Recent developments in the nomenclature,presence, isolation,detection and clinical impact of extracellular vesicles

The research field of extracellular vesicles (EVs), such as microparticles and exosomes, is growing exponentially. The goal of this review is to provide an overview of recent developments relevant to the readers of the Journal of Thrombosis and Haemostasis. We will discuss nomenclature, the presence of EVs in fluids, methods of isolation and detection, and emerging clinical implications. Although research on EVs has been performed within the ISTH for over a decade, most of the recent research on EVs has been brought together by the International Society on Extracellular Vesicles (ISEV). To achieve an overview of recent developments, the information provided in this review comes not only from publications, but also from latest meetings of the ISEV (April 2015, Washington, DC, USA), the International Society on Advancement of Cytometry (June 2015, Glasgow, UK), and the ISTH (June 2015, Toronto, Canada).     

Single vs. swarm detection of microparticles and exosomes by flow cytometry

See also Harrison P, Gardiner C. Invisible vesicles swarm within the iceberg. This issue, pp 916‐8.

Summary.

Background:  Microparticles and exosomes are cell‐derived vesicles and potential biomarkers for disease. Recently, the Scientific Standardization Committee collaborative workshop of the ISTH initiated standardization of vesicle detection by flow cytometry with polystyrene beads. Because polystyrene beads have different optical properties from biological vesicles, and because the mechanisms causing the detection signal are incompletely understood, there are contradictions between expected and observed results. Objectives:  To develop a model with which to relate the detection signal of a flow cytometer to the diameter of vesicles and clarify observed discrepancies. Methods:  We combined measurements of polystyrene and silica beads with an estimated refractive index of vesicles and performed Mie calculations of light scattering. Results:  We established the relationship between measured light scattering and the diameter of vesicles. The Megamix gating strategy proposed by the Scientific Standardization Committee selects single vesicles and cells with diameters between 800 and 2400 nm when applied on the forward‐scattering detector of regular flow cytometers. Nevertheless, we demonstrated that, irrespective of the applied gating, multiple vesicles smaller than 220 nm or multiple 89‐nm silica beads were counted as a single event signal at sufficiently high concentrations. Conclusions:  Vesicle detection by flow cytometry is attributed to large single vesicles and swarm detection of smaller vesicles; that is, multiple vesicles are simultaneously illuminated by the laser beam and counted as a single event signal. Swarm detection allows the detection of smaller vesicles than previously thought possible, and explains the finding that flow cytometry underestimates the concentration of vesicles.     

Tracking of Tumor Cell–Derived Extracellular Vesicles In Vivo Reveals a Specific Distribution Pattern with Consecutive Biological Effects on Target Sites of Metastasis

Purpose

Extracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis.

Procedures

Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell–derived versus extracellular vesicles from healthy serum.

Results

In contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV.

Conclusions

In conclusion, distribution of tumor cell–derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.

     

Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH

Background: Light transmission aggregometry (LTA) is the most common method used in clinical and research laboratories to assess platelet function. However, the method has never been standardized. Objectives: As the first step towards development of methodological guidelines, the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH) undertook a large, detailed, global survey of LTA practices. Methods: Members of ISTH and of External Quality Assurance in Thrombosis and Haemostasis organizations were invited to complete a 129 item, online questionnaire. Results were analyzed anonymously to participant identities. Results: The online supplement for this article ( http://www.isth.org/Publications/OfficialCommunications/PlateletPhysiology/LightTransmissionAggregometry/tabid/201/Default.aspx ) contains the full details of the study findings. 359 (244 clinical, 115 research) laboratories from 48 countries participated in the survey. LTA was widely used to assess inherited or acquired bleeding disorders. Common practices were identified in sample collection, processing and analysis and although some are generally considered acceptable, others are not ideal. The agonist concentrations used for LTA varied, and many laboratories used ADP, collagen, epinephrine and Ristocetin, at more than one concentration, in addition to arachidonic acid. The parameters commonly used to assess LTA responses were maximal amplitude or % aggregation, which was considered particularly important, in addition to the presence of a ‘secondary wave’, deaggregation, shape change and a measure of the lag phase. However, many laboratories did not have appropriate reference intervals. Conclusions: This is the largest and most detailed survey of LTA practices ever undertaken. It shows a very high variability in LTA practices worldwide, and, as a consequence, methodological standardization is necessary. The information gathered in this survey will be helpful in the development of ISTH methodological guidelines for LTA.     

