a normal and reduced factor VIII (FVIII) from 218 laboratories were sent to the EQA providers who analyzed the data set using their method of evaluation for aPTT and FVIII, determining the performance for each laboratory record in the data set. Providers also summarized their statistical approach to assignment of target values and laboratory performance. Each laboratory record in the data set was graded pass/fail by all EQA providers for each of the four analytes. There was a lack of agreement of pass/fail grading among EQA programs. Discordance in the grading was 17.9 and 11% of normal and prolonged aPTT results, respectively, and 20.2 and 17.4% of normal and reduced FVIII results, respectively. All EQA programs in this study employed statistical methods compliant with the International Standardization Organization (ISO), ISO 13528, yet the evaluation of laboratory results for all four analytes showed remarkable grading discordance.

Hermansky-Pudlak syndrome subtype 5 (HPS-5) novel mutation in a 65 year-old with oculocutaneous hypopigmentation and mild bleeding diathesis: The importance of recognizing a subtle phenotype.

Hermansky-Pudlak syndrome (HPS) - characterized by the distinct clinical phenotypes of both oculocutaneous albinism and mild bleeding diathesis-is caused by mutations in genes that have crucial roles in the assembly of cellular organelles (skin melanosomes, platelet delta [dense] granules, lung lamellar bodies, and cytotoxic T-cell lymphocyte granules). Immunodeficiency, pulmonary fibrosis and granulomatous colitis are associated with some, but not all subtypes of HPS, with varying degrees of clinical severity. We describe a patient diagnosed with platelet dense granule storage pool deficiency (DG-SPD) at age 38 years after he presented with spontaneous intracranial hemorrhage. His mild oculocutaneous hypopigmentation was subtle. In the following 27 years, he did not develop severe bleeding nor pulmonary or gastrointestinal complications. A novel homozygous c.1960A>T; p.Lys654* mutation in the HPS-5 protein gene (HPS5) was identified through next generation sequencing, (NGS) which is consistent with the patient's clinical and laboratory phenotypes. This case underscores the importance of recognizing the mild clinical phenotype of HPS-5 and utilization of both laboratory and molecular testing for diagnosis, prognostication, and surveillance for end organ damage in patients affected with HPS.

Comprehensive Platelet Phenotypic Laboratory Testing and Bleeding History Scoring for Diagnosis of Suspected Hereditary Platelet Disorders: A Single-Institution Experience.

OBJECTIVES: Patients with hereditary/congenital platelet disorders (HPDs) have a broad range of clinical manifestations and laboratory phenotypes. We assessed the performance characteristics of the International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) and clinically validated platelet laboratory tests for diagnosis of HPDs. METHODS: The records of 61 patients with suspected HPDs were reviewed and ISTH-BAT scores calculated. RESULTS: Nineteen (31%) patients had thrombocytopenia, and 46 (75%) had positive ISTH-BAT scores. Thirteen and 17 patients had prolonged PFA-100 (Dade Behring, Miami, FL) adenosine diphosphate and epinephrine closure times, respectively. Twenty-two had abnormal platelet light transmission aggregation. Twenty-four had platelet transmission electron microscopy (PTEM) abnormalities (10 dense granule deficiency, 14 other ultrastructural abnormalities). Positive ISTH-BAT scores were associated with thrombocytopenia (P < .0001) and abnormal PTEM (P = .002). Twenty-three patients had normal results. CONCLUSIONS: ISTH-BAT identified patients with suspected HPDs but lacked a robust association with laboratory abnormalities. Despite comprehensive laboratory testing, some patients may have normal results.

From vitamin K antagonists to liver international normalized ratio: a historical journey and critical perspective.

Adoption of international normalized ratio (INR) to harmonize prothrombin time has greatly improved the safety and effectiveness of vitamin K antagonists (VKA) oral anticoagulant therapy. INR is also a major laboratory variable in calculating the widely used Model for End-Stage Liver Disease (MELD) score for liver transplant organ prioritization. However, since the conventional INR (INRVKA) is calibrated specifically for VKA patients, its interlaboratory variation has a significant impact on the accuracy of MELD score. Though still requiring further clinical validation in large numbers of waitlisted patients, the alternative liver INR calibrated by using plasma from liver disease patients instead of VKA patients may harmonize the differences and thus more suitable for MELD score calculation. The objective of this article is to review the history of INR, MELD score, and liver INR, and discuss the challenges and solutions of liver INR implementation.

Operative and nonoperative management of chronic disseminated intravascular coagulation due to persistent aortic endoleak.

Disseminated intravascular coagulation (DIC) due to endoleak is a rare complication following endovascular aneurysm repair. Two of the four previously reported cases occurred in patients with cirrhosis. We describe three patients with normal liver function who developed DIC due to delayed high-flow (type Ia or III) endoleaks. Two patients underwent successful surgical repair, and the third was managed medically. All three patients had chronic thrombocytopenia prior to developing an endoleak as did the four reported cases in the literature. We propose that thrombocytopenia, like cirrhosis, be considered a risk factor for DIC due to endoleaks in patients undergoing endovascular aneurysm repair.

Hypertrophic obstructive cardiomyopathy, bleeding history, and acquired von Willebrand syndrome: response to septal myectomy.

Bleeding with severe aortic stenosis is linked to acquired von Willebrand syndrome and loss of high-molecular-weight multimers of von Willebrand factor. Valve replacement resolves bleeding tendency and loss of high-molecular-weight multimers. We report outcomes in 5 patients with symptomatic obstructive hypertrophic cardiomyopathy and spontaneous gastrointestinal, mucosal, or excessive postsurgical bleeding in whom acquired von Willebrand syndrome was documented. All 5 patients underwent surgical septal myectomy with resolution of acquired von Willebrand syndrome.

Plasma von Willebrand factor multimer quantitative analysis by in-gel immunostaining and infrared fluorescent imaging.

INTRODUCTION: Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps. MATERIALS/METHODS: VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed. RESULTS: The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5% and 6% respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas. CONCLUSIONS: The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification.

Diagnosis and management of von Willebrand disease: guidelines for primary care.

Von Willebrand disease is an inherited condition characterized by deficiency of von Willebrand factor, which is essential in hemostasis. The National Heart, Lung, and Blood Institute has released new evidence-based guidelines for the diagnosis and management of the disease. There are three major subtypes of von Willebrand disease, classified as partial quantitative deficiency (low levels) of von Willebrand factor (type 1), qualitative deficiency (type 2), or virtually complete deficiency (type 3). Diagnosis is usually made by reviewing the patient's personal and family history of bleeding and by clinical evaluation for more common reasons for bleeding, supplemented with laboratory tests. Assessment may be used to determine bleeding risk before surgery and other invasive procedures, and to diagnose reasons for unexplained hemorrhaging. Von Willebrand factor levels of 30 IU per dL or lower are required for the definite diagnosis of inherited von Willebrand disease. Persons with levels of 30 to 50 IU per dL may not have the disease, but may need agents to increase von Willebrand factor levels during invasive procedures or childbirth. Treatment is tailored to the subtype of the disease: increasing plasma concentration of von Willebrand factor by releasing endogenous stores with desmopressin or replacing nonexistent or ineffective von Willebrand factor by using human plasma-derived, viral-inactivated concentrates; treatment is often combined with hemostatic agents that have mechanisms other than increasing von Willebrand factor. Regular prophylaxis is seldom required, and treatment is initiated before planned invasive procedures or in response to bleeding.

Von Willebrand disease: key points from the 2008 National Heart, Lung, and Blood Institute guidelines.

UNLABELLED: Von Willebrand disease (VWD) is the most common inherited bleeding disorder and may affect as many as one in 100 women. The condition results from a deficiency, dysfunction, or absence of von Willebrand factor (VWF). In women, the most common symptom of VWD is menorrhagia. Of women with menorrhagia, 5-20% have been found to have previously undiagnosed VWD. Besides menorrhagia, women with VWD are more likely to experience other conditions that manifest with abnormal reproductive tract bleeding. The patient with a suspected bleeding disorder should be referred to a hemophilia treatment center or hematologist with expertise in bleeding disorders for definitive diagnosis. After diagnosis, the first choice of therapy for the management of menorrhagia in adolescents or adult females who do not desire child bearing is still hormonal contraceptives. Women who fail hormonal contraceptives, yet desire future child bearing, and women who desire pregnancy are candidates for hemostatic therapy, which is generally reserved for patients with VWF levels less than 50 international units/dL. During pregnancy, VWF levels rise, frequently obviating the need for hemostatic therapy at the time of delivery. Minor procedures can be managed with 1-desamino-8-D-arginine vasopressin, antifibrinolytic medication, or both, but major surgery or childbirth requires replacement with VWF and should be conducted in a center with available hematologists, anesthesiologists, pharmacists, and laboratory support experienced in the management of bleeding disorders. LEVEL OF EVIDENCE: III.

Transient neonatal acquired von Willebrand syndrome due to transplacental transfer of maternal monoclonal antibodies.

Although typically a disorder of adults, acquired von Willebrand syndrome (AVWS) is increasingly being recognized in the pediatric population in association with congenital cardiac diseases, certain neoplasia, and hypothyroidism. Transplacental transfer of maternal immunoglobulin G (IgG) antibodies as a cause of neonatal disorders in infants born to mothers with autoimmune conditions has been reported. We describe the diagnosis and peripartum clinical management of AVWS due to monoclonal gammopathy of undetermined significance (MGUS) and the first reported case of transient neonatal AVWS due to transplacental transfer of maternal IgG antibodies.