Hollow organosilica beads as reference particles for optical detection of extracellular vesicles

Essentials

• Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement.
• Hollow organosilica beads were prepared, characterized, and tested as reference particles.
• Light scattering properties of hollow beads resemble that of platelet‐derived EVs.
• Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs.

Summary

Background

The concentration of extracellular vesicles (EVs) in body fluids is a promising biomarker for disease, and flow cytometry remains the clinically most applicable method to identify the cellular origin of single EVs in suspension. To compare concentration measurements of EVs between flow cytometers, solid polystyrene reference beads and EVs were distributed in the first ISTH‐organized interlaboratory comparison studies. The beads were used to set size gates based on light scatter, and the concentration of EVs was measured within the size gates. However, polystyrene beads lead to false size determination of EVs, owing to the mismatch in refractive index between beads and EVs. Moreover, polystyrene beads gate different EV sizes on different flow cytometers.

Objective

To prepare, characterize and test hollow organosilica beads (HOBs) as reference beads to set EV size gates in flow cytometry investigations.

Methods

HOBs were prepared with a hard template sol‐gel method, and extensively characterized for morphology, size, and colloidal stability. The applicability of HOBs as reference particles was investigated by flow cytometry with HOBs and platelet‐derived EVs.

Results

HOBs proved to be monodisperse with a homogeneous shell thickness. Two‐angle light‐scattering measurements by flow cytometry confirmed that HOBs have light‐scattering properties similar to those of platelet‐derived EVs.

Conclusions

Because the structure and light‐scattering properties HOBs resemble those of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads with which to standardize optical measurements of the EV concentration within a predefined size range.

     

弥散性血管内凝血诊断评分标准在产科的应用评价

目的 采用已有的4种国际非孕弥散性血管内凝血（DIC）诊断评分标准对产科DIC的诊断进行评估，探索更适合产科DIC诊断的“金标准”。 方法 选择2009年6月－2012年6月期间产科拟诊DIC的孕产妇为研究对象，用日本卫生福利部（JMHW）提出的JMHW、日本危重病协会（JAAM）提出的JAAM、国际血栓与止血委员会（ISTH）提出的ISTH显性和ISTH非显性4种诊断评分标准联合诊断和构建“金标准”，以此评价4种诊断标准对产科DIC诊断的特性。 结果 受试者工作特征（ROC）曲线分析显示ISTH非显性标准、ISTH显性标准、JMHW、JAAM的ROC曲线下面积分别为0.939、0.865、0.867、0.867，ISTH非显性标准灵敏度和特异度与“金标准”在不同诊断界值时较一致，同时优于其他3种诊断标准。 结论 ISTH非显性标准较适合作为临床产科DIC诊断，其对产科这一特殊发病人群的DIC诊断具有更科学的临床诊断价值。     

Value assignment of the WHO 2nd International Standard von Willebrand factor,concentrate (09/182)

To cite this article: Hubbard AR, Hamill M, Beeharry M, Bevan SA, Heath AB, on behalf of the SSC sub‐committee on von Willebrand factor of ISTH. Value assignment of the WHO 2nd International Standard von Willebrand factor, concentrate (09/182). J Thromb Haemost 2011; 9 : 1638–40.DOI: 10.1111/j.1538‐7836.2011.04365.x .     

中性粒细胞胞外诱捕网标志物游离DNA水平与脓毒症血小板活化的相关性

目的研究中性粒细胞胞外诱捕网（NETs）标志物游离DNA（cfDNA）水平与血小板（PLT）活化之间的相关性。 方法根据纳入标准、排除标准将2018年9至12月中国医科大学附属第一医院ICU收治的51例患者分为脓毒症组（29例）和对照组（22例）。用酶联免疫吸附测定（ELISA）方法检测患者外周血浆的cfDNA、血小板因子4（PF4）、P-选择素浓度。采用SPSS 25.0进行统计学分析，比较2组患者入ICU 24 h内生化指标、急性生理学与慢性健康状况（APACHEⅡ）评分、序贯器官衰竭评估（SOFA）评分、国际血栓与止血委员会（ISTH）评分、日本危重病协会（JAAM）评分、cfDNA以及PLT标志物（PF4、P-选择素）的差异性。同时将脓毒症组cfDNA和PLT、PLT标志物（PF4、P-选择素）、炎症指标［中性粒细胞计数（NE）、C反应蛋白（CRP）、降钙素原（PCT）］、凝血指标［凝血酶原时间（PT）、活化部分凝血酶时间（APTT）、D-二聚体（D-D）、抗凝血酶Ⅲ活性（AT-Ⅲ）］、疾病严重程度（APACHEⅡ评分、SOFA评分、ISTH评分、JAAM评分）等分别进行相关性分析。 结果脓毒症组患者cfDNA水平明显高于对照组，差异有统计学意义［（174.499±76.596）ng/ml vs （114.354±19.319）ng/ml，P＜0.05］，但2组P-选择素、PF4水平比较，差异无统计学意义（P＞0.05）。脓毒症组中cfDNA水平与P-选择素/PLT、D-D、CRP、APACHEⅡ评分、JAAM评分之间具有相关性（r=0.3849、0.3872、0.6211、0.4542、0.4451，P＜0.05）；与NE、PLT计数、PLT标志物（PF4、P-选择素）、ISTH评分等无明显相关性（P＞0.05）。 结论脓毒症组患者cfDNA明显升高。脓毒症组中cfDNA与D-D之间具有明显正相关性，提示NETs可能与继发性纤溶过程关系更加密切。尽管脓毒症组中cfDNA与P-选择素/PLT之间有正相关性，但因为P-选择素并不是特异性的PLT活化标志物，所以NETs与脓毒症PLT活化的关系仍需进一步探究。     

Treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is a serious condition associated with sepsis. Clinical management of DIC is hampered by lack of clear diagnostic criteria. The International Society on Thrombosis and Haemostasis (ISTH) has proposed a diagnostic scoring algorithm for overt DIC based on routine laboratory tests. The objective was to assess a modified version of the ISTH scoring system and determine the effect of drotrecogin alfa (activated) (DrotAA, recombinant human activated protein C) on patients with DIC. The large database from the PROWESS clinical trial in severe sepsis was retrospectively used to assess a modified ISTH scoring system. Baseline characteristics and treatment effects of DrotAA were evaluated. At baseline, 29% (454/1568) of patients had overt DIC. Overt DIC was a strong predictor of mortality, independent of APACHE II score and age. Placebo-treated patients with overt DIC had higher mortality than patients without (43 vs. 27%). DrotAA-treated patients with overt DIC had a trend towards greater relative risk reduction in mortality than patients without (29 vs. 18%, P = 0.261) but both groups had greater relative risk reduction than placebo-treated patients. Serious bleeding rates during DrotAA infusion in patients with and without overt DIC were slightly increased (P = 0.498), compared with placebo, while clinically overt thrombotic events during the 28-day period were slightly reduced (P = 0.144). Modified ISTH overt DIC scoring may be useful as an independent assessment for identifying severe sepsis patients at high risk of death with a favorable risk/benefit profile for DrotAA treatment. Patients without overt DIC also received significant treatment benefit.     

SSC/ISTH classification of hemophilia A: can hemophilia center laboratories achieve the new criteria?

Summary.  To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL⁻¹ were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1–29 U dL⁻¹ and 1–33 U dL⁻¹ respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1–5 U dL⁻¹. The median FVIII:C for the third sample was 5.8 U dL⁻¹ with a range of 1.5–36 U dL⁻¹. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL⁻¹; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.     

多指标协同应用在危重症合并血小板减少患者诊断DIC中的作用

目的通过对危重症治疗过程中出现血小板减少的患者进行观察,并进行有关凝血功能的检查,以争取早期发现、诊断弥漫性血管内凝血（DIC）。方法纳入出现血小板减少的危重症患者56例,进行DIC全套检查,并根据国际血栓与出血性疾病协会DIC诊断评分标准诊断DIC26例,根据四格表法分别对凝血酶原时间（PT）、部分活化凝血酶原时间（APTT）、凝血酶时间（TT）、纤维蛋白原总量（Fg）、抗凝血酶Ⅲ（ATⅢ）以及出血（包括皮下出血及淤斑）的敏感性、特异性、准确性、似然比及预测值进行诊断。结果PT延长、Fg降低的诊断特异性为87．0％;ATⅢ降低以及出现PT延长、Fg降低或出血三者之一诊断DIC的敏感性为96．0％。结论以血小板减少为基础的多指标联合诊断DIC可提高敏感性和特异性。     

Incidental venous thromboembolism in oncology patients

Khorana AA, O’Connell C, Agnelli G, Liebman HA, Lee AYY, On Behalf of the Subcommittee on Hemostasis and Malignancy of the SSC of the ISTH. Incidental venous thromboembolism in oncology patients. J Thromb Haemost 2012; 10: 2602–4.     

Definition of major bleeding in clinical investigations of antihemostatic medicinal products in surgical patients

Summary.  The definition of major bleeding varies between studies on surgical patients, particularly regarding the criteria for surgical wound-related bleeding. This diversity contributes to the difficulties in comparing data between trials. The Scientific and Standardization Committee (SSC), through its subcommittee on Control of Anticoagulation, of the International Society on Thrombosis and Haemostasis has previously published a recommendation for a harmonized definition of major bleeding in non-surgical studies. That definition has been adopted by the European Medicines Agency and is currently used in several non-surgical trials. A preliminary proposal for a parallel definition for surgical studies was presented at the 54^th Annual Meeting of the SSC in Vienna, July 2008. Based on those discussions and further consultations with European and North American surgeons with experience from clinical trials a definition has been developed that should be applicable to all agents that interfere with hemostasis. The definition and the text that follows have been reviewed and approved by relevant co-chairs of the subcommittee and by the Executive Committee of the SSC. The intention is to seek approval of this definition from the regulatory authorities to enhance its incorporation into future clinical trial protocols.     

Venous thromboembolism in cancer clinical trials: recommendation for standardized reporting and analysis

Carrier M, Khorana AA, Zwicker JI, Lyman GH, Le Gal G and Lee AYY on behalf of the subcommittee on Haemostasis and Malignancy for the SSC of the ISTH. Venous thromboembolism in cancer clinical trials: recommendation for standardized reporting and analysis. J Thromb Haemost 2012; 10: 2599–601.     

2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: medical indications

The clinical indications for diagnostic flow cytometry studies are an evolving consensus, as the knowledge of antigenic definition of hematolymphoid malignancies and the prognostic significance of antigen expression evolves. Additionally the standard of care is not routinely communicated to practicing clinicians and diagnostic services, especially as may relate to new technologies. Accordingly there is often uncertainty on the part of clinicians, payers of medical services, diagnostic physicians and scientists as to the appropriate use of diagnostic flow cytometry. In an attempt to communicate contemporary diagnostic utility of immunophenotypic flow cytometry in the diagnosis and follow-up of patients with hematolymphoid malignancies, the Clinical Cytometry Society organized a two day meeting of international experts in this area to reach a consensus as to this diagnostic tool. This report summarizes the appropriate use of diagnostic flow cytometry as determined by unanimous approval of these experienced practitioners.     

Treatment effects of high-dose antithrombin without concomitant heparin in patients with severe sepsis with or without disseminated intravascular coagulation

BACKGROUND: Disseminated intravascular coagulation (DIC) is a serious complication of sepsis that is associated with a high mortality. OBJECTIVES: Using the adapted International Society on Thrombosis and Haemostasis (ISTH) diagnostic scoring algorithm for DIC, we evaluated the treatment effects of high-dose antithrombin (AT) in patients with severe sepsis with or without DIC. PATIENTS AND METHODS: From the phase III clinical trial in severe sepsis (KyberSept), 563 patients were identified (placebo, 277; AT, 286) who did not receive concomitant heparin and had sufficient data for DIC determination. RESULTS: At baseline, 40.7% of patients (229 of 563) had DIC. DIC in the placebo-treated patients was associated with an excess risk of mortality (28-day mortality: 40.0% vs. 22.2%, P < 0.01). AT-treated patients with DIC had an absolute reduction in 28-day mortality of 14.6% compared with placebo (P = 0.02) whereas in patients without DIC no effect on 28-day mortality was seen (0.1% reduction in mortality; P = 1.0). Bleeding complications in AT-treated patients with and without DIC were higher compared with placebo (major bleeding rates: 7.0% vs. 5.2% for patients with DIC, P = 0.6; 9.8% vs. 3.1% for patients without DIC, P = 0.02). CONCLUSIONS: High-dose AT without concomitant heparin in septic patients with DIC may result in a significant mortality reduction. The adapted ISTH DIC score may identify patients with severe sepsis who potentially benefit from high-dose AT treatment.     

不同DIC诊断评分标准对严重脓毒症患者病情评价的比较

目的:探讨不同DIC评分标准判断严重脓毒症和脓毒症休克的患者DIC和MODS发生和预后的价值。方法:收集246例严重脓毒症和脓毒症休克患者入院第1、3、7天的各项生理参数和实验室指标,分别使用ISTH显性DIC评分标准、非显性DIC评分标准和JAAMDIC评分标准进行诊断评分,同时进行SOFA评分,观察不同DIC评分标准之间确诊率、诊断时间点的差异;比较DIC确诊组间的病死率、SOFA分值的差异,绘制三种DIC评分标准的ROC曲线,计算曲线下面积,衡量各个评分系统对危重患者的病情严重程度和预后的判断准确性。结果:三种评分标准均能在一定程度上准确反映危重病患者多脏器功能衰竭的发生和预后,使用ISTH显性标准确诊的患者病死率和SOFA分值最高,诊断时间点同时或晚于ISTH非显性标准和JAAM标准;在DIC确诊组间的病死率和SOFA分值的比较上,JAAM标准与ISTH显性标准之间差异无统计学意义(P>0.05),ISTH非显性标准与ISTH显性标准之间差异有统计学意义,JAAM标准和ISTH非显性标准之间,病死率差异无统计学意义、SOFA分值差异有统计学意义;3个评分标准的ROC曲线下面积分别为0.739、0.724和0.778,相互之间比较差异均有统计学意义。结论:ISTH显性标准诊断DIC特异性最高,敏感性差;ISTH非显性标准诊断DIC敏感性最高,特异性差;JAAM标准诊断DIC敏感性和特异性均较高,对危重病患者的MODS发生和预后判断更为准确,可作为早期干预治疗的首选诊断标准。     

Bayesian analysis of the epidemiology of bleeding in critically ill children

PurposeWe updated our findings on the epidemiology of clinically relevant bleeding (CRB) in critically ill children. We also determined the concordance of CRB as defined by the International Society of Thrombosis and Haemostasis, i.e., ISTH definition, and characteristics identified by pediatric intensivists in a recent survey, i.e., survey definition.MethodsIn a prospective cohort study, we included children <18 years old who were admitted to the pediatric intensive care unit for >1 day. We followed them daily for bleeding. Bayesian inference was used as the primary analytic tool to incorporate our prior findings.ResultsUsing the ISTH definition, the estimated frequency of CRB was 10.0% (95% credible interval, CrI: 7.6%, 12.8%) from 41 of 405 children who had CRB. The estimated frequency from 4 of 12 adolescents >13 years old who received mechanical ventilation or vasopressor support and had CRB was 32.9% (95% CrI: 12.0%, 58.8%). Using the survey definition, the estimated frequency of CRB for the entire cohort was 10.8% (95% CrI: 8.3%, 13.8%). Concordance between definitions for each bleeding event was 0.40 (95% confidence interval: 0.27, 0.52).ConclusionsOur updated findings highlight the high frequency of CRB regardless of definition used for CRB.     

Characteristics and outcomes of reversed patients admitted to an emergency department for VKA-related intramuscular hematoma

Background

According to the International Society on Thrombosis and Haemostasis (ISTH), intramuscular hematoma without other severity criteria is not considered a major bleeding. Objectives: In a large cohort of reversed vitamin K antagonist (VKA) patients admitted to the emergency unit for muscular hematoma, we assess frequency, severity, and anticoagulation management based on whether ISTH criteria were met or not.

Modified score for disseminated intravascular coagulation in the critically ill

Objective To assess the value of the diagnosis of overt disseminated intravascular coagulation (DIC) according to the International Society on Thrombosis and Haemostasis (ISTH) criteria and that of the parameters included in the ISTH score for overt DIC in predicting day 28 mortality in intensive care patients. Also, to assess the value of the components of the score in the diagnosis of overt DIC.Design and setting Retrospective clinical study in a university hospital intensive care unit.Patients and participants 494 consecutive patients admitted in the ICU between January 2002 and October 2003.Measurements and results Clinical and laboratory data, including hemostatic parameters, were collected from computerized databases and patient files. Altogether 19% (95/494) of the patients fulfilled the criteria for overt DIC. Their day 28 mortality rate was higher than that of patients without overt DIC (40% vs. 16%). The lowest platelet count (area under curve, AUC, 0.910), highest plasma D-dimer (AUC 0.846), lowest antithrombin (AUC 0.823), and Owren-type prothrombin time activity (AUC 0.797) discriminated well the patients with and without overt DIC, whereas plasma fibrinogen (AUC 0.690) had poor discriminative power. No patient with the diagnosis of overt DIC had decreased plasma fibrinogen. Day-1 SOFA and APACHE II score, the first CRP measurement, and the lowest antithrombin were independent predictors of day 28 mortality.Conclusions The diagnosis of overt DIC was not an independent predictor of day 28 mortality. In ICU patients plasma antithrombin seems a promising candidate in the panel of indicators for overt DIC whereas the value of plasma fibrinogen is in doubt